IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF
SIDEROPHORES PRODUCED BY HALOPHILIC
AND ALKALIPHILIC BACTERIA
By
Abigail Marie Richards
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
Department of Chemical Engineering
AUGUST 2007 To the Faculty of Washington State University
The members of the Committee appointed to examine the dissertation of ABIGAIL
MARIE RICHARDS find it satisfactory and recommend that it be accepted.
______Chair
______
______
ii ACKNOWLEDGEMENT
I would like to especially thank my committee members Dr. Brent Peyton, Dr. William
Apel, Dr. James Petersen, and Dr. David Yonge and especially Dr. Richard Zollars who was willing to fill in at the last minute. It was through their encouragement that I decided to embark on my Ph. D. and I would like to commend them for their efforts in providing such an excellent, well balanced education. They have each been excellent mentors to me and I would like to thank them for all of their advice and input. I would also like to thank the Chemical Engineering
Department at WSU for all of the support throughout both my undergraduate and graduate education. Special thanks to Jo Ann McCabe for helping me to remotely order supplies while I was at the CBE.
I was able to perform the work for this project at a number of locations and would like to thank my gracious hosts at each of those sites: Special thanks to Dr. Antonio Ventosa for allowing me to work in his laboratory in Sevilla, Spain and to all of his students who were fantastic hosts and gave me my first taste of molecular biology. Thank you to those at the INL, in particular, Dr. William Apel, Dr. Vicki Thompson, Dr. Gary Groenewold and Dr. Garold
Gresham for generously providing me with time on their mass spec. instrumentation at the Idaho
National Laboratory throughout my Ph. D. work and thoughtful discussions about my results. I’d like to thank Dr. Anne Camper, my host at the Center for Biofilm Engineering and all of the people in her lab and throughout the CBE who made my experience at Montana State University so enjoyable including Mark Shirtliff, Mark Burr, Stewart Clark, Ben Klayman, Jennifer
Faulwetter and Erin Field. Everyone at the CBE there immediately made me feel at home and I
iii feel privileged to have had this experience. Dr. Robin Gerlach was infinitely helpful with the identification of all of my siderophores by allowing me to use his LC-MS system and the time that he spent helping me developing my LC-MS methods. John Newman, also at the Center for
Biofilm Engineering, was also instrumental in methods development, particularly with HPLC.
I’d like to thank my family for their support throughout all of my schooling, and my husband Lee for helping with the editing of this document and encouragement.
This work was supported almost entirely by the Inland Northwest Research Alliance through a three year research grant as well as a two year individual fellowship which provided my support for the past two years. Through the INRA program I was able to continue my interdisciplinary education and this work could not have been accomplished without their generous financial support. The LC-MS instrument used for siderophore identification was provided by the Defense University Research Instrumentation Program (DURIP) Contract
Number: W911NF0510255.
iv IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF
SIDEROPHORES PRODUCED BY HALOPHILIC
AND ALKALIPHILIC BACTERIA
Abstract
By Abigail Marie Richards, Ph. D.
Washington State University
August 2007
Chair: Brent M. Peyton
The first two chapters of the present dissertation focus on a description of two main topics. The first addresses siderophore production by plants and microbes as a means of acquiring ferric iron. Also described is the ability of siderophores to coordinate metals other than ferric iron, such as heavy metals and radionuclides, which potentially alters their speciation and mobility. The second chapter give an overview of the biology of halophilic and alkaliphilic microorganisms.
The third part of this dissertation involves the identification and characterization of siderophores produced by the halophilic and alkaliphilic bacterium Halomonas campisalis.
Several desferrioxamine siderophores including desferrioxamines G1, G1t, X3, X7, D2, and E were isolated from low-iron, culture supernatant and structurally characterized by ESI-MS and ESI-
MS/MS. This work represents the first documentation of ferrioxamine production by a halo- alkaliphilic bacterium.
v The fourth part of this dissertation is an assessment of siderophore production in a naturally saline and alkaline environment, the soda lake Soap Lake, located in eastern
Washington State, USA. Eight siderophore producing halo-alkaliphiles were isolated from Soap
Lake. Of these isolates, several were found to belong to the genus Halomonas. The isolate SL28, most closely related to Halomonas pantelleriense, was found to produce a new family of six of amphiphilic siderophores, named the sodachelins. The sodachelin siderophores are of particular interest because, when exposed to UV light, they facilitate a photolytic reduction of Fe(III) to
Fe(II) along with a cleavage of the ligand located at the b-hydroxyaspartate residue. To my knowledge, this is the first characterization of amphiphilic siderophores produced by a bacterium from a soda lake environment that is capable of reducing Fe(III).
The final portion of this dissertation contains suggestions for future work. Much of this work focuses on the identification of the siderophores produced by other halophilic and alkaliphilic isolates obtained in an earlier portion of this work. Siderophore production in halo- alkaliphiles (and extremophiles in general) is poorly characterized and some of the isolates appear to produce siderophores that may constitute new compounds.
vi TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS…………………………………………………………………… iii
ABSTRACT………………………………………………………………………………….... iv
LIST OF TABLES…………………………………………………………………………….. xii
LIST OF FIGURES…………………………………………………………………………… xiii
CHAPTER ONE: A BRIEF OVERVIEW OF MICROBIAL IRON
TRANSPORT AND SIDEROPHORE PRODUCTION………………...……………………. 1
1.0 Introduction…………...………………………………………………………………….... 1
1.1 Microbial siderophore production ………………………………………………………… 4
1.2 Siderophore based iron acquisition………………………………………………………... 5
1.3 Iron, siderophores and disease causing microorganisms………………………………….. 8
1.4 General siderophore structural traits……………………………………………………... 10
1.5 Siderophore production by marine microorganisms………………………………………. 13
1.6 Siderophore affinity for divalent heavy metal cations and radionuclides…………………. 14
1.7 Concluding remarks………………………………………………………………………... 15
1.8 References………………………………………………………………………………….. 15
CHAPTER TWO: HALOPHILIC AND ALKALIPHILIC
MICROORGANISMS………………………………………………………………………… 30
2.0 Extremophiles……………………………………………………………………………… 30
2.1.0 Halophiles……………………………………………………………………………….. 30
vii 2.1.1 Mechanisms of halotolerance……………………………………………………...... 32
2.1.2 Saline and hypersaline environments……………………………………………………. 32
2.2.0 Alkaliphiles………………………………………………………………………………. 34
2.2.1 Specific mechanisms of alkaline tolerance………………………………………………. 34
2.3.0 Soda lakes.……………………………………………………………………………….. 35
2.3.1 Soap Lake………………………………………………………………………………... 36
2.5 Concluding remarks………………………………………………………………………... 38
2.4 References………………………………………………………………………………….. 39
CHAPTER THREE: IDENTIFICATION AND CHARACTERIZATION
OF A SUITE OF NATURAL FERRIOXAMINE SIDEROPHORES
PRODUCED BY A HALO-ALKALIPHILC BACTERIUM…………………………………. 42
3.0 ABSTRACT………………………………………………………………………………... 43
3.1 INTRODUCTION………………………………………………………………………….. 44
3.2 MATERIALS AND METHODS…………………………………………………………….50
3.2.1 Growth conditions………………………………………………………………………… 50
3.2.2 Siderophore detection………………………………………………………………...... 50
3.2.3 Siderophore isolation……………………………………………………………………. 51
3.2.4 Siderophore characterization…………………………………………………………….. 52
3.3 RESULTS………………………………………………………………………………….. 52
3.4 DISCUSSION……………………………………………………………………………… 53
3.5 CONCLUSION……………………………………………………………………………. 58
3.6 ACKNOWLEDGEMENTS……………………………………………………………….. 58
3.7 LITERATURE CITED……………………………………………………………………. 59
viii CHAPTER FOUR: NOVEL AMPHIPHILIC SIDEROPHORES PRODUCED
BY A BACTERIUM ISOLATED FROM A SODA LAKE………………………………….. 79
4.0 ABSTRACT………………………………………………………………………………… 80
4.1 INTRODUCTION………………………………………………………………………….. 81
4.2 MATERIALS AND METHODS………………………………………………………….. 85
4.2.1 Sample collection…………………………………………………………………………. 85
4.2.2 16S rRNA sequencing…………………………………………………………………..... 86
4.2.3 Growth medium……………………………………………………………………………86
4.2.3.1 Initial enrichment and growth medium…………………………………………………. 86
4.2.3.2 Growth medium for Halomonas strains………………………………………………… 87
4.2.3.3 Iron removal from complex media components………………………………………... 88
4.2.3.4 Iron limit medium for Halomonas strain SL28…………………………………………. 88
4.2.4 Siderophore detection and characterization………………………………………………. 88
4.2.5 Siderophore isolation………………………………………………………………………89
4.2.6 Structure determination…………………………………………………………………… 90
4.2.7 Photochemical experiments………………………………………………………………. 90
4.2.8 Fatty acid analysis………………………………………………………………………… 92
4.3 RESULTS……………………………………………………………………………………92
4.3.1 Isolate identification………………………………………………………………………. 92
4.3.2 Siderophore isolation………………………………………………………………………93
4.3.3 Structure determination…………………………………………………………………… 93
4.3.4 Photochemical experiments………………………………………………………………. 95
ix 4.4 DISCUSSION………………………………………………………………………………..96
4.4.1 Siderophores from saline and alkaline environments…………………………………….. 96
4.4.2 Iron cycling in aquatic environments…………………………………………………….. 97
4.4.3 Siderophore mediated iron cycling……………………………………………………….. 98
4.4.4 Amphiphilicity in siderophores…………………………………………………………...101
4.5 CONCLUSIONS…………………………………………………………………………. 102
4.6 ACKNOWLEDGEMENTS………………………………………………………………. 103
4.7 References……………………………………...…………………………………………. 103
CHAPTER FIVE: SUGGESTIONS FOR FUTURE WORK..……………………………...... 124
APPENDIXES
A. Growth and production of siderophores with respect to pH for Halomonas campisalis…...127
B. Additional HPLC chromatograms and mass spectra of ferrioxamine siderophores isolated from Halomonas campisalis…………………………………………………………………… 134
C. Table of masses, structural information and fragmentation patterns for ferrioxamine siderophores…………………………………………………………………………………….143
D. 16S rDNA sequences and closest BLAST search matches for isolates from Soap Lake….. 145
E. Siderophore production with respect to growth for Soap Lake isolates
SL01; SL11 and SL28…………………………………………………………………………..156
F. MALDI-TOF MS/MS data for sodachelin siderophres……………………………………..162
G. Exact mass data for sodachelin siderophores……………………………………………….169
H. Fatty acid analysis results for sodachelin siderophores…………………………………… 180
I. Preliminary mass spectral data for SL01 siderophores…………………………………….. 193
x J. UV-Visible spectral data for Sodachelin E and photolytic reduction of Fe(III)……………. 198
xi LIST OF TABLES
Table 3.1 Fragmentation details of unidentified “ferrioxamine-like” compounds isolated from
low-iron culture supernatant of H. campisalis………………………………………… 78
Table 4.1 Closest match of BLAST search on a segment of the 16S rRNA gene for siderophore
producing isolates in Soap Lake………………………………………………………. 110
Table 4.2 Mass data for siderophores produced by Halomonas sp. SL28 in the desferri and ferri
form……………………………………………………………………………………. 111
Table 4.3 Y fragment m/z values observed by ESI-MS/MS spectrometry of the
sodachelins…………………………………………………………………………….. 112
Table 4.4 B fragment m/z values observed by ESI-MS/MS spectrometry of the
sodachelins……………………………………………………………………………. 113
xii LIST OF FIGURES
Figure 3.1 Examples of siderophores: (a) the hydroxamate siderophore desferrioxamine E; (b)
the catecholate siderophore, enterobactin; and the carboxylate siderophores (c) aerobactin
and (d) rhizoferrin………………………………………………………………………. 69
Figure 3.2 Siderophore production by H. campisalis with respect to cell growth at pH 10 and
10% NaCl……………………………………………………………………………….. 70
Figure 3.3 HPLC Chromatogram of desferrioxamine siderophore produced by H. campisalis
grown at pH 10 and 10% NaCl…………………………………………………………. 71
Figure 3.4 Mass spectral data for m/z = 619.5………………………………………………….. 72
Figure 3.5 Mass spectral data for m/z = 573.4…………………………………...……..………. 73
Figure 3.6 Mass spectral data for m/z = 587.4…………………..……………………………… 74
Figure 3.7 Mass spectral data for m/z = 601.4……………………………………….…………. 75
Figure 3.8 Mass spectral data for m/z = 615.4………………………………………………….. 76
Figure 3.9 Mass spectral data for m/z = 519.5………………………………………………….. 77
Figure 4.1 Siderophores representing hydroxamate, catecholate and a-hydroxy carboylic acid
based structures: (a) desferrioxamine E, (b) enterobactin, (c) aerobactin, (d)
rhizoferrin………..……………………………………………………………………..114
Figure 4.2 Amphiphilic siderophores isolated from marine environments: (a) marinobactins, (b)
aquachelins, and (c) amphibactins…………………………………………………….. 115
Figure 4.3 Siderophore production by Halomonas sp. strain SL28 with respect to time….….. 116
Figure 4.4 HPLC/UV chromatogram of sodachelin siderophores eluted from a C8 column. (a)
Shows the elution of siderophores in the desferri form while (b) shows the earlier
retention time of siderophores as the elute in the ferri form……………………………117
xiii Figure 4.5 ESI-MS/MS fragmentation spectrum of sodachelin F……………………………..118
Figure 4.6 The assignment of y and b fragments as determined by MS/MS data for sodachelin F.
The y fragments are conserved for each sodachelin siderophore while the b fragments
differ depending on the nature of each fatty acid tail. Fragments corresponding to the
fatty acid appendages were seen in very low abundance while those corresponding to the
peptidic headgroup were not observed…………………………………………………119
Figure 4.7 UV-Vis spectra of Fe(III)-sodachelin E prior to and following UV exposure..…….120
Figure 4.8 MS spectrum of sodachelin E (a) prior to and (b) after UV exposure…………….. 121
Figure 4.9 Schematic of the potential photolytic reaction pathways of Fe(III)-sodachelin
complexes and reduction of Fe(III) to Fe(II)…………………………………………...122
Figure 4.10 Production of Fe(II) during the siderophore mediated photochemical reduction of
Fe(III) in the Fe(III)-sodachelin F complex…………………………………………….123
xiv CHAPTER ONE
A BRIEF OVERVIEW OF MICROBIAL IRON TRANSPORT
AND SIDEROPHORE PRODUCTION.
1.0 Introduction
Iron is the fourth most abundant element of the earth’s crust and amongst metals, it is
second only to aluminum. While iron is widespread in the environment, it is often considered
biologically unavailable as it is often only found in the form of highly insoluble Fe(III)
(oxyhydr)oxides. Under anaerobic conditions, Fe(II) is soluble, readily available and may be
taken up by anaerobic bacteria without the help of iron chelators. Fe(III) has a solubility of 10-8
M at pH 3, and as such, acid-tolerant bacteria may find close to enough iron to satisfy their
nutritional requirements. Under aerobic conditions, Fe(II) is readily soluble and solutions of up
to 100 mM can be obtained at physiological pH (Neilands, 1991), but it is quickly oxidized to
Fe(III) and forms a complex of precipitated Fe(III) minerals, such as amorphous ferrihydrite,
-38 goethite and hematite. The solubility product of Fe(OH)3 is approximately 10 so by
calculation, the concentration of Fe3+ at neutral, aerobic conditions is 10-17 – 10-18 M in the absence of any external Fe(III) chelators. Most microbial life requires between 10-8 to 10-6 M for optimal growth, such that, without chelators, most microbes inhabiting aerobic, neutral or alkaline environments would live in a state of permanent iron deficiency.
For most forms of life, including those in the microbial realm, iron is a versatile and necessarily nutrient. Iron is a component of electron transport proteins such as cytochromes, ferredoxines and iron-sulfur proteins. Iron plays a vital role in oxygen transport in both
1 hemoglobin and myoglobin in which oxygen is bound to the Fe(II)-heme. Other enzymes which
use iron at the active site include peroxidases, catalases and are heme containing proteins
(Lippard and Berg, 1994). Non-heme containing proteins include ribonecleotide reductase and
methane monooxygenase which contain oxygen bridged di-iron centers at the active sites. Iron
is also key in nitrogen fixation in which the nitrogenase enzyme utilizes iron alone, or
molybdenum or vanadium together with iron to reduce atmospheric nitrogen to ammonia.
Nearly all living organisms utilize iron in some capacity with the exception of the Lactobacilli
and Borellia bergdorferii, the lyme disease pathogen (Archibald, 1983; Posey et al. 2000).
It is thought that prior to the introduction of O2 into the Earth’s atmosphere, which began approximately 3 x109 years ago, iron was abundant. Because of this, many organisms evolved utilizing iron in various biological functions (Beinert, et al. 1997). Iron can adopt two readily convertible redox states Fe(III) and Fe(II) and because of its readily accessible reduction potential of (Fe(III)/Fe(II) = 0.770 V) iron can be adapted by the enzyme environment to encompass a wide range of reduction potentials. Insertion of iron into specific proteins can allow for the control of the reduction potential which ranges from +300 mV in some compounds to -
490 in certain iron sulfur proteins (Payne, 1988; Andrews et al., 2003). Both Fe(II) and Fe(III) form six-coordinate octahedral complexes with either O, N, or S as donating electrons. In coordination configurations involving only oxygen around the iron, the reduction potential will be low, while schemes with only nitrogen in the coordination sphere will result in a high reduction potential. Thus, all-oxygen ligands will have a greater propensity to bind Fe(III) while all-nitrogen ligands will favor complexes with Fe(II).
2 The slow but steady introduction of oxygen into the earth’s atmosphere by photosynthetic organisms gradually decreased the availability of iron. The predominant form of iron in the aerobic environment, ferric iron, is extremely insoluble at 10-18 M at pH 7. Furthermore, iron can be extremely toxic under aerobic conditions due to its involvement in harmful Fenton type reactions (Touati, 2000), leaving the bacteria in an environment in which a vital nutrient was becoming essentially insoluble and potentially toxic. Even though iron was becoming increasingly scarce, the dependence on iron metallo-enzymes was so significant and entrenched, that no viable substitute was selected by bacteria as evidenced by its inclusion in many vital enzymes. During the transition to an aerobic environment, microorganisms developed highly sophisticated strategies to obtain iron from their surroundings and manage it within their cells.
These strategies include 1) the reduction of iron; 2) use of iron chelating agents to solubilize iron and active transport of the Fe(III)-chelator complex; 3) the acquisition of iron from host iron sources such as transferrin, lactoferrin and heme.
Because iron is a necessary but often toxic nutrient for almost all forms of bacterial life, bacteria have adopted a series of controls to obtain and manage this vital nutrient. The first control is a high-affinity transport system that can successfully scavenge iron in various forms from the surrounding environment. To control excess iron within the cell, iron is often deposited within the cells in the form of intercellular iron stores, such as bacterioferritin, to provide a source of iron that can be utilized when iron is scarce (Yariv et al.,1981; Andrews et al., 1991;
Harrison et al., 1991). To combat redox stresses induced by ferric iron in the aerobic environment, bacteria have adopted specific redox stress resistance systems such as the
3 degradation of iron-induced reactive oxygen species (Lushchak, 2002). Iron consumption is tightly controlled by the down-regulation of iron-containing proteins when iron limiting conditions exist. Finally, bacteria have developed an interconnected system which coordinates the above-mentioned controls for iron uptake and regulation according to the availability of iron
(Andrews et al., 2003).
1.1 Microbial siderophore production
One of the most common strategies for iron sequestration in an aerobic environment is through the synthesis and excretion of low molecular weight chelators, with a very high and
30 specific affinity for Fe(III), typically greater than Ksp = 10 , known as siderophores. These siderophores are able to solubilize iron prior to transport into the cell (Winkelmann, 2001). The term siderophores is derived from the Greek which means simply, “iron carriers.” Over 500 different siderophores have been identified and are produced by various organisms ranging from microbes to plants. Although most siderophores are excreted into the extracellular environment, some remain within the cell envelope, such as the mycobactins, synthesized by the mycobacteria and the amphibactins synthesized by Vibrio sp. R-10 (Martinez et al., 2003; De Voss et al., 1999;
Ratledge and Dover, 2000). Most siderophores are approximately 600 Da in size, but have been observed as small as 200 Da in the case of PDTC and as large as 2000 Da (Budzikiewicz, 2003;
Budzikiewicz, 2005; Scott, 2003). Common precursors for siderophore biosynthesis include citrate, amino acids, dihydroxybenzoate and N5 -acyl-N5 -hydroxyornithine (Winkelmann, 2002).
Siderophores can extract iron from insoluble hydroxides or iron bound to surfaces. It can also facilitate extraction from numerous compounds such as ferric-citrate and ferric phosphate, as
4 well as iron bound to other biological materials such as transferrin and plant flavone pigments
(Winkelmann, 2002).
While iron is biologically necessary to many organisms it is also quite toxic in excessive
quantities. Because of this propensity for inducing cell damage, free iron is tightly regulated in
biological systems by coordination with transfer proteins like lactoferrin and transferrin. Any
excess iron is stored in ferritin (Crichton, 1982). In some bacterial strains, excess iron is stored
in bacterioferritin which is related to ferritin, but contains heme (Yariv et al., 1981). The
Lactobacilli contain only a few atoms of iron per cell (Archibald, 1983) and have evolved to live
in highly iron restricted environments such as dairy products which contain high levels of
lactoferrin and glycoprotein which tightly complex iron. These organisms can tolerate high
H2O2 environments and acidic environments. In such an environment, bacteria that contain a
great deal of iron would experience harmful Fenton type reactions. Instead the Lactobacilli
utilize the vitamine B12 which is a cobalt containing reductase for the generation of deoxynucleotide precursors for DNA synthesis (Archibald, 1983).
1.2 Siderophore based iron acquisition.
In many bacteria, iron concentrations are approximately 1.8% dry weight and in E. coli
they have been estimated between 105 – 106 atoms per cell depending on the growth conditions
(Abdul-Tehrani et al. 1999; Rouf, 1964). To accumulate appropriate levels of iron, many bacteria synthesize and secrete siderophores to solubilize iron. These complexes are then taken up via outer membrane receptors for Gram negative bacteria, which have very high affinity for
5 their corresponding Fe(III)-siderophore complexes (Braun et al., Stintzi et al., 2000). These
outer membrane receptors are used because the Fe(III) siderophore complex is too large to
diffuse into the cells through the porins. The siderophore specific outer membrane receptors are
generally only induced under iron starved conditions and are typically not present if iron is
sufficient. In Gram positive bacteria, these receptor proteins are anchored in the cytoplasmic
membrane because Gram positive bacteria lack an outer membrane.
Quite often, bacteria will possess more than one type of outer membrane receptor,
typically producing three to nine outer membrane protein receptors for Fe(III)-siderophores
under conditions of iron stress (Guerinot, 1994). These receptors may recognize exogenous
siderophores, such as E. coli K-12, which produces at least six known outer membrane receptors with specificity for multiple siderophores including coprogen, rhodotorulic acid, ferrioxamine B and D1, ferrichrome, dicitrate, enterobactin, dihydroxybenzoic acid and dihydroxybenzoyl serine.
Of these siderophores, only enterobactin and its breakdown products, dihydroxybenzoic acid, and dihydroxybenzoyl serine, are actually produced by E. coli. An even more extreme case is
Pseudomonas aeruginosa, which is thought to contain up to 34 outer membrane siderophore receptors based on genomic analysis while it produces only a few of its own (Stover, 2000;
Koster, 2001). The ability of bacteria to utilize the siderophores of their neighbors is likely quite common as it permits cooperation within a microbial community for the purpose of scavenging iron. Also, the ability to utilize the siderophores of other neighboring bacteria prevents any inhibition of growth due to complexation by an unrecognizable ligand (Andrews et al., 2003).
6 Internally, the process of taking up iron by E. coli is driven by the cytosolic membrane potential and mediated by the TonB-ExbB-ExbD complex system (Larsen et al., 1994; Higgs et al., 1998). The TonB system is thought to span the periplasmic space, enabling contact with
TonB-dependent receptors in the outer membrane (Higgs et al., 2002). It is thought that ExbB and ExbD use the membrane electrochemical charge gradient to produce an energized form of
TonB that mediates a conformational change in the contacted outer membrane receptors. The conformational change, in turn, leads to the translocation of the Fe(III) to the periplasm
(Reynolds et al., 1980; Wooldridge et al., 1992). The transport of the Fe(III)-siderophore complexes across the periplasmic space and cystoplasmic membrane is mediated by periplasmic binding proteins and associated cytoplasmic membrane transporters (Clarke, et al., 2002; Clarke et al., 2000; Koster et al., 2001; Bruns et al., 1997). The binding protein collects the Fe(III)- siderophore complex as it is released from the outer membrane receptors and shuttles it to the appropriate permease located on the inner membrane. In E. coli, the shuttling protein, FhuD can recognize hydroxamate siderophores by interacting with the iron-hydroxamate center. Since the hydroxamate backbone does not directly interact with FhuD, it is able to recognize different types of hydroxamate siderophores (Koster, 2001).
ATP-binding cassette (ABC) transporters utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes. ABC-systems also facilitate the transport of the siderophores across the cytoplasmic membrane and into the cytostol (Koster, 1991;
Mietzner et al., 1998; Boos, 1996). While E. coli contains six outer membrane siderophore transporters, it contains only three associated binding-protein-dependent ATP-binding cassette
(ABC) systems suggesting that outer membrane receptors play a much larger role in the
7 specificity of Fe(III)-siderophore acquisition than do the interior iron transport mechanisms of the cell. Again, in P. aeruginosa, this is even more extreme as the genome sequence suggested the presence of up to 34 different TonB-dependent outer membrane receptors for Fe(III)- siderophores and only four potentially associated ABC transporters (Stover, 2000; Koster, 2001).
In P. aeruginosa, when more than one siderophore is present, the system of outermembrane receptors is up-regulated such that one that is most successful in delivering iron to the cell will be expressed (Dean and Poole, 1993). Generally, the hierarchy of the preferred iron transport system reflects the strength and stability of the siderophore iron complex (Guerinot, 1994).
ATP-binding cassette transporters finally deliver the Fe(III)-siderophores to the cytostol where the iron removal may be facilitated by reduction of the ferric iron. The release of the Fe(III) from the siderophores once within the cytoplasmic membrane is an energy intensive process (Braun and Killmann, 1999). After release within the cell, iron is either incorporated into ferri-proteins or stored for future use.
