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LIFE AT THE LIMITS: DIVERSITY, PHYSIOLOGY AND BIOENERGETICS OF

HALOALKALITHERMOPHILES

KAREN JEAN BOWERS

(Under the Direction of Juergen Wiegel)

ABSTRACT

Haloalkalithermophiles are poly-extremophiles adapted to grow at high salt concentrations, alkaline pH values and temperatures greater than 50ºC. Halophilic alkalithermophiles are of interest from physiological perspectives as they combine unique adaptive mechanisms and cellular features that enable them to grow under extreme conditions.

Water and sediment samples from the lakes of the Wadi An Natrun, Egypt and from Lake

Magadi, Kenya were investigated for the presence of novel haloalkalithermophiles. These athalassohaline lakes are noted for their high temperature, alkaline pH and high Na+ concentrations. Two novel bacterial species, Natranaerobius jonesii and Natranaerobius grantii, and one novel archaeal species, Natronolimnobius aegyptiacus, were isolated and characterized.

Furthermore, the adaptive mechanisms and bioenergetic properties of the species belonging to the order Natranaerobiales were investigated. Collectively, these microorganisms display a ΔpH homeostasis, rather than an intracellular pH homeostasis; a growth requirement for Cl-; and are moderately UV resistant.

INDEX WORDS: Halophile, Alkaliphile, Thermophile, Extremophile, Alkalithermophile,

Haloalkalithermophile, Natranaerobiales, Natranaerobius jonesii,

Natranaerobius grantii, Natronolimnobius aegyptiacus, Wadi An Natrun,

Kenya Rift Valley, Adaptive mechanisms, Intracellular pH, Proton motive force, Membrane potential

LIFE AT THE LIMITS: DIVERSITY, PHYSIOLOGY AND BIOENERGETICS OF

HALOALKALITHERMOPHILES

KAREN J. BOWERS

B.A., University of Georgia, 2003

M.Ed., Troy University, 2008

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2010

Karen Jean Bowers

LIFE AT THE LIMITS: DIVERSITY, PHYSIOLOGY AND BIOENERGETICS OF

HALOALKALITHERMOPHILES

KAREN J. BOWERS

Major Professor: Juergen Wiegel

Committee: Lawrence J. Shimkets William B. Whitman

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia May 2010

DEDICATION

To my mother, who always said I could.

ACKNOWLEDGEMENTS

There are numerous people who deserve thanks for making this thesis possible, whether by providing academic advice and support, personal encouragement, a willing ear or a swift kick, when needed. First and foremost, I need to thank my advisor, Juergen Wiegel, for all of his support and guidance. I would have been hard-pressed to find a better mentor.

The assistance of my committee members, Larry Shimkets and Barny Whitman has also been extremely helpful and is most appreciated. I also owe an enormous debt of gratitude to my former labmate, Noha Mesbah, who helped me in more ways than I can ever express. My labmates Isaac Wagner and Elizabeth Burgess, along with undergraduate lab members Jacob

Gilleland and Litty Varghese also deserve thanks for assistance with keeping my perspective, sense of humor, with lab work and for a fresh eye when needed. Other friends at the

University of Georgia, Dana Cook, Noreen Lyell, Chandra Carpenter, Dawn Adