Siderophores from Neighboring Organisms Promote the Growth Of

Home , Parvularcula, Parvularcula bermudensis, Winogradskyella

Siderophores From Neighboring Organisms Promote the Growth of

Uncultured Bacteria

A dissertation presented

Anthony D’Onofrio

to The Department of Biology

In partial fulfillment of the requirements for the degree of Doctor of Philosophy

in the field of

Biology

Northeastern University Boston, Massachusetts November, 2008

Siderophores From Neighboring Organisms Promote the Growth of

Uncultured Bacteria

Anthony D’Onofrio

ABSTRACT OF DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology in the Graduate School of Arts and Sciences of Northeastern University, November, 2008

ABSTRACT

The majority of bacterial cells in an environmental sample will not grow on synthetic media and are known as “unculturable”. As high as 99.9% of total cells counted by microscopy will not form colonies on standard Petri dishes. This phenomenon is known as the Great Plate Count Anomaly and is a significant unsolved problem in microbiology. The data presented in this study tests the hypothesis that some unculturable bacteria fail to grow on synthetic media because they are lacking specific growth factors from neighboring species, which signal a suitable environment to grow.

In order to test this hypothesis, environmental bacteria were isolated from intertidal sediment and examined for helper-dependent relationships where an unculturable bacteria would only grow in the presence of a culturable helper species. Several such pairs were identified and a model unculturable, M. polysiphoniae KLE1104, was chosen for further study with model helper strain, M. luteus KLE1011, isolated from the same environment. Filtered spent supernatant of M. luteus KLE1011 as well as E. coli was capable of inducing growth of M. polysiphoniae KLE1104. E. coli knockout strains deficient in production of the iron chelating siderophore, enterobactin, were unable to induce growth. Testing purified enterobactin confirmed the compound was necessary and sufficient to induce growth of macrocolonies of M. polysiphoniae KLE1104. Five siderophores were purified from M. luteus KLE1011, and each was capable of inducing growth of the unculturable. Structure elucidation of the siderophores revealed that they were novel acyl-desferrioxamines with variable terminal modifications to increase hydrophobicity. Several species were then isolated from the same environment, which were dependent on M. luteus KLE1011. Six of these isolates along with two others 3

previously isolated from the same environment were tested for growth induction by a panel of 16 commercial siderophores and the five M. luteus KLE1011 siderophores.

Each unculturable isolate was helped by a different set of siderophores, ranging from 6 to all 21 siderophores tested. This growth dependence on a varying set of siderophores suggests a strategy of only growing in the presence of a suitable environment, which is signalled by the presence of siderophores from appropriate neighbors.

ACKNOWLEDGEMENTS

I would like to thank my advisor Kim Lewis for being an insightful, fair and patient mentor. His dedication to investigate challenging scientific questions was inspiring and provided a wonderful atmosphere to conduct research.

I would also like to thank my thesis committee, Slava Epstein, Veronica Godoy-Carter,

Jon Clardy and Eric Stewart for their support and helpful advice.

Thanks to Eric Stewart for his excellent guidance. The success of the project would not have been possible without him.

Thanks also to Kathrin Witt for her many contributions to the project, Jason Crawford for his excellent chemical isolation and structure elucidation work and Ekaterina Gavrish for her 16S sequencing and helpful suggestions.

Thanks of course to the rest of the Lewis Lab past and present who provided stimulating conversation, advice and humor.

I would like to thank my wife for her love and support.

I would also like to thank my parents for always encouraging me to get a higher education and providing me with the resources to do so. 5

DEDICATION

I would like to dedicate this thesis to my wife, Melanie D’Onofrio and my parents,

Mario and Alison D’Onofrio for their love and support.

TABLE OF CONTENTS

Abstract 3

Acknowledgements 5

Dedication 6

Table of Contents 7

List of Figures 8

List of Tables 9

Chapter 1. Introduction 10

Chapter 2. Methods 17

Chapter 3. Results 21

Chapter 4. Discussion 31

Figures 40

Tables 55

Appendix I 57

Appendix II 58

Appendix III 59

References 60

LIST OF FIGURES

Figure 1. Sand Biofilm Community on Canoe Beach Intertidal Sediment Samples.

Figure 2. Growth of M. polysiphoniae KLE1104 is Induced by M. luteus KLE1011.

Figure 3. Growth Induction of M. polysiphoniae KLE1104 by Enterobactin from E. coli.

Figure 4. Siderophores Produced by Helper Strain M. luteus KLE1011.

Figure 5. Isolation of Bacteria Dependent on M. luteus KLE1011.

Figure 6. Verification of M. luteus KLE1011 Dependent Isolates.

Figure 7. Growth Induction of Unculturable Isolates By the Five M. luteus KLE1011

Siderophores.

Figure 8. Commercial Siderophores Tested For Growth Induction of Unculturables.

Figure 9. Growth Induction of Unculturables By Different Siderophores.

Figure 10. Complementation of Siderophore Dependence With Soluble Iron (II).

Figure 11. Increased Recovery Adding Soluble Fe(II) to R2Asea Plates.

Figure 12. Fe(II) Induction of Rubritalea sp. KLE1210 and Parvularcula sp. KLE1250.

Figure 13. Viable Cells Recovered From a Single Intertidal Pebble.

Figure 14. Iron Dependence of Isolates Re-Suspended From Pebble Biofilm.

Figure 15. Recovery of Intertidal Sediment Biofilm Bacteria From a Single Pebble.

LIST OF TABLES

Table 1. Closest Relatives of 15 M. luteus KLE1011 Dependent Isolates.

Table 2. Closest Relatives of Iron Dependent Isolates From Canoe Beach.

CHAPTER 1: INTRODUCTION

The “Great Plate Count Anomaly” is an important unsolved problem in microbiology referring to the large discrepancy between cells counted in an environmental sample and the number of colonies formed on solid media (Butkevich,

1932; Staley and Konopka, 1985). It is generally accepted that less than 1% of cells in any given environmental sample will form colonies on synthetic media (Barer and

Harwood, 1999; Giovannoni, 2000). Those that fail to grow in the lab are commonly referred to as “unculturable” or “uncultivable” bacteria. Advances in culture independent sequencing and phylogenetic comparison of 16s rDNA have revealed that several deep branching clades of bacteria can be detected but have never been cultured

(Colwell and Grimes, 2000; Handelsman, 2004). In 1987 Carl Woese proposed a phylogenetic tree of life based on the comparison of 16s and 18s rRNA sequences

(Woese, 1987). In his original tree, three domains were proposed, the Eubacteria

(Bacteria), the Archaeabacteria (Archaea) and the Eukaryotes. The Bacteria were divided into 11 phyla based on distinct groups that clustered together when comparing the sequence similarity of 16s rDNA from cultured species (Appendix I, pg. 57). Based on the amplification and sequencing of 16s rDNA directly from the environment, numerous candidate phyla have been added which have no cultured representatives. By

1998, 36 bacterial phyla had been proposed, of which 13 were candidate phyla with no cultured members (Appendix II, pg. 58) (Hugenholtz et al., 1998). By 2003, this number had risen to 52 phlya; 26 being candidate phyla (Appendix III, pg. 59) (Rappe and

Giovannoni, 2003). It therefore appears that the large unculturable fraction of cells, which fail to grow on synthetic media are in part comprised of members of species and 10

entire phlya which have never been cultured. Large scale DNA sequencing studies have provided valuable data about the abundance and diversity of biosynthetic operons in various marine environments (Rusch et al., 2007). Bacteria grown in pure culture however, can provide physiological insights that cannot be obtained by sequence data alone and the search for novel secondary metabolites will be greatly aided by the ability to culture new species.

Several studies have shown that increased recovery or culturing of rare isolates can be achieved by growing cells in conditions that simulate the natural environment.

Pelagibacter ubique, a member of the previously uncultured SAR11 phylum, was grown in pure culture by “extinction culturing” which involves the inoculation of single cells from seawater into small volumes (2 mL) of autoclaved seawater amended with very low carbon sources (Rappe et al., 2002). While growth in liquid media yielded concentrations up to 106 cells/mL, the strain did not grow on solid synthetic media. The authors state that this limitation may suggest the presence of an unusual growth factor in the ecology of the organism. The complete genome of P. ubique was subsequently sequenced, revealing interesting details that would not have been possible without a successful culturing method (Giovannoni et al., 2005). P. ubique has the smallest genome, and the fewest number of predicted open reading frames for any free living organism. It also has the smallest intergenic regions of any organism sequenced to date.