1.3 Iron, siderophores and disease causing microorganisms
The mammalian body is an environment in which iron is tightly regulated and unavailable to invading microorganisms. Iron availability is critical to the virulence of many pathogenic bacteria (Expert et al. 1996; Genco and Desai, 1996; Mietzer et al., 1998; Vasil et al. 1999). If the invading microbes are unable to obtain iron within the host system, the bacteria are not able to multiply. Iron is tightly regulated in the mammalian host system; nearly 99.99% of the iron present is held intracellularly in ferritin (an iron storage protein) or present in heme, while the remaining iron is tightly bound to iron binding proteins transferrin, the iron carrier in the blood,
8 lactoferrin which complexes iron in secretory fluids. This reduces the concentration of free
extracellular iron in mammals to around 10-18 M (Bullen et al., 1978).
The detection of low levels of environmental iron by pathogens often trigger the
induction of virulence genes (Litwin et al., 1993; Payne, 1988; Payne, 1993) In some situations,
bacteria have developed mechanisms in which they are able to utilize the iron found in host
ferritin, hemoglobin or free heme directly. Disease causing bacteria such as Serratia marcescens
and E. coli 0157 use heme; Neisseria gonorrhoeae and Haemophilus influenzae use hemoglobin and also possess lactoferrin and transferrin receptors which allow them to utilize iron from those sources as well (Gray-Owen et al., 1996; Genco and Desai, 1996). In other cases, the pathogenic bacteria are able to reduce the Fe(II) contained in transferrin and take it up in the form of Fe(II) (Otto et al., 1992). Some pathogens produce siderophores which are capable of competing with the host’s iron chelating compounds. One commonly produced siderophore is enterrobactin, so named because of its production by enteric bacteria. This siderophore is a tri- catecholate siderophore with an iron stability constant of 1052 which allows it to compete with lactoferrin, transferrin and heme for iron. Siderophores have also been detected in the sputum of cystic fibrosis patients (Hass et al., 1991). In P. aeruginosa biofilm development, it has been recently found that when iron is limited by extracellular compounds such as the host defense system (lactoferrin etc.) that biofilm development is limited. The iron difficiency results in a twitching motility of the colonizing bacterium upon the potential attachment sites and the development of a mature biofilm is prevented (Singh et al., 2002; Banin et al., 2005).
Overcoming iron deficiency may be an important first step in biofilm formation and colonization by pathogens such as P. aeruginosa.
9 1.4 General siderophore structural traits
The selectivity of siderophores for iron depends upon the optimal selection of number and type of metal binding groups in addition to the steriochemical arrangement. To date, siderophores have incorporated hydroxamate, catecholate and or a-hydroxycarboxilic acid binding subunits arranged in various configurations including linear, tripodal, endocyclic and exocyclic, and these ligand types comprise the most efficient iron-binding ligands in nature
(Winkelmann, 2002). The number of iron binding functional groups, or denticity, is an important component of the siderophore architecture. The overwhelming majority of siderophores are hexadentate, which optimally satisfies the six coordination sites available on
Fe(III), however, tetradentate and even bidentate siderophores have been identified (Boukhalfa and Crumbliss, 2002). The actual organization or architecture of the iron binding moieties will affect complex stability. Cyclic structures such as ferrioxamine E and alcaligin both show a higher Fe(III) affinity than their linear analogues ferrioxamine B and Rhodotorulic Acid
(Anderegg et al. 1960; Spasojevic et al., 1999; Bickel et al., 1960; Carron et al., 1979; Hou et al.,
Cooper et al., 1978). It is thought that the increased stability constants of cyclic siderophores are due in part to a preorganization of the molecule in a form which easily binds iron. In terms of concentration, hexadentate ligands are more favored than tetradentate ligands (Albrecht-Gary and Crumbliss, 1998). However, from the standpoint of energy expenditure, it is possible that molecules of lower denticity are more efficient to produce because they are generally smaller molecules with lower complexity.
10 Many of the hexadentate siderophores are based on hydroxamate and/or catecholate binding
subunits and have a very high affinity for Fe(III) in part because they completely satisfy Fe(III)’s
six coordination sites in a single molecule. In general, hexadentate siderophores have a much
lower affinity for Fe(II). The hexadentate hydroxamate siderophores desferrioxamine B and
desferrioxamine E have high stability constants with Fe(III) of 10 30.6 and 10 32.5, respectively, but only complex Fe(II) with stability product constants of 20 orders of magnitude less (10 10.0 and 10 12.1 for desferrioxamine B and E, respectively) (Spasojovec et al., 1999). Tetradentate siderophores, such as rhodotorulic acid, on the other hand, cannot achieve full Fe(III) coordination saturation with a single molecule but assemble two or three molecules of the ligand to a single Fe(III) atom. This yields complex species of various stoichiometry depending on the
+ ligand configuration, pH and metal to ligand ratio (Fe2L3, FeL(LH), Fe(LH3), Fe(OH)2 ,
2+ Fe2L2(OH2)4 ) (Spasojevic et al. 2001). Alcaligin, on the other hand, is preorganized to form monomeric complexes (Hou et al., 1996; Hou et al. 1998)
Several classes of siderophores have been identified including catecholate-type siderophores, hydroxamates and citric acid based siderophores. The majority of siderophores may be divided into three main structural classes depending on their functional groups. Hydroxamate siderophores include examples such as ferrioxamines, ferrichromes and coprogens. Siderophores containing catecholate iron coordinating groups include the enterobactins, vibriobactins and yersiniabactin, while carboxylate and mixed ligand a-hydroxamates include pyoverdines, azotobactins and ferribactins. Catecholate siderophores were originally thought to be characteristic of bacteria whereas hydroxamates were thought to be prevalent only in fungi, but
11 with the discovery of many hydroxamate producing bacteria, this criterion is obsolete. Bacteria
have been found to not only produce hydroxamate siderophores, but also oxazoline nitrogen, a-
hydroxycarboxylates, and even hydrazine.
One prominent and well studied class of hydroxamate siderophores is the ferrioxamines,
which are a group of natural, iron-chelating siderophores. The ferrioxamines were first found to
be secreted in the desferri form under iron limiting conditions by Gram-positive Streptomyces
and Nocardia species (Bickel et al., 1960; Keller-Schierlein and Prelog, 1961; Keller-Schierlein and Prelog, 1962; Keller-Schierlein et al., 1965), but have since been identified in several other genera including Gram-negative Pseudomonads, Arthrobacter, Chromobacterium, Erwinia herbicola and amylovora, and a marine Vibrio (Muller and Zahner, 1968; Berner et al., 1988;
Feistner et al., 1993; Martinez et al., 2001; Feistner and Ishimaru, 1996; Zawadzka et al.. 2006).
Many ferrioxamines have been identified and characterized to date, including ferrioxamine A, B,
C, D1, D2, E, F, G1, G2a-c, H, I, T1-8 and X1-7 (Winkelmann, 1991; Fiestner et al., 1993). A characteristic feature of the ferrioxamines is a repeating motif of an a-amine-w-hydroxyamino alkane with succinate or acetate. These siderophores are either linear or cyclic, and generally fall within a size of about 500-600 Da. With the exception of the dihydroxamic acids, such as desferrioxamine H, alcaligin and bisucaberin, the ferrioxmaines are hexidentate ligands that contain three hydroxamate groups that facilitate the chelation of ferric iron. The best studied of the ferrioxamine siderophores, desferrioxamine B (DFB), known by the trade name Desferal, is produced industrially by fermentation of Streptomyces pilosus and is used to treat a variety of medical disorders such as iron overload disease and aluminum chelation during dialysis (Schupp et al., 1988).
12 1.5 Siderophore production by marine microorganisms.
In marine environments, iron is quite often severely limited (Martin and Fitzwater, 1988;
Martin et al., 1994; Johnson et al., 1997; Morel and Prince, 2003). Much of the iron present in
surface ocean waters is complexed with organic ligands (Gledhill and Vandenberg, 1994; de
Baar et al., 1995; Rue and Bruland, 1995; Wu and Luther, 1995) and this has been suggested to
be of biological origin (Rue and Bruland, 1997). During an experiment to supplement oceanic
waters with ferric iron to stimulate photosynthetic organisms and carbon sequestration, it was
found that iron chelating ligands detected in marine waters increased significantly in a short
amount of time. Siderophores have been suggested to be a source of these iron complexing
ligands and increased studies into marine siderophore production resulted.
While the number of siderophores isolated from marine bacteria is dwarfed by the
hundreds of siderophores identified from terrestrial bacteria, several prominent structural
features have been identified in marine siderophores (Butler, 2005). One class of siderophores
facilitates the photoreduction of chelated Fe(III) in natural sunlight present in the mixed layer of
the upper ocean (Barbeau et al, 2001, 2002; Bergeron et al., 2003). This photoreactivity is
provided by a-hydroxycarboxylic acid moieties, in the form of either b-hydroxyaspartate or citric acid. Another class, some of which induce Fe(III) photoreduction, are the amphiphilic siderophores that contain unique peptidic headgroups appended by one of a series of fatty acid tails (Martinez et al. 2000, Martinez et al. 2003). The fatty acid chain length varies in length from C12 to C18. Some are secreted extracellularly like the aquachelins and the marinobactins
(Martinez et al., 2000) while others, such as the amphibactins, contain longer C18 fatty acid tails
13 and remain cell associated. Iron cycling in the upper ocean could be significantly affected by
the siderophores produced by marine microorganisms. The ornibactins are amphiphilic
siderophores that were isolated from the terrestrial bacterium Burkholderia cepacia, but these
siderophores contain much shorter fatty acid appendages of C4, C6 and C8 (Stephan et al., 1993;
Meyer et al., 1995). Other than Burholderia, the only other amphiphilic terrestrial siderophores are those produced by the Mycobacteria (Ratledge and Dale, 1999). Some amphiphilic siderophores contain a citrate backbone such as acinetoferrin and rhizobactin 1021 (Okujo et al.,
1994; Persmark et al. 1994)) with a single C8 and C10 fatty acid appendage, respectively.
1.6 Siderophore affinity for divalent heavy metal cations and radionuclides
Although highly specific for iron, siderophores have been shown to bind other metals such as actinides and heavy metals (Brainard et al., 1992; Whisenhunt et al., 1996; Neubauer et al., 2002) The production of different siderophores with varying affinity for Fe(III) and other transition metals in order to supply the cells with essential trace elements has been suggested by several authors (Visca et al., 1992; Duhme et al., 1998; Kalinowski et al. 2004). Because of their ability to chelate metals other than Fe(III), siderophores have potential for applications in metal recovery and remediation strategies, but also may contribute to the unexpected mobility and leaching of contaminants thought to be immobilized based on existing chemical models.
Siderophores from the ferrioxamine family, in particular the siderophores DFB and DFE, have been shown to coordinate a variety of heavy metals such as Cu(II), Ni(II), Pb(II) and Zn(II)
(Farkas et al., 1995; Hepinstall et al., 2005; Kraemer et al., 1999; Neubauer et al., 2000) as well as tetravalent actinides such as Pu(IV), U(IV) and Th(IV) (Brainard et al., 1992; Whisehunt et
14 al., 1996; Neu et al., 2000). Some metal siderophore complexes approach the stability of the
Fe(III) complex, as seen DFB complexed with Th(IV) and Pu(IV) which are reported to have
stability constants of 1026.6 and 1030.8, respectively, while that for iron is 1030.6 (Whisenhunt et al., 1996).
1.7 Concluding remarks
Siderophore production, utilization, or both, is a trait common to mainly aerobic bacteria and fungi due to their specific requirements for iron. Most studies involving siderophore production have focused on terrestrial microbes from near neutral environments and pathogenic bacteria. Only recently has the study of siderophores begun to focus on other environments such as marine systems. Because the requirement for iron appears to be common to nearly all known microbial life, there are many exciting prospects for the study of iron acquisition systems and siderophore production in particular by microbes that inhabit environments that are considered to be “extreme.” The following chapter addresses halophiles and alkaliphiles, both classes of microorganisms that are considered to be “extremophilic.”
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29 CHAPTER TWO
HALOPHILIC AND ALKALIPHILIC MICROOGANISMS
2.0 Extremophiles
Extremophiles possess physiological traits that permit them to inhabit environments that are hostile to other organisms. These environments may include hot springs or high pressure hydrothermal vents on the ocean floor, saline and alkaline soda lakes, low temperature environments located at the earth’s poles or subsurface aquatic environments, and acidic environments created by geothermal activity or mine debris. Many of the organisms found in these environments not only tolerate their surroundings, but require one or more extremes to function and reproduce. Extremophiles have developed unique biochemical features and enzymes which can function at the extremes of pH, temperature or osmotic strength necessary for their survival.
2.1.0 Halophiles
Halophiles are microorganisms that are capable of surviving in saline environments.
These organisms flourish in locations such as the Dead Sea, the Great Salt Lake, embedded in rock salt mines, and cold temperature hypersaline lakes such as those found in Antarctica (Grant et al. 1998). The terms halo-tolerant, moderate halophile and extreme halophile have been defined by Kushner (1978, 1993). In general, extreme halophiles require, at minimum, 1 M
NaCl (~60 g L-1) for growth and in many cases structural integrity of the cell membrane.
Extreme halophiles are almost exclusively members of the Archaea and will often tolerate levels of salt that are near or at saturation, and grow optimally above 3 M NaCl (~ 180 g L-1). Extreme
30 halophiles can be found in hypersaline habitats that contain salinities near that of halite
precipitation and are predominately Archaea (Rodriguez-Valera et al. 1981). Moderate
halophiles are generally members of the bacterial domain and require at least 0.4 M NaCl (~ 25 g
L-1) for growth with growth optima occurring between 0.5 – 2.0 M NaCl ( 30 – 180 g L-1), however this definition is not necessarily as rigid as that for extreme halophiles. Finally, halo- tolerant and halo-versatile microbes are defined as those that can grow in the absence of salt, but can tolerate up to 1.0 and 3.0 M salt, for halo-tolerant and halo-versatile microbes, respectively
(Grant et al., 1998). The specific salt tolerance of these microbes, however, is dependent on other environmental conditions such as temperature, pH and medium composition. Marine microbes constitute a class of slightly halophilic organisms. While most marine microbes require 2-3% salt for optimal growth, many are inhibited at salt concentrations that are only slightly higher (Larsen, 1986).
While not garnering as much attention extreme halophiles, moderately halophilic bacteria may be more environmentally significant than extreme halophiles, due to their ability to thrive under a wide range of salinities. Moderate halophiles are widely distributed in saline environments, and may be found in hypersaline lakes, desert and saline soils, saltern ponds, salt mines, salted foods and the oceans (Ventosa, 1988; Ventosa et al. 1998). Salinovibrio costicola and Halomonas halodenitrificans are perfect examples of the adaptability of many moderate halophiles as these organisms can grow in water activities near that of freshwater (0.98) to near saturation of NaCl (0.86) (Kustner, 1978). Converse to this adaptability, many extremely halophilic archaea require at least 1.5 M NaCl to retain the structural integrity of their cell walls, which differ from bacteria by the possession of ether-linked isopranoid lipids (Ross et al. 1981).
31 2.1.1 Mechanisms of halotolerance
Survival in environments of high salinity requires a set of tools to combat the osmotic stresses realized between the interior of the microbial cell and the high ionic strength exterior.
Halophilic archaea maintain an osmotic balance by accumulating high levels of intracellular salt, typically potassium, from their environment (Oren, 1999; Eisenburg et al., 1992). Halophilic bacteria, in general, rely on the synthesis or accumulation of organic compounds, termed compatible solutes, to maintain an osmotic balance across the membrane. Because many of these compounds can be synthesized, rather than scavenged from the environment, microorganisms utilizing this method of maintaining an osmotic balance are more adaptable to changes in extracellular salt concentrations and may tolerate and thrive over a wider range of salinities than halophilic archaea (Oren, 1999). Another mechanism for tolerating high salt environments is adaptations made to cell wall composition (Russell et al., 1985) in which a hydrophilic outer layer is present along with a hydrophobic interlayer. In contrast to halophiles, non-halophilic bacteria, when subjected to high ionic strength environments experience plasmolysis, which is an outward recession of the cytoplasm caused by the outward flow of water from within the cell in an attempt to maintain an osmotic balance. (Kargi and Uygur, 1996).
2.1.2 Saline and hypersaline environments
Hypersaline waters are loosely defined as those that contain salt concentrations that are greater than that found in seawater (greater than 3.5%). These types of systems can be separated into two environments. Thalassohaline environments are derived from marine waters and become concentrated through evaporation so that at the earlier stages of this process, they have a
32 mineral composition that is similar to seawater. As evaporation progresses, the similarity ceases
as the minerals become more concentrated and eventually precipitate in the following order:
calcite (CaCO3), gypsum (CaSO4 2H2O), halite (NaCl), sylvite (KCl) and lastly carnallite (KCl,
MgCl2 6H2O). The final concentration of the thalassohaline brine is dominated by magnesium and chloride ions. Often, this brine is slightly more acidic than the oceanic waters from which it was derived (Grant and McGenity, 1998). Athalassohaline brines tend to develop as a result of local geography and geology, but may be influenced by seawater to some extent. However, one of the most substantial differences between thalassohaline and athalassohaline brines is the pH.
As mentioned previously thalassohaline brines are typically slightly more acidic than the seawater from which they were derived. This is predominantly due to the precipitation of carbonate with excess calcium in the form of calcite. In athalassohaline systems, the waters are typically deficient in both Ca2+ and Mg2+ with respect to the concentration of carbonate and thus, the system tends to generate an alkaline pH (Grant and McGenity, 1998).
One potential source of halophilic bacteria and archaea are from within subsurface geological salt formations. Recent isolations from subsurface salt formations and underground brines have yielded a variety of halophilic and halo-tolerant bacteria (Vreeland et al., 1998). One such subsurface salt formation is the Waste Isolation Pilot Plant (WIPP), an underground repository built by the US Department of Energy (DOE) for the storage of defense-related radioactive wastes. The WIPP facility lies 650 m below the ground surface in a geologically stable, bedded salt formation in New Mexico. A survey of the microbial life contained within the WIPP site yielded nearly 150 isolates including various known extreme halophiles such as
Haloarcula, Halobacterium, Halococcus and Haloferax among many previously undescribed
33 isolates (Vreeland et al., 1998). Other surveys have isolated moderate halophiles, in addition to
extreme halophiles, from the WIPP site such as members of the genus Halomonas (Francis et al.
2004; Gillow et al., 2000).
2.2.0 Alkaliphiles
Alkaliphiles constitute a class of extremophilic microorganisms that realize optimal
growth at or above a pH of 9. Facultatively alkaliphilic microorganisms are those that may grow
at high pH, but grow optimally at near neutral conditions while obligate alkaliphiles require at
least a pH of 8.5 or 9.0 for growth. Alkaliphiles are ubiquitous and have been isolated from
neutral environments, alkaline environments such as thermal hot springs in volcanically active
areas and saline-alkaline environments such as the soda lakes of East Africa (Horikoshi, 2004).
Alkaliphiles have also been found in deep sea sediments collected from the Marinas Trench at
depths reaching nearly 11000 meters below the surface (Takami et al., 1997). Alkaliphiles make
up a fraction of the microbial population in “typical” neutral soils, with counts of 102-105 per
gram of soil, which roughly translates to approximately 1/100 of the total population. (Horikoshi,
K., 1991).
2.2.1 Specific mechanisms of alkaline tolerance
Many alkaliphiles grow optimally at a pH of 10. This is significantly higher than the pH
at which most neutrophilic organisms thrive. To prevent degradation of DNA, alkaliphilic
microorganisms maintain a cytoplasmic pH of approximately 2 pH units lower than what is
present outside the cell wall. Alkaliphiles use an electrochemical gradient of Na+ rather than of
H+ for solute transport and flagella rotation. The plasma membrane maintain the differential pH
34 by using a Na+/H+ antiporter system, K+/H+ antiporter and ATPase-driven H+ explusion. A proton motive force is generated in the cells, either by the excretion of H+ from ATP metabolism or by the electron transport chain. The hydrogen ions are then reincorporated into the cells through the co-transport of various substances – often Na+ or K+ by the electron transport chain.
In the Na+ dependent transport systems, H+ is exchanged for Na+ by specific Na/H tranporters which then generate a sodium motive force, driving substrate that accompany the Na into the cells (Kaieda, 1998;Krulwich, 1983; Krulwich, 2001). These transport systems have been found to maintain in internal pH that is approximately 2-2.3 units lower than the external pH
(Horikoshi, 2004).
2.3.0 Soda lakes
Soda Lakes are athalassohaline environments that contain high concentrations of sodium carbonate and sodium bicarbonate fraction among the soluble salts and thus represent a very specific type of saline lake. In general, soda lakes are located inland, in areas with drier climates that contribute to the accumulation of salts lakes present in closed drainage basins (John et al.
1998). Quite frequently, the local geology contributes to the development of their aqueous chemisty as sodium is leached from the sodium-rich mineral formations by the carbonate rich groundwater that is deficient in Ca2+ or Mg2+. This deficiency of Ca2+ and Mg2+ permits the presence of high, stable concentrations of sodium carbonate. The very high buffering capacity of the sodium carbonate allows for the maintenance of a very high pH, often hovering around pH =
10, a situation rarely encountered in most other natural ecosystems. Alkaline hot springs, located in volcanic and geologically active areas may also harbor extremes of pH and the alkalinity generated is likely a result of silicate decomposition and often lacks the stability found in soda
35 lakes (Hensel et al 1997; Jones et al. 1998). Because of the stable conditions of alkaline pH and
frequently elevated dissolved solids concentration, these environments allowed for the
development of a consortium of obligately alkaliphilic and often halophilic microorganisms.
Many of the well known hypersaline soda lakes are located in very arid regions such as the
Eastern African Rift Valley in Kenya and Tanzania, the Libyan Desert in Egypt, and in the rain-
shadowed deserts of the western United States. Because of the high alkalinity of soda lakes,
often in conjunction with high salinity, they harbor a diversity of alkaliphilic and often halophilic
microbial species.
Soda lakes are very productive environments with respect to microbial life. There are
dense populations of cyanobacteria as well as alkaliphilic anoxygenic photorophic bacteria.
Bacterial counts often reach 107 to 108 cells per ml (Grant et al., 1990). Based on studies that
surveys of the microbial diversity of these soda lakes, it is becoming apparent that the
Halomonas/Deleya group often constitutes a major bacterial grouping (Jones et al. 1994;
Duckworth et al., 1996; Grant et al. 1998) and a quick survey of the literature yields many new
species of Halomonas isolated in the past decade. These results, of course, should be taken with a grain of salt, because what is cultured is quite often a reflection of the sampling methods and culture conditions. It has been reported that the use of a nutrient rich medium will favor the growth of members of the g division of the g-Proteobacteria (of which the Halomonadaceae are members) at the expense of other groups (Wagner et al. 1994).
2.3.1 Soap Lake
36 Soap Lake is a soda lake located in Grant County in central Washington State, USA in
the Grand Coulee Basin. This is a semi-arid region located in the rain shadow of the Cascade
Mountains. Soap Lake is the terminal lake in a series of lakes that are characterized by
increasing salinity and alkalinity. It is just over 300 hectares in area and has a maximum depth of
27 m (Anderson, 1858). This lake was created during the end of the last ice age when the Glacial
Lake Missoula ice dam, located on the Clarkfork river, burst sending floodwaters at up to 40 to
60 cubic kilometers per hour (9.5 to 15 cubic miles per hour) through eastern Washington State.
This flooding was estimated to have occurred periodically every 55 years over a 2000 year
period between 13,000 and 15,000 years ago. The channeled and rippled scablands of eastern
Washington were formed as a result. Soap Lake formed within one of these ripples in a closed
basin left behind from the erosion of the Missoula floods. Because the lake has no surface inlet
(other than runoff) and no outlet streams, its only losses of moisture are due to evaporation and
over the years, it has gradually increased in dissolved solids concentration and alkalinity.
Like many soda lakes, Soap Lake is characterized by high concentrations of sodium carbonate
(6870 mg/L) and sodium bicarbonate (5209 mg/L). This has resulted in high alkalinity and the
maintenance of a pH that averages approximately 9.9 throughout the water column. In addition
to its high alkalinity, a unique feature of Soap Lake is that it is meromictic, as it possesses two
distinct layers which do not intermix. The upper layer of the lake, termed the mixolimnion layer
is brackish, containing approximately 15 g liter-1 dissolved solids and is aerobic. The lower layer of the lake contains a much higher dissolved solids concentration, reaching 140 g liter-1, is much colder (6 to 8oC) and anaerobic (Sorokin et al., 2007). Probably one of the most remarkable features of the lake it its extraordinarily high sulfide content in the monimolimion layer of up to
37 200 mM sulfide, which is one of the highest concentrations ever recorded in natural waters
(Sorokin et al., 2007). The monomolimnion and mixolimnion layers are separated by an abrupt
chemocline located at about 20-23 m in depth. At this chemocline, the oxygen concentration
plummets from near saturation to zero, total dissolved solids transition from approximately 15 g
liter-1 to over 140 g liter -1 and sufide increases from trace quantities to over 100 mM, all in a
mater of about a meter or less (Sorokin et al. 2007;Anderson, 1958, Walker 1975). The
mixolimnion and monimolimnion layers are estimated to not have mixed for upwards of 2000
years (Patel et al. in preparation; Oremland, 1993; Rice, 1988).
Many novel bacteria have been isolated from Soap Lake, including Nitrincola
lacisaponensis, which marks a novel genus isolated from decomposing wood taken from the
shore of the lake (Dimitriu et al., 2005). The chemocline in Soap Lake is home to a dense
population of sulfur-oxidizing Thioalkalimicrobium, which reached population densities of up to
107 cells ml-1, and Thioalkalivibrio and a new species of Thioalkalimicrobium was recently isolated from the lake (Sorokin, et al., 2007). Recently, a representative of a novel genus of iron reducing bacteria was isolated from the lake (Patel et al., in preparation).
2.4 Concluding Remarks
To date, few studies have investigated siderophore production in halophilic and alkaliphilic bacteria. Gascoyne et al. (1991) report siderophore production in an alkaline environment while Dave et al. (2006) recently detected siderophore production in a number of halophilic Archaea. Neither of these reports includes a complete structural characterization of these siderophores. Studies of siderophores produced by bacteria from marine environments
38 have shown the production of unique amphiphilic siderophores with a peptidic iron chelating head group and a fatty acid tail of various carbon number by Marinobacter sp. and Halomonas aquamarina (Martinez et al., 2000). It is unknown if there is much similarity between aquatic marine siderophores and those produced by halophiles and alkaliphiles. Halophilic and alkaliphilic bacteria are likely producers of siderophores. Microoganisms inhabiting soda lakes, such as Soap Lake may be a source of novel siderophore structures and this dissertation attempts to not only detect siderophore producing halophilic and alkaliphilic bacteria within the lake, but to characterize the siderophore structures.
2.4 REFEREENCES
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Eisenberg, Henryk; Mevarech, Moshe; Zaccai, Giuseppe. Biochemical, structural, and
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Grant, W. D.; Mwatha, W. E.; Jones, B. E. Alkaliphiles : ecology, diversity and applications.
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Hensel, R.; Matussek, K.; Michalke, K. Tacke, L. Tindall, B.J.; Kohloff, M. Siebers, B.;
Dielenschneider, J. Sulfophobococcus zilligii gen. nov., spec. nov. a novel
39 hyperthermophlic archaeum isolated from hot alkaline springs of Iceland. Systematic and
Applied Microbiology. (1997), 20 102-110.
Horikoshi, K. Microoganisms in Alkaline Environments. (1991) Kodansha-VCH, Tokyo-
Weinheim-New-York-Cambridge-Basel.
Jones, B.E.; Grant, W.D.; Duckworth, A.W.; Owenson, G.G. Microbial diversity of soda lakes.
Extremophiles. (1998) 2, 191-200
Kargi, F.; Uygur, A.. Biological treatment of saline wastewater in an aerated percolator unit
utilizing halophilic bacteria. Environmental Technology (1996), 17(3), 325-30.
Koyama, Noriyuki; Nosoh, Yoshiaki. Effect of potassium and sodium ions on the cytoplasmic
pH of an alkalophilic Bacillus. Biochimica et Biophysica Acta, Biomembranes
(1985), 812(1), 206-12.
Koyama, Noriyuki; Wakabayashi, Kunitoshi; Nosoh, Yoshiaki. Effect of potassium on the
membrane functions of an alkalophilic Bacillus. Biochimica et Biophysica Acta,
Biomembranes (1987), 898(3), 293-8.
Krulwich, Terry Ann; Ito, Masahiro; Gilmour, Ray; Guffanti, Arthur A. Mechanisms of
cytoplasmic pH regulation in alkaliphilic strains of Bacillus. Extremophiles (1997),
1(4), 163-169.
Oremland, R. S.; Miller, L. G. (1993) Biogeochemistry of Natural Gases in Three Alkaline,
Permanently Stratified (Meromictic) Lakes. USGS Paper 1570. pp. 439-452.
Oren, Aharon; Litchfield, Carol D. A procedure for the enrichment and isolation of
Halobacterium. FEMS Microbiology Letters (1999), 173(2), 353-358.
40 Rice, C. A.; Tuttle, M. L.; Briggs, P. H. (1988) Sulfur Speciation, Sulfur Isotopy, and Elemental
Analysis of Water-Column, Pore Water, and Sediment Samples from Soap Lake,
Washington. USGS Open File Report. 88-22.
Russell, N. J.; Kogut, M.; Kates, M. Phospholipid biosynthesis in the moderately halophilic
bacterium Vibrio costicola during adaptation to changing salt concentrations. Journal of
General Microbiology (1985), 131(4), 781-9.
Sorokin, D.Y.; Foti, M.; Pinkart, H.C; Muyzer, G. Sulfur-oxidizing bacteria in Soap Lake
(Washington State), a meromictic, haloalkaline lake with an unprecedented high sulfide
content. Applied and Environmental Microbiology. (2007), 73(2), 451-455.
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Trench. FEMS microbiology letters (1997), 152(2), 279-85.
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42 CHAPTER THREE
IDENTIFICATION AND CHARACTERIZATION OF A SUITE OF FERRIOXMAINE
SIDEROPHORES PRODUCED BY A HALO-ALKLALIPHILIC BACTERIUM
ABIGAIL M. RICHARDS1, ROBIN GERLACH2, BRENT M. PEYTON2* and WILLIAM A. APEL3
To be submitted to Applied and Environmental Microbiology
1 Department of Chemical Engineering, Washington State University, Dana Hall 118, Spokane
St. Pullman, WA 99164-2710, USA
2 Department of Chemical and Biological Engineering, Montana State University, 303 Cobleigh
Hall, PO Box 173920, Bozeman, MT 59717-3920
3 Department of Biological Sciences, Idaho National Laboratory, 2351 N. Boulevard, PO Box
1625, Idaho Falls, ID USA, 83415
* Corresponding author phone: (406) 994-2221
FAX: (406) 994-5308
Email: [email protected]
43 3.0 ABSTRACT
Desferrioxamine siderophores are produced by a wide variety of terrestrial and aquatic gram-
positive and gram-negative bacteria. In addition to iron, desferrioxamine B and desferrioxamine
E have been shown to coordinate radionuclides, such as Pu(VI), with stability constants rivaling
that for ferric iron and thus could impact radionuclide speciation and mobility. In many cases,
radionuclides exist in conjunction with high salinity and pH, but siderophore production in these
environments is poorly characterized. Siderophore production by microorganisms indigenous to
saline and alkaline environments could contribute to enhanced mobility of radionuclide and
metals stored in such locations. Siderophore production was identified in the moderately
halophilic, alkaliphile, Halomonas campisalis. Several desferrioxamine siderophores including desferrioxamines G1, G1t, X3, X7, D2, and E were isolated from low-iron culture supernatant and structurally characterized by ESI-MS and ESI-MS/MS. This work represents the first documentation of ferrioxamine siderophore production by a halo-alkaliphilic bacterium. These results suggest that if ferrioxamine production is common to other halo-alkaliphiles, radionuclide speciation and mobility could be affected by siderophores produced in saline and alkaline environments.
44 3.1 INTRODUCTION
All microorganisms, except the Lactobacilli and Borrelia burgdorferi, have nutritional requirements for ferric iron that are often not met in aqueous, aerobic environments due to the low solubility of iron at circumneutral pH (Archibald, 1983; Posey et al., 2000). To overcome the scarcity of iron in such environments, many microorganisms secrete small, organic iron chelating molecules, called siderophores, that have a very high affinity for ferric iron (K=1025-
1050). These iron chelating agents are secreted under iron-starved conditions and have the primary role of scavenging iron from the environment. The dissolution rates of iron oxides and the soluble fraction of ferric iron are increased in the presence of siderophores, resulting in an overall concentration of Fe(III)-siderophore complexes that help meet the nutritional demands of the siderophore producing microbes (Ruggiero et al., 2002; Kraemer, 2004; Cheah et al., 2003;
Romheld, 1991). The Fe(III)-siderophore complex is recognized by the host through specific membrane-embedded receptors on the cell surface and is actively transported into the cells
(Koster, 2001; Winkelmann, 2001; Andrews et al., 2003). Because of widespread iron deficiency, siderophores are common in soil and marine environments reaching concentrations of approximately 0.1 – 0.01 mM in soils (Powell et al., 1980). While the exact concentration of siderophores in marine environments is not determined, siderophore production has been detected in a number of marine isolates (Wilhelm and Trick, 1994; Trick, 1989, Guan et al.,
2001). It is suggested that siderophores comprise a significant portion of the iron-binding ligands present in marine environments (Butler, 2005; Macrellis et al., 2001).
45 The majority of siderophores may be divided into three main structural classes depending
on their functional groups (Figure 1). Hydroxamate siderophores include the ferrioxamines,
ferrichromes and coprogens. Siderophores containing catecholate iron coordinating groups
include the enterobactins, vibriobactins and yersiniabactin, while carboxylate and mixed ligand
a-hydroxamates include pyoverdines, azotobactins and ferribactins. One prominent and well
studied class of hydroxamate siderophores is the ferrioxamines, which are a group of natural,
iron-chelating siderophores. The ferrioxamines were first found to be secreted in the desferri
form under iron limiting conditions by Gram-positive Streptomyces and Nocardia species
(Bickel et al., 1960; Keller-Schierlein and Prelog, 1961; Keller-Schierlein and Prelog, 1962;
Keller-Schierlein et al., 1965), but have since been identified in several other genera including
Gram-negative Pseudomonads, Arthrobacter, Chromobacterium, Erwinia, and a marine Vibrio
(Muller and Zahner, 1968; Berner et al., 1988; Feistner et al., 1993; Martinez et al., 2001;
Feistner and Ishimaru, 1996; Zawadzka et al.. 2006). Many distinct ferrioxamines have been identified and characterized to date, including ferrioxamine A, B, C, D1, D2, E, F, G1, G2a-c, H, I,
T1-8 and X1-7 (Winkelmann, 1991; Fiestner et al., 1993). A characteristic feature of the ferrioxamines is a repeating of an a-amine-w-hydroxyamino alkane motif with either succinate or acetate. These siderophores are either linear or cyclic, and generally fall within a size range of about 500-600 Da. With the exception of the dihydroxamic acids, such as desferrioxamine H, alcaligin and bisucaberin, the ferrioxmaines are hexidentate ligands that contain three hydroxamate groups that facilitate the chelation of ferric iron. The best studied of the ferrioxamine siderophores, desferrioxamine B (DFB), known by the trade name Desferal, is produced industrially by fermentation of Streptomyces pilosus and is used to treat a variety of medical disorders such as iron overload disease and aluminum chelation during dialysis (Schupp
46 et al., 1988). Desferrioxamine E (DFE), identical to the antibiotic nocardamine, is a cyclic sideramine that consists of 5-succinyl-1-amino-5-hydroxyaminopentane, which is derived from
L-lysine (Keller-Schierlein and Perlog, 1961).
Although highly specific for iron, siderophores have been shown to bind other metals such as actinides and heavy metals (Brainard et al., 1992; Bouby et al., 1998a; Bouby et al.,
1998b; Crumbliss, 1991; Dubbin and Ander, 2003; Enyedy et al., 2004; Groenewold et al., 2004;
Hepinstall et al., 2005; Keith-Roach et al., 2005; Kraemer et al., 1999; Kraemer et al., 2002;
MacCordick et al., 1995; Neu et al., 2000; Neubauer and Gerhard, 1999; Neubauer et al., 2000;
Neubauer et al., 2000; Neubauer et al., 2002; Renshaw et al., 2002; Whisenhunt et al., 1996;
Yoshida et al., 2004). The production of different siderophores with varying affinity for Fe(III) and other transition metals in order to supply the cells with essential trace elements has been suggested by several authors (Visca et al., 1992; Duhme et al., 1998; Kalinowski et al.. 2004).
Because of their ability to chelate metals other than Fe(III), siderophores have potential for applications in metal recovery and remediation strategies, but also may contribute to the unexpected mobility and leaching of contaminants thought to be immobile based on existing chemical models. Siderophores from the ferrioxamine family, in particular the siderophores DFB and DFE, have been shown to coordinate a variety of heavy metals such as Cu(II), Ni(II), Pb(II) and Zn(II) (Farkas et al., 1995; Hepinstall et al., 2005; Kraemer et al., 1999; Neubauer et al.,
2000) as well as tetravalent actinides such as Pu(IV), U(IV) and Th(IV) (Brainard et al., 1992;
Whisehunt et al., 1996; Neu et al., 2000). Some actinide siderophore complexes approach the stability of the Fe(III) complex, as seen DFB complexed with Th(IV) and Pu(IV) which are
47 reported to have stability constants of 1026.6 and 1030.8, respectively, while that for iron is 1030.6
(Whisenhunt et al., 1996).
Because of these high stability constants, siderophores may have the potential to significantly alter the mobility of metal contaminants in subsurface environments. In many cases, radionuclide and heavy metal wastes are located in environments of high ionic strength or alkalinity. Many of the nuclear waste tanks at the DOE Hanford reservation are characterized by high salinity and alkalinity (Fredrickson et al., 2004; Deng et al., 2006). Various halophilic microorganisms, including members of the genus Halomonas have been isolated from highly
saline environments such as the Waste Isolation Pilot Plant, a repository for nuclear wastes
located within deep geological salt formations (Gillow et al., 2000; Vreeland et al., 1998).
Microorganisms in environments that contain stored radionuclide wastes could be siderophore
producers and thus have an effect on contaminant speciation and mobility, but to date,
siderophores from halo-alkaliphiles have not been characterized
Extremophiles are organisms that thrive under conditions that are considered hostile to
most organisms. These conditions include extremes of temperature, pH, osmotic strength and
pressure. Halophiles include members of the bacterial, archaeal and fungal domains. Extreme
halophiles are almost exclusively composed of members of the archaea and many grow
optimally at salt concentrations near saturation (Kushner and Kamenkura, 1988). Moderate
halophiles grow optimally between 0.5 to 2.5 M salt, while halotolerant microbes may tolerate
up to 2.5 M salt but grow optimally at concentrations below 0.5 M (Kushner, 1978; Larsen,
1986, Grant et al., 1998). The term “alkaliphile” is used to describe microorganisms which grow
48 optimally at pH values greater than 9 while they grow very slowly, or not at all, at the near
neutral pH value of 6.5. Quite frequently, alkaliphilic microbes will have optimum growth at a
pH above 10 (Horikoshi, 2004). Halo-alkaliphiles require both elevated salt concentrations and
high pH for optimal growth.
To date, only a handful of studies have investigated the production of siderophores in
saline or alkaline environments. Gascoyne et al. (1991) report siderophore production in an
alkaline environment but do not go on to conduct any structural characterizations while Dave et
al. (2006) recently detected siderophore production in a number of halophilic Archaea. Neither
of these reports includes a complete structural characterization of these siderophores. Studies of
siderophores produced by bacteria from marine environments (Marinobacter sp. and Halomonas aquamarina) have shown the production of unique amphiphilic siderophores with a peptidic iron chelating head group and a fatty acid tail of various carbon number (Martinez et al., 2000). With relatively few examples of siderophore produced by marine organisms and halophiles and alkaliphiles, one cannot necessarily draw comparisons between them.
It can be seen that very little is known about the nature of siderophores produced by extremophiles, in particular, halophiles or alkaliphiles. To examine this area of research, we selected Halomonas campisalis, a moderately halophilic alkaliphile, as a potential producer of siderophores. A suite of ferrioxamine siderophores was identified by H. campisalis when grown in iron limited conditions. This is the first report which identifies the production of ferrioxamine siderophores by a halo-alkaliphile.
49 3.2 MATERIALS AND METHODS
3.2.1 Growth conditions. Halomonas campisalis ((ATCC# 700597, American Type Culture
Collection, Manassas, VA) was grown with lactate as a carbon source in a medium that consisted of the following: NaCl, 100 g/L; C3H5O3Na, 10g/L; Na2B4O7, 4g/L; NH4Cl, 1 g/L; NaNO3, 0.2 g/L; KH2PO4, 0.5 g/L; yeast extract, 1 g/L. The pH of the medium was adjusted to 10 (or the desired value for pH specific experiments) with NaOH. The yeast extract was deferrated using
Chelex 100 resin (Sigma Chemical) following a previously published method (Domingue et al.,
1990). Cultures were grown aerobically at 30 oC, shaking at 150 rpm in acid washed flasks with extra deep baffles.
3.2.2 Siderophore Detection. Halomonas campisalis was initially screened for siderophore production using the CAS agar plate method (Schwyn and Neilands 1987). The growth medium described above was used for the CAS assay, in place of the nutrient composition described by
Schwyn and Neilands (1987), and adjusted to pH 10 to account for the pH and salinity required by H. campisalis for growth. Because of the elevated pH, the plates had a greenish hue, rather than the blue typically seen near pH 7. In spite of this difference, a distinct orange halo, indicative of siderophore production, surrounded microbial colonies after a few days.
Siderophore production was monitored with respect to growth at pH 8, 9, 10 and 11 using the
CAS liquid assay. The siderophores were tested for the presence of hydroxamate moieties through the Csáky assay and desferrioxamine B (Sigma Chemical) was used as a hydroxamate standard (Csáky, 1948). The Arnow assay was used to detect catecholate structural moieties and
2,3-dihydroxybenzoic acid (Sigma Chemical) was used as a standard for catecholates (Arnow,
1937).
50 3.2.3 Siderophore Isolation. A one liter culture of H. campisalis was grown in the saline and
alkaline growth medium for 5 days. Cells were removed from the growth medium by
centrifugation at 6000 rpm using a Sorvall floor model centrifuge for 20 minutes at 4oC and the
cell pellet was discarded. To initially separate the siderophores from the high ionic strength
growth medium, the cell free supernatant was passed through Bond Elut solid phase extraction
C18 cartridges (Varian Inc., Palo Alto, CA). After passing spent growth media through them, the cartridges were rinsed with deionized water and then siderophores were eluted with methanol.
The crude siderophore extract was evaporated to dryness in a rotary evaporator. This material was then redissolved in water/0.1 % trifluoroacetic acid (TFA) and purified on an Alltech C-18 reverse-phase column (25 x 1 cm) using a Dionex DX500 HPLC system. The mobile phase consisted of water/acetonitrile with 0.01% TFA (A = 99.99 % Water/0.01 % TFA; B = 80 % acetonitrile/19.99 % water/0.01 % TFA). Siderophores were eluted using a gradient 0% B to
60% B for 40 minutes followed by 60% B to 100%B for 10 minutes, then 100% B for 5 minutes followed by 0% B for 5. The flowrate was maintained at 1 ml/min and the elution of compounds was monitored at an absorbance of 210 nm using a Dionex AD20 absorbance detector. Iron binding fractions were identified using the CAS assay and pooled and evaporated to dryness in a centrifugal evaporator. Crude mixtures of H. campisalis siderophores were also purified as the
Fe(III)-complex by adding FeCl3 to crude solutions of siderophore obtained from the C18 cartridges. When these Fe(III)- siderophore preparations were purified by HPLC, the absorbance at 435 nm was monitored because hydroxamate siderophores, such as the ferrioxamines, have a secondary absorbance maxima at or near 425-435 nm.
51 3.2.4 Siderophore Characterization. Electrospray mass spectrometry (ESI-MS) was performed using a 6300 series Agilent SL ion trap mass spectrometer equipped with an electrospray ionization source. This instrument was operated in positive mode for all experiments. An
Agilent 1100 liquid chromatography system was configured in line with the ESI-MS system and liquid chromatography mass spectrometry (LC-MS), so experiments could be run in tandem.
This system included a diode array absorbance detector which allowed the entire UV-Visible spectrum of each peak to be recorded. Single mass spectra were generated online by MS analysis during LC runs while MS/MS analyses were obtained with a direct injection ESI-MS/MS method using collected fractions that contained a single siderophore. Chromatography conditions were identical to those described earlier for LC-MS experiments. LC-MS analyses were conducted using siderophores in the iron-free form as well as the ferrated form.
Desferrioxamine B (Sigma Chemical) and desferrioxamines E and G1 (EMC Micro-collections,
Tubingen, Germany) were used as standards in HPLC purification and comparison of mass spectra.
3.3 RESULTS
Using the CAS assay, Halomonas campisalis was found to produce siderophores.
Siderophore production was monitored with respect to cell growth in liquid medium that contained deferrated yeast extract. As shown in Figure 2, siderophore production reached a maximum after 120 hours and lagged cell growth. Siderophores were produced at all pH values tested ranging from 8 to 11 and reached a maximum concentration equivalent to approximately
300-400 mM DFB. If standard yeast extract was used, siderophores were still detected in the culture supernatant, but at a concentration approximately half that obtained in cultures grown
52 with deferrated yeast extract. A negative response to the Arnow assay and positive response to
the Csáky assay indicated that the siderophores produced contained hydroxamate moieties rather
than catecholate moieties.
The hydroxamate siderophores from H. campisalis were isolated and purified by HPLC.
Both the absorption and mass spectra obtained in this study permitted siderophore identification.
For tandem LC-MS experiments, both MS and MS/MS spectra were collected. The major
ferrioxamines found to be produced by H. campisalis were the linear ferrioxamines G1, G1t and
the cyclic ferrioxamines E, D2, X3 and X7. The LC chromatogram of siderophores produced by
H. campisalis is shown in Figure 3 and the identity of each peak is assigned. Collision induced dionization (CID) mass spectra for the iron-free ferrioxamine siderophores yielded spectra characteristic of ferrioxamine siderophores, since breakages typically occur at the location of the hydroxamate and amide bonds. The total ion spectra and CID spectra of G1, G1t, E, D2, X3 and
X7, including the assignment of fragment ions to specific portions of the parent ion are shown in
Figures 4 through 9. A comparison of the fragmentation patterns obtained from H. campisalis siderophores with desferrioxamine E and G1 standards and with previously published data for ferrioxamines showed similar patterns (Fiestner et al, 1993; Feistner and Hsieh, 1995; Zawadzka et al., 2007). The composition of the ferrioxamine suite produced by H. campisalis did not change with respect to pH (data not shown) as indicated by LC chromatograms and LC-ESI/MS spectra. In addition to the ferrioxamines that were identified, there were several unknown compounds which eluted that did not correspond to previously identified ferrioxamine siderophores including parent ion masses ([M+H]+) of 501, 585, 617 and 599. The fragment ions of the CID spectra are listed for each compound in Table 1. The ferrioxamines G1, E, D2 and X7,
53 were purified as the Fe(III)-siderophore complex and showed absorption maxima at 425- 435 nm, which is characteristic of trihydroxamate ferric siderophores. Furthermore, the addition of
53 amu to the original mass of each parent ion suggested the combination with ferric iron along with the loss of three hydrogen atoms during iron coordination (data not shown).
3.4 DISCUSSION
Many phenomena, ranging from simple to complex interactions, control the behavior of toxic metals and radionuclides in the environment (Gadd, 1996; Gadd, 2004). Microorganisms can alter the solubility of metals by facilitating a) intra- or extracellular accumulation, b) direct or indirect reduction or oxidation, c) the production of organic acids which enhance the soluble fraction of these metals d) the alteration of local pH levels, e) biomineralization, and f) biocolloid formations; all of which impact the speciation, solubility and ultimately the mobility of these metals in the environment. Siderophores, although highly specific for iron, have been shown to bind other metals such as actinides and heavy metals (Hernlem et al., 1999;
Whisenhunt et al., 1996). Because of their ability to chelate metals other than Fe(III), siderophores have potential for applications in metal recovery and remediation strategies. As mentioned earlier, stability constants of DFB complexes with Pu(IV) or Th(IV) approach that with iron. Because of the ability to coordinate these compounds, siderophores may significantly alter the mobility of metal contaminants in subsurface environments.
Tetravalent actinides are similar to Fe(III) in several ways that determine their coordination chemistry including ionic radius ratio and first hydrolysis constants. In the case of
Pu(IV) and Fe(III), both metal ions have strong Lewis acidities, hydrolyze at relatively low pH
54 and form resulting hydroxides that are highly insoluble, providing very low concentrations of the
free metal (Brainard et al., 1992). Because siderophores contain binding groups rich in hard
oxygen donors, such as hydroxamate, catecholate and carboxylate functional groups, they can
harbor significant binding affinity for hard metals ions like Fe(III) and Pu(IV) (Crumbliss, 1991;
Neu et al., 2000).
In addition to the extracellular mobilization of metals, the siderophores DFB and DFE,
when bound to Pu(IV), have been shown to compete with Fe(III)-siderophore complexes and are
actively taken up by Microbacterium flavescens (John et al., 2001). The authors speculate that this phenomena is not unique to M. flavescens, and the active uptake of Pu(IV)-siderophore complexes is possible in other species of bacteria. This would imply yet another mechanism of contaminant transport via motile cells. Frazier et al. (2005) found that with uraninite (UO2), siderophores dramatically increase both its solubility and dissolution kinetics over a pH range of
3-10, potentially increasing UO2 migration and mobility. This study was particularly remarkable in that siderophores, while specifically designed to mobilize Fe(III), were even more effective at promoting UO2 dissolution than they were at mobilizing Fe from goethite. Furthermore, the presence of additional Fe(III) did not decrease the rate of DFB promoted UO2 dissolution. Alum shales found in Ranstad, Sweden represent one of the largest known uranium deposits in the world. In spite of measures taken to abate the leaching of uranium, metals are still found to leach from the site and this has been attributed in part to mobilization by pyoverdine siderophores
(Kalinowski et al. 2004). In the case of Pu(IV) dissolution by DFE and DFB, it was found that
Pu(IV) hydroxide solubilization was only slightly enhanced by either siderophore while chelating agents such as EDTA, NTA and citrate did enhance solubilization. It was found that
55 DFE allows Pu(IV) dissolution rates that are an order of magnitude greater than that of DFB
(Ruggiero et al., 2002).
Significant heavy metal and radionuclide contamination exists at many US Department of
Energy (DOE) sites. Plutonium migration which far exceeds that predicted by existing models
has been detected at the Nevada Test Site, Los Alamos National Laboratory, and in the
groundwater at the Hanford Site (Kersting et al. 1999; Penrose et al. 1990; Dai et al. 2005). This
contamination may occur under typical aquifer conditions, but is also frequently in conjunction
with high ionic strength and/or pH conditions, such as at the WIPP facility, which is located
within a geological salt formation or at the DOE Hanford Site where much of the radioactive
waste is stored in tanks containing extremely alkaline, high ionic strength liquids (Buck and
McNamara, 2004). Microbial life most likely exists under the conditions in both locations
(Fredrickson et al., 2004; Vreeland at al., 1998). It is essential to understand the effect these
conditions have on the production of siderophores by microorganisms that thrive at high pH and
salinity. The halophilic and alkaliphilic bacterium, Halomonas campisalis, was originally isolated from soil beneath a dried salt flat located in Eastern Washington State. This bacterium grows in salt concentrations ranging from 0.2 M to 4.5 M with an optimum of 1.5 M. While H. campisalis has a pH optimum of 9.5, it can replicate over a wide range of pH conditions with growth detected at pH values as low as 6 and as high as 11 (Mormile et al. 1999). This organism is a gram negative member of the g-proteobacteria and is a facultative anaerobe, able to reduce
nitrate and nitrite. It can grow on a wide variety of carbon sources (Mormile et al., 1999)
including aromatic compounds such as phenol, benzoate, catechol and salycilate (Oie et al.,
2007; Alva and Peyton, 2003). Because H. campisalis can utilize a number of organic
56 substrates, thrive under a variety of both aerobic and anaerobic conditions and proliferate over a
wide range of pH and salinity, it was selected as a model organism for halo-alkaliphilic
siderophore producers.
A suite of at least six primary ferrioxamine siderophores, including desferrioxamine G1,
G1t, X3, X7, D2 and E, were found to be produced by Halomonas campisalis. Desferrioxamine
G1 and E were found at the highest concentration relative to total siderophore production, while trace amounts of other ferrioxamine siderophores such as desferrioxamine G1t, and D2 were also detected. Described here, the ability of H. campisalis to produce ferrioxamine siderophores is the first report of ferrioxamine production by a halophilic alkaliphile. Siderophore production, particularly ferrioxamine siderophores, by halophilic and alkaliphilic microorganisms could significantly impact the mobility of metal contaminants present in saline or alkaline environments.
In addition to the ferrioxamine siderophores identified, there were several unidentified compounds that were found in extracts of spent growth medium from H. campisalis. The mass of the parent ion for each compound as well as the predominant fragment ions found in CID spectra are reported in Table 1. While it has not been determined if these compounds can bind ferric iron, the fragment ions appear to be somewhat related to those typical of ferrioxamine siderophores, such as m/z 201 which corresponds to a 5-succinyl-1-amino-5- hydroxyaminopentane in many ferrioxamine siderophores. Additional fragment ions typical of ferrioxamines present in the unknowns are m/z 401 which may correspond to two 5-succinyl-1-
57 amino-5-hydroxyaminopentane groups. Additional experimentation to determine the configuration and iron binding ability of these siderophores is underway.