The interesting “streamlining” of this organism would not have been discovered without the ability to sequence genomic DNA from a cultured strain. A method for genome sequencing from a single cell has been reported, but complete assembly of the genome was not achieved (Marcy et al., 2007). 11

Another method has been developed to incubate samples in a diffusion chamber, which simulates the natural environment. The technique involves inoculating samples in agar sandwiched between two semi-permeable membranes (0.03 µm) that restrict the movement of cells. The diffusion chamber is then incubated on top of sediment submerged in natural seawater. Recovery up to 40% was reported using this method

(Kaeberlein et al., 2002). The diffusion chamber also successfully increased recovery of isolates from rarely cultivated groups such as Deltaproteobacteria, Verrucomicrobia,

Spirochaetes and Acidobacteria (Bollmann et al., 2007). The method is successful presumably because cells incubating in their natural environment have access to the chemical factors that are produced by other members of the community. One limitation of the diffusion chamber, however, is the difficulty in maintaining growth of many strains once they are removed from seawater tanks and plated on Petri dishes. A better understanding of the compounds inducing growth in the natural environment and in diffusion chambers will aid in maintaining growth and studying such uncultivable organisms. In a similar strategy, environmental samples have also been grown on filter membranes placed directly on soil to allow the diffusion of factors from the soil. Using this method, FISH analysis revealed microcolonies of members of the uncultured TM7 phylum (Ferrari et al., 2005). Techniques simulating natural environmental conditions have been successful most likely due to the availability of natural factors required for growth. While such advances are important and provide practical culturing methods, a better understanding of the signals involved will further improve culturing strategies.

Other studies have shown increased recovery using a variety of media additions, including cAMP and homoserine lactone (Bruns et al., 2002; Bruns et al., 2003). 12

Increased recovery has also been reported from freshwater sediment by plating samples on media solidified with gellan gum instead of agar (Tamaki et al., 2005). While these studies provide valuable insights, none have achieved recoveries higher than 10% and do not verify that any of the media amendments are actually signals provided by helpers in the natural environment. Purifying compounds from helper strains can provide a more direct way of identifying growth factors as opposed to screening media additions without any prior indication of their efficacy. One interesting example of an unculturable species helped by an environmental isolate has been preliminarily described. The

Alphaproteobacterium, Catellibacterium nectariphilum, is induced in liquid culture by the spent supernatant of several Sphingomonas species isolated from the same activated sludge (Tanaka et al., 2004). Ten-percent spent supernatant was added to nutrient media and growth was measured by optical density showing clear growth induction. E. coli

DH5α also was effective, while Pseudomonas putida and Bacillus subtilus failed to induce growth. Efforts by the group to isolate and identify the growth factor were unsuccessful although they report it being heat-stable, a non-peptide and having low molecular weight (below 1,000 Da). Several additions to the media were tested including vitamins, short-chained fatty acids, amino acids, alcohols, norepinephrine, dopamine, isoprotenol, 5-hydroxytryptomine, homoserine lactones and cAMP, all of which failed to induce growth. Such reports of helper strains, which aid growth of an unculturable, typically fall short of identifying a specific compound that induces growth.

One recent exception is the discovery of a short 5 amino acid peptide, which induces the growth of microcolonies of a marine sediment unculturable isolate (Nichols et al., 2008).

The peptide, LQPEV, at 3.5 nM induces growth of Psychrobacter sp. strain MSC33. It 13

was also shown that a relative of Cellulophaga lytica, isolated from the same environment, was able to induce growth of the unculturable when grown in co-culture separated by a semi-permeable membrane. The discovery of additional growth factors will provide a more complete understanding of the interactions in microbial communities, and provide signals to help induce the growth of unculturable bacteria on synthetic media.

The results in the present study describe the discovery of novel siderophores from a culturable marine bacterium, which induce the growth of several otherwise unculturable bacterial species from the same environment. Siderophores are low molecular weight compounds which have a high binding affinity for insoluble Fe(III).

Bacteria and fungus release siderophores to sequester Fe(III) and then transport the ferric form back into the cell (Neilands, 1995). Iron is required by almost all living organisms to form the catalytic center for redox reactions in numerous enzymes involved in electron transport, photosynthesis and amino acid, nucleoside and DNA synthesis

(Wandersman and Delepelaire, 2004). Iron is essential for growth, but in aerobic conditions it is oxidized to the insoluble Fe(III) state and is bound to organic ligands.

Iron is also thought to be the growth-limiting factor for microorganims in the marine environment (Street and Paytan, 2005; Tortell et al., 1999). Controversial ‘ocean fertilization’ experiments have been conducted which result in microbial blooms after the addition of iron (Boyd et al., 2007; Chisholm et al., 2001; Coale et al., 2004; Coale et al., 1996; Martin et al., 1994). Microorganims in aquatic environments produce siderophores to scavenge for iron, which is both scarce and essential for growth.

Members of the Lactobacillus have long been thought to be the exception to the rule of 14

requiring iron, although one study showed an iron growth requirement in a specific defined media (Elli et al., 2000). An early report of this phenomenon was entitled,

“Lactobacillus plantarum, an organism not requiring iron,” but the paper describing the full genome sequence of Lactobacillus plantarum WCFS1, had no mention of iron and it seems unclear if the organism’s iron independence is conclusive (Archibald, 1983;

Kleerebezem et al., 2003).

The structures of numerous siderophores have been determined from bacteria such as Escherichia coli (enterobactin), Acinetobacter calcoaceticus (acinetobactin),

Mycobacterium tuberculosis (mycobactin), Pseudomonas aeruginosa (pyoverdine and pyochelin) and Yersinia pestis (yersiniabactin) (Crosa and Walsh, 2002). Siderophores isolated from marine bacteria are commonly amphiphilic, such as marinobactin

(Marinobactin sp. DS40M6), aquachelin (Halomonas aquamarina), amphibactin (Vibrio sp.), ochrobactin (Ochrobacter sp. SP18) and synechobactin (Synechococcus PCC7002)

(Butler and Vraspir, 2009). These amphiphilic marine siderophores consist of a peptidic iron chelating head region with a variable fatty acid tail. In the absence of Fe(III), these siderophores are thought to spontaneously form micelles in aqueous solution (Martinez et al., 2000). It seems that marine organisms commonly produce multiple variants of a siderophore with altered hydrophobicities to decrease loss to open water in the aquatic environment. The novel siderophores discovered in the present study also have terminal modifications, resulting in five versions with various hydrophobicities, presumably to help retain them within the sand biofilm. These siderophores were able to induce the growth of several otherwise unculturable species from the same intertidal environment.

This discovery will help design novel methods for enriching and isolating members of specific bacterial clades based on their particular siderophore specificity.

CHAPTER 2: METHODS

Scanning Electron Micrography of Intertidal Sediment

Samples of intertidal marine sediment were fixed by immersion in 3.0% glutaraldhyde in

0.1 M cacodylate buffer (pH 7.4) containing 0.35 M sucrose, for 1 hour at 4 °C. After rinsing in buffer the samples were post-fixed in 1.0% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at room temperature. Following postfixation the samples were rinsed with buffer then dehydrated through a graded series of ethanol. The specimens were then critical point dried with CO2 using a Smadri PVT-3 critical point dryer (Tousimis Corp. Rockville, MD). The specimens were mounted on aluminum specimen mounts with colloidal graphite, coated with 10-15 nm platinum/palladium

(80:20) using a Denton DV502 vacuum evaporator, and examined with a

Hitachi S-4800 field emission SEM (Hitachi, Ltd. Tokyo, Japan).

Isolation of Unculturable Bacteria and Helpers

Samples of intertidal sediment were collected from Canoe Beach at the Northeastern

University Marine Science Center in Nahant, MA. Sediment (5 g) was resuspended in 5 mL of filtered, autoclaved seawater and vortexed for 1 min. Samples were diluted and plated on solid R2Asea medium (R2A (Difco) diluted in 50% filtered, autoclaved seawater) in ten-fold dilutions to obtain plates of varying colony density. Plates were incubated at 30 °C for 5 days. Pairs of colonies growing within a 2 cm distance were cross-streaked in the pattern shown in Figure 2B on fresh plates to screen for helper- dependent patterns. Potential unculturable and helper isolates were resuspended in sterile seawater with 15% glycerol and frozen at 80 °C. Dependence on a helper was verified by spreading a plate with 103 to 104 cells of the potential 17

unculturable and spotting the potential helper species. Plates were incubated at 30 °C for

3-7 days and observed for growth of the potential unculturable near the helper spot. To isolate species dependent on M. luteus KLE1011, environmental samples were plated and spotted with KLE1011. After incubation for 5 days at 30 °C, colonies were screened that grew within 2 cm of KLE1011.