3.5 CONCLUSION
Siderophores, in particular the ferrioxamine siderophores, have been shown to form stable complexes with a plurality of toxic heavy metals and radionuclides. Complexation of environmental contaminants with microbial exudates such as siderophores could potentially affect equilibrium concentrations and speciation beyond what is predicted by existing models.
Production of ferrioxamine siderophores has been documented in a very diverse group of bacteria, such as Gram-positive Streptomyces, and now includes a halophilic alkaliphile from the genus Halomonas. This work is the first report of ferrioxamine siderophore production by a microorganism that thrives under conditions of high pH and salinity. Because the ability to synthesize ferrioxamines is widespread amongst very diverse bacterial species, it is likely that ferrioxamine production is not unique to H. campisalis and that other halophilic and alkaliphilic bacteria may produce ferrioxamine siderophores. Further characterization of siderophore production by halophiles and alkaliphiles is needed to adequately address the impact that these organisms could have on metal contaminant mobility in saline and alkaline environments.
3.6 ACKNOWLEDGEMENTS
The authors would like to acknowledge the Inland Northwest Research Alliance for providing the funding for this research through both a research grant (FHDGSKA) and a graduate fellowship. The LC-MS instrument used for siderophore identification was provided by
58 the Defense University Research Instrumentation Program (DURIP) Contract Number:
W911NF0510255.
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68 List of Figures
O O N NH OH HO O NH OH NH O O HO O N OH O O O N OH O OH O H NH NH O N O HO O O
HO
O OH O O NH N NH OH OH ONH O OH O HO O OH OH HO NH N OH O O OO O HO OH O OH
Figure 1. Examples of siderophores: (a) the hydroxamate siderophore desferrioxamine E; (b) the catecholate siderophore, enterobactin; and the carboxylate siderophores (c) aerobactin and (d) rhizoferrin.
69 3 600
2.5 500
2 400
1.5 300 OD 600 nm
1 200 M Siderophore m 0.5 100 (desferrioxamine equivalent)
0 0 0 20 40 60 80 100 120 140 160 180 200 Time, hours
OD600 deferrated medium OD600, standard medium siderophore production, deferrated medium siderophore production, standard medium
Figure 2. Siderophore production by H. campisalis with respect to growth at pH 10 and 10%
NaCl.
70 DFE
2000 1
1750 DFG
1500 3 Unknown m/z = 599.8 7 1250 DFX DFX 1000 Unknown m/z = 501.4 2 750 DFD Unknown m/z = 617.8 1 t 500 Unknown m/z = 585.5 Absorbance, 210 nm [mAU] DFG 250
0 25 30 35 40 45
Time, minutes
Figure 3. HPLC Chromatogram of desferrioxamine siderophores produced by H. campisalis grown at pH 10 and 10% NaCl.
71 a) x108 619.4
1.5
1.0
0.5 319.3 310.3 0.0 519.4 x106 319.3
6 401.3 Intensity, counts Intensity,
4 201.2
2 301.2 501.3 619.4 419.4 183.2 283.1 483.3 0 150 200 250 300 350 400 450 500 550 600
m/z
b) 501 419 301 219 101 = y O OH O O
NH N OH H2N N NH N
OH O O OH O b = 119 201 319 401 519
Figure 4. a) Mass spectral data for m/z = 619.5. The total mass spectrum is shown at an elution time of 29.5 minutes (top) and the CID spectrum (bottom) of m/z = 619.4 shows the fragment ions of this species. b) Structure of desferrioxamine G1 showing the fragment analysis from CID mass spectrometry.
72 a) x108
1.5 573.4
1.0
0.5 601.3 0.0 x106 373.2 401.2 8 Intensity, counts Intensity, 6
4 173.1 201.1
283.1 419.3 2 355.2 183.1 255.2 473.3 555.2 219.1 319.3 455.2 0 150 200 250 300 350 400 450 500 550 600
m/z a m/z = 173 : c-e’; d-f’ f NH N b) c) b’ O O HO m/z = 201 : a-c’; b-d’; e-a’; f-b’ f’ O a’ NH O m/z = 255 : c-f’ b e m/z = 283 : a-d’; e-b’ N e’ OH c’ OH m/z = 373 : a-e’; c-a’; d-b’ O N d’ m/z = 401 : e-c’ N c m/z = 455 : a-f’; c-b’ H d O
Figure 5. a) Mass spectral data for m/z = 573.4. The total mass spectrum is shown at an elution time of 34.7 minutes (top) and the CID spectrum (bottom) of m/z = 573.4 shows the fragment ions of this species. b) List of the fragment ions as they correspond to specific locations on the structure of desferrioxamine X7. Fragments resulting from breakages at c and e’ and d and f’
(m/z = 173) indicate the presence of a succinyl-amino-hydroxyaminopropane, indicative of desferrioxamine X7 not desferrioxamine X1. c) Fragmentation points of ferrioxamine X7.
73 a) x107 587.4 4 3
2 601.3
1 399.3 283.2 0 x105 387.2 201.2 401.2 Intensity, counts 2 187.2
1 283.2 569.2 501.2 301.2 469.2 169.2 219.2 369.2 0 150 200 250 300 350 400 450 500 550 600 m/z
f O b) NH c) a N f’ HO m/z = 187 : e-a’; f-b’ m/z = 287 : e-c’ O a’ b’ e O m/z = 201 : a-c’; b-d’; m/z = 387 : c-a’; f-d’ O HN N e’ b m/z = 401 : a-e’; b-f’; OH c-e’ ; d-f’ c’OH m/z = 269 : e-b’ m/z = 469 : c-b’; e-d’ O H N m/z = 283 : a-d’; c-f’ m/z = 483 : a-f’ N d d’ c O
Figure 6. a) Mass spectral data for m/z = 587.4. The total mass spectrum was taken at an elution time of 36.1 minutes (top) and the CID spectrum (bottom) of m/z = 587.4 shows the fragment ions of this species. b) List of the fragment ions as they correspond to specific locations on the structure of desferrioxamine D2. Fragments resulting from breakages at e-a’ and f-b’ (m/z = 187) indicate that a portion of the molecule contains a succinyl-amino-hydroxyaminobutane, indicative of desferrioxamine D2. Other fragments suggest succinyl-amino- hydroxyaminopentane fragments like those seen in DFE
74 8 a) x10 601.4 4 3 2 1 0 401.2 x107 201.1 1.5 Intensity, counts
1.0 283.1 301.2 0.5 166.0 219.1 319.3 383.3 183.1 419.3 483.3 583.3 339.2 501.3 0.0 200 300 400 500 600 m/z b) c) a O b’ m/z = 201 : a-c’; b-d’; c-e’; d-f’; e-a’; f-b’ N NH b f HO O m/z = 283 : a-d’; c-f’; e-b’ NH a’ O O HO m/z = 319 : b-e’; d-a’; f-c’ c’ N f e’ m/z = 401 : a-e’; b-f’; c-a’; d-b’; e-c’; f-d’ e ’ N OH O d’ c m/z = 483 : a-f’; c-b’; e-d’ H N m/z = 519 : b-a’: d-c’; f-e’ O d
Figure 7. a) Mass spectral data for m/z = 601.4. The total mass spectrum was taken at an elution time of 38.2 minutes (top) and the CID spectrum (bottom) of m/z = 601.4 shows the fragment ions of this species. b) List of the fragment ions as they correspond to specific locations on the structure of desferrioxamine E, which is shown graphically in c).
75 a) x108 615.4 2.0
1.5
1.0
0.5 401.4 601.4 0.0 415.1 6 x10 215.2 Intensity, counts. 1.5 401.2
1.0 483.3 201.3 0.5 333.4 283.2 0.0 100 200 300 400 500 600 m/z f c) NH b) m/z = 201 : c-e’; d-f’; e-a’; f-b’ O a HO N m/z = 215 : a-c’; b-d’ f’ O a’ O e O b’ m/z = 283 : c-f’; e-b’ NH b e’ c’ N OH m/z = 333 : b-e’; f-c’ OH O N m/z = 401 : c-a’; d-b’; H d’ m/z = 415 : a-e’; b-f’; e-c’; f-d’ N c
m/z = 483 : c-b’ d O
Figure 8. a) Mass spectral data for m/z = 615.4. The total mass spectrum was taken at an elution time of 46.4 minutes (top) and the CID spectrum (bottom) of parent ion m/z = 615.4. b) A list of the fragment ions as they correspond to specific locations on the structure of desferrioxamine X3.
Fragments resulting from breakages at a-c’ and b-d’ (m/z = 215) indicate that a portion of the molecule contains a succinyl-amino-hydroxyaminohexane, indicative of desferrioxamine X3.
Other fragments suggest two additional succinyl-amino-hydroxyaminopentane fragments like those seen in DFE.
76 7 a) x10 3 519.3
2
619.4 1 401.3 589.3 573.4 0 5 X10 419.3 8 301.2
Intensity, counts Intensity, 6 201.2
4 219.2 283.2 2 154.2 319.3 401.2 501.2 0 200 300 400 500 600 m/z b) 401 319 201 119 OH O O OH
NH N NH NH H NH N
O OH O 119 201 319 401
Figure 9. a) Mass spectral data for m/z = 519.5. The total mass spectrum is shown at an elution time of 33.3 minutes (top) and the CID spectrum (bottom) of m/z = 519.4 shows the fragment ions of this species. b) Structure of desferrioxamine G1t showing the fragment analysis from
CID mass spectrometry. This molecule is similar to desferrioxamine G1 but lacks the c-terminal succinyl group, which corresponds to a mass difference of 100 amu.
77 Table 1: Fragmentation details of unidentified “ferrioxamine-like” compounds isolated from low-iron culture supernatant of H. campisalis.
Parent Ion Retention Daughter fragments for iron free form m/z Time [min] 483, 401, 385, 367, 303, 283, 267, 219, 585.4 32.4 201, 185, 168 584, 501, 483, 419, 399, 316, 301, 283, 617.4 37.3 219, 201
501.4 37.6 483, 401, 301, 283, 201, 165
566, 483, 401, 399, 366, 316, 383, 219, 599.7 41.5 201
78 CHAPTER FOUR
Novel Amphiphilic Siderophores Produced by a Bacterium Isolated from a Soda Lake
ABIGAIL M. RICHARDS1, ROBIN GERLACH2, BRENT M. PEYTON2* and WILLIAM A. APEL3
To be submitted to Extremophiles
1 Department of Chemical Engineering, Washington State University, Dana Hall 118, Spokane
St. Pullman, WA 99164-2710, USA
2 Department of Chemical and Biological Engineering, Montana State University, 303 Cobleigh
Hall, PO Box 173920, Bozeman, MT 59717-3920
3 Department of Biological Sciences, Idaho National Laboratory, 2351 N. Boulevard, PO Box
1625, Idaho Falls, ID USA, 83415
* Corresponding author phone: (406) 994-2221
FAX: (406) 994-5308
Email: [email protected]
79 4.0 ABSTRACT
There are few published reports of siderophore production by halophilic or alkaliphilic
microorganisms. Eight siderophore producing halo-alkaliphiles were isolated from Soap Lake, a
soda lake located in eastern Washington State, USA. Of these isolates, several were found to
belong to the genus Halomonas. The isolate SL28, most closely related to Halomonas
pantelleriense, produces a new family of six amphiphilic siderophores that we have named
sodachelins. The sodachelins are composed of a common, iron-coordinating peptidic head group
consisting of seven amino acids linked to fatty acid carbon chains that range in length from 10 to
14 carbons. The iron coordinating groups include two hydroxylated and acetylated ornithine
residues and one b-hydroxyaspartate residue. When exposed to UV light, these siderophores facilitate a photolytic reduction of Fe(III) to Fe(II) along with a cleavage of the ligand located at the b-hydroxyaspartate residue. To our knowledge, this is the first characterization of this novel amphiphilic siderophore structure or any siderophore produced by a bacterium from a soda lake environment. With the low Fe(III) availability at pH 9-10, we suggest that siderophore production may be very prevalent in saline and alkaline environments, such as soda lakes, and furthermore, may be an important component in the biogeochemical cycling of iron in these systems.
80 4.1 INTRODUCTION
With the exception of the Lactobacilli and Borellia bergdorferi, iron is essential for the growth of all known microorganisms. Iron is necessary for growth and multiplication, and it is a key component in numerous enzymes as well as synthesis of DNA precursors (Harrison and
Morel, 1986; Raven, 1990; Andrews et al., 2003). While it is the fourth most abundant element in the earth’s crust, iron is a limiting nutrient in many aerobic environments due to the formation
-38 of highly insoluble ferric hydroxides. The solubility product of Fe(OH)3 is approximately 10 so by calculation, the concentration of Fe(III) at neutral, saturated aerobic conditions is 10-18 M in the absence of any external Fe(III) chelators. Dissolved iron in freshwater is found in two predominant forms: either as various types of colloidal Fe(III)-(oxy)hydrides or complexed to dissolved organic matter. To overcome the scarcity of iron under iron-limited conditions, many aerobic microorganisms secrete siderophores, which are low molecular weight, chelators with high specificity for ferric iron. These siderophores competitively sequester ferric iron from the environment to support microbial growth. The siderophore complex, once formed, is recognized by its cognate receptor expressed on the bacterial outer membrane, which catalyzes the internalization of the Fe(III) siderophore complex.
Typical Fe(III)-coordinating groups found in siderophores include hydroxamate and catecholate moieties as well as a-hydroxy carboyxlic acid groups, such as citrate or b-hydroxy
aspartate as shown in Figure 1. In early research, siderophores containing hydroxamate groups
were thought to be produced only by fungi, such as the siderophore coprogen, while bacteria
were thought to produce only catecholate based siderophores, such as enterobactin. This
delineation has been disproved by the discovery of multiple bacteria that synthesize hydroxamate
81 based siderophores. The a-hydroxy carboyxlic acid functional groups are found in siderophores
produced by both marine and terrestrial organisms, and include siderophores such as rhizoferrin
and aerobactin (Butler, 2005). In aquatic environments such as the ocean, dissolved iron occurs
almost entirely in the form of complexes with strong organic ligands, most of which are
presumed to be of biological origin (Glehill and Van den Berg, 1994; Rue and Bruland, 1995;
Powell and Donat, 2001; Gress et al., 2004).
Several unique siderophores have recently been identified in marine isolates, a number of
which contain a-hydroxy carboyxlic acid moieties such as b-hydroxyaspartic acid or citric acid
(Butler et al. 2005). The unique marine siderophores recently isolated include suites of amphiphilic siderophores that have various fatty acids appended to peptidic head groups which contain the iron coordinating functional groups such as hydroxamates and b-hydroxyaspartic
acid (Figure 2). A number of siderophores with a-hydroxy carboyxlic acid moeities produced by marine organisms have been found to undergo a light induced ligand to metal charge transfer that results in the reduction of Fe(III) to Fe(II) and decarboxylation of the ligand (Barbeau et al.,
2001; Barbeau et al., 2002; Bergeron et al., 2003). While these a-hydroxy carboyxlic acid groups are present in siderophores produced by terrestrial microorganisms, their habitats, primarily enteric environments or subsurface soils, preclude them from much exposure to sunlight or UV radiation, and thus the photoreduction of iron involving these siderophores under such conditions insignificant, although photoreduction is quite possible if exposed to sunlight. In marine environments, where the euphotic zone extends up to 40 m, siderophores with a-hydroxy carboyxlic acid moeities may play a significant role in the photochemically mediated redox cycling of iron in ocean surface waters (Barbeau et al., 2003).
82 Historically, siderophore research has been focused on the iron sequestration mechanisms of pathogenic bacteria, but has recently expanded to examine siderophore production by microbes in the rhizosphere and aquatic environments. While the number of identified siderophores from marine environments has increased, very little is known of iron accumulation strategies utilized by halophilic and alkaliphilic microorganisms that may inhabit saline and alkaline environments such as soda lakes. Halophiles include members of the bacterial, archaeal and fungal domains. Extreme halophiles are almost exclusively composed of members of the archaea and many grow optimally at salt concentrations near saturation (Kushner and
Kamenkura, 1988). Moderate halophiles grow optimally between 0.5 to 2.5 M salt; halotolerant microbes may tolerate up to 2.5 M salt, but grow optimally at concentrations below 0.5 M
(Kushner, 1978; Larsen, 1986; Grant et al., 1998). Alkaliphiles grow optimally at pH values greater than 9 and grow very slowly, or not at all, at the near neutral pH value of 6.5. Quite frequently, alkaliphilic microbes may have optimum growth at a pH between 10 and 12
(Horikoshi, 2004). Haloalkaliphiles require both elevated salt concentrations and high pH for optimal growth.
Soda lakes represent a specific type of saline lake, in which sodium carbonate and sodium bicarbonate are a dominant fraction of the soluble salts. The high buffering capacity of this system maintains a very stable, high-to-extremely high pH of approximately 9.5-10.5. These conditions are rarely found in other natural ecosystems (Sorokin and Kuenen, 2005). Soda lakes are typically located inland, in arid locations, such as the East African Rift Valley, Libyan Desert
83 and in the western mountain rain-shadowed desert of the United States. Quite frequently, the salt
concentration in soda lakes far exceeds that found in oceanic environments (Hammer, 1986).
Soap Lake is a soda lake located in Grant County in central Washington State, USA. It is
the terminal lake in a series of lakes that are characterized by increasing salinity and alkalinity.
This is a meromictic lake which possesses two distinct, permanently stratified layers as a result
of subsurface topography and a high dissolved solids concentration in the lower depths of the
lake. These two layers are estimated to have not mixed for upwards of 2000 years (Patel et al. in
preparation). Soap Lake is fed by surface water runoff and has no outlet, which, over time, has
resulted in high concentrations of sodium carbonate (6870 mg/L), sodium bicarbonate (5209
mg/L) and sulfate (12800 mg/L) depending on depth, contributing to a high alkalinity and a pH
averaging 9.9 throughout the lake. The monimolimnion layer, or the bottom layer of the lake, is
anoxic and is characterized by cold temperatures of 6 to 8oC, high dissolved solids reaching 140 g liter-1 and some of the highest concentrations of sulfide (12800 mg/L) ever recorded in natural
waters (Sorokin et al., 2007). The upper layer of the lake, termed the mixolimnion layer, is
aerobic and can be classified as a brackish environment because it contains approximately 15 g
liter-1 dissolved solids. These two layers are separated by an abrupt chemocline located at a
depth of approximately 20-23 m. The dissolved iron concentration throughout Soap Lake is low
enough to limit the growth of its microbial community in the aerobic mixolimnion layer, which
raises the questions regarding the mechanisms that these microbes utilize for iron acquisition.
Little is known about the methods used by halophilic and alkaliphilic organisms to
acquire iron in environments where it is scarce. The production of siderophores by several
84 halophilic archaea was recently reported, but a detailed structural characterization of these
siderophores, other than chemical tests was not provided (Dave et al., 2006). Of the archaea
tested by Dave et al., five haloarchaea, including Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterringena turkmenica, Halogeometricum sp. and an alkaliphilic Natrialba sp. were reported to produce siderophores, all of which contained carboxylate groups.
Halomonas aquamarina, a marine isolate also of the Halomonadaceae, was found to produce a suite of amphiphilic siderophores known as the aquachelins (Martinez, 2000) and a marine
Vibrio was found to the siderophore ferrioxamine G1 (Martinez et al., 2001). The aquachelins are photoreactive when complexed with Fe(III). In addition to Halomonas aquamarina, several species of Halomonas have been isolated from marine environments (Fuiimoto, 2006; Kaye et al., 2004; Romanenko et al. 2002). Halomonas species are ubiquitous in soda lakes (Jones et al.,
1998; Ventosa at al., 1998) and it is possible that some of the traits found in the aquachelins, may be mirrored in siderophores produced by microorganisms inhabiting soda lakes.
We investigated siderophore production by microorganisms inhabiting Soap Lake by enriching sediment and water samples taken from both the monimolimnion and mixolimnion layers under aerobic, saline and alkaline conditions. One isolate, SL28, was found to produce a suite of six amphiphilic siderophores. In this paper, we report the structural characterization of this new siderophore family, named the sodachelins, and their ability to mediate the photochemical reduction of iron.
4.2 MATERIALS AND METHODS
4.2.1 Sample collection. Four samples were obtained from Soap Lake (Washington State) in the spring of 2004. These consisted of sediment and water samples from the mixolimnion and the
85 monimolimnion layers. Five strains of Halomonas were obtained from Dr. Russell Vreeland of
Westchester University (Westchester, PA) which included H. elongata, H. halmophila, H.
magadiensis, H. meridiana, and H. variablis.
4.2.2 16S rRNA sequencing. DNA was extracted from siderophore producing colonies on plates using a Bio 101 DNA extraction kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. The 16S rRNA genes were amplified by the polymerase chain reaction (PCR) using 8f and 1492r primers as described in Amann et al. (1995) and Lui et al.
(2002). The PCR conditions were: approximately 10 ng DNA added to the appropriate amount of
Eppendorf MasterMix (Eppendorf North America, Westbury NY) which contained 0.06 U/mL
Taq DNA polymerase, and 2.5x Taq reaction buffer with 125 mM KCl, 75 mM Tris-HCl pH 8.4,
4 mM Mg2+, 0.25% Nonidet-P40 and 500 µM each of dNTP. The PCR was run in an Eppendorf
Mastercylcler thermal cycler. The temperature profile was 94oC for 3 minutes followed by 30 cycles of 90oC for 1 min, 50oC for 1 min, 72oC for 1 min followed by a final step at 72oC for 10 minutes. The PCR products were sequenced at Laragen Inc. using standard bacterial primers.
4.2.3 Growth Medium
4.2.3.1 Initial enrichment and growth medium preparation. Lake sediment and water samples were initially enriched using an Enrichment Medium (EM) which contained the following
. . components (g/L): NaCl, 50.0; Na2B4O7, 1.12; NH4Cl, 1.0; CaCl2 2H2O, 0.06; MgCl2 6H2O,
0.05; NaNO3, 0.85; KH2PO4, 0.50; KCl, 0.01; tryptic soy broth, 0.50; a-ketoglutaric acid, 0.50;
C3H5O3Na, 0.79; C2H3O2Na, 0.58; sodium pyruvate, 0.50. A trace metals solution was added to
. the enrichment medium at 10ml/L which contained the following (mg/L): H3BO4, 6.0; CoCl2
86 . . . . 6H2O, 12.0; CuCl2 2H2O,1.50; MnCl2 4H2O, 10.0; NiCl2 6H2O, 2.50; Na2MoO4 2H2O, 2.50;
ZnCl, 7.0. The pH of the medium was adjusted to 9.0 with 10 N NaOH. Solid medium was prepared with the addition of 15 g/L technical agar. For initial enrichments from sediments, approximately 20 g of mixolimnion and monimolimnion sediment were supplemented with 80 ml of the EM and shaken aerobically at 30oC in 250 ml baffled flasks. For the monimolimnion and mixolimnion water samples, 50 ml of EM was added to 50 ml of the respective Soap Lake water samples and these were also shaken aerobically at 30oC in 250 ml baffled flasks. After approximately one week, all liquid and sediment enrichments were markedly turbid. The enrichments were then streaked for isolation on chrome azurol S (CAS) agar plates to assay for siderophore production (Schwyn and Neilands, 1987) described below. The plates were incubated at 30oC and monitored for the development of orange halos surrounding the colonies which indicates siderophore production. Colonies that were positive for siderophore production were removed and maintained aerobically in either solid or liquid medium using EM that had not been subjected to deferration.
4.2.3.2 Growth medium for Halomonas strains. The five known strains of Halomonas species, H. elongata, H. halmophila, H. magadiensis, H. meridiana, and H. variablis, used in this study were maintained on a growth medium consisting of the following (g/L): casamino acids with vitamins, 7.5; proteose peptone #3, 5.0; yeast extract, 1.0; tri-sodium citrate, 3.0; MgSO4·7H2O,
20.0; K2HPO4 0.5; KCl, 2.0; NaCl 80.0. The pH was adjusted to 8.0 using 10 M NaOH and then the medium was then sterilized by filtration. Solid medium was prepared by adding agar at a concentration of 15 g/L to the nutrient medium. Siderophore production was determined using the CAS agar plate assay. For this, undefined components (proteose peptone and yeast extract)
87 were deferrated (Domingue et al. 1990) and sodium citrate was eliminated from the medium
because it interfered with the CAS assay.
4.2.3.3 Iron removal from complex media components. Iron was removed from all undefined
portions of growth media, such as yeast extract or TSB, used for siderophore studies using the
method of Domingue et al. (1990) which results in up to 98% removal of iron in the case of TSB.
Chelex 100 resin was purchased from Sigma Chemical and used according to the manufacture’s
instructions.
4.2.3.4 Iron limited medium for Halomonas strain sp. SL28. Isolate SL28 was grown in an iron
limited medium which contained the following in g/L: NaCl, 50.0 g; Na2B4O7, 1.12 g; NH4Cl,
. . 1.0 g; CaCl2 2H2O, 0.06 g; MgCl2 6H2O, 0.05 g; NaNO3, 0.85 g; KH2PO4, 0.50 g; KCl, 0.01 g; deferrated yeast extract, 0.25 g; sodium pyruvate, 5g. This was an iron limited medium which stimulated siderophore production, and it also permitted high cell densities, which enhanced the iron-stress seen by the cells and maximized siderophores concentrations detected in the medium supernatant.
4.2.4 Siderophore detection and characterization. The presence and quantification of siderophores was determined using both the CAS liquid and agar plate methods (Schwyn and
Neilands, 1987). The Arnow method was used to assay for the presence of catecholate groups, and 1,3-dihydroxybenzoate was used as a positive control (Arnow, 1937). The Csaky method was used to determine the presence of hydroxamate moieties and for this, the trihydroxamate desferrioxamine B (Sigma Chemical, St. Louis MO) was used as a positive control (Csáky,
88 1948). In experiments where siderophore production was monitored with respect to bacterial
growth, liquid samples were removed at regular intervals, the optical density was measured at
600 nm, and the siderophore concentration in the cell free supernatant was quantified by the CAS
assay relative to standards prepared using desferrioxamine B (Schwyn and Neilands, 1987).
4.2.5 Siderophore isolation. Two liters of deferrated high cell growth medium in acid washed
flasks with extra deep baffles were inoculated with a 24 hr culture of Halomonas sp. strain SL28.
These cultures were shaken aerobically at 150 rpm at a temperature of 30 oC. After approximately 90 hours of incubation the presence of siderophores was confirmed using the CAS liquid assay. Cells were removed by centrifugation at 6000 g for 20 minutes, the cell pellet was discarded, and the supernatant was retained. To remove the siderophores from the high ionic strength medium, the supernatant was first passed through Bond Elut solid phase extraction C2 cartridges (Varian Inc, Palo Alto, CA) which were conditioned following the manufacturers instructions. Siderophores were then eluted with 100% methanol. The media supernatant which passed through the C2 cartridges was assayed for siderophore activity using the CAS liquid assay. Supernatant still retaining siderophore activity was reapplied to a fresh C2 Bond Elut cartridge and eluted with 100% methanol. The crude siderophore extract was dried in a rotary evaporator and resuspended in nanopure water (ddH2O) and 0.01% trifluoroacetic acid (TFA).