Testing Spent Media of M. luteus KLE1011 and E. coli Strains

To test the helping ability of M. luteus KLE1011 spent media, 5 mL cultures of liquid

R2NP media (0.5 g yeast extract, 0.5 g casamino acids, 0.5 g dextrose, 0.3 g sodium pyruvate and 0.3 g dipotassium phosphate dissolved in 50% filtered, autoclaved seawater) were inoculated from a glycerol stock and incubated at 30 °C with shaking for

2 days. Cultures were then centrifuged and filtered (0.22 mm). M. polysiphoniae

KLE1104 was spread on R2Asea plates followed by the addition of a tissue insert

(Nunc) with a 0.02 mm bottom membrane. 500 µL of spent media was added to the insert and plates were incubated at 30 °C for 5 days. E. coli BW25113 and the ΔluxS,

ΔtnaA, ΔentB and ΔentC knockouts from the Keio Collection knockout library (Baba et al., 2006) were tested using the same protocol.

Structure Elucidation of KLE1011 Acyl-desferrioxamine Siderophores

NMR experiments were performed on desferric siderophores in deuterated dimethylsulfoxide with a symmetrical NMR microtube susceptibility-matched with dimethylsulfoxide (Shigemi, Inc.) on a Varian INOVA 600 MHz NMR. The ferric and desferric forms of each purified siderophore were also separated over HPLC and detected using either a linear quadrupole ion trap / Fourier transform ion cyclotron resonance hybrid MS (Thermo Electron) or a linear quadropole MS (Agilent 6130). The 18

+1 ion of the desferric and ferric siderophores were selected for fragmentation analysis by MS/MS.

Growth Induction by Siderophores

The siderophores desferrifusigen, desferriaerobactin, desferricrocin, desferriarthrobactin, desferrischizokinen, desferrichrome, desferrirhizoferrin, desferrirhodin, desferrirhodotorulic acid, desferritriacyetylfusarinine C, desferrioxamine G, desferriornibactin-C6, desferrichrysin, desferricoprogen, desferrirubin, desferrioxamine

B (EMC Microcollections, Germany) and enterobactin (Genaxxon Bioscience,

Germany) were dissolved in DMSO at 0.01 mg/mL. To test growth induction, 10 mL of siderophore (0.01 mg/mL) was spotted on R2Asea plates seeded with an unculturable isolate. Plates were incubated for 3-7 days at 30 °C and observed for growth around the spotted siderophore.

Screening For Iron Dependent Unculturable Bacteria

Samples of intertidal sediment (5 g) from Canoe Beach in Nahant, MA were suspended in 5 mL of filtered, autoclaved seawater and vortexed for 1 minute. After allowing the bulk of sand to settle, samples were plated in 10-fold dilutions on R2Asea plates with added iron (0.001% iron sulfate added after autoclaving) to obtain plates with a low density of environmental colonies (10 – 50). After incubation for 7 days at 30 °C, colonies were picked with a sterile toothpick and stabbed onto both R2Asea and R2Asea

Iron plates. Plates were incubated for 14 days at 30 °C and observed for isolates which only grew on plates supplemented with iron.

Iron Dependence of Bacterial Isolates From a Single Intertidal Pebble

Intertidal sediment was collected from Canoe Beach in Nahant, MA. A single pebble, 19

approximately 5 mm in diameter, was selected randomly and washed seven times in sterile seawater. A 100 µL sample was plated to confirm the absence of viable cells from residual seawater. The sample was then sonicated for 30 seconds on a 40% power setting. The sample was diluted 10-fold and plated in triplicate on R2A ASsalts (R2A,

2% Artificial Sea Salts (Sigma)) with and without added iron(II) sulfate (0.001%). After incubation for 5 days at 30 °C, colonies were counted at 12x magnification.

Identification of Isolates by 16S rRNA Gene Sequencing

The 16S rRNA genes of the isolates were amplified using universal eubacterial PCR primers. Sequencing of the 16S rRNA gene was performed with an ABI3730XL automatic DNA sequencer by Macrogen Corp. (http://www.macrogen.com). The identification of phylogenetic neighbors and the calculation of pairwise sequence similarity were carried out using the EzTaxon server (http://www.eztaxon.org/; (Chun et al., 2007)). Nucleotide sequences for model helper M. luteus KLE1011, as well as the 8 unculturable isolates tested with the siderophore panel, have been deposited in the

GenBank database; KLE1009: FJ229465, KLE1011: FJ229461, KLE1063: FJ229467,

KLE1078: FJ229459, KLE1080: FJ229460, KLE1104: FJ229466, KLE1111: FJ229462,

KLE1122: FJ229464, KLE1123: FJ229463.

CHAPTER 3: RESULTS

Isolation of Unculturable Bacteria Dependent on Helper Growth Induction

Samples of intertidal sediment were collected from Canoe Beach in

Massachusetts Bay near Nahant, MA. Sediment was re-suspended in sterile seawater and vortexed to disrupt cells from the biofilm. Representative sand particles were imaged by Scanning Electron Microscopy to check for the presence of a biofilm community on sediment from the same environment (Figure 1). The SEM images confirmed the presence of a biofilm with a variety of shapes that appeared to be microscopic cells. The most obvious of these cells were bacilli shaped bacteria (1.5 x

0.5 µm), which appeared to produce long, thin structures for attachment to the biofilm

(Figure 1E). Re-suspended samples were plated in ten-fold dilutions on R2Asea media

(dissolved in 50% sterile seawater). After incubation at 30° C for 5 days, colonies of varied size, morphology and color appeared on the plates (Figure 2A). In previous experiments it was observed that the number of colonies formed seemed to be disproportionately higher on plates with a greater density of colonies. It was hypothesized that some species forming colonies only grew due to a helping factor provided by a colony of a nearby culturable species. Therefore, pairs of colonies growing within 2 cm of each other were picked and each re-streaked on one half of an

R2Asea plate. Each pair was streaked twice for dilution on the plate, and cross-streaked through the center. The plating pattern allowed for areas of increasing distance between the pairs tested. In many cases, after incubation, one of the isolates appeared to grow 21

well in areas of the plate in close proximity to the second isolate, but colony size of the isolate gradually decreased as the distance increased from the second isolate. In these cases it appeared that the first isolate was receiving a growth-inducing factor from the other “helper” species and would be unculturable if plated in isolation. In many cases, upon re-inoculation, these dependent strains would indeed only grow on solid R2Asea media in the presence of a culturable helper species. One such unculturable isolate,

Maribacter polysiphoniae KLE1104 (99.9% similar to Maribacter polysiphoniae LMG

23671(T) based on 16s rDNA comparison) was studied further to identify the growth- inducing factor. A model helper strain isolated from the same environment,

Micrococcus luteus KLE1011 (99.5% identical to Micrococcus luteus DSM 200030 (T) by 16s rDNA comparison), was cross-streaked with M. polysiphoniae KLE1104 (Figure

2B). The colony size of Micrococcus luteus KLE1011 remained consistent throughout the plate, which is typical for culturable bacteria. The colony size of M. polysiphoniae

KLE1104, in contrast, gradually decreased as distance from the culturable helper species increased. Plates were then spread evenly with M. polysiphoniae KLE1104 and spotted with M. luteus KLE1011 to confirm the dependent nature of the unculturable isolate.

The same test was done with several potential unculturable isolates and Figure 2C shows a typical example of the helping effect. Unculturable species formed a distinct ring of growth around the spotted helper, but their colony size gradually decreased as distance from the helper increased. Many of the unculturable strains isolated and tested, including M. polysiphoniae KLE1104, did not form colonies visible at 160x magnification unless they were grown in the presence of a helper strain. Other strains isolated were clearly helped by M. luteus KLE1011 (colony size was larger near the 22

helper) but were still capable of forming small colonies without help.

Filtered spent supernatant of M. luteus KLE1011 grown in liquid R2NP media

(R2A without agar, diluted in 50% sterile seawater) was tested for helping ability. M. polysiphoniae KLE1104 was spread evenly on a plate and 500 µL of filtered spent supernatant of M. luteus KLE1011 was added to a tissue insert with a 0.22 µm membrane (Figure 2D). The same pattern of growth induction was observed indicating the growth-induction factor was diffusible and present in the culture supernatant. Media alone was not capable of inducing growth.

Identification of Genes From Escherichia coli Coding for a Growth Factor

In order to identify genes responsible for production of the growth factor by testing helper knockout strains, we tested Escherchia coli BW25113 for helping ability.

Filtered spent supernatant of E. coli grown in liquid R2NP media was able to induce growth of many of the unculturable strains tested including M. polysiphoniae KLE1104

(Figure 3A). E. coli strains deficient in production of known secreted metabolites were tested for helping ability. Supernatant from a strain deficient in AI-2 production (Δ luxS) was capable of inducing growth (Figure 3B) as well as supernatant from a strain that does not secrete indole (ΔtnaA) (Figure 3C). However, strains deficient in genes coding for the first two steps in the enterobactin biosynthetic pathway (ΔentB and

ΔentC) were not capable of inducing growth of M. polysiphoniae KLE1104 (Figure

3D,E). This indicated that the growth induction factor was enterobactin, the iron sequestering siderophore produced by E. coli. To verify that enterobactin alone was

capable of inducing growth, a purified sample of enterobactin was spotted on a spread plate of M. polysiphoniae KLE1104. Enterobactin was capable of the same strong growth induction seen by the helpers (Figure 3E).

Structure Elucidation of Novel Desferrioxamine Siderophores From M. luteus

KLE1011

Since it was verified that the E. coli factor inducing growth of M. polysiphoniae

KLE1104 was the siderophore enterobactin, it was of interest to determine if the environmental helper M. luteus KLE1011 was also inducing growth through a siderophore. Since siderophore production is known to be upregulated in iron-limited conditions, M. luteus KLE1011 was grown in Artificial Seawater media with 100 pM

Fe(III). The spent culture supernatant was acidified (pH = 2) and the metabolites were removed by solid phase extraction (Amberlite XAD-2). Five different siderophores were purified over size exclusion chromatography (LH-20) and reversed-phase (C18) high- pressure liquid chromatography (HPLC). In addition to exhibiting iron binding properties in the chromazurol S assay, both the desferric and ferric forms of each siderophore were observed by HPLC-mass spectrometry (MS) coupled with UV-visible spectroscopy analysis, confirming their role in iron acquisition (Figure 4A). Nuclear magnetic resonance (NMR) spectroscopy was carried out to elucidate the structure of the

5 purified siderophores, revealing structural similarity to the desferrioxamine class with a central core with alternating N-hydroxycadaverine and succinic acid units. The structures differed from the known siderophore desferrioxamine B in their terminal acyl

attachments. Three of the siderophores (3-5) terminate with branched chain fatty acids while two (1-2) terminate with an atypical phenylacetyl moiety. Additional modifications were observed in acyl-desferrioxamines 1 and 3, beginning with a primary amine acetyl-cap. The five siderophores discovered were each capable of inducing growth of M. polysiphoniae KLE1104, demonstrating that they were the growth factors responsible for the helping activity (Figure 4C,D).

Isolation of Bacteria Dependent on M. luteus KLE1011 For Growth

After verifying that siderophores from M. luteus KLE1011 could induce growth of M. polysiphoniae KLE1104, it was of interest to determine whether this was a unique phenomenon or if several species of bacteria could be isolated from the same environment that would also be dependent on the novel desferrioxamine siderophores from M. luteus KLE1011. A screen was conducted to isolate new strains from Canoe

Beach that were dependent on M. luteus KLE1011. Samples of intertidal sediment were collected and plated on R2Asea plates in ten fold dilutions. M. luteus KLE1011 was spotted on each plate and incubated at 30 °C for 5 days (Figure 5). After incubation, colonies growing within 2 cm of M. luteus KLE1011 were cross-streaked with the same helper (KLE1011) to test for dependence. Stocks were prepared of isolates that appeared to be dependent and further verified by spread plates of the potential unculturables spotted with M. luteus KLE1011 (Figure 6). A total of 185 colonies were screened, of which 46 (25%) were helped by M. luteus KLE1011. Twenty of these isolates were helped, but were capable of weak growth when plated alone, while the

remaining 26 isolates (14% of 185 screened) had no growth in the absence of the helper.

16s rDNA was amplified and sequenced for 15 of these isolates showing the dependent bacteria isolated were members of the Proteobacteria or Bacteroidetes and had 16s rDNA similar to typed culturable bacteria (Table 1). Six of the dependent isolates from the screen were tested with each of the 5 purified siderophores from M. luteus KLE1011.

All of the isolates tested showed strong growth induction by at least one of the KLE1011 siderophores. Examples of induction by the M. luteus KLE1011 siderophores are shown in Figure 7.

Siderophore Specificity of M. luteus KLE1011 Dependent Isolates

After isolating several bacterial species dependent on M. luteus KLE1011 for growth induction, it was of interest to determine the specificity of representative isolates for various siderophores. Sixteen commercial siderophores from bacterial and fungal sources (Figure 8) and the 5 siderophores from M. luteus KLE1011 were tested for growth induction of six isolates from the screen. The model unculturable M. polysiphoniae KLE1104 was added to the screen as well as one other strain,

Cyclobacterium amurskyense KLE1009, isolated in initial cross-streak experiments.

KLE1009 showed the widest range of siderophore induction and grew in the presence of all 21 siderophores tested, while other strains such as Sulfitobacter sp. KLE1123 only grew in the presence of 6 of the siderophores (Figure 9). Each strain was induced by a particular set of siderophores. The Bacteroidetes on average were capable of using a wider range of siderophores than the Proteobacteria. The Bacteroidetes were induced by

an average of 18.0%+/-2.9% of the siderophores, while the Proteobacteria were induced by 9.0%+/-4.1% of the siderophores tested.

Complementation of Siderophore Dependence With Soluble Fe(II)

Siderophores provide bacteria with a source of iron. Therefore, we tested whether adding soluble Fe(II) to media in the form of iron(II) sulfate would allow growth of M. polysiphoniae KLE1104. Iron Sulfate was added to media over a range of concentrations after autoclaving and it was found that 0.001% iron sulfate was the ideal concentration allowing growth of large colonies. It was also found that spotting 1% iron sulfate on spread plates of M. polysiphoniae KLE1104 or other helper dependent unculturable isolates would produce a concentration gradient allowing growth of the unculturable in a distinct ring (Figure 10). Since this 0.001% optimum concentration exceeds reported concentrations of soluble iron in seawater by six orders of magnitude, it is likely that these unculturable isolates are relying on siderophores from neighboring helper species for growth induction in the environment (Johnson et al., 1997). The ability to complement this need however, by adding Fe(II) after autoclaving media, led us to test relative recovery from an environmental sample adding soluble iron at the optimum concentration. Samples of intertidal sediment were collected from Canoe

Beach and cells from the biofilm were dispersed by vortexing. Samples were plated in ten-fold dilutions in triplicate on R2Asea and R2Asea with added iron(II) sulfate (final concentration of 0.001%; added after autoclaving). The plates were incubated at 30° C and colonies were counted after 10 days at 12x magnification. The soluble iron resulted

in a 10.5 fold higher recovery (Figure 11). The increase in recovery was even greater on plates where 10-fold less cells were plated. At the 1000 fold dilution from the vortexed sample, a 25.8-fold increase in recovery was observed. This higher recovery is apparently due to a lack of siderophore helping effects on non-iron supplemented plates with a low number of colonies. Plates with added soluble iron had a characteristic qualitative difference from the control plates. The plates appeared similar except for the presence of numerous small colonies (< 0.5 mm in diameter), which appeared on the

R2Asea Iron plates after 3-5 days. A total of 107 small colonies were picked from

R2Asea Iron plates and re-inoculated on plates with and without added iron to test for iron dependence. Filtering out plates that were contaminated and those where no growth appeared on either plate, 20 out of 95 colonies only grew on plates with added iron. An additional 53 colonies grew better with iron (larger colony size) but were capable of forming microcolonies without iron. In a second trial of the same experiment, 11 of 16 colonies tested showed strong iron dependence. 16s rDNA was amplified and sequenced from 15 of these iron dependent isolates (from both isolation sets), revealing the presence of 2 strains from rarely cultured groups (Table 2).

One of the isolates, Parvularcula sp. KLE1250, has a 16s rDNA sequence most similar to Parvularcula bermudensis (Proteobacteria, Alphaproteobacteria,

Parvularculales, Parvularculaceae). While members of the Alphaproteobacteria are commonly cultured, only 1 typed strain and 3 cultured isolates within the order

Parvularculales are reported in the Ribosomal Database (Michigan State University).

Parvularcula bermudensis became the first member of this novel 7th order of the

Alphaproteobacteria when it was reported in 2003 (Cho and Giovannoni, 2003). 28

The second rare iron dependent strain isolated, Rubritalea sp. KLE1210 is a member of the Verrucomicrobia phylum. Members of the Verrucomicrobia are frequently found in high numbers from environmental 16s rDNA sequencing but tend to be rarely cultured in isolation (Hugenholtz et al., 1998). The Ribosomal Database contains 16s rDNA sequences from only 10 typed strains and 94 cultured isolates within this entire phylum. Both Parvularcula sp. KLE1250 and Rubritalea sp. KLE1210 grew well when 1% iron sulfate was spotted (Figure 12), but were not induced when tested with the 16 commercial and 5 KLE1011 siderophores.