This was applied to a 15cm x 4.6 mm Supelcosil LC-8 column (Supelco, Bellefonte, PA) in 200 mL aliquots and the siderophores were purified using a gradient which began at 80/20 (%A/B) for one minute and increased linearly to 40/60 (% A/B) by 60 minutes. For this method, A=
99.99% ddH2O and 0.01% TFA and B=80% acetonitrile (ACN), 0.01% TFA, 19.99% ddH2O.
The flowrate was 1.0 ml/min. This was followed by column regeneration at 100% B for 10
89 minutes and then reconditioning at 80/20 (%A/B) for 20 minutes. The absorbance of the eluent was monitored at 210 nm and siderophore activity was monitored by the CAS liquid assay. The siderophores eluted as six fractions over 40 to 55 minutes. Siderophore active fractions were labeled SL28 A-F and later designated sodachelins A, B, C, D, E and F. A second HPLC separation for the purpose of final purification of each siderophore prior to MS analysis on the same column used a gradient of 100% A to 60% B over 25 minutes.
4.2.6 Structure determination. Electrospray mass spectrometry (ESI-MS) was performed using an Agilent Something 6300 series Agilent SL ion trap mass spectrometer. An Agilent
1100 liquid chromatography system was attached to the ESI-MS system and LC-MS experiments could be run in tandem. Single mass spectra were generated online by MS analysis during LC runs while MS/MS analyses were obtained with directly injected ESI-MS/MS using collected fractions that contained a single siderophore. For LC-MS experiments, samples were loaded and run using the same column and gradient described previously. LC-MS analyses were conducted on the siderophores in the iron-free form as well as the ferrated form. High resolution MS/MS experiments were performed using an Applied Biosystems QSTAR XL Hybrid LC/MS/MS
System, and exact mass determinations were made with a Bruker Microtof (ESI-TOF).
4.2.7 Photochemical Experiments. Ferric nitrate was added to individual sodachelins purified in the iron-free form and the Fe(III)-sodachelin complexes were repurified via HPLC using the short LC method described in section 2.4. The individual ferric sodachelins were evaporated to dryness in a rotary evaporator and resuspended in water buffered to pH=9.9 with a buffer containing 6870 mg/L sodium carbonate and 5209 mg/L sodium bicarbonate to represent the
90 alkalinity of Soap Lake. These solutions were placed in 30 ml quartz tubes and exposed to
simulated sunlight in an environmental chamber equipped with a solar simulator that used a
metal-halide lamp (MHG) (K.H. Steuemagel Light Systems (KHS), Germany) to provide the
source of simulated solar radiation. The lamp was controlled by a computer program which
could vary the lamp irradiance by adjusting the power input from 0-1140 W/m2, measured one
meter from the lamp. For the photochemical experiments performed here, lamp power was
2 adjusted to produce 565 W/m , which is the average of the total global irradiation measured
between the hours of 09:00 to 16:00 at a solar monitoring station located in Cheney, Washington
over the months of June through August. This location is near Soap Lake and similar in
elevation and weather patterns so solar radiation is likely comparable. The temperature within
the chamber was computer controlled such that the Fe(III)-sodachelin samples within the quartz
tubes remained at 20oC.
The complexes of Fe(III)-sodachelin C through F were exposed to the simulated sunlight for 25 hours at an irradiance of 565 W/m2. Completion of the reaction was determined by monitoring the decrease in the original parent ion of the Fe(III) siderophore using mass spectrometry. For experiments to determine quantitative Fe(II) production with sodachelin C, the Fe(II) chelator bathophenanthroline disulfonate (BPDS) was added to a 15 mM solution of sodachelin C in the sodium bicarbonate buffer (pH = 9.9) such that the BPDS was at a final concentration of 150 mM. Fe(II) reduction and release during the photolysis of the Fe(III)-sodachelin complexes in the simulated sunlight could be determined by the absorbance of the Fe(II)-BPDS complex at 536 nm which increased as the reaction progressed. Light-free and siderophore-free controls were also analyzed for Fe(II) chelation.
91 4.2.8 Fatty acid analysis. Fatty acid analysis of the sodachelin siderophores was completed by
Microbial ID, a division of Midi Laboratories, Inc. (Newark, DE). Concentrated preparations of each purified sodachelin were submitted to Microbial ID for direct fatty acid methyl ester analysis (FAME). Fatty acids were identified by gas chromatography using Midi Labs Sherlock
Identification System and Eukary peak naming software.
4.3.0 RESULTS
4.3.1 Isolate identification. Siderophore producing isolates were enriched via CAS agar plates from sediment and water samples taken from both the mixolimnion and monimolimnion of Soap
Lake. In total, 30 isolates were obtained, and of these, nine were unique as determined by 16S rRNA gene sequencing. The closest match as identified by a BLAST search for each isolate is shown in Table 1. Since a number of these Soap Lake isolates belonged to the genus
Halomonas, five additional strains of Halomonas including, H. elongata, H. halmophila, H. magadiensis, H. meridiana and H. variablis were obtained and assayed for siderophore production on CAS agar plates. Each of the Halomonas strains showed an orange halo surrounding bacterial growth indicating siderophore production. The Csáky and Arnow assays were used to determine the presence of hydroxamate or catecholate moieties. Unfortunately, some of the Soap Lake isolates and Halomonas strains used in this study did not generate measurable amounts of siderophore activity in the iron limited, liquid media in which they were grown. Of the several strains that did produce significant amounts of siderophores in liquid medium, all were found to contain hydroxamate functionality by means of the Csáky assay as shown in Table 1, while catecholate moieties were not detected. Isolate SL28, most closely related to Halomonas pantelleriense, produced a significant amount of siderophore (~ 120 mM
92 equivalence of DFB) when grown in an iron limited liquid medium and was selected for further
structural characterization. As shown in Figure 3, siderophore production by SL28 reached a
maximum after approximately 96 hours of growth in mid-stationary phase. This siderophore was
found to contain hydroxamate groups according a positive response to the Csáky assay. The
Arnow assay indicated that this siderophore did not contain catecholate groups.
4.3.2 Siderophore isolation. Figure 4(a) contains an HPLC/UV chromatogram that shows the
elution of six peaks over an acetonitrile concentration of approximately 40-50%, which indicated
the production of a suite of compounds. Fractions collected for each of these peaks showed
siderophore activity by the CAS assay and by the development of a red color following the
addition of Fe(NO3). The retention time of these siderophores was much greater than those of the hydroxamate ferrioxamine siderophores, indicating the siderophore suite produced by SL28 was a larger molecular weight or possessed more non-polar characteristics. As shown in Figure
4(b), the addition of iron to the crude siderophore extract showed the elution of the same number of peaks, but at earlier retention times. Absorbance was lower in this case because after the addition of iron to the crude extract, it was repurified with bond elut cartridges to remove free iron from the mixture. The final concentration of siderophores was slightly diluted after this process. For the culturing conditions in this study, the siderophores isolated from the spent cell free medium were predominantly in the iron-free form.
4.3.3 Structure determination. LC/ESI-MS analysis of the siderophore showed the presence of
singly protenated [M+H]+ and doubly protenated [M+2H]2+ siderophores. The simultaneous LC-
MS analysis showed a series of siderophores with mass to charge ratios, for the singly charged
93 form, of 1078.5, 1122.5, 1104.5, 1106.5, 1132.5 and 1134.5, in order of elution. There was a 2 amu difference between m/z=1104.5 and m/z=1106.5 as well as m/z=1132.5 and m/z=1134.5 suggesting the possible presence of an unsaturated bond. A 28 amu difference between m/z=1078.5, m/z=1106.5 and m/z=1134.5 as well as m/z=1104.5 and m/z=1132.5, which suggested the presence of 2 additional alkyl groups in the molecules of greater mass. The mass data for each of the siderophores in both the desferri and ferri form as determined by LC/ESI-MS are shown in Table 2. Both the singly protenated and doubly protented forms were subjected to
ESI-MS/MS analysis. As seen in Table 3 all six compounds yielded an identical major series of y fragments: 793, 665, 578, 450, 278 and 191. The major b fragments of each parent ion followed a similar pattern amongst all 6 compounds and reflected the same mass differences of 2 amu and
28 amu seen in the parent compounds, suggesting a common head group attached to a hydrocarbon chain of increasing carbon number or varied level of saturation. The siderophore of m/z=1122.5 differs from the siderophore m/z=1106.5 by 16 amu suggesting the addition of an oxygen atom. By examining the b-fragments it appears that this additional oxygen atom is located on the carbon chain. The collisionally induced disassociation spectrum for sodachelin F is shown in Figure 5. The assignments of y and b fragments as determined by MS/MS data
(shown in Figure 5 for sodachelin F) with respect to the structure are shown in Figure 6.
The fragmentation analysis determined the presence of seven amino acid residues in the following sequence beginning at the N-terminus: N-OH, N-OAc-ornithine, serine, N-OH, N-
OAc-ornithine, glutamine, serine, glutamine and threonine-b-OH-apartate. The isobaric amino acid glutamine was distinguished from lysine using a high resolution MS/MS/TOF analysis shown in Figure 5. A fragment mass of 128.095 was present in all siderophores which indicates
94 glutamine residues. Lysine residues on the other hand would have a fragment mass of 128.059.
The presence of two glutamine residues in the sodachelins is unique. In the aquachelin
siderophores, only one glutamine is present and a serine residue takes the place of the second
glutamine residue in the sodachelins (Martinez et al., 2000). The fatty acid methyl ester analysis
showed the presence of the following fatty acids for sodachelins A-F, respectively: 10:0; 12:0
3OH; 12:1 w7c; 12:0; 14:1 w7c; 14:0. These were consistent with the predicted compositions of each fatty acid as determined by mass spectral data.
4.3.4 Photochemical Experiments. Sodachelin E was purified by LC in the iron complexed form. Fe(III)-siderophore complexes were evaporated to dryness and resuspended in sodium carbonate bicarbonate buffered water at a pH of 9.9. Siderophores were exposed to sunlight for
24 hours. UV-Vis spectra of the sodachelin E before and after light exposure are shown in
Figure 7. ESI-MS analysis of Fe(III)-siderophores shielded from light had an m/z value of
1185.5. This species were no longer present in the light exposed samples. In the mass spectra of light exposed siderophore an intense peak which corresponded to the cleavage of the fatty acid
+ tail (Figure 8). In the case of siderophore E, m/z= 226.3 may correspond to [C12H25NO + H] indicating a singly unsaturated fatty acid tail of 12 carbons and the retention of the amide group from the b-hydroxyasparatic acid residue. The peak at m/z = 846.3 was representative of the peptidic headgroup of the original siderophore complexed with iron, minus the b- hydroxyasparatic acid residue and fatty acid tail. A schematic of the predicted cleavage products is shown in Figure 9. Other cleavage products were also visible with m/z values of 1093.5,
966.4, 918.3 and 874.5. Photochemical experiments were repeated with the inclusion of the
95 Fe(II) chelator BPDS and showed the steady release of Fe(II) with prolonged exposure to
sunlight (Figure 10).
4.4.0 DISCUSSION
4.4.1 Siderophores from saline and alkaline environments. In spite of high abundance in the
earth’s crust, and in many soils and sediments, iron is considered to be a trace element in aquatic
habitats (Schroder et al., 2003). In soda lakes, where the pH is often above 9.0, soluble iron is
generally unavailable (Zavarinza et al., 2006). The saline and alkaline lake, Soap Lake, has iron
concentrations ranging from 0.11 mg L-1 to 0.5 mg L-1 in the water column and 0.08 mg L-1 to
0.5 mg/L-1 in the sediments (Patel et al., in preparation). A significant portion of this iron is
likely in the form of biologically unavailable ferric iron hydroxides. Microbial mechanisms of
iron acquisition in extreme environments may involve the production of unique siderophores or
unique iron transport mechanisms. In the past, the majority of siderophore-based studies have
focused on disease causing or terrestrial microorganisms. Only recently has the focus shifted to
marine organisms, which has resulted in the identification of novel siderophores (Martinez et al.
2000; Martin et al., 2006; Yusia et al., 2005; Hickford et al., 2004; Barbeau et al., 2002). Few
studies have investigated siderophore production by extremophilic microorganisms and to date,
none have sought to identify the structure of siderophores or prevalence of siderophore producers
in a natural soda lake environment, such as Soap Lake.
A number of halo-alkaliphilic isolates obtained from Soap Lake water and sediment
samples produced siderophores as determined by the CAS agar plate assay. Many of these
organisms were very closely related to members of the genus Halomonas. This genus along with
96 Chromohalobacter is included in the family Halomonadaceae which constitutes a diverse group of moderately halophilic microorganisms. Represented in this group are both aerobes and facultative anaerobes. Due to the aerobic conditions employed during the enrichment for siderophore production, it is not surprising that the bulk of the isolates were closely related to members of the genus Halomonas which are ubiquitous moderately halophilic bacteria and are frequently isolated from a wide variety of saline environments throughout the world (John et al.,
1998; Ventosa et al., 1998). The confirmation of siderophore production in five randomly selected Halomonas species, along with previously reported siderophore production by H. campisalis and H. aquamarina suggests that siderophore production may be a trait that is common to many, if not all, Halomonas species. While siderophore production may be a common trait, the siderophores produced apparently may vary greatly from ferrioxamine siderophores produced by H. campisalis to include amphiphilic siderophores such as the aquachelins produced by H. aquamarina and the sodachelins produced by a Halomonas strain closely related to H. pantelleriense isolated from Soap Lake. Currently, the siderophores of another Halomonas sp. isolated in this study, SL01, are under investigation and show no similarity to either the ferrioxamines produced by H. campisalis, H. aquamarina or Halomonas strain SL28 based on both HPLC chromatograms, molecular mass and prelimnary mass spectral fragmentation data (data not shown).
4.4.2 Iron cycling in aquatic environments. Iron reduction has been widely detected in soda lakes and alkaliphilic iron reducing microorganisms were found to reduce amorphous ferric hydroxides. The intensity of this process is thought to be independent of alkalinity (Zavarinza et al., 2006). In fact, numerous iron reducing bacteria have been identified in Soap Lake and recent
97 efforts have resulted in the identification of several novel iron reducing bacteria (Patel et al., in
preparation). Dissimilatory Fe(III) reduction plays a significant role in the biogeochemical
cycling of iron in many aquatic environments. The Fe(II) secreted by iron reducing bacteria
under anaerobic conditions reacts with a number of anions to produce a variety of minerals such
as siderite (FeCO3) , which results from a reaction of Fe(II) with carbonate or viviante
. (Fe3(PO4)2 8H2O), which is the product of Fe(III) reacting with phosphate (Lovley et al., 2001;
Lovley, 1991). Microorganisms are thought to play a predominant role in the biogeochemical cycling of iron. In marine environments, the speciation of dissolved iron has been shown to be dominated by complexation with strong organic ligands (Glehill and Van den Berg, 1994; Rue and Bruland, 1995; Powell and Donat, 2001; Gress et al., 2004). It has been suggested that photochemistry is likely to greatly affect ligand-Fe(III) complex cycling either by the direct photochemical reactions of the free ligands or the reactions of the Fe(III)-ligand complexes which can lead to the photolysis of the ligand and simultaneous reduction of Fe(III) to Fe(II).
4.2.3 Siderophore mediated iron cycling. Many marine and aquatic organisms that produce siderophores are thought to be involved in increasing the soluble fraction of ferric iron in the environment through the production of siderophores. Many of these siderophore-Fe(III) complexes are then directly involved with the cycling of iron through the photomediated reduction of ferric iron in many siderophore-Fe(III) complexes (Barbeau et al., 2001; Barbeau et al., 2003; Martin et al., 2006). This photochemically mediated redox cycling of iron has been shown to involve ligands that contain either a citrate moiety based system, mixed catecholate/a- hydroxy carboxylate ligands or mixed hydroxamate/a-hydroxy carboxylate functional groups
(Barbeau et al., 2003). In the case of a-hydroxy carboxylate siderophores such as marinobactin
98 or aquachelin, this photolytic process involves an oxidative cleavage of the ligand at the site of
the b-hydroxyaspertate residue, resulting in two primary ligand products and the release of Fe(II)
(Barbeau et al., 2001). Generally, one of the ligand products possesses significant ferric iron binding capabilities by retaining a portion of the original iron coordination sites and remains a fully functional siderophore (Barbeau et al., 2001; Barbeau et al., 2002; Kupper et al., 2006).
Ferrous iron has an estimated half-life of two to ten minutes in aquatic environments (Sung and
Morgan, 1980), and while it may be available only fleetingly, it could be directly taken up by the microorganisms or reooxidized to Fe(III) and chelated by another siderophore or siderophore photoproduct (Barbeau et al., 2001; Bergeron et al., 2003; Hickford et al., 2004).
Like the aquachelin and marinobactin siderophores, the sodachelin siderophores also contain an a-hydroxy carboxylate functional group in the form of a b-hydroxyaspertate residue
that is immediately adjacent to the fatty acid chain. The Fe(III)-sodachelin complexes readily
undergo photolysis when exposed to simulated sunlight. This reduction was mediated by a
ligand-to-metal change transfer reaction demonstrated by the UV-VIS spectrum of the
photolysed Fe(III)-sodachelin C complex (Figure 7). The loss of an electronic transition in the
near-ultraviolet centered near 300 nm is thought to correspond to the charge transfer from the b- hydroxyaspertate residue to Fe(III) as seen in Figure 7. The absorbance maxima seen at approximately 430 nm in the UV-VIS spectra of the Fe(III)-photoproduct is indicative of the coordination of iron with hydroxamate functional groups. The photoproducts were found to still retain siderophore binding activity as shown in the MS analysis which indicated the presence of a photoproduct-Fe(III) complex (Figure 8). A schematic of the predicted photoproducts of the sodachelin siderophores are shown in Figure 9. There were a number of photoproducts that were
99 not expected and a more detailed analysis of the photoproducts, including ESI-MS/MS of the photoproducts is currently under investigation.
The aquachelins, which are very similar in structure to the sodachelins, also undergo this photolytic siderophore cleavage and reduction of iron (Barbeau et al., 2001). The photoproduct, in the case of the aquachelins, had an Fe(III) stability constant of 1011.5 whereas the intact siderophore has a conditional stability constant of 1012.5 indicating that the photoproduct remains a viable siderophore. Further experimentation involving the uptake of aquachelin-59Fe(III) and the photolysed aquachelin-59Fe(III) complex by a natural assemblage of planktonic marine organisms showed that the photolysed aquachelin- 59Fe(III) increased the biovailablilty of
59Fe(III) over the intact aquachelin-59Fe(III) complex by twofold. There is a significant similarity of the peptides incorporated in the photoproduct retaining siderophore activity in the aquachelins, sodachelins and marinobactins. This may increase the universal nature of these compounds as a siderophore usable by the surrounding microbial community as a whole.
Since ferrous iron is likely quickly re-oxidized in the aerobic zone of Soap Lake, it is unclear why these microbes would expend extra energy synthesizing a siderophore that may ultimately be cleaved by sunlight. One explanation is that the fatty acid tails are somewhat cell associated and make these types of siderophores more resistant to diffusion away from the cell.
The reduction of iron, if it takes place near the cell surface may allow sufficient time for the bacteria to uptake Fe(II) into their cells via diffusion. It may also be possible that a loosely symbiotic relationship has developed between bacteria and phytoplankton in the euphotic zone of the water column. Iron limitation has been documented in the high-nutrient, low-chlorophyll
100 regions of the open ocean (Behrenfeld and Kolber, 1999; Brand et al., 1983; Fitzwater et al,
1996; LaRoche et al., 1996; Martin et al., 1994). Bacteria producing iron photo-reductive siderophores would increase soluble iron during the photoperiod through the reduction of Fe(III) in the Fe(III)-siderophore complex and subsequent release of Fe(II). Phytoplankton may benefit from the enhanced soluble iron and realized enhanced growth rates. In turn, bacteria may benefit from increased levels of organic matter within the euphotic zone of the water column due to enhanced phytoplankton growth. Furthermore, the siderophore- Fe(III) photoproduct appears to increase biologically available Fe(III) to microbial communities and may be taken up by phytoplankton as well.
4.4.4 Amphiphilicity in siderophores. Amphiphilic siderophores such as the aquachelins, marinobactins and amphibactins, contain a peptidic head that coordinates ferric iron as well as a series of fatty acids that are appended at the amine terminus. The degree of amphiphilicity of the siderophores is affected by both the number of peptides in the headgroup and the length of the fatty acid chain which, in these siderophores, ranges from C-12 to C-18. Some amphiphilic siderophores have been found to be bound to the cell membrane, while others are released into the environment (Martin et al., 2006; Xu et al., 2002). The amphibactins, with a short peptidic head of only four amino acids and long fatty acid chain consisting of C-14 to C-18 fatty acids, are primarily cell associated (Martin et al. 2006). It is possible that these cell associated siderophores have developed over time to address the issue of siderophore diffusion in marine environments. The sodachelins, on the other hand, have a peptidic headgroup which contains seven amino acids and contain shorter fatty acid chains of varied lengths from C10 to C14. The number of amino acid residues in the peptidic portion and the length of the fatty acid chains
101 suggest a chemical nature which remains soluble in polar environments, such as that present in
Soap Lake. While these are indeed extracellular compounds, the sodachelins may have some
limited association with the cells due to the extremely polar nature of the alkaline waters of Soap
Lake which would favor a closer association of the relatively non-polar fatty acid side chains
with the cellular membrane. This affinity could maintain a higher concentration of siderophores
in the vicinity of the microbial growth and prevent siderophore loss due to diffusion into the
environment.
4.5 CONCLUSIONS
To the best of our knowledge, this work is the first documentation of siderophore production in a
soda lake. Siderophore production was detected in a number of bacterial isolates from sediment
and water samples from Soap Lake, a soda lake. Many of the siderophore producing isolates
were of the genus Halomonas, and five other Halomonas species were also found to produce
siderophores. This suggests that siderophore production may be a trait common to the genus
Halomonas. A new family of six amphiphilic siderophores, the sodachelins, is produced by the isolate Halomonas sp. strain SL28. These siderophores bear strong structural resemblance to the
marinobactin, aquachelin and amphibactin siderophores. Furthermore, the sodachelins mediate
the photochemical reduction of Fe(III) which produces Fe(II) and a photoproduct which still
retains Fe(III) binding activity. To date, siderophore production has not been well documented
in saline and alkaline environments and this work demonstrates the presence of siderophores in
such environments and the potential for the discovery of novel siderophores that may be similar
to those found within marine environments.
102 4.6 ACKNOWLEDGEMENTS
The authors would like to thank the Biosciences Department at the Idaho National Laboratory for
the generous use of their Applied Biosystmes QSTAR mass spectrometer for accurate mass
determination of amino acid residues. We also thank our funding source, the Inland Northwest
Research Alliance for providing both project and student support.
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109 List of Tables
Table 1. Closest match of BLAST search on a segment of 16s rRNA gene for siderophore producing isolates in Soap Lake.
% Isolate Closest Match in Blast Search Consensus Location found in Lake Identity
mixolimnion water, SL01 Halomonas variablis strain HTG7 99% 969 mixomolimnion sediment
SL02 b-proeobacterium HTCC 525 99% 713 mixolimnion sediment
SL04 Antartic seawater bacterium R7375 99% 740 mixolimnion sediment
SL11 Halomonas desiderata 99% 740 monimolimnion sediment
SL15 Halomonas nitrotophilus 99% 785 monimolimnion water
SL17 Pseudoalteromonas sp. 95% 372 monimolimnion water
SL28 Halomonas muralis/pantelleriense 97% 785 mixolimnion water
mixolimnion water, SL29 Halomonas sp. Lake Bogoria isolate 8B1 99% 790 monimolimnion sediment, monimolimnion water
110 Table 2. Mass data for siderophores produced by Halomonas sp. SL28 in the desferri and ferri form.
m/z Siderophore desferri ferri sodachelin A 1078.5 1131.5 sodachelin B 1122.5 1175.5 sodachelin C 1104.5 1157.5 sodachelin D 1106.5 1159.5 sodachelin E 1132.5 1185.5 sodachelin F 1134.5 1187.5
111 Table 3. Y fragment m/z values observed by ESI-MS/MS spectrometry of the sodachelins. All fragments include the addition of two protons to create a positive charge on the amine. No y7 fragments were observed under the experimental conditions employed, indicating that a cleavage between the fatty acid tails and peptidic headgroups were somewhat rare. The y fragments also showed a loss of water which likely arises from the serine side-chains.
Assignment Sodachelin A Sodachelin B Sodachelin C Sodachelin D Sodachelin E Sodachelin F
y6 793 793 793 793 793 793
y6 - H2O 775 775 775 775 775 775
y5 665 665 665 665 665 665
y5 - H2O 647 647 647 647 647 647
y4 578 578 578 578 578 578
y4 - H2O 560 560 560 560 560 560
y3 450 450 450 450 450 450
y3 - H2O 432 432 432 432 432 432
y2 278 278 278 278 278 278
y2 - H2O 260 260 260 260 260 260
y1 191 191 191 191 191 191
112 Table 4. B fragment m/z values observed by ESI-MS/MS spectrometry of the sodachelins.
These fragments also typically saw a loss of water, again likely arising from the serine side chains. m/z values corresponding to the fatty acid tails were observed but at very low intensity, indicating that these were not favored fragmentation points under the experimental conditions.
Assignment Sodachelin A Sodachelin B Sodachelin C Sodachelin D Sodachelin E Sodachelin F
b7 888 932 914 916 942 944
b7 - H2O 870 914 896 898 924 926
b6 801 845 827 829 855 857
b6 - H2O 783 827 809 811 837 839
b5 629 673 655 657 683 685
b5 - H2O 611 655 637 639 665 667
b4 501 544 527 529 555 557
b4 - H2O 483 526 509 511 537 539
b3 414 458 440 442 468 470
b3 - H2O ------422 424 450 452
b2 286 330 312 314 340 342
b2 - H2O ------
113 List of Figures
O O N NH OH HO O NH OH NH O O HO O N OH O O O N OH O OH O H NH NH O N O HO O O (a) (b) HO
O OH O O NH N NH OH OH ONH O OH O HO O OH OH HO NH N OH O O OO O HO OH O OH (c) (d)
Figure 1. Siderophores representing hydroxamate, catacholate and a-hydroxy carboxylic acid based structures: (a) desferrioxamine E, (b) enterobactin, (c) aerobactin, and (d) rhizoferrin.