Analysis of Siderophore Dependence on a Single Pebble Biofilm Community

Originally, our intertidal sediment samples were obtained by vortexing 5 g of sediment and plating cells that remained in suspension after allowing the bulk sand to settle. From this method, it was unclear if the unculturable species isolated were loosely associated with the sediment or attached members of the sand biofilm community. In order to verify that the same iron/siderophore dependence applied to members of an intertidal sand biofilm community, a single pebble (approximately 5 mm in diameter) was washed with sterile seawater and sonicated in 900 µL of sterile seawater to re- suspend cells from the biofilm. Samples (100 µL) were plated on R2A ASsalts Iron before and after sonication to verify that all colony-forming cells were re-suspended from the biofilm and not from un-rinsed seawater. After 4 days of incubation at 30° C several colonies of diverse color, size and morphology could be seen growing from the sonicated sample (Figure 13A). The control plated before sonication in contrast showed

no growth, verifying that the cells plated were disrupted from the natural sand biofilm

(Figure 13B). After 10 days of incubation, 1288 colonies were counted, viewed at 12x magnification. This gave an estimate of 11,592 viable, culturable cells that were dislodged from the pebble. Several colonies (75) were picked and re-streaked on R2A

ASsalts with and without added soluble iron to screen for isolates dependent on iron. A mix of colony sizes was picked, attempting to avoid replicates of colonies that appeared similar. After incubation for one week, plates were observed and several strains only formed colonies on the plates supplemented with soluble iron (Figure 14). Twelve of the isolates did not re-grow on either plate when re-inoculated. Most of the strains formed large colonies on iron plates but had no growth or only very small microcolonies without iron. Of those that formed microcolonies, growth of single colonies was usually not visible further away from the large initial inoculum. These iron dependent isolates accounted for 56% (35 of 63) of the colonies screened which grew upon re-inoculation.

Increased recovery from the same sample was also tested by plating in triplicate on plates with and without 0.001% iron sulfate. A 4.9-fold increase in recovery was observed on the iron-supplemented media (Figure 15).

CHAPTER 4: DISCUSSION

The majority of bacterial cells in an environmental sample are unculturable and do not form colonies on synthetic media. The unculturable fraction has been estimated to be as high as 99.9%, presenting a major limitation in our ability to study the full scope of bacterial physiological diversity. The results presented in this study reveal that a significant fraction of unculturable bacteria, from intertidal sediment, fail to grow on synthetic media due to a lack of siderophores provided by neighboring culturable organisms. While the siderophores are providing iron, an element necessary for growth, the varying siderophore specificity of different unculturable siderophore dependent species suggests a complex strategy of only growing in the presence of suitable neighboring organisms.

Previous studies have shown that recovery from environmental samples in a simulated natural environment was much higher compared to standard Petri dish methods (Kaeberlein et al., 2002). The reasons for this increased recovery are thought to be partly a result of otherwise unculturable species receiving proper chemical factors produced by other organisms in the environment. It was observed that recovery on densely seeded plates was disproportionably higher than recovery on lower dilution plates with fewer colonies (Figure 11 shows this effect for the R2Asea non-iron plates).

Based on these observations it was hypothesized that some of the colonies that formed on crowded plates, would be unculturable in isolation and only grew initially due to a growth-inducing factor provided by a nearby colony of a culturable species. Therefore, several pairs of colonies recovered from an intertidal sediment sample were re-streaked in a cross-streak pattern to screen for helper-dependent relationships. Several 31

unculturable isolates were identified that did not form colonies unless grown in the presence of a helper recovered from the same environment. A model unculturable, M. polysiphoniae KLE1104, was unculturable in isolation but grew in the presence of filtered spent-supernatant from model helper M. luteus KLE1011 as well as E. coli. E. coli entB and entC mutants defective in the production of the siderophore enterobactin lacked growth-induction ability. A purified sample of enterobactin was capable of inducing the same growth of large macrocolonies, confirming that the compound was responsible for growth-induction by E. coli.

The model helper, M. luteus KLE1011 was then examined for production of siderophores capable of inducing growth. Marine isolates of M. luteus have previously been reported to show iron-binding activity, but the structure of any siderophores produced has remained unknown (Cabaj and Kosakowska, 2007). Spent supernatant of

M. luteus KLE1011, grown in low iron media to induce production of siderophores, was fractionated and analyzed by MS and NMR to determine the structure of the purified compounds. Five new acyl-desferrrioxamine siderophores were identified. Each was capable of inducing growth of the model unculturable, M. polysiphoniae KLE1104. The novel desferrioxamines produced by M. luteus KLE1011 contained various terminal modifications that would reduce their aqueous solubility. Desferrioxamines 2 and 5 are capped with an acetyl group on the primary amine, and desferrioxamines 3-5 terminate with branched chain fatty acids. Desferrioxamines 1 and 2 terminate with an atypical phenylacetyl moiety. These modifications to the basic structure of desferrioxamine B, the terrestrial counterpart of these compounds, would reduce solubility of the siderophores and help retain them within the biofilm community. An examination of the 32

sand biofilm SEM images reveals an extracellular matrix of organic material mixed with bacterial cells. It is likely that this matrix retains hydrophobic siderophores such as those produced by M. luteus KLE1011. The particular mix of siderophores present could strongly influence the resulting structure of the community by only inducing the growth of unculturable bacteria with proper siderophore receptors. The initial colonization of a sand grain by siderophore-producing bacteria would therefore dictate the resulting colonization of siderophore dependent species.

To determine how common siderophore dependent unculturable bacteria were in the intertidal sediment community, colonies growing near spots of M. luteus KLE1011 were screened for dependence. Fourteen percent of the colonies screened, growing near

M. luteus KLE1011, from a marine intertidal sediment sample appeared to be unculturable on standard R2Asea media, but grew in the presence of M. luteus

KLE1011. The relative ease of isolating these unculturable isolates illustrates the commonality of siderophore dependence in the intertidal sediment community.

Sequences of 16s rDNA were obtained for 15 of the M. luteus KLE1011 dependent isolates, showing they belonged to a variety of bacterial genera from both the

Proteobacteria and Bacteroidetes (Table 1). Two of the species, related to Sulfitobacter delicatus and Simiduia agarivorans were isolated multiple times. Six unique species, which were not capable of forming colonies without help were chosen for further study;

Maribacter sp. KLE1063, Winogradskyella sp. KLE1078, Hyphomonas sp. KLE1080,

Aurantimonas sp. KLE1122, Simiduia sp. KLE1111 and Sulfitobacter sp. KLE1123. To investigate the siderophore specificity of the model unculturables, a panel of 16 commercial siderophores from bacterial and fungal sources, as well as the 5 KLE1011 33

siderophores were tested for growth induction of each isolate. The model unculturable,

M. polysiphoniae KLE1104, and one other strain, Cyclobacterium sp. KLE1009, isolated in initial cross-streak experiments were added to the siderophore panel test. Each unculturable isolate showed a particular pattern of siderophores which could induce their growth. Cyclobacterium sp. KLE1009 showed the widest range of helping and was induced by all 21 siderophores tested. Others were helped by far less, such as

Sulfitobacter sp. KLE1123, which was only induced by 6 of the siderophores. All 8 unculturables tested were induced by at least one of the M. luteus KLE1011 siderophores. KLE1011 Sid1 induced growth of all the isolates tested and was therefore the compound responsible for the relatively high rate of recovery of dependent isolates from the environmental sample. On average, the Bacteroidetes were capable of being induced by a greater number of siderophores than the Proteobacteria. This variable range and pattern of siderophore induction between phyla suggests ways of enriching recovery of particular groups by supplementing low iron media with a specific siderophore. For example, Desferriornibactin-C6 induced the 4 Bacteroidetes tested while not inducing the growth of the 4 Proteobacteria. A low-iron media supplemented with this siderophore could possibly increase the relative recovery of Bacteroidetes from an environmental sample. The identification of siderophores inducing the growth of members of under-represented groups could be particularly valuable in recovering additional isolates from the same group.

Since the presence of siderophore dependent unculturables was common in the intertidal sediment samples, recovery was tested adding soluble Fe(II) to R2Asea media.

Vortexed intertidal sediment, from the same environment originally sampled, was plated 34

on media with and without 0.001% iron sulfate added after autoclaving. Recovery was increased 10.5-fold compared to the control without added iron. Colonies growing on the iron plates were then re-innoculated on plates with and without added iron to screen for colonies with strong soluble iron dependence. Twenty-one percent of colonies picked from iron supplemented plates only formed colonies on plates with the same high level of iron. Since this high level of soluble Fe(II) is not commonly present in the marine sediment, it is likely that these isolates are dependent on siderophores in the natural environment. Fifteen of the iron-dependent isolates were identified by 16s rDNA sequencing revealing the presence of two isolates, from under-represented groups;

Rubritalea sp. KLE1210 and Parvularcula sp. KLE1250. This unusual level of novelty within a small set of isolates suggests that protocols designed to recover

Fe(II)/siderophore dependent bacteria may provide a high hit rate of species from under- represented groups.

Rubritalea sp. KLE1210 is a member of the phylum Verrucomicrobia which is widespread in the environment and present at up to 10% of total cells in soil (Hugenholtz et al., 1998). Despite being a common member of soil environments, members of the phylum are rarely cultured. Only 94 sequences of 16s rDNA from cultured representatives are reported in the Ribosomal Database. The phylum was proposed in

1997 when three species of Prosthecobacter were reassigned to form the division based on 16s rDNA sequences (Hedlund et al., 1997). Since then, additional cultured isolates have slowly been reported and the complete genome sequence of the methanotrophic

Verrucomicrobium, Methylacidiphilum infernorum, was recently reported (Hou et al.,

2008). The presence of a Verrucomicrobia representative in the identification of a small 35

set of soluble iron-dependent isolates suggests that the culturing of additional members of this phylum may be possible using techniques aimed at the isolation of siderophore dependent bacteria from intertidal sediment. Interestingly, none of the 21 siderophores tested were successful in inducing growth of Rubritalea sp. KLE1210. Spotting 1% iron sulfate, however, does induce growth, consistent with the rest of the siderophore dependent unculturables tested. From these observations, it reasonable to propose that

Rubritalea sp. KLE1210 may be induced by a novel siderophore from a neighboring organism in the intertidal sediment. Identification of such a siderophore could be used to selectively increase recovery of isolates from this phylum, and would be an exciting future direction of research based on these observations.

Parvularcula sp. KLE1250 is a member of the poorly represented

Parvularculales order within the Alphaproteobacteria. Only 3 cultured isolates from this group are reported in the Ribosomal Database. The order was proposed when two strains of Parvularcula bermudensis were isolated in 2003, cultured by the high- throughput dilution to extinction method (Cho and Giovannoni, 2003). Since then, only two other cultured strains from this order have been reported; Parvularcula sp. CC-

MMS-1 and Parvularculaceae bacterium P33. Similar to Rubritalea sp. KLE1210,

Parvularcula sp. KLE1250 was also induced by spotting 1% iron sulfate but did not grow in the presence of any of the 21 siderophores from the panel. Identification of a siderophore from an environmental helper of this organism might also be used to selectively enrich and culture representatives from the same order.

In order to confirm that siderophore/iron dependent unculturable bacteria comprise part of the sand biofilm community, a sample of cells was disrupted from a 36

small intertidal pebble by sonication. The sample was plated on R2Asea media with and without additional iron, resulting in a 4.9-fold higher recovery on iron-supplemented plates. Several colonies were picked and screened for iron dependence. Fifty-six percent of the isolates screened showed strong dependence on iron, verifying that the biofilm community on intertidal sediment is comprised of a significant fraction of iron dependent unculturable bacteria. These unculturable isolates are most likely depending on the presence of siderophores from neighboring culturable isolates in the low-iron marine environment. These results are consistent with the findings of Guan and Kamino who reported that 60% of marine bacterial isolates out of a total of 421 tested did not produce siderophores (Guan et al., 2001). They also reported the growth induction of an

Alphaproteobacterium by the addition of an exogenous siderophore produced by a

Vibrio species. The growth assays were performed on defined media treated with

Chelex-100 to completely remove soluble iron, and it is therefore unclear if the isolate was unculturable and incapable of forming growth on standard media. Interestingly, 12 of the 75 colonies re-streaked from the high iron plates did not grow on either medium.

These colonies could represent species that are dependent on a factor other than a siderophore, which was provided by a colony present on the initial recovery plate.

Considering the high percentage of iron-dependent isolates on the original plate, the helper could be an isolate which itself needed help to grow in the form of iron which complemented an environmental need for siderophores. This type of two-step dependent phenomenon is an exciting possibility to be tested in the future.

The reasons behind the Great Plate Count Anomaly are unclear. Why would the majority of bacterial cells in the environment fail to grow on nutrient media? Perhaps 37

for some of these bacteria it not a matter of improper nutrient conditions but the lack of a proper signal to exit from a state of dormancy. Non-growing, stationary and dormant persister cells are resistant to noxious conditions such as exposure to antibiotics, while growing cells become vulnerable (Lewis, 2007). For these reasons it could be an advantageous strategy to remain dormant and only grow when specific factors signal a safe environment. From the results in this study, it seems that a significant portion of bacteria from intertidal sediment will only grow in the presence of specific siderophores and are otherwise in a state of dormancy. In their natural environment it is unlikely they are exposed to levels of soluble iron that would otherwise allow growth. The unculturable isolates are close relatives of culturable typed strains, which presumably are capable of producing siderophores. The siderophore dependent strains therefore seem to have gained a selective advantage by losing the ability to produce their own siderophores. The selective advantage conferred seems to be the ability to remain dormant unless they are in the presence of favorable neighboring species, signaled by the presence of particular siderophores. This ability of only growing when there is a higher probability of neighbors present, which pose no threat, could greatly increase their survival. While these findings are interesting from the perspective of understanding the fundamental interactions within a biofilm community, they also provide valuable insights to improve culturing methods. Bacteria from under-represented groups were isolated which have strong growth dependence for elevated levels of soluble iron. The isolates can most likely be induced by a siderophore, which could be used for enrichment and culturing of additional related species. Identifying the siderophore, which induces growth of Rubritalea sp. KLE1210 for instance, would allow testing of 38

this hypothesis. The siderophore could be loaded with iron and added to defined media, devoid of soluble Fe(II) and tested for increased recovery of Verrucomicrobia. The hypothesis is supported by the observation that three of the siderophores from the panel, desferriornibactin-C6, desferrirubin and desferrichrome only induced growth of

Bacteroidetes and not the Proteobacteria tested. The results presented reveal that siderophore dependent unculturable bacteria, from a variety of clades, are common members of intertidal sediment biofilms and methods to increase recovery of unculturables can be designed based on these findings.

Figure 1. Sand Biofilm Community on Canoe Beach Intertidal Sediment Samples. (A)

A representative sample of sand from Canoe Beach. (B-E) Scanning electron micrographs of the biofilm community. Scale bars: A = 3 mm; B = 50 µm; C = 10 µm;

D = 10 µm; E = 3 µm.

Figure 2. Growth of M. polysiphoniae KLE1104 is Induced by M. luteus KLE1011.

(A) Isolation spread plate of bacteria from intertidal sediment. (B) Environmental helper

M. luteus KLE1011 cross-streaked (right side of plate) with unculturable isolate M. polysiphoniae KLE1104 (left side of plate). Colonies of the unculturable strain are larger when in closer proximity to the helper. (C) A spot of M. luteus KLE1011 (white circle) induces growth of M. polysiphoniae KLE1104. No growth is seen on the side of the plate opposite M. luteus KLE1011 (D) Filtered spent supernatant of M. luteus

KLE1011 induces growth of M. polysiphoniae KLE1104.

Figure 3. Growth Induction of M. polysiphoniae KLE1104 by Enterobactin from E. coli.

Filtered spent supernatant of E. coli BW25113 induces growth of M. polysiphoniae

KLE1104 but spent culture media from enterobactin mutant strains does not. (A)

Supernatant from the parental E. coli BW25113 strain induces growth, as does supernatant from strains deficient in (B) ΔluxS and (C) ΔtnaA. Supernatant of strains deficient in enterobactin synthesis (D) ΔentB and (E) ΔentC do not induce growth of the unculturable. (F) Purified enterobactin spotted on a plate evenly spread with M. polysiphoniae KLE1104 induced growth of the isolate. Colony size decreased with increasing distance from the enterobactin source and no growth of the unculturable was seen further away from the source.

Figure 4. Siderophores Produced by Helper Strain M. luteus KLE1011. Five novel siderophores were isolated and characterized from the model helper, and shown to induce the growth of the model unculturable. (A) Reversed-phase C18 HPLC-MS / UV- visible spectroscopic analysis. The ferric and desferric siderophores were separated over

C18 HPLC, demonstrating a range of hydrophobic interactions with the nonpolar resin.

The more polar ferric forms eluted faster than the desferric forms, showed masses indicative of iron complexation (MS), and exhibited characteristic iron-ligand charge transfer absorption (UV-visible). (B) Acyldesferrioxamine siderophore structures. (C) M. luteus KLE1011 acyl-desferrioxamine 3 was spotted on a plate evenly spread with M. polysiphoniae KLE1104. The purified siderophore induced growth of the unculturable isolate. (D) A control plate without help showed no growth of the unculturable. 43

Figure 5. Isolation of Bacteria Dependent on M. luteus KLE1011. Intertidal samples from Canoe Beach were spread on R2Asea plates followed by spotting of the environmental helper, M. luteus KLE1011. In this example, two spots of M. luteus

KLE1011 were applied as well as a spot of environmental mix to investigate their relative helping abilities. The four colonies marked with asterisks represent examples of colonies that were screened for M. luteus KLE1011 growth dependence.