114 O O O O O H N N N N (a) N HO HO (b) HO HO HO O O O O R NH NH NH OH NH NH OH N NH NH R NH NH O H O O O O O OH OH OH
H2N O R= O R= E O I O O D2 H O
D1 OH O G O C OH O O F B O E O A (c) O D O O OH OH O N N C HO HO HO O OH O O O OH O NH NH NH OH B R NH NH NH NH O O O O OH OH
H2N O O D R= O C
O B
O A
Figure 2. Amphiphilic siderophores isolated from marine environments: (a) marinobactins, (b) aquachelins, and (c) amphibactins.
115 1.2 200
180 1.0 160 M
140 m 0.8 120
0.6 100
80 OD 600 nm 0.4 60 equivalent to DFB 40 0.2 Siderophore Concentration, 20
0 0 0 0 20 40 60 80 100 120 140 160 Time, Hrs
OD 600nm Siderophore Concentration, mM
Figure 3. Siderophore production by Halomonas sp. strain SL28 with respect to time. Data points are averages of three replicate experiments and error bars represent the standard deviation.
Error bars are not visible in locations where they do not exceed the data points.
116 1500
1250
1000
750
500
250
0 Absorbance 210 nm [mAU] 10 20 30 40 50 60
Time [minutes]
250
200
150
100
50
0 Absorbance 210 nm [mAU] 10 20 30 40 50 60
Time [minutes]
Figure 4. HPLC/UV chromatograms of sodachelin siderophores eluted from a C8 column. (a)
Shows the elution of siderophores in the desferri form while (b) shows the earlier retention time of the siderophores as they elute in the ferri form.
117 Figure 5. ESI-MS/MS fragmentation spectrum of sodachelin F.
118 793 665 578 450 278 191
CH3 CH3 O O
N N OH HO HO
HO OH O O O O RR NH NH NH O NH NH NH NH
O O O OH OH
H2N O H2N O
344342 470 557 685 857 944
N-OH- N-OH- Thr-b- Gln Ser Gln Ser OH-Asp N-OAC- N-OAC- Orn Orn O R: O A D H3C H3C
OH O O
B H3C E H3C O O
H3C C H3C F
Figure 6. The assignment of y and b fragments as determined by MS/MS data for Sodachelin F.
The y fragments are conserved for each siderophore while the b fragments differ depending on the nature of each fatty acid tail. Fragments corresponding to the fatty acid appendages were seen in very low abundance while those corresponding to the peptidic headgroup (m/z=924) were not observed.
119 0.05
0.045
0.04
0.035
0.03
0.025
0.02 Absorbance
0.015
0.01
0.005
0 240 290 340 390 440 490 wavelength, nm
Figure 7. UV-Vis spectra of Fe(III)-sodachelin F (______) prior to UV exposure and following (------) after UV exposure.
120 x106 593.4 8
6 1185.5
4 Intensity, counts
2 1159.5 620.3 529.5 331.4 0 x10
6 200.3
5 918.3
1093.5 4 226.3 547.3 645.4 966.4 3 676.4 Intensity, countsIntensity,
2 483.8 846.3 719.4 1024.4 423.8 135.2 1 344.3 278.3 0 200 400 600 800 1000 1200 1400 m/z
Figure 8. MS spectrum of sodachelin E (a) prior to UV exposure and after (b) UV exposure.
121 3+ Fe
O O O O
- -N N O O O hn N N - HO HO O OH H O O O O OH H H H O O O R N N N O NH N N N H NH NH NH O NH NH NH O H O H O H OH OH O O O OH OH
H2N O H2N O H2N O H2N O
2+ + Fe + R R=
O O OH O
O O O
Figure 9. Schematic of the potential photolytic reaction pathways of Fe(III)-sodachelin
complexes and reduction of Fe(III) to Fe(II).
122 7
6
5
4
3
2
1 M Fe(II) captured by BPDS m 0
-1 0 200 400 600 800 1000 1200 1400 1600 Time, Minutes
Light exposed sample control
Figure 10. Production of Fe(II) during the siderophore mediated photochemical reduction of
Fe(III) in the Fe(III)-sodachelin F complex.
123 CHAPTER FIVE
Suggestions for future work.
1. Both modified D and L amino acids are often found in bacterial siderophores, which has been proposed as a strategy to avoid peptidase digestion (Teintze et al. 1981). Because of the potential for both L and D amino acids, the true structure of the sodachelins technically is incomplete. Partial hydrolysis of the siderophore can yield peptide fragments with only a few amino acid residues. Chiral amino acid analysis may be performed using HPLC analysis using
Marfey’s chiral reagent, N-a-(2,4-dinitro-5-fluoro-phenyl)-L-alainaminde, (Marfey, 1984) or another method may be to use GC analysis with a chiral column such as HP-Chiral ß columns from Agilent Technologies.
2. Several other siderophore producing bacteria were isolated from Soap Lake. Several of this isolates, including SL01, SL11, SL18 produced significant amounts of siderophore in liquid medium. Preliminary experimentation with at least two siderophores produced by SL01, have shown that the siderophores produced are of a large molecular weight, ~ 1100 Da, and elute at an acetonitrile concentration of approximately 40-50%, like the sodachelins. It is possible that these siderophores are also amphiphilic. Collisionally induced MS/MS data shows no similarity to the siderophores produced by SL28, or other amphiphilic siderophores documented in the literature, suggesting that these may be a new siderophore at well. An initial survey of the MS/MS data left the author struggling for any hints towards the structure. A more global approach towards structure determination is suggested, which would include 1H and 13C NMR, direct and chiral analysis of amino acid hydrolysis products, and additional MS/MS experiments. Prior to these
124 specific structural experiments, it might be possible to determine if this siderophore contains any
a-hydroxycarboxylic acid moieties, in the form of either b-hydroxyaspartate or citric acid, by investigating the potential for this siderophore to photoreduce ferric iron in the siderophore complex. The reduction of ferric iron, to date has been detailed in many bacterial siderophores and appears to be dependent on iron chelating functional groups (Barbeau et al. 2003). An analysis of the photoproducts may yield information on first, if there are any a- hydroxycarboxylic ligands present and then it may be possible to determine if they are amino acid based as in b-hydroxyaspartate or citric acid based.
3. Amphiphilic siderophores are increasing common in marine environments. Prior to recent the discovery of marine siderophores, it was thought, by some, that siderophores were insignificant and expensive for bacteria to produce due to the likelihood that these molecules would be lost in the bulk surrounds by diffusion and convection. The fatty acid portion of amphiphilic siderophores may provide a closer association of the siderophores with the bacterial cell. This association may be even more pronounced in environments that are highly polar – such as soda lakes. A series of membrane partitioning experiments following the procedures outlined by Luo et al. (2005) and Xu et al. (2002), with adjustments made during experiments to account for the higher dissolved solids that may be present in more saline environments.
4. It is somewhat unusual for bacteria to expend energy in situations where they don’t realize
“return” on their investment. On first glance, it might appear very strange for these bacteria to be synthesizing amphiphilic siderophores. When exposed to sunlight, siderophores like the sodachelins, aquachelins and marinobactins cleave at the b-hydroxyaspartate residue, resulting in
125 the loss of that residue and the fatty acid . This results in iron reduction and release, as well as
production of a smaller ligand that still retains some binding affinity for Fe(III). It was shown
that the ligand photoproduct L* is more biologically available to other microorganisms in the
community (Martinez et al. 2001). One question is if the photoproduct which retains siderophore
activity is recycled by the bacteria. Question: Are the fatty acid tail, b-hydroxyaspartate residue and the sodachelin photoproduct reassembled? Method to test question: use purified and labeled Fe(III) sodachelin photoproduct to supplement early stationary phase cultures of
SL28. The early stationary phase cultures will be grown in an iron-limited medium for approximately 2-3 days, centrifuged and resuspended fresh iron-limited medium supplemented with sodachelin photoproduct. After a set period of time, siderophores will be purified using the methods detailed in Chapter 4 and will be analyzed to determine if fatty acid tails and b- hydroxyaspartate residues have been reattached to the labeled siderophore photoproducts.
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126 APPENDIX A: Growth and production of siderophores with respect to pH for Halomonas
campisalis
127 Table A1: Raw data for siderophore production at pH 8 using the optical density at 600nm to track cell growth and the CAS assay determined at 630 nm to determine siderophore production. date time hours pH 8 OD600 pH 8 OD630 OD600 Blank OD 630 Blank 1/3/2003 14:00 81 0.009 1 1.236 0.002 1.166 2 0.01 2 1.15 3 0.007 3 1.199 F 0.01 F 1.273 1/3/2003 22:30 16.51 0.018 1 1.272 -0.001 1.193 2 0.017 2 1.174 3 0.018 3 1.361 F 0.015 F 1.223 1/4/2003 6:00 241 0.046 1 1.258 -0.001 1.184 2 0.044 2 1.145 3 0.044 3 1.291 F 0.047 F 1.212 1/4/2003 16:30 34.51 0.181 1 1.246 0.001 1.168 2 0.175 2 1.099 3 0.189 3 1.266 F 0.194 F 1.191 1/4/2003 22:00 401 0.36 1 1.228 0.033 1.13 2 0.358 2 1.145 3 0.392 3 1.256 F 0.407 F 1.18 1/5/2003 6:00 481 0.767 1 1.207 0.001 1.151 2 0.728 2 1.098 3 0.807 3 1.241 F 0.771 F 1.139 1/5/2003 18:00 601 1.291 1 0.931 -0.001 1.144 2 1.297 2 0.591 3 1.385 3 0.622 F 1.464 F 1.122 1/6/2003 6:00 721 1.644 1 0.05 0.002 1.16 2 1.664 2 0.049 3 1.673 3 0.049 F 1.826 F 0.178 1/6/2003 14:00 801 1.745 1 0.986 0 1.172 2 1.75 2 0.966 3 1.762 3 0.952 F 1.94 F 1.078 1/6/2003 22:00 881 1.831 1 0.918 0 1.186 2 1.836 2 0.905 3 1.839 3 0.931 F 2.047 F 1.02 1/7/2003 6:00 961 1.907 1 0.821 0 1.186 2 1.901 2 0.864 3 1.904 3 0.815 F 2.133 F 0.922 1/7/2003 14:00 104 1 1.948 1 0.751 0 1.147 2 1.94 2 0.73 3 1.948 3 0.71 F 2.187 F 0.858 1/7/2003 22:00 112 1 1.99 1 0.643 0 1.151 2 1.976 2 0.632 3 1.986 3 0.669 F 2.24 F 0.734 1/8/2003 8:00 122 1 2.037 1 0.567 0 1.16 2 2.013 2 0.55 3 2.033 3 0.519 F 2.286 F 0.745 1/8/2003 2:00 128 1 2.058 1 0.5 1.155 2 2.035 2 0.487 3 2.059 3 0.435 F 2.284 F 0.646 1/9/2003 18:00 156 1 2.139 1 0.387 1.155 2 2.108 2 0.397 3 2.142 3 0.344 F 2.245 F 0.647 1/13/2003 18:00 252 1 2.11 0.475 1.167 2 2.108 2 0.444 3 2.177 3 0.355 F 2.154 F 0.792
128 Table A2: Raw data for siderophore production at pH 9 using the optical density at 600 nm to track cell growth and the CAS assay determined at 630 nm to determine siderophore production. date time hours pH 9 OD600 pH 9 OD630 1/14/2003 6:00 0 inoculation!!! 1/14/2003 14:00 81 0.031 1.266 2 0.027 1.084 3 0.029 1.124 F 0.026 1.125 1/14/2003 22:00 161 0.253 1.128 2 0.242 1.072 3 0.267 1.132 F 0.244 1.117 1/15/2003 6:00 24 1 got stuck 2 3 F 1/15/2003 14:00 321 1.615 0.049 2 1.6 0.047 3 1.607 0.046 F 1.687 0.798 1/15/2003 22:00 401 1.826 0.904 2 1.805 0.887 3 1.841 0.884 F 1.928 0.999 1/16/2003 6:00 481 1.959 0.792 2 1.967 0.785 3 1.929 0.772 F 2.105 0.894 1/16/2003 14:00 561 2.051 0.668 2 2.024 0.696 3 2.057 0.67 F 2.226 0.728 1/17/2003 6:00 721 2.201 0.254 2 2.172 0.354 3 2.208 0.305 F 2.375 0.332 1/17/2003 14:00 801 2.239 0.5 2 2.221 0.583 3 2.256 0.55 F 2.358 0.614 1/17/2003 22:00 881 2.27 0.507 2 2.266 0.592 3 2.286 0.554 F 2.337 0.71 1/18/2003 22:00 112 1 2.27 0.309 2 2.278 0.408 3 2.273 0.368 F 2.308 0.63 1/20/2003 14:00 150 1 2.219 0.3 2 2.217 0.429 3 2.2 0.413 F 2.268 0.649 1/21/2003 14:00 174 1 2.204 0.322 2 2.189 0.417 3 2.176 0.405 F 2.261 0.659 1/22/2003 14:00 198 1 2.179 0.412 2 2.162 0.496 3 2.252 0.461 F 2.142 0.735
129 Table A3: Raw data for siderophore production at pH 10 using the optical density at 600nm to track cell growth and the CAS assay determined at 630 nm to determine siderophore production. Date Time Hours pH 10 OD600 pH 10 OD630 pH 10 600 Blank pH 10 OD630 Blank 12/9/2002 11:15 81 0.025 1 0.94 0 1.005 2 0.025 2 0.97 3 0.027 3 0.977 F 0.027 F 1.008 12/10/2002 7:15 161 0.31 1 0.953 0 1.254 2 0.346 2 0.932 3 0.322 3 0.931 F 0.384 F 0.946 12/10/2002 3:15 241 1.356 1 0.851 0 1.109 2 1.407 2 0.835 3 1.364 3 0.746 F 1.384 F 0.892 12/10/2002 11:15 321 1.811 1 0.084 0 1.064 2 1.818 2 0.077 3 1.779 3 0.063 F 1.813 F 0.475 12/11/2002 7:15 401 1.999 1 0.355 -0.011 0.945 2 1.991 2 0.334 3 1.99 3 0.307 F 2.027 F 0.66 12/11/2002 3:15 481 2.104 1 0.554 -0.01 1.03 2 2.093 2 0.547 3 2.087 3 0.503 F 2.159 F 0.735 12/12/2002 7:15 641 2.25 1 0.18 -0.01 1.002 2 2.233 2 0.163 3 2.21 3 0.124 F 2.339 F 0.503 12/12/2002 3:15 721 2.298 1 0.543 -0.002 0.998 2 2.292 2 0.548 3 2.266 3 0.492 F 2.389 F 0.719 12/12/2002 11:15 801 2.327 1 0.485 -0.001 0.99 2 2.319 2 0.415 3 2.303 3 0.558 F 2.38 F 0.768 12/13/2002 7:15 881 2.354 1 0.481 0.001 0.982 2 2.353 2 0.503 3 2.336 3 0.417 F 2.371 F 0.742 12/13/2002 2:15 951 2.342 1 0.488 0 0.99 2 2.347 2 0.497 3 2.345 3 0.39 F 2.364 F 0.771 12/14/2002 3:15 120 1 2.313 1 0.458 0 0.998 2 2.315 2 0.486 3 2.315 3 0.376 F 2.364 F 0.754 12/16/2002 3:15 168 1 2.272 1 0.534 -0.003 0.988 2 2.305 2 0.509 3 2.274 3 0.542 F 2.315 F 0.776 12/17/2002 3:15 192 1 2.256 1 0.611 0 0.993 2 2.292 2 0.589 3 2.263 3 0.532 F 2.324 F 0.813
130 Table A4: Raw data for siderophore production at pH 11 using the optical density at 600nm to track cell growth and the CAS assay determined at 630 nm to determine siderophore production. date time hours pH 11 OD600 pH 11 OD 630 OD600 Blank OD 630 Blank 1/3/2003 14:00 81 0.022 1 0.915 0.002 0.974 2 0.021 2 0.926 3 0.021 3 0.929 F 0.019 F 0.964 1/3/2003 22:30 16.51 0.049 1 0.944 -0.001 0.964 2 0.046 2 0.963 3 0.045 3 0.956 F 0.041 F 0.986 1/4/2003 6:00 241 0.417 1 0.929 -0.001 0.977 2 0.493 2 0.927 3 0.441 3 0.917 F 0.409 F 0.945 1/4/2003 16:30 34.51 1.528 1 0.851 0.001 0.965 2 1.637 2 0.263 3 1.569 3 0.509 F 1.553 F 0.881 1/4/2003 22:00 401 1.783 1 0.04 0.033 0.955 2 1.791 2 0.041 3 1.808 3 0.041 F 1.785 F 0.238 1/5/2003 6:00 481 1.943 1 0.676 0.001 0.948 2 1.942 0.712 3 1.983 0.704 F 1.971 F 0.815 1/5/2003 18:00 601 2.062 1 1.011 -0.001 0.962 2 2.063 2 1.023 3 2.098 3 0.99 F 2.167 F 1.006 1/6/2003 6:00 721 2.173 1 0.043 0.002 0.97 2 2.194 2 0.046 3 2.226 3 0.054 F 2.286 F 0.245 1/6/2003 14:00 801 2.211 1 0.524 0 0.974 2 2.231 2 0.479 3 2.262 3 0.532 F 2.319 F 0.761 1/6/2003 22:00 881 2.258 1 0.459 0 0.971 2 2.272 2 0.562 3 2.305 3 0.489 F 2.358 F 0.735 1/7/2003 6:00 961 2.292 1 0.45 0 0.991 2 2.32 0.541 3 2.335 3 0.48 F 2.38F 0.74 1/7/2003 14:00 1041 2.319 1 0.424 0 0.959 2 2.327 2 0.556 3 2.363 0.573 F 2.379 F 0.793 1/7/2003 22:00 1121 2.337 1 0.474 0 0.897 2 2.346 2 0.516 3 2.352 3 0.425 F 2.368 F 0.718 1/8/2003 8:00 1221 2.331 1 0.401 0 0.963 2 2.347 2 0.515 3 2.337 3 0.634 F 2.36F 0.746 1/8/2003 2:00 1281 2.331 0.366 0 0.967 2 2.338 2 0.507 3 2.334 3 0.452 F 2.36F 0.717 1/9/2003 18:00 1561 2.314 1 0.457 0 0.967 2 2.331 2 0.554 3 2.317 3 0.511 F 2.355 F 0.772 1/13/2003 18:00 2521 2.248 1 0.606 0 0.964 2 2.322 0.667 3 2.313 0.66 F 2.259 F 0.835
131 Siderophore Expression by Halomonas campisalis at pH 8
2.5 600
500 2
400 1.5
600 300 OD 1 M Siderophore 200 m
OD600, deferrated medium (desferrioxamine equivalent) 0.5 OD600 standard medium 100 siderophore production, deferrated medium
siderophore production, standard medium 0 0 0 50 100 150 200 250 Time, hours
Figure A1: Siderophore production with respect to growth by Halomonas campisalis at pH 8.
Siderophore Expression by Halomonas campisalis at pH 9
2.5 600
500 2
400 1.5
600 300 OD 1 OD600, deferrated meduim
200 M siderophore m (DEF equivalent) OD600, standard medium 0.5 siderophore production, deferrated medium 100
siderophore production, standard medium 0 0 0 50 100 150 200 Time, hours Figure A2: Siderophore production with respect to growth by Halomonas campisalis at pH 9.
132 Siderophore Expression by Halomonas campisalis at pH 8,9,10, and 11
3 600
2.5 500
2 400
1.5 300 OD600
1 200 pH 10 OD600
Series1 0.5 100 pH 10 siderophore production
Series2 0 0 0 50 100 150 200 250 Time, hours Figure A3: Siderophore production with respect to growth by Halomonas campisalis at pH 10.
Siderophore Expression by Halomonas campisalis at pH 8,9,10, and 11
2.5 600
500 2
400 1.5
300 OD600 1 200 pH 11 OD600
Series1 0.5 pH 11 siderophore production 100
Series2
0 0 0 50 100 150 200 250 300 Time, hours Figure A4: Siderophore production with respect to growth by Halomonas campisalis at pH 11.
133 APPENDIX B: Additional HPLC chromatograms and
mass spectra of ferrioxamine siderophores
134 DAD1 B, Sig=435,16 Ref=360,100 (ABBIE\CRUDE002.D) 28.278
30
23.512 20 mAU
10 22.961
25.078
Area: 27.857 46.366 2.665 47.266 0 2.758
-10 0 10 20 30 40 50 60 min Figure B1: HPLC chromatogram of ferrated H. campisalis siderophores.
135 +MS, 28.1-28.6min #(1469-1509) x10 7
4
m/z 654 654.3 3 Ferrioxamine E Intens. 2
1
681.8 0 100 200 300 400 500 600 700 800 900 1000
m/z
Figure 2B: Mass spectrum of ferrioxamine E
+MS, 23.5-23.7min #(1056-1076) x10 7 2.5
672.3 2.0
Intens. 1.5
1.0
0.5
336.6 104.4 224.6290.9 543.3 880.6 999.5 0.0 100 200 300 400 500 600 700 800 900 1000
m/z
Figure B3: Mass spectrum of ferrioxamine G1
136 x106 +MS, 22.7-22.8min #(984-1001)
5 626.2
4
Intens. 3
2
1 149.9 229.2 359.9 537.8 648.2 494.2 721.5 863.5 0 100 200 300 400 500 600 700 800 900 1000
m/z
Figure B4: Mass spectrum of ferrioxamine X7
+MS, 25.0-25.1min #(1191-1200) x10 6
3 640.2
2 Intens.
1
937.2 662.2 460.1 893.2 407.6 213.5 284.4 489.0 573.7 697.3 839.9 965.8 0 100 200 300 400 500 600 700 800 900 1000
Figure B5: Mass spectrum of ferrioxamine D2
137 DAD1 C, Sig=210,8 Ref=360,100 (ABBIE\07050904.D) mAU E
2000 G1 pH 8 1750
1500
1250
1000
750 X7 D2
500 X7
250
0
0 10 20 30 40 50 60 min DAD1 C, Sig=210,8 Ref=360,100 (ABBIE\07051001.D) mAU E 2000 G1 pH 9 1750
1500
1250
1000 X7 X7 D2 750
500
250
0
0 10 20 30 40 50 60 min DAD1 C, Sig=210,8 Ref=360,100 (ABBIE\07051003.D) E mAU pH 10 2000
1750 G1 1500 X7 1250 X7 D2
1000
750
500
250
0
0 10 20 30 40 50 60 min
Figure B6: Siderophore profiles of H. campisalis with respect to pH.
138 x108 +MS, 41.5min #2729 599.4 1.0
0.5
483.3
Intens. 0.0 7 483.2 x10 401.3 2.0
1.5
1.0 566.3 201.2 0.5 316.2 219.2 283.2 0.0 150 200 250 300 350 400 450 500 550 600 m/z
Figure B7: Unknown ferrioxamine 599.5
139 +MS, 37.8min #1848 x108 2.0 501.3 1.5 1.0 601.3 0.5 533.3
Intens. 0.0 6 x10 201.2 4 302.1 283.2 401.2 2
165.1183.2 220.3 483.3 0 200 300 400 500 600 m/z
Figure B8: Unknown ferrioxamine 501.3
140 +MS2(585.7), 32.4min #1498 x108 1.00 585.4
0.75
0.50
0.25 625.3 293.3 0.00 6 267.2 x10 201.2 Intens. 3
402.3 2
386.3 1 185.2 303.3 168.2 283.2 367.2 585.3 219.1 483.2536.5 0 200 300 400 500 600 m/z
Figure B9: Unknown ferrioxamine siderophore 585.3.