Figure 6. Verification of M. luteus KLE1011 Dependent Isolates. Potential M. luteus

KLE1011 dependents were verified by spreading the potential unculturable evenly on an

R2Asea plate and spotting KLE1011. Shown are six isolates from the screen. The white spots are KLE1011. 45

Figure 7. Growth Induction of Unculturable Isolates By the Five M. luteus KLE1011

Siderophores. Unculturable isolates were spread evenly on R2Asea plates and spotted with each of the 5 novel acyl-desferrioxamine siderophores. Representative plates are shown. (A) KLE1009 induced by KLE1011 Sid1. (B) KLE1078 induced by KLE1011

Sid2. (C) KLE1104 induced by KLE1011 Sid3. (D) KLE1104 induced by KLE1011

Sid4. (E) KLE1104 induced by KLE1011 Sid5.

Figure 8. Commercial Siderophores Tested For Growth Induction of Unculturables.

The structures of 16 commercially available siderophores are shown which were each tested for growth induction of 8 unculturable species. The siderophores are arranged based on three structural classes: (A) cyclic and linear trihydroxamates, (B) dihydroxamates, and (C) carboxylic acid-type.

Figure 10. Complementation of Siderophore Dependence With Soluble Iron (II). The siderophore dependent unculturables isolated could also be induced to grow if soluble

Fe(II) was spotted. 1% iron sulfate was spotted (10 µL) on spread plates of KLE1009,

KLE1104 and KLE1078 inducing a distinct ring of growth around the concentration gradient. In the natural environment these isolates are most likely depending on siderophores for growth since this high concentration of soluble iron is not typically available in intertidal sediment.

Figure 11. Increased Recovery Adding Soluble Fe(II) to R2Asea Plates. Samples collected from Canoe Beach were plated in 10-fold dilutions on R2Asea plates with and without added iron(II) sulfate (0.001% added after autoclaving). Colony counts increased 10.5-fold. Each data point is the average of three replicates with standard deviation error bars.

Figure 12. Fe(II) Induction of Rubritalea sp. KLE1210 and Parvularcula sp. KLE1250.

The growth of two iron-dependent strains from under-represented bacterial groups was induced by spotting 1% iron sulfate on spread plates of each isolate. The closest relative of Rubritalea sp. KLE1210 is Rubritalea spongiae (Phylum Verrucomicrobia) with

91.7% 16s rDNA identity (A). The closest relative of Parvularcula sp. KLE1250 is

Parvularcula bermudensis with 94.0% 16s rDNA identity (B). Iron is spotted on a filter disc in panel B. 51

Figure 13. Viable Cells Recovered From a Single Intertidal Pebble. A pebble, approximately 5 mm in diameter, collected from Canoe Beach, was washed seven times to remove excess seawater from the surface. Panel A shows the variety of bacterial growth resulting from the biofilm dislodged by sonication. Panel B shows a control plated before sonication, verifying that the cells were obtained from the biofilm community growing on the pebble. 52

Figure 14. Iron Dependence of Isolates Re-Suspended From Pebble Biofilm. Bacterial strains isolated from a biofilm growing on a small intertidal pebble (approximately 5 mm in diameter) were screened for iron dependence to verify that the iron/siderophore dependent phenomenon existed for members of a sand biofilm community. Isolates were re-innoculated on R2A ASsalt plates with (B, D) and without (A, C) added 0.001% iron(II) sulfate. The same isolate was streaked on plates A and B. A second isolate, was streaked on plates C and D.

Figure 15. Recovery of Intertidal Sediment Biofilm Bacteria From a Single Pebble. To verify that iron dependent unculturable bacteria are part of intertidal sediment biofilm communities, a sample of bacteria resuspended from a small pebble (approximately 5 mm in diameter) was plated on R2A ASsalt plates with and without added iron sulfate.

A 4.9-fold increase in recovery was seen on plates supplemented with soluble iron. Data is the average of three replicates for each condition with standard deviation error bars.

KLE Isolate Number Closest Relative Identity Phylum Class Order Sulfitobacter delicatus KMM M23 KLE1123 3584(T) 99.9 Proteobacteria Alphaproteobacteria Rhodobacterales Sulfitobacter delicatus KMM M57 none 3584(T) 99.9 Proteobacteria Alphaproteobacteria Rhodobacterales Sulfitobacter delicatus KMM M106 none 3584(T) 99.8 Proteobacteria Alphaproteobacteria Rhodobacterales Sulfitobacter delicatus KMM M128 none 3584(T) 99.6 Proteobacteria Alphaproteobacteria Rhodobacterales Maribacter arcticus M63 KLE1063 KOPRI 20941(T) 96.5 Bacteroidetes Flavobacteria Flavobacteriales Winogradskyella thalassocola KMM M78 KLE1078 3907(T) 97.8 Bacteroidetes Flavobacteria Flavobacteriales Aurantimonas M22 KLE1122 coralicida WP1(T) 99.3 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomonas M80 KLE1080 johnsonii MHS-2(T) 99.2 Proteobacteria Alphaproteobacteria Rhodobacterales Simiduia agarivorans M11 KLE1111 SA1(T) 97.4 Proteobacteria Gammaproteobacteria unclassified Simiduia agarivorans M50 none SA1(T) 97.5 Proteobacteria Gammaproteobacteria unclassified Simiduia agarivorans M16 none SA1(T) 97.5 Proteobacteria Gammaproteobacteria unclassified Simiduia agarivorans M65 none SA1(T) 97.5 Proteobacteria Gammaproteobacteria unclassified Simiduia agarivorans M30 none SA1(T) 97.4 Proteobacteria Gammaproteobacteria unclassified Reinekia blandensis M9 none MED 297(T) 96.5 Proteobacteria Gammaproteobacteria unclassified Pseudoalteromonas M83 none marina Mano4(T) 99.9 Proteobacteria Gammaproteobacteria Alteromonadales

Table 1. Closest Relatives of 15 M. luteus KLE1011 Dependent Isolates. Colonies were screened for strong M. luteus KLE1011 growth dependence. The closest relatives of 15 of the dependent isolates are shown with 16s rDNA percent identity, as well as the phylum, class and order of each isolate. The temporary isolate number is given along with the KLE number for those that were studied further.

Closest Isolate Relative Identity Phylum Class Order Sulfitobacter mediterraneus FeD204 DSM 12244T 99.6 Proteobacteria Alphaproteobacteria Rhodobacterales Parvularcula bermudensis KLE1250 KCTC 12087T 94.0 Proteobacteria Alphaproteobacteria Parvularculales

Brevundimonas FeD202 alba CB88T 98.7 Proteobacteria Alphaproteobacteria Caulobacterales Sphingopyxis flavimaris KCTC FeD208 12232T 99.8 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingopyxis flavimaris KCTC FeD75 12232T 99.7 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacter gaetbuli KCTC FeD207 12227T 95.7 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacter citreus RE35F FeD200 1T 96.3 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacter aquimaris SW- FeD206 110T 98.0 Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacter aquimaris SW- FeD201 110T 98.0 Proteobacteria Alphaproteobacteria Sphingomonadales

Flaviramulus FeD203 basaltis H35T 96.8 Bacteroidetes Flavobacteria Flavobacteriales

Flaviramulus FeD205 basaltis H35T 96.9 Bacteroidetes Flavobacteria Flavobacteriales

Flaviramulus FeD209 basaltis H35T 96.8 Bacteroidetes Flavobacteria Flavobacteriales Psychroserpens burtonensis FeD56 ATCC 700359T 96.8 Bacteroidetes Flavobacteria Flavobacteriales Gaetbulibacter marinus IMCC FeD66 1914T 95.6 Bacteroidetes Flavobacteria Flavobacteriales Rubritalea spongiae YM21 KLE1210 132T 91.7 Verrucomicrobia Verrucomicrobiae Verrucomicrobiaceae

Table 2. Closest Relatives of Iron Dependent Isolates From Canoe Beach. Twenty percent of colonies screened from high iron plates did not form colonies without added iron (0.001% iron sulfate). 16s rDNA sequences were obtained for 15 of these isolates.

The closest relative of each isolate is shown with percent identity based on 16s rDNA sequence, as well as the phylum, class and order of each strain. 56

Appendix I. Phylogentic Tree of the 11 Original Bacterial Phyla Proposed. The figure is taken from the 1987 review by Carl Woese, and shows the orginal phylogentic tree of the Eubacteria (Bacteria) based on 16s rDNA sequences from cultured bacteria (Woese,

1987). The Gram-positive bacteria were later split into two separate phyla, the

Firmicutes and Actinobacteria.

Appendix II. Phylogenetic Tree of Bacteria Published in 1998. The figure is taken from the review by Hugenholtz, Goebel and Pace, showing the 36 bacterial phyla recognized at the time (Hugenholtz et al., 1998). The 13 candidate phyla with no known cultured representatives are shown in outline.