141 7 x10 +MS2(617.8), 37.6min #1790 6 617.4 601.4 4 Intens. 2
501.4 573.4 6 x10 419.3 3 501.2
2
584.3 1 301.2 399.3 219.2 316.2 201.2 283.2 483.3 0 200 300 400 500 600 m/z
Figure B10: Unknown ferrioxamine siderophore m/z = 617.4
142 APPENDIX C: Table of masses, structural information and
fragmentation data for ferrioxamine siderophores
143 Daughter Fragments for Iron Free PFO MW R1 m n o p R2 m/z m/z +Fe form
G2bt 504.6 H 5 4 5 0 H 505.6 558.6 401, 387, 319, 305, 283, 201, 187
G1t 518.7 H 5 5 5 0 H 519.7 572.7 401, 319, 301, 283, 219, 201, 183
X8 530.7 531.7 584.7 531, 442, 393, 345, 305, 227, 201, 128
A2 532.6 H 5 4 4 0 COCH3 533.6 586.6
X9 544.7 545.7 598.7 545, 475, 428, 345, 319, 227, 201
A1 546.7 H 5 5 4 0 COCH3 547.7 600.7
X2 558.6 cyclic 4 4 4 0 CO(CH2)2CO- 559.6 612.6 373, 287, 269, 187, 169, 154
B 560.7 H 5 5 5 0 COCH3 561.7 614.7
X1 572.7 cyclic 4 4 5 0 CO(CH2)2CO- 573.7 626.7 387, 373, 287, 269, 201, 187, 169, 154
X7 572.7 cyclic 3 5 5 0 CO(CH2)2CO- 573.7 626.7 573,419, 401, 373, 283, 201, 173, 154
401, 387, 301, 283, 269, 201, 187, 183, D 586.7 cyclic 4 5 5 0 CO(CH ) CO- 587.7 640.7 2 2 2 154
E 600.7 cyclic 5 5 5 0 CO(CH2)2CO- 601.7 654.7 401, 383, 301, 283, 201, 183, 165
D1 602.7 CH3CO 5 5 5 0 COCH3 603.7 656.7
505, 487, 405, 387, 319, 287, 269, 201, G 604.7 H 5 5 4 0 CO(CH ) COOH 605.7 658.7 2a 2 2 187 505, 419, 405, 401, 387, 319, 305, 301, G 604.7 H 5 4 5 0 CO(CH ) COOH 605.7 658.7 2b 2 2 283, 219, 201, 187, 183, 165, 101 505, 419, 401, 319, 305, 301, 283, 201, G 604.7 H 4 5 5 0 CO(CH ) COOH 605.7 658.7 2c 2 2 187, 183
X3 614.7 cyclic 5 5 6 0 CO(CH2)2CO- 615.7 668.7
519, 501, 419, 401, 319, 301, 283, 219, G 618.7 H 5 5 5 0 CO(CH ) COOH 619.7 672.7 1 2 2 201, 187
T4 622.7 623.7 676.7 623, 605, 505, 423, 319, 305, 206, 201
X4 628.7 cyclic 5 6 6 0 CO(CH2)2CO- 629.7 682.7
720, 702, 571, 534, 520, 469, 387, 334, T 719.7 720.7 773.7 5 268, 201, 187 734, 716, 585, 534, 483, 416, 401, 385, T 733.7 734.7 787.7 6 334, 283, 201
T8 758.9 cyclic 4 4 4 1 CO(CH2)2CO- 759.9 812.9
T3 772.9 cyclic 3 5 5 1 CO(CH2)2CO- 773.9 826.9 773, 601, 573, 401, 373, 201, 173
T7 772.9 cyclic 4 4 5 1 CO(CH2)2CO- 773.9 826.9
787, 769, 601, 587, 483, 401, 301, 283, T 786.9 cyclic 4 5 5 1 CO(CH ) CO- 787.9 840.9 2 2 2 201, 166 801, 783, 601, 483, 401, 301, 283, 201 , T 800.9 cyclic 5 5 5 1 CO(CH ) CO- 801.9 854.9 1 2 2 166
144 APPENDIX D: 16S rDNA sequences and closest BLAST
search matches for isolates from Soap Lake
145 SL01 Sequence
GAAAGACATCACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTA ATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGG CTTGATAAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACT GTCAAGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCG TAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACAC TGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGATGTCGACCAGCCGTTGGGTGCCTAGCGCACTTTGTGGCGAAGTTAACGCG ATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACG GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCT TACCTACTCTTGACATCTACAGAAGCCGGAAGAGATTCTGGTGTGCCTTCGGGAACT GTAAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGT CCCGTAACGAGCGCAACCCTTGTCCTTATTTGCCAGCGCGTAATGGCGGGAACTCTA AGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATG GCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCCGGTACAAAGGGTTGCGAG CTCG
Description Max score Max ident
Halomonas variabilis strain HTG7 16S ribosomal RNA gene, partial sequence 1456 99%
Halomonas sp. MN12-2a 16S ribosomal RNA gene, partial sequence 1445 99%
Uncultured bacterium clone rRNA057 16S ribosomal RNA gene, partial sequence 1434 99%
Uncultured soil bacterium clone PK_VIII 16S ribosomal RNA gene, partial sequence 1423 99%
Halomonas sp. M6-20C 16S ribosomal RNA gene, partial sequence 1423 99%
Halomonas variabilis SW04 16S ribosomal RNA gene, partial sequence 1423 99%
Halomonas sp. B-1055 16S ribosomal RNA gene, partial sequence 1423 99%
Halomonas sp. MAN K9 gene for 16S rRNA, partial sequence 1419 98%
Halomonas sp. DG1230 16S ribosomal RNA gene, partial sequence 1417 98%
146 SL02 Sequence
GGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTATATAAGTCAGATGTGAAATCC CCGGGCTCAACCTGGGAACTGCATTTGAGACTGTATGGCTAGAGTGTGTCAGAGGG GGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGATATGTGGAGGAATACCGATG GCGAAGGCAGCCCCCTGGGATAACACTGACGCTCATGCACGAAAGCGTGGGGAGCA AACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGG ACTTAATTGTCTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACG GTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGAT GTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTACGGAATT CCGAAGAGATTTGGAAGTGCTCGCAAGAGAACCGTAACACAGGTGCTGCATGGCTG TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGT CATTAGTTGCTACATTTAGTTGAGCACTCTAATGAGACTGCCGGTGACAAACCGGAG GAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCA
Description Max score Max ident
Beta proteobacterium HTCC525 16S ribosomal RNA gene, partial sequence 1205 99%
Uncultured bacterium clone 221ds20 16S ribosomal RNA gene, partial sequence 1188 98%
Beta proteobacterium Wuba70 16S ribosomal RNA gene, partial sequence 1188 98% uncultured betaproteobacterium partial 16S rRNA gene, clone A9 1188 98%
Uncultured bacterium clone 227ds5 16S ribosomal RNA gene, partial sequence 1177 98%
Undibacterium sp. CCUG 49012 partial 16S rRNA gene, strain CCUG 49012 1171 98% uncultured betaproteobacterium partial 16S rRNA gene, clone C10 1171 98%
Glacier bacterium FXS9 16S ribosomal RNA gene, partial sequence 1164 97%
Beta proteobacterium A1020 16S ribosomal RNA gene, partial sequence 1155 97%
Uncultured beta proteobacterium partial 16S rRNA gene, clone SW15 1151 97%
147 SL04 Sequence
TGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGT AAAGCGCGCGTAGGTGGCTAAGTAAGATGGGTGTGAAATCCCCGGGCTCAACCTGG GAACTGCATCCATAACTGCTTGGCTAGAGTACGGTAGAGGGTAGTGGAATTTCCTGT GTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCT GGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC CTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAACCTTGAGTTCTTAG TGGCGCAGCTAACGCACTAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAA CTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA GCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATT GGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTG AGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGT TATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGA CGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGGGGGT ACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCTCTCGTAGTCC GGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAAT
Description Max score Max ident
Pseudomonas sp. 1_C16_29 16S ribosomal RNA gene, partial sequence 1522 99%
Antarctic seawater bacterium R7375 16S rRNA gene 1519 99%
Arctic seawater bacterium R7078 16S rRNA gene 1517 99%
Antarctic saline lake bacterium 33 strain 33 16S ribosomal RNA gene, partial sequence 1507 99%
Uncultured bacterium clone ANTLV9_D02 16S ribosomal RNA gene, partial sequence 1500 99%
Pseudomonas sp. gap-f-57 16S ribosomal RNA gene, partial sequence 1500 99%
Pseudomonas sp. ice-oil-327 16S ribosomal RNA gene, partial sequence 1500 99%
Pseudomonas sp. D5044 16S ribosomal RNA gene, partial sequence 1489 99%
Pseudomonas sp. ice-oil-516 16S ribosomal RNA gene, partial sequence 1487 98%
Pseudomonas sp. 18III/A01/067 16S ribosomal RNA gene, partial sequence 1483 99%
148 SL11 Sequence GTGGCGCAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCCTCGGGTTGTAA AGCACTTTCAGTGGGGAAGAAAGCCTTCCGGTTAATACCCGGGAGGAAGGACATCA CCCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGT GCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCC GGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGA GTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGG AGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAA AGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTC GACTAGCCGTTGGGTCCTTCGCGGACTTTGTGGCGCAGTTAACGCGATAAGTCGACC GCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCAC AAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTG ACATCCTCGGAATCCGCCAGAGATGGCGGAGTGCCTTCGGGAACCGAGAGACAGGT GCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAG CGCAACCCTTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCG GTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTA GGGCTACACACGTGCTACAATGGTCAGTACAAAGGGTT
Description Max score Max ident
H.desiderata 16S ribosomal RNA 1604 99%
Halomonas sp. IB-I6 partial 16S rRNA gene, strain IB-I6 1537 98%
Halomonas nitritophilus isolate WST 3 16S ribosomal RNA gene, partial sequence 1537 98%
Halomonas sp. AIR-2 16S ribosomal RNA gene, partial sequence 1528 97%
Halomonas nitritophilus isolate WST 7 16S ribosomal RNA gene, partial sequence 1528 97%
Halomonas daqingensis strain DQD2-30T 16S ribosomal RNA gene, partial sequence 1522 97%
Bacterial sp. 16S rRNA gene (Lake Bogoria isolate WB4) 1509 97%
Halomonas phoceae strain CCUG 5096 16S ribosomal RNA gene, partial sequence 1502 97%
Bacterial sp. 16S rRNA gene (Lake Elmenteita isolate 35E2) 1498 97%
Halomonas sp. IB-O7-1 partial 16S rRNA gene, strain IB-O7-1 1495 97%
149 SL15 Sequence GACATCACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATAC GGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTG ATAAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCA GGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGA GATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAG GTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAA CGATGTCGACTAGCCGTTGGGTCCCTCGCGGACTTTGTGGCGCAGTTAACGCGATAA GTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGG CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCT ACCCTTGACATCCTGCGAACCCTTCGGAGACGAAGGGGTGCCTTCGGGAACGCAGA GACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG TAACGAGCGCAACCCTTGTCCTTATTTGCCAGCGGGTAATGCCGGGAACTCTAAGGA GACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCT TACGGGTAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAACTCGC GAGAGTGCGCTAATCCCATAAA
Description Max score Max ident
Halomonas sp. C17 16S ribosomal RNA gene, partial sequence 1474 99%
Halomonas sp. G7 16S ribosomal RNA gene, partial sequence 1447 98%
Halomonas sp. Ap-5 16S ribosomal RNA gene, partial sequence 1437 98%
Halomonas sp. IB-O7-1 partial 16S rRNA gene, strain IB-O7-1 1435 98%
Halomonas nitritophilus partial 16S rRNA gene, strain IB-Ar4 1435 98%
Halomonas sp. G-AMM5 16S ribosomal RNA gene, partial sequence 1406 98%
Halomonas nitritophilus isolate WST 3 16S ribosomal RNA gene, partial sequence 1406 98%
Unidentified Hailaer soda lake bacterium F16 16S ribosomal RNA gene, partial sequence 1402 97%
Bacterial sp. 16S rRNA gene (Lake Elmenteita isolate 44E3) 1399 97%
Halomonas nitritophilus strain MSU4010 16S ribosomal RNA gene, partial sequence 1393 98%
150 SL17 Sequence AGCCCATCCCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTA TCACCGGCAGTCTCCCTAGAGTTCCCGCCATGACGCGCTGGCAACTAAGGATAGGG GTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACAACACGAGCTGACGACAGCC ATGCAGCACCTGTCTTACAGTTCCCGAAGGCACAGTCTTATCTCTAAGACCTTCTGT AGATGTCAAGGGATGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCA CCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCC AGGCGGTCGACTTAGTGCGTTAGCTGCGTCACTC
Description Max score Max ident
Uncultured bacterium clone MB-A2-149 16S ribosomal RNA, partial sequence 647 97%
Idiomarina sp. JK38 16S ribosomal RNA gene, partial sequence 632 97%
Idiomarina sp. JK17 16S ribosomal RNA gene, partial sequence 632 97%
Idiomarina sp. JK4 16S ribosomal RNA gene, partial sequence 632 97%
Uncultured Idiomarina sp. clone DS071 16S ribosomal RNA gene gene, partial sequence 632 97%
Colwellia rossensis 16S ribosomal RNA gene, partial sequence 606 95%
Colwellia sp. BSi20399 16S ribosomal RNA gene, partial sequence 604 96%
Antarctic bacterium SIDMSP4C5 16S ribosomal RNA gene, partial sequence 604 96%
Uncultured Antarctic sea ice bacterium clone AntCL3G12 16S ribosomal RNA gene, partial sequence 604 95%
151 SL28 Sequence GCCTACACATGCAAGTCGAGCGGCAGCACGGGAAGCTTGCTTCCTGGTGGCGAGCG GCGGACGGGTGAGTAATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGA AACTCAGGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCT TGCGCTATCGGATGAGCCTATGCCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCA AGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACATCGGGACTGAGACA CGGCCCGAACTCCTACGGGAGGCAGCASTGGGGAATATTGGACAATGGGCGCAAGC CTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAG TGAGGAAGAAGGCCTTGGGCTTAATACGTCCGAGGAAGGACATCACTCACAGAAGA AGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAA TCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAG CCCTGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAG GAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAG TGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAG CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTG GGAGCCTCGAGTTCTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGT ACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAG CATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCTTCGGA AGCCGAGAGAGATCTTGGTGTGCCTTCGGGAACCGAAAGACAGGTGCTGCATGGCT GTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCCTG TCCCTATTTGCCAGCACGTAATGGTGGGAACTCTAGGGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACA CGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGCGACGTGGAGCTAATCCCA TAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATC GCTAGTAATCGTGAATCAG
152 SL28 Closest matches by BLAST search with above sequence Description Max score Max ident
H.pantelleriense 16S rRNA gene 2320 98%
Halomonas muralis partial 16S rRNA gene, specimen voucher LMG 20971 2165 96%
Halomonas muralis partial 16S rRNA gene, specimen voucher LMG 20970 2165 96%
Halomonas muralis partial 16S rRNA gene, strain LMG-19418 2161 96%
Halomonas muralis partial 16S rRNA gene, type strain LMG 20969T 2159 96%
Halomonas phoceae strain CCUG 5096 16S ribosomal RNA gene, partial sequence 2154 96%
Halomonas sp. EF11 16S ribosomal RNA gene, partial sequence 2132 95%
Halomonas campaniensis 16S rRNA gene, type strain 5AG 2121 95%
Halomonas sp. IB-O18 partial 16S rRNA gene, strain IB-O18 2115 95%
Halomonas sp. 3019 partial 16S rRNA gene 2115 95%
153 SL29 Sequence GAGGAAGGACATCACCCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCG GTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGG CGGTCTGATAAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGA ACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAAT GCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGA CGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACG CCGTAAACGATGTCGACTAGCCGTTGGGGTCCTTGAGACCTTTGTGGCGCA:GTTAAC GCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTG ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAA CCTTACCTACCCTTGACATCGAGAGAACTTGGCAGAGATGCCTTGGTGCCTTCGGGA ACTCTCAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTA AGTCCCGTAACGAGCGCAACCCTTGTCCTTATTTGCCAGCGCGTAATGGCGGGAACT CTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCA TGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGACGGTACAAAGGGTTGCA AAGCCGCGAGGTGGAGCTAATCCCATAAAGCTGTTCTCAGTCCGGATCGGAGTCTGC AA
154 SL29 Closest match of blast search
Description Max score Max ident
Halomonas campisalis strain LL6 16S ribosomal RNA gene, complete sequence 1565 100%
Halomonas campisalis strain LL5 16S ribosomal RNA gene, complete sequence 1565 100%
Halomonas campisalis strain LL4 16S ribosomal RNA gene, complete sequence 1565 100%
Halomonas campisalis strain LL3 16S ribosomal RNA gene, complete sequence 1565 100%
Halomonas campisalis strain LL2 16S ribosomal RNA gene, complete sequence 1565 100%
Halomonas campisalis strain LL1 16S ribosomal RNA gene, complete sequence 1565 100%
Bacterial sp. 16S rRNA gene (Lake Bogoria isolate 8B1) 1548 99%
Bacterial sp. 16S rRNA gene (Lake Bogoria isolate 25B1) 1541 99%
Halomonas sp. Z-7009 16S ribosomal RNA gene, partial sequence 1526 99%
Bacterial sp. 16S rRNA gene (Lake Bogoria isolate WB2) 1513 98%
155 APPENDIX E: Siderophore production with respect to growth for Soap Lake isolates SL01,
SL11 and SL28
156 Table E1: Raw data for blank growth medium for optical density and CAS assay. date Time Hours Blank 0D600Blank 0d630 9-Sep 3:00 PM 0 0.033 1.143 9-Sep 9:00 PM 6 0 1.02 10-Sep 5:30 AM 14.5 0.000 1.018 10-Sep 9:00 AM 18 0.000 1.014 10-Sep 3:00 PM 24 0.000 1.003 10-Sep 9:00 PM 30 0.000 1.028 11-Sep 3:00 AM 36 0.000 0.987 11-Sep 9:00 AM 42 0.000 1.012 11-Sep 3:00 PM 48 0.000 1 11-Sep 9:00 PM 54 0.000 1.008 12-Sep 3:00 AM 60 0.000 1.018 12-Sep 9:00 AM 66 0.000 1.002 12-Sep 3:00 PM 72 0.000 1.005 12-Sep 9:00 PM 78 0.000 1.017 13-Sep 6:00 AM 87 0.000 1.009 13-Sep 9:00 AM 90 0.000 1.01 13-Sep 3:00 PM 96 0.000 1.002 13-Sep 9:00 PM 102 0.000 1.054 14-Sep 9:00 AM 114 0.000 1.009 14-Sep 3:00 PM 120 0.000 1.012 14-Sep 9:00 PM 126 0.000 1.010 15-Sep 3:00 PM 144 0.000 1.007 15-Sep 9:00 PM 150 0.000 1.006
Table E2: Raw data for isolate SL01 growth
157 SL1 SL1 SL1 SL1 SL1 SL1 SL1 date Time Hours 0D600 1 0D600 2 0D600 3 Dillution 9-Sep 3:00 PM 0 0.000 0.002 0.001 1.000 9-Sep 9:00 PM 6 0.054 0.057 0.031 1.000 10-Sep 5:30 AM 14.5 0.521 0.557 0.144 1 10-Sep 9:00 AM 18 0.603 0.628 0.26 1 10-Sep 3:00 PM 24 0.813 0.737 0.613 2 10-Sep 9:00 PM 30 0.812 0.789 0.657 5 11-Sep 3:00 AM 36 0.827 0.887 0.744 5 11-Sep 9:00 AM 42 0.962 0.964 0.8 5 11-Sep 3:00 PM 48 0.975 1.012 0.924 10 11-Sep 9:00 PM 54 1.028 1.022 0.977 20 12-Sep 3:00 AM 60 1.031 1.058 0.941 20 12-Sep 9:00 AM 66 1.092 1.12 1.021 20 12-Sep 3:00 PM 72 1.058 1.095 1.059 20 12-Sep 9:00 PM 78 1.086 1.084 1.052 20 13-Sep 6:00 AM 87 1.071 1.101 1.044 20 13-Sep 9:00 AM 90 1.017 1.029 1.028 20 13-Sep 3:00 PM 96 1.023 1.026 0.997 20 13-Sep 9:00 PM 102 0.992 1.056 0.986 20 14-Sep 9:00 AM 114 0.985 1.14 0.975 20 14-Sep 3:00 PM 120 0.943 0.9071 0.93 20 15-Sep 9:00 AM 138 0.929 1.014 0.959 20 15-Sep 3:00 PM 144 0.932 0.953 0.913 20 Table E2: Raw data for isolate SL01 siderophore production
SL1 SL1 SL1 SL1 SL1 SL1 SL1 OD630 1 OD630 2 OD 630 3 OD600 AVEST DEV OD 630 AVEST DEV uM eq DFB error 1.218 1.140 1.270 0.001 0.001 1.179 0.055 -0.65 -0.030193 1.126 1.108 1.115 0.056 0.002 1.117 0.013 -1.95 -0.022205 0.952 0.893 1 0.539 0.025 0.923 0.042 1.92 0.086937 0.755 0.594 0.988 0.616 0.018 0.675 0.114 6.86 1.158006 0.569 0.372 0.934 0.775 0.054 0.471 0.139 21.76 6.441996 0.81 0.76 0.978 0.801 0.016 0.785 0.035 24.22 1.090809 0.635 0.585 0.919 0.857 0.042 0.610 0.035 39.14 2.268295 0.29 0.299 0.768 0.963 0.001 0.295 0.006 72.64 1.569762 0.538 0.533 0.772 0.994 0.026 0.536 0.004 95.18 0.628437 0.727 0.75 0.862 1.025 0.004 0.739 0.016 109.57 2.413075 0.698 0.725 0.784 1.045 0.019 0.712 0.019 123.39 3.311058 0.691 0.674 0.749 1.106 0.020 0.683 0.012 130.68 2.301678 0.685 0.661 0.739 1.071 0.021 0.673 0.017 135.39 3.413999 0.69 0.679 0.709 1.074 0.019 0.685 0.008 133.99 1.522597 0.718 0.697 0.733 1.072 0.029 0.708 0.015 122.46 2.570302 0.753 0.652 0.703 1.025 0.007 0.703 0.071 124.78 12.6851 0.735 0.71 0.719 1.015 0.016 0.723 0.018 114.32 2.797122 0.71 0.69 0.728 1.011 0.039 0.700 0.014 137.65 2.780928 0.681 0.726 0.685 1.033 0.093 0.704 0.032 124.09 5.612594 0.672 0.655 0.713 0.927 0.018 0.664 0.012 141.05 2.555365 0.696 0.672 0.731 0.967 0.043 0.684 0.017 132.38 3.284332 0.707 0.655 0.831 0.933 0.020 0.681 0.037 132.77 7.168687
158 SL1 Growth and Siderophore Production
1.20 200
160 0.90 M DFB m
120
0.60 OD 600 80
0.30 40 Siderophore Concentration
0.00 0 0 20 40 60 80 100 120 140 160 Time, Hours
Cell Growth siderophore production Figure E1: SL01 graph of siderophore production with respect to growth.
Table E3: Raw data for growth of isolate SL11 SL11 SL11 SL11 SL11 SL11 SL11 SL11 date Time Hours 0D600 1 0D600 2 0D600 3 Dillution 9-Sep 3:00 PM 0 -0.019 -0.001 -0.022 1 9-Sep 9:00 PM 6 -0.009 -0.007 -0.012 1 10-Sep 5:30 AM 14.5 -0.001 0.008 -0.007 1 10-Sep 9:00 AM 18 0.003 -0.018 -0.002 1 10-Sep 3:00 PM 24 0.05 0.008 0.04 1 10-Sep 9:00 PM 30 0.121 0.076 0.11 1 11-Sep 3:00 AM 36 0.314 0.237 0.313 1 11-Sep 9:00 AM 42 0.56 0.423 0.57 1 11-Sep 3:00 PM 48 0.787 0.699 0.831 1 11-Sep 9:00 PM 54 1.02 1.087 1.052 1 12-Sep 3:00 AM 60 1.232 1.381 1.244 1 12-Sep 9:00 AM 66 1.445 1.615 1.455 1 12-Sep 3:00 PM 72 1.581 1.754 1.585 1 12-Sep 9:00 PM 78 1.742 1.667 1.735 1 13-Sep 6:00 AM 87 1.746 1.802 1.737 1 13-Sep 9:00 AM 90 1.748 1.78 1.727 1 13-Sep 3:00 PM 96 1.759 1.766 1.744 1 13-Sep 9:00 PM 102 1.739 1.753 1.732 1 14-Sep 9:00 AM 114 1.713 1.733 1.703 1 14-Sep 3:00 PM 120 1.673 1.696 1.665 1 15-Sep 9:00 AM 138 1.627 1.666 1.622 1 15-Sep 3:00 PM 144 1.606 1.638 1.606 1
159 Table E5: Raw data for siderophore production of isolate SL11 using the CAS assay SL11 SL11 SL11 SL11 SL11 SL11 SL11 OD630 1 OD630 2 OD 630 3 OD600 AVESTDEV OD 630 AVEST DEV uM eq DFB error 1.3 1.301 1.115 -0.014 0.011 1.239 0.107 -1.715120309 -0.148296 0.986 1.131 0.976 -0.009 0.003 1.031 0.087 -0.220990035 -0.018594 0.997 1.13 0.974 0.000 0.008 1.034 0.084 -0.31536174 -0.025693 0.991 1.128 0.988 -0.006 0.011 1.058 0.099 -0.889190675 -0.0832 0.988 1.153 1.027 0.033 0.022 1.056 0.086 -1.082817122 -0.088428 0.99 1.114 0.968 0.102 0.023 1.024 0.079 0.079734643 0.006129 0.994 1.071 0.987 0.288 0.044 1.017 0.047 -0.629771732 -0.028852 0.935 0.986 0.937 0.518 0.082 0.953 0.029 1.201429836 0.036427 0.904 0.972 0.888 0.772 0.067 0.921 0.045 1.612021858 0.078038 0.881 0.979 0.857 1.053 0.034 0.906 0.065 2.080351722 0.148463 0.81 0.937 0.758 1.286 0.083 0.835 0.092 3.683693517 0.406228 0.694 0.894 0.653 1.505 0.095 0.747 0.129 5.214979876 0.9002 0.905 0.625 0.763 1.640 0.099 0.694 0.098 5.209607699 0.732503 0.726 0.813 0.516 1.715 0.041 0.685 0.153 6.689556233 1.491097 0.694 0.874 0.597 1.762 0.035 0.722 0.141 5.835459011 1.136558 0.619 0.897 0.511 1.752 0.027 0.676 0.199 6.783260293 1.999253 0.602 0.844 0.531 1.756 0.011 0.659 0.164 7.014659206 1.746752 0.652 0.914 0.602 1.741 0.011 0.723 0.168 6.441762321 1.493742 0.692 0.94 0.598 1.716 0.015 0.743 0.177 5.395430199 1.28245 0.704 0.947 0.634 1.678 0.016 0.762 0.164 5.063872309 1.092169 0.72 0.643 0.95 1.638 0.024 0.771 0.160 4.8542114 1.005646 0.743 0.958 0.674 1.617 0.018 0.792 0.148 4.387225917 0.82086
SL11 Growth and Siderophore Production
2.000 200
1.800 180
1.600 160
1.400 140 M DFB m
1.200 120
1.000 100 OD 600 0.800 80
0.600 60
0.400 40 Siderophore concentration
0.200 20
0.000 0 0 20 40 60 80 100 120 140 160 Time, Hours
Cell growth OD 600 Siderophore concentration Figure E2: Graphic of siderophore production with respect to growth for Soap Lake isolate SL11.
Table: E6: Raw data for growth of isolate SL28 (Graphic shown in Chapter 4).
160 SL28 SL28 SL28 SL28 SL28 SL28 SL28 date Time Hours 0D600 1 0D600 2 0D600 3 Dillution 9-Sep 3:00 PM 0 0.031 0.03 0.032 1 9-Sep 9:00 PM 6 -0.006 -0.013 -0.007 1 10-Sep 5:30 AM 14.5 0.017 0.015 0.016 1 10-Sep 9:00 AM 18 0.03 0.032 0.033 1 10-Sep 3:00 PM 24 0.139 0.128 0.123 1 10-Sep 9:00 PM 30 0.172 0.17 0.175 1 11-Sep 3:00 AM 36 0.296 0.28 0.302 1 11-Sep 9:00 AM 42 0.471 0.473 0.459 1 11-Sep 3:00 PM 48 0.619 0.675 0.714 1 11-Sep 9:00 PM 54 0.719 0.727 0.717 2 12-Sep 3:00 AM 60 0.744 0.755 0.79 5 12-Sep 9:00 AM 66 0.811 0.949 0.867 5 12-Sep 3:00 PM 72 0.887 0.868 0.965 10 12-Sep 9:00 PM 78 0.896 0.84 0.971 20 13-Sep 6:00 AM 87 0.861 0.79 0.993 20 13-Sep 9:00 AM 90 0.819 0.794 0.89 20 13-Sep 3:00 PM 96 0.848 0.962 0.835 20 13-Sep 9:00 PM 102 0.891 0.845 0.816 20 14-Sep 9:00 AM 114 0.836 0.945 0.813 20 14-Sep 3:00 PM 120 0.892 0.878 0.798 20 15-Sep 9:00 AM 138 0.862 0.772 0.76 20 15-Sep 3:00 PM 144 0.874 0.978 0.725 20
Table E7: Raw data for siderophore production with respect to growth of Soap Lake isolate SL28 using the CAS assay (Graphic shown in Chapter 4) SL28 SL28 SL28 SL28 SL28 SL28 SL28 OD630 1 OD630 2 OD 630 3 OD600 AVEST DEV OD 630 AVEST DEV uM eq DFB error 1.303 1.268 1.088 0.031 0.001 1.220 0.115 -1.374486659 -0.130005 0.987 0.98 0.965 -0.009 0.004 0.977 0.011 0.85717347 0.009858 0.978 0.983 0.962 0.016 0.001 0.974 0.011 0.878986978 0.009896 0.98 0.997 0.97 0.032 0.002 0.982 0.014 0.639947834 0.008893 0.986 0.999 0.999 0.130 0.008 0.995 0.008 0.170254265 0.001285 0.993 1.003 0.976 0.172 0.003 0.991 0.083 0.744190002 0.062093 0.994 1.009 0.949 0.293 0.011 0.984 0.031 0.062285116 0.001976 0.972 0.978 0.987 0.468 0.008 0.979 0.008 0.668210976 0.005153 0.678 0.531 0.67 0.669 0.048 0.626 0.083 7.657103825 1.010517 0.577 0.64 0.651 0.721 0.005 0.623 0.040 15.66701362 1.004662 0.669 0.715 0.71 0.763 0.024 0.698 0.025 32.20715643 1.164573 0.475 0.496 0.51 0.876 0.069 0.494 0.018 51.97937458 1.854861 0.69 0.663 0.724 0.907 0.051 0.692 0.031 63.75227687 2.814695 0.805 0.812 0.817 0.902 0.066 0.811 0.006 82.8806465 0.615753 0.748 0.731 0.767 0.881 0.103 0.749 0.018 105.7423083 2.543643 0.752 0.706 0.749 0.834 0.050 0.736 0.026 111.3185089 3.894257 0.767 0.703 0.738 0.882 0.070 0.736 0.032 108.7987959 4.737307 0.724 0.719 0.733 0.851 0.038 0.725 0.007 127.798343 1.250016 0.705 0.732 0.732 0.865 0.071 0.723 0.016 116.1676063 2.504666 0.7 0.747 0.743 0.856 0.051 0.730 0.026 114.1059227 4.073054 0.702 0.692 0.724 0.798 0.056 0.706 0.016 123.4511144 2.862581 0.766 0.689 0.708 0.859 0.127 0.721 0.040 116.495427 6.481143
161 APPENDIX F. MALDI –TOF MS/MS data for sodachelin siderophores.
162 Determination of lysine or glutamine residue
Table F1: Amino acid isotopic residue masses
Amino Acid Residue Isotopic Mass Glycine 57.02145 Alanine 71.03711 Serine 87.03203 Proline 97.05276 Valine 99.06841 Threonine 101.04768 Cystein 103.00919 Leucine 113.08406 Isoleucine 113.08406 Asparagine 114.04293 Aspartic Acid 115.02694 Glutamine 128.05858 Lysine 128.09496 Glutaminc acid 129.04259 Methionine 131.04049 Histidine 137.05891 Phenylalanine 147.06841 Arginine 156.10111 Tyrosine 163.06333 Tryptophan 186.07931
163 Table F2: worksheet to determine identity of residues of 128 amu in size. These were taken from mass spectral data shown in Figures F1 through F4 and selected to include the fragments that would contain the residue in question. The errors determined between the calculated residue mass and the predicted mass for either glutamine or lysine suggest that glutamine residues are present in sodachelins C, D, E and F.
Fragment mass Parent Fragments Calculated • ppm containing potential • ppm lysine Mass (m/z) Subtracted Residue Mass glutamine glutamine or lysine (m/z) 1104.4615 793.3425 793-665 128.0674 68.9 215.2 665.2751 793-578-ser 128.0506 62.5 346.5 578.2599 (793-450-ser)/2 128.0503 64.8 348.8 450.2099 578-450 128.0500 67.0 351.0 665-450-ser 128.0332 198.4 482.4 average residue mass 128.0503 64.8 348.8
1106.5673 793.3820 793-665 128.0587 0.9 283.1 665.3233 793-578-ser 128.0670 65.5 218.5 578.2380 (793-450-ser)/2 128.0620 27.0 257.0 450.2259 578-450 128.0571 11.6 295.6 665-450-ser 128.0654 53.0 231.0 average residue mass 128.0620 27.0 257.0
1132.5761 793.3735 793-665 128.0302 221.6 505.6 665.3433 793-578-ser 128.0568 14.1 298.1 578.2847 (793-450-ser)/2 128.0517 53.8 337.8 450.2381 578-450 128.0466 93.6 377.5 128.0732 113.9 170.1 average residue mass 128.0517 53.8 337.8
1134.6018 793.3384 793-665 128.0152 338.7 622.6 665.3232 793-578-ser 128.0674 68.6 215.4 578.2847 (793-450-ser)/2 128.0614 22.3 261.7 450.2265 578-450 128.0582 3.0 287.0 665-450-ser 128.0647 47.6 236.5 average residue mass 128.0534 40.6 324.6
164 +TOF Product (1104.0): 180 MCA scans from Jan24-2007-SL28-01-F1b-MSMS1.wiff Max. 116.0 counts. a=3.57144452181778130e-004, t0=-1.61204597255007690e+001
450.2277 115
110
105
100
95
90
85
80
75
70
65 827.4205 60 637.3216 914.4530 55 1104.5350 Intensity, counts Intensity, 793.3740 50 655.3429 45 422.2336 301.1572 388.1917 40 509.2704 527.2821 35 665.3049 1086.5428 30 809.4147 440.2449 25 578.2926
20 216.1035 278.1383 344.1543 432.2163 499.2190 896.4606 15 619.3194 775.3683 10
5
0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z, amu Figure F1: Maldi-TOF MS/MS fragmentation data for sodachelin C
165 +TOF Product (1106.0): 180 MCA scans from Jan24-2007-SL28-01-F2a-MSMS1.wiff Max. 335.0 counts. a=3.57144452181778130e-004, t0=-1.61204597255007690e+001
450.2259 335
320
300
280
260
240
220
200
180 916.4698 829.4375 160
Intensity, counts 639.3420 140 793.3820 1106.5673
120 301.1598 516.2453 424.2492 657.3545 100 1088.5549 388.1894 529.2945 511.2803 811.4294 80 665.3233 578.2830
60 442.2622
278.1425 344.1607 898.4504 493.2736 40 326.1469 621.3296 776.3731
20
0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z, amu
Figure F2: Maldi-TOF MS/MS fragmentation data for sodachelin D
166 +TOF Product (1132.0): 180 MCA scans from Jan24-2007-SL28-01-F3a-MSMS1.wiff Max. 364.0 counts. a=3.57144452181778130e-004, t0=-1.61204597255007690e+001
450.2381 360
340
320
300
280
260
240
220 665.3433 200
180 855.4512
Intensity, counts Intensity, 160 1132.5761 942.4892 793.3735 140 516.2473 683.3641 120 301.1550 537.3000 100 388.1879 1114.5636 555.3117 837.4341 80 578.2847
344.1603 60 647.3385 924.4795 432.2132 468.2744 776.3546 40 278.1428
20
0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z, amu Figure F3: Maldi-TOF MS/MS fragmentation data for sodachelin E
167 +TOF Product (1134.0): 180 MCA scans from Jan24-2007-SL28-01-F4-MSMS1.wiff Max. 404.0 counts. a=3.57144452181778130e-004, t0=-1.61204597255007690e+001
450.2265 400
380
360
340
320
300
280
260
240
220 857.4775
200 1134.6018 944.5099
Intensity, counts Intensity, 180 667.3732 793.3841 160
140 516.2507 301.1568
120 685.3832 1116.5904 388.1875 839.4621 100 539.3128 578.2847 80 557.3301
60 344.1609 470.2936 926.4988 499.2180 649.3613 40
20
0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z, amu
Figure F4: Maldi-TOF MS/MS fragmentation data for sodachelin F.
168 APPENDIX G: Exact Mass Data for Sodachelin Siderophores
This appendix contains micro-TOF mass spectral data obtained at the Proteomics Facility at
Montana State University. Data was obtained for both the iron-free and Fe(III)-siderophore complex for sodachelins C-F. Data for sodachelin B was only obtained in the Fe(III) form and sodachelin A was not detected above the baseline in these experiments.
169 Figure G1. MicroTOF spectral data for ferri-Sodachelin B. The top spectrum is the experimental sample while the middle and bottom are the predicted isotopic ratios and mass for the iron- siderophore complex and desferri-siderophore. In this sample, the desferri form of Sodachelin B was not present. This yields a mass error of 2.3 ppm for Fe(III)-sodachelin B
170 [M+H]+
[M+Fe- [M+Na] + + 3H]
Figure G2. MicroTOF spectral data for desferri and ferri Sodachelin C. The top spectrum is the experimental sample while the middle and bottom are the predicted isotopic ratios and mass for the iron-siderophore complex and apo-siderophore. This yields a mass error of 2 ppm for sodachelin C and 7.5 ppm for Fe(III)-sodachelin C
171 Figure G3. Additional MicroTOF spectral data for the Fe(III)-Sodachelin C complex. The top spectrum is the experimental sample while the middle is a zoom of 1157.4502 and the bottom is the predicted mass and isotopic distribution of sodachelin C. This was for a sample of sodachelin C purified as the Fe(III)-sodachelin C complex. This yielded an error of 2.85 ppm.
172 [M+H]+ [M+Na + ] [M+K]+ [M+Fe-3H]+
Figure G4. MicroTOF spectral data for desferri and ferri Sodachelin D. The top spectrum is the experimental sample while the middle and bottom are the predicted isotopic ratios and mass for the iron-siderophore complex and apo-siderophore. This gave an error of 2.26 for sodachelin D and 4.5 for Fe(III)-sodachelin D.
173 Figure G5. Additional MicroTOF spectral data for the Fe(III)-Sodachelin D complex. The top spectrum is the experimental sample while the middle is a zoom of 1159.4644 and the bottom is the predicted mass and isotopic distribution of sodachelin D. This gave an error of 4.13 ppm for
Fe(III)-sodachelin D.
174 [M+H]+
[M+Na [M+K]+ [M+Fe-3H]+ ]+
Figure G4. MicroTOF spectral data for desferri and ferri Sodachelin E. The top spectrum is the experimental sample while the middle and bottom are the predicted isotopic ratios and mass for the iron-siderophore complex and apo-siderophore. This gave an error of 1.41 ppm for sodachelin E. No data was available for Fe(III)-sodachelin E.
175 Figure G5. Additional MicroTOF spectral data for the Fe(III)-Sodachelin E complex. The top spectrum is the experimental sample while the middle is a zoom of 1159.4644 and the bottom is the predicted mass and isotopic distribution of sodachelin E. This gave an error of 1.86 ppm for
Fe(III)-sodachelin E.
176 [M+H]+
[M+Na [M+K]+ [M+Fe-3H]+ ]+
Figure G5. MicroTOF spectral data for desferri- and ferrated Sodachelin F. The top spectrum is the experimental sample while the middle and bottom are the predicted isotopic ratios and mass for the iron-siderophore complex and desferri-siderophore. This gave an error in the mass of the sodachelin F sample of 2.82 ppm and 0.08 ppm for Fe(III)-sodachelin F.
177 APPENDIX H. Fatty acid analysis results for sodachelin siderophores
This appendix contains fatty acid analysis data obtained by Microbial ID, Midi Inc. using a standard procedure for fatty acid esterification and methylation. Samples of the sodachelins A-F were collected using the standard HPLC purification method and were concentrated 20 fold prior to submission to Midi Labs. An estimated 50 mM of siderophore was present in each sample.
178 FID1 A, (E07502.624\A0045924.D) pA 0.740 2.096
12
10 1.639
8
6 1.682 4.113 3.801 3.390 2.879 3.514
4 2.743 1.199 1.252
0.5 1 1.5 2 2.5 3 3.5 4 min
Figure H1: GC data for Sodachelin B fatty acid methyl ester analysis performed by Microbial ID
(Midi Labs).
179 Table H1: Fatty acid methyl ester peak identification for Sodachelin B.
RT Response Ar/Ht ECL Peak Name Percent Comment1 Comment2 0.740 1.194E+9 0.018 6.665 SOLVENT PEAK ---- < min rt 1.199 393 0.010 9.654 ---- 1.252 326 0.009 10.002 10:0 1.65 ECL deviates 0.002 Reference 0.004 1.639 5740 0.008 11.815 unknown 11.825 26.36 ECL deviates -0.010 1.682 3132 0.010 11.997 12:0 14.26 ECL deviates -0.003 Reference -0.001 2.096 10944 0.009 13.484 12:0 3OH 46.78 ECL deviates 0.001 2.743 374 0.009 15.577 16:0 N alcohol 1.50 ECL deviates 0.003 2.879 582 0.009 16.001 16:0 2.31 ECL deviates 0.001 Reference 0.001 3.390 724 0.011 17.607 18:3 w6c (6,9,12) 2.82 ECL deviates 0.007 3.514 488 0.010 18.000 18:0 1.89 ECL deviates 0.000 Reference 0.000 3.801 627 0.009 18.926 19:0 cyclo w8c 2.42 ECL deviates -0.006 4.113 816 0.010 19.961 ----
The peak at a retention time of 2.096 minutes is the fatty acid chain extracted from the
Sodachelin B sample and was assigned as b-hydroxy dodecanoic acid (12:0 3OH). There was also a large amount of ECL 11.815 at a retention time of 1.639 minutes and a lesser quantity of
ECL 11.997 at 1.682 minutes (12:0) which is due to some overlap of Sodachelin B and C and D in the separation process. Sodachelin B was present only in small quantities relative to
Sodachelins C-F and required significant concentration. Thus, some of Sodachelin C and D were present in detectable amounts for this analysis. ECL=Equivalent chain length. RT= Retention time. Ar/Ht = peak area/peak height.
180 FID1 A, (E07502.624\A0055926.D) pA 0.740 1.682 2.200 7.5
7
6.5
6
5.5
5 2.398 3.479 4.5 4.113 1.640 3.801
4 3.389 1.199 2.663 2.744 2.880
3.5
0.5 1 1.5 2 2.5 3 3.5 4 min
Figure H2: GC data for Sodachelin D fatty acid methyl ester analysis performed by Microbial ID
(Midi Labs).
181 Table H2: Fatty acid methyl ester peak identification for Sodachelin D.
RT Respons Ar/Ht ECL Peak Name Percent Comment1 Comment2 0.740 1.19E+ 0.018 6.666 SOLVENT PEAK ---- < min rt 1.199 489 0.010 9.657 ---- 1.640 697 0.008 11.820 unknown 11.825 1.31 ECL deviates -0.005 1.682 48989 0.008 12.000 12:0 91.43 ECL deviates 0.000 Reference 0.001 2.200 4610 0.008 13.835 ---- 2.398 1509 0.009 14.481 Sum In Feature 1 2.56 ECL deviates 0.007 15:1 iso H/13:0 3OH 2.663 377 0.009 15.322 ---- 2.744 365 0.009 15.576 16:0 N alcohol 0.60 ECL deviates 0.002 2.880 324 0.009 16.000 16:0 0.53 ECL deviates 0.000 Reference 0.003 3.389 498 0.010 17.602 18:3 w6c (6,9,12) 0.79 ECL deviates 0.002 3.479 1275 0.010 17.886 18:1 w6c 2.03 ECL deviates 0.002 3.801 477 0.009 18.923 19:0 cyclo w8c 0.76 ECL deviates -0.009 4.113 717 0.011 19.958 ------1509 ------Summed Feature 1 2.56 15:1 iso H/13:0 3OH 13:0 3OH/15:1 iso H
This table shows the names assigned to each of the fatty acid peaks for a sample of sodachelin D.
The primary peak in this analysis is at an ECL of 12.000 and constitutes 91.4% of total peak area. This was assigned as 12:0 which is consistent with the predicted fatty acid chain based on mass spectral data. There were trace amounts of other fatty acids, likely due to the concentration techniques employed. ECL=Equivalent chain length. RT= Retention time. Ar/Ht = peak area/peak height.
182 FID1 A, (E07502.624\A0065928.D) pA 0.740 2.200 2.249 4.105 4.4
4.2 2.880 3.055 1.684
4 3.515 0.982
3.8 1.215
3.6
3.4
0.5 1 1.5 2 2.5 3 3.5 4 min
Figure H3: GC data for Sodachelin F fatty acid methyl ester analysis performed by Microbial ID
(Midi Labs).
183 Table H3: Fatty acid methyl ester peak identification for sodachelin F.
RT Response Ar/Ht ECL Peak Name Percent Comment1 Comment2 0.740 1.193E+9 0.018 6.666 SOLVENT PEAK ---- < min rt 0.982 475 0.011 8.242 ---- < min rt 1.215 321 0.009 9.760 ---- 1.684 725 0.010 12.001 12:0 2.28 ECL deviates 0.001 Reference 0.006 2.200 1271 0.009 13.834 ---- 2.249 31997 0.008 13.998 14:0 92.98 ECL deviates -0.002 Reference 0.003 2.880 693 0.009 16.001 16:0 1.91 ECL deviates 0.001 Reference 0.004 3.055 609 0.010 16.550 17:1 anteiso w9c 1.66 ECL deviates -0.002 3.515 435 0.010 18.000 18:0 1.17 ECL deviates 0.000 Reference 0.003 4.105 1265 0.012 19.928 ----
The peak at a retention time of 2.249 minutes is the fatty acid chain extracted from the sodachelin F sample and was assigned as tetradecanoic acid (14:0). It constituted 92% of the total peak area. ECL=Equivalent chain length. RT= Retention time. Ar/Ht = peak area/peak height.
184 FID1 A, (E07521.485\A0046222.D) pA 1.689 4.747 4.939 6.765 7.269 7.528 10.785
5.25 9.565
5 13.993 3.262
4.75
4.5 2.363 1.995 14.303 4.25
4 4.457 1.866 3.149 4.388
3.75 10.478 9.923
3.5
5 10 15 20 25 30 35 min Figure H4: Total fatty acid content of crude sodachelin siderophore extract
185 Table H4: Total fatty acid composition of crude sodachelin siderophore mix
RT Response Ar/Ht ECL Peak Name Percent Comment1 Comment2 1.689 3.647E+8 0.023 7.013 SOLVENT PEAK ---- < min rt 1.866 903 0.020 7.349 ---- < min rt 1.995 1936 0.017 7.594 ---- < min rt 2.363 2365 0.019 8.293 ---- < min rt 3.149 891 0.021 9.786 ---- 3.262 4435 0.024 10.000 10:0 2.82 ECL deviates 0.000 Reference 0.007 4.388 1233 0.031 11.422 10:0 3OH 0.73 ECL deviates -0.001 4.457 1813 0.029 11.495 C12 Primary Alcohol 1.07 ECL deviates 0.005 4.747 29448 0.029 11.799 12:1 w7c 17.05 ECL deviates -0.007 4.939 34872 0.031 12.000 12:0 20.01 ECL deviates 0.000 Reference 0.005 6.765 19742 0.036 13.456 12:0 3OH 10.70 ECL deviates 0.001 7.269 36961 0.036 13.816 14:1 w7c 19.79 ECL deviates 0.004 7.528 16461 0.037 14.000 14:0 8.76 ECL deviates 0.000 Reference 0.004 9.565 9026 0.041 15.278 Unknown 15.273 "D" 4.65 ECL deviates 0.005 9.923 1534 0.043 15.490 Sum In Feature 2 0.79 ECL deviates 0.000 14:0 3OH/16:1 ISO 10.478 1492 0.043 15.818 16:1 w7c 0.76 ECL deviates 0.001 10.785 10826 0.042 16.000 16:0 5.50 ECL deviates 0.000 Reference 0.003 13.993 9718 0.051 17.825 Sum In Feature 8 4.83 ECL deviates 0.000 18:1 w9t 14.303 5124 0.046 18.000 18:0 2.55 ECL deviates 0.000 Reference 0.002 ---- 1534 ------Summed Feature 2 0.79 12:0 ALDE Unknown 10.928 ------16:1 ISO I/14:0 3OH 14:0 3OH/16:1 ISO ---- 9718 ------Summed Feature 8 4.83 18:1 w9t. 18:1 w9t
This is the total fatty acid composition of crude sodachelin siderophore mix. In order to name
the unsaturated fatty acids, the EUKARY peak naming table was used because it contains data
on more fatty acids than the previously used method for sodachelin B, D and E. In this case, the
fatty acid that should correspond to sodachelin C was assigned a double bond in the w7 cis position and sodachelin E was also assigned an w7 cis double bond. There were longer fatty acids in this sample, but it is unknown if the are derived from siderophores or present in the crude sample as an artifact.
186 FID2 B, (E07521.485\B0046223.D) pA 6.2 1.693 4.747
6 2.003 25.609 2.371
5.8 3.268 1.874
5.6 6.756
5.4
5.2
5
5 10 15 20 25 30 35 min Figure H5: Fatty acid methyl ester peak identification for sodachelin A
187 Table H5: Fatty acid methyl ester peak identification for sodachelin A.
RT Respons Ar/H ECL Peak Name Percen Comment1 Comment2 1.693 4.859E+ 0.02 7.010 SOLVENT PEAK ---- < min rt 1.874 1142 0.02 7.354 ---- < min rt 2.003 1918 0.01 7.600 ---- < min rt 2.371 1598 0.02 8.301 ---- < min rt 3.268 2005 0.02 10.005 10:0 31.06 ECL deviates 0.005 Reference 4.747 3677 0.03 11.803 12:1 w7c 52.09 ECL deviates -0.003 6.756 1267 0.03 13.458 12:0 3OH 16.85 ECL deviates 0.003 25.609 6082 0.25 24.908 ---- > max ar/ht
This is the assignment of fatty acid methyl esters present in a very concentrated sample of sodachelin A. Because sodachelin A is produced in the lowest concentration of all sodachelins, there is some overlap in fatty acids from sodachelin B and C. The peak eluting at a retention time of 3.268 minutes corresponds to 10:0, which matches what was expected based on mass spectral data.
188 FID1 A, (E07521.485\A0056224.D) pA 8 1.689 4.748
7.5 3.149 7
6.5 2.364 6
5.5 4.540 4.940 5 1.995 6.592
4.5 25.919 6.766 28.060 23.866 9.042 21.671 30.613 19.353 33.758 11.683 16.922 14.360 4 37.722 1.866
3.5
5 10 15 20 25 30 35 min Figure H6: Fatty acid methyl ester peak identification for sodachelin C
189 Table H6: Fatty acid methyl ester peak identification for sodachelin C.
RT Respons Ar/H ECL Peak Name Percen Comment1 Comment2 1.689 3.567E+ 0.02 7.019 SOLVENT PEAK ---- < min rt 1.866 585 0.02 7.355 ---- < min rt 1.995 2692 0.01 7.601 ---- < min rt 2.364 5936 0.01 8.300 ---- < min rt 3.149 9536 0.02 9.792 ---- 4.540 6668 0.02 11.584 ---- 4.748 17473 0.02 11.803 12:1 w7c 50.28 ECL deviates -0.003 4.940 6712 0.03 12.003 12:0 19.14 ECL deviates 0.003 Reference 0.006 6.592 4479 0.03 13.334 ---- 6.766 4548 0.03 13.459 12:0 3OH 12.24 ECL deviates 0.004 9.042 3783 0.03 14.967 Unknown 14.967 9.75 ECL deviates 0.000 11.683 3135 0.04 16.514 ---- 14.360 2975 0.04 18.031 ---- 16.922 3032 0.04 19.494 ---- 19.353 3482 0.04 20.919 21:1 w5c 8.58 ECL deviates -0.009 21.671 4027 0.04 22.332 ---- 23.866 4443 0.04 23.726 ---- 25.919 4769 0.04 25.073 ---- 28.060 4615 0.05 26.272 ---- 30.613 4440 0.06 27.703 ---- 33.758 4243 0.07 28.987 ---- 37.722 3812 0.08 30.486 ---- > max rt
This is the assignment of fatty acid methyl esters present in a very concentrated sample of sodachelin C. There is some overlap in fatty acids from sodachelin B and D, but the peak at
4.748 comprises the largest percentage of the samples. This peak corresponds to a 12:1 w7c fatty acid. The length of this fatty acid and single unsaturated bond matches what was expected from mass spectral data.
190 FID2 B, (E07521.485\B0056225.D) pA
9 1.694 7.258
8.5
8
7.5
7
6.5
6 2.004 2.372 7.515 4.938
5.5 1.875 6.757
5 10 15 20 25 30 35 min
Figure H7: Fatty acid methyl ester peak identification for sodachelin E
191 Table H7: Fatty acid methyl ester peak identification for sodachelin E.
RT Respons Ar/H ECL Peak Name Percen Comment1 Comment2 1.694 4.81E+8 0.02 7.010 SOLVENT PEAK ---- < min rt 1.875 616 0.02 7.353 ---- < min rt 2.004 1490 0.01 7.599 ---- < min rt 2.372 1507 0.02 8.300 ---- < min rt 4.938 1870 0.03 12.000 12:0 7.90 ECL deviates 0.000 Reference 0.007 6.757 1284 0.03 13.457 12:0 3OH 5.14 ECL deviates 0.002 7.258 19642 0.03 13.816 14:1 w7c 77.76 ECL deviates 0.004 7.515 2337 0.03 14.000 14:0 9.20 ECL deviates 0.000 Reference 0.006
The peak which elutes at 7.258 minutes comprises 77 % of the total peak area and matches with
the fatty acid 14:1 w7c. The length of this fatty acid matched what was expected from MS data for sodachelin E. Low amounts of 12:0, 12:0 3OH and 14:0 are also present and likely a result of some carryover during the purification of sodachelin E.
192 APPENDIX I. Preliminary mass spectral data for SL01 siderophores.
193 Figure I1: MALDI-MS/MS data for SL01 siderophore. This is the total MS/MS spectrum.
194 Figure I2: Zoom of fragment ions from SL01 MALDI-MS/MS spectrum from 80-400 amu.
195 Figure I3: Zoom of fragment ions from MALDI-MS/MS of siderophore from SL01 from 400 – 800 amu
196 Figure I4: Zoom of fragment ions from MALDI-MS/MS from siderophore from SL01 from 800- 1200 amu.
197 APPENDIX J: UV-Vis spectrum of Sodachelin F This contains raw data for the UV-Vis spectrum of Sodachelin F before and after UV irradiation at 565 W/m2
1 Table J1: Raw data for UV-Vis spectrum of the Fe(III)-sodachelin F prior to UV exposure in a sodium bicarbonate buffer at pH 9.9. The blank is of a siderophore free and iron free solution of sodium bicarbonate.
2 Table J1: Continued
3 Table J2: Raw data for UV-Vis spectrum of the Fe(III)-sodachelin F after exposure to simulated sunlight in a sodium bicarbonate buffer at pH 9.9. The blank is of a siderophore free and iron free solution of sodium bicarbonate.
4 Table J2: Continued
5 Table J3: Data for production of Fe(II) form the Fe(III)-sodachelin F complex exposed to simulated sunlight with the bathophenanthroline disulfonate chelator (BPDS)
6 Table J4: Data for the production of Fe(II) from the Fe(III)-sodachelin F complex. Control without BPDS exposed to sunlight
7 Table J5: Data for the production of Fe(II) from the Fe(III)-sodachelin F complex. Control with BDPS shielded from UV light.
8