Appendix III. Phylogentic Tree of Bacteria Published in 2003. The figure is taken from the review by Rappe and Giovannoni (Rappe and Giovannoni, 2003). By 2003, 52 bacterial phyla were recognized. The 26 candidate phyla are shown in gray. Phyla with cultured representatives are in white. The original phyla proposed by Woese are in black.

REFERENCES

Archibald, F. (1983). Lactobacillus plantarum, an organism not requiring iron. In FEMS Microbiology Letters, pp. 29-32.

Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2, 2006 0008.

Barer, M.R., and Harwood, C.R. (1999). Bacterial viability and culturability. Adv Microb Physiol 41, 93-137.

Bollmann, A., Lewis, K., and Epstein, S.S. (2007). Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol 73, 6386-6390.

Boyd, P.W., Jickells, T., Law, C.S., Blain, S., Boyle, E.A., Buesseler, K.O., Coale, K.H., Cullen, J.J., de Baar, H.J., Follows, M., et al. (2007). Mesoscale iron enrichment experiments 1993-2005: synthesis and future directions. Science 315, 612-617.

Bruns, A., Cypionka, H., and Overmann, J. (2002). Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea. Appl Environ Microbiol 68, 3978-3987.

Bruns, A., Nubel, U., Cypionka, H., and Overmann, J. (2003). Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton. Appl Environ Microbiol 69, 1980-1989.

Butkevich, V.S. (1932). Zür Methodik der bakterioloschen Meeresuntersuchungen und einige Angaben über die Verteilung der Bakterien im Wasser und in den Büden des Barents Meeres. Trans Oceanogr Inst Moscow 2: 5-39 (in Russian with German summary) 2, 5-39.

Butler, A., and Vraspir, J. (2009). Chemistry of Marine Ligands and Siderophores. Annual Review of Marine Science 1.

Cabaj, A., and Kosakowska, A. (2007). Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea. Microbiol Res.

Chisholm, S.W., Falkowski, P.G., and Cullen, J.J. (2001). Oceans. Dis-crediting ocean fertilization. Science 294, 309-310.

Cho, J.C., and Giovannoni, S.J. (2003). Parvularcula bermudensis gen. nov., sp. nov., a marine bacterium that forms a deep branch in the alpha-Proteobacteria. International journal of systematic and evolutionary microbiology 53, 1031-1036.

Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K., and Lim, Y.W. (2007). EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57, 2259-2261.

Coale, K.H., Johnson, K.S., Chavez, F.P., Buesseler, K.O., Barber, R.T., Brzezinski, M.A., Cochlan, W.P., Millero, F.J., Falkowski, P.G., Bauer, J.E., et al. (2004). Southern Ocean iron enrichment experiment: carbon cycling in high- and low-Si waters. Science 304, 408-414.

Coale, K.H., Johnson, K.S., Fitzwater, S.E., Gordon, R.M., Tanner, S., Chavez, F.P., Ferioli, L., Sakamoto, C., Rogers, P., Millero, F., et al. (1996). A massive phytoplankton bloom induced by an ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean. Nature 383, 495 - 501.

Colwell, R.R., and Grimes, D.J. (2000). Nonculturable Microorganisms in the Environment (Washington, DC, American Society for Microbiology).

Crosa, J.H., and Walsh, C.T. (2002). Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiology and Molecular Biology Reviews 66, 223-+.

Elli, M., Zink, R., Rytz, A., Reniero, R., and Morelli, L. (2000). Iron requirement of Lactobacillus spp. in completely chemically defined growth media. Journal of applied microbiology 88, 695-703.

Ferrari, B.C., Binnerup, S.J., and Gillings, M. (2005). Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria. Appl Environ Microbiol 71, 8714-8720.

Giovannoni, S.J. (2000). Evolution, Diversity and Molecular Ecology of Marine Prokaryotes. In Microbial Ecology of the Oceans, D. Kirchman, ed. (New York, NY, Wiley-Liss, Inc.), pp. 47-84.

Giovannoni, S.J., Tripp, H.J., Givan, S., Podar, M., Vergin, K.L., Baptista, D., Bibbs, L., Eads, J., Richardson, T.H., Noordewier, M., et al. (2005). Genome streamlining in a cosmopolitan oceanic bacterium. Science 309, 1242-1245.

Guan, L.L., Kanoh, K., and Kamino, K. (2001). Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions. Appl Environ Microbiol 67, 1710-1717.

Handelsman, J. (2004). Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68, 669-685.

Hedlund, B.P., Gosink, J.J., and Staley, J.T. (1997). Verrucomicrobia div. nov., a new division of the bacteria containing three new species of Prosthecobacter. Antonie van Leeuwenhoek 72, 29-38.

Hou, S., Makarova, K.S., Saw, J.H., Senin, P., Ly, B.V., Zhou, Z., Ren, Y., Wang, J., Galperin, M.Y., Omelchenko, M.V., et al. (2008). Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia. Biology direct 3, 26.

Hugenholtz, P., Goebel, B.M., and Pace, N.R. (1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180, 4765- 4774.

Johnson, K.S., Gordon, R.M., and Coale, K.H. (1997). What controls dissolved iron concentrations in the world ocean? Marine Chemistry 57, 137-161.

Kaeberlein, T., Lewis, K., and Epstein, S.S. (2002). Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment. Science 296, 1127- 1129.

Kleerebezem, M., Boekhorst, J., van Kranenburg, R., Molenaar, D., Kuipers, O.P., Leer, R., Tarchini, R., Peters, S.A., Sandbrink, H.M., Fiers, M.W., et al. (2003). Complete genome sequence of Lactobacillus plantarum WCFS1. Proceedings of the National Academy of Sciences of the United States of America 100, 1990-1995.

Lewis, K. (2007). Persister cells, dormancy and infectious disease. Nat Rev Microbiol 5, 48-56.

Marcy, Y., Ouverney, C., Bik, E.M., Losekann, T., Ivanova, N., Martin, H.G., Szeto, E., Platt, D., Hugenholtz, P., Relman, D.A., et al. (2007). Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proceedings of the National Academy of Sciences of the United States of America 104, 11889-11894.

Martin, J.H., Coale, K.H., Johnson, K.S., Fitzwater, S.E., Gordon, R.M., Tanner, S.J., Hunter, C.N., Elrod, V.A., Nowicki, J.L., Coley, T.L., et al. (1994). Testing the Iron Hypothesis in Ecosystems of the Equatorial Pacific-Ocean. Nature 371, 123-129.

Martinez, J.S., Zhang, G.P., Holt, P.D., Jung, H.T., Carrano, C.J., Haygood, M.G., and Butler, A. (2000). Self-assembling amphiphilic siderophores from marine bacteria. Science 287, 1245-1247.

Neilands, J.B. (1995). Siderophores - Structure and Function of Microbial Iron Transport Compounds. Journal of Biological Chemistry 270, 26723-26726.

Nichols, D., Lewis, K., Orjala, J., Mo, S., Ortenberg, R., O'Connor, P., Zhao, C., Vouros, P., Kaeberlein, T., and Epstein, S.S. (2008). Short peptide induces an "uncultivable" microorganism to grow in vitro. Applied and Environmental Microbiology 74, 4889- 4897.

Rappe, M.S., Connon, S.A., Vergin, K.L., and Giovannoni, S.J. (2002). Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418, 630-633.

Rappe, M.S., and Giovannoni, S.J. (2003). The uncultured microbial majority. Annu Rev Microbiol 57, 369-394.

Rusch, D.B., Halpern, A.L., Sutton, G., Heidelberg, K.B., Williamson, S., Yooseph, S., Wu, D., Eisen, J.A., Hoffman, J.M., Remington, K., et al. (2007). The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 5, e77.

Staley, J.T., and Konopka, A. (1985). Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39, 321-346.

Street, J.H., and Paytan, A. (2005). Iron, phytoplankton growth, and the carbon cycle. Met Ions Biol Syst 43, 153-193.

Tamaki, H., Sekiguchi, Y., Hanada, S., Nakamura, K., Nomura, N., Matsumura, M., and Kamagata, Y. (2005). Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake by molecular and improved cultivation-based techniques. Appl Environ Microbiol 71, 2162-2169.

Tanaka, Y., Hanada, S., Manome, A., Tsuchida, T., Kurane, R., Nakamura, K., and Kamagata, Y. (2004). Catellibacterium nectariphilum gen. nov., sp. nov., which requires a diffusible compound from a strain related to the genus Sphingomonas for vigorous growth. Int J Syst Evol Microbiol 54, 955-959.

Tortell, P.D., Maldonado, M.T., Granger, J., and Price, N.M. (1999). Marine bacteria and biogeochemical cycling of iron in the oceans. Fems Microbiology Ecology 29, 1-11.

Wandersman, C., and Delepelaire, P. (2004). Bacterial iron sources: from siderophores to hemophores. Annu Rev Microbiol 58, 611-647.

Woese, C.R. (1987). Bacterial evolution. Microbiological reviews 51, 221-271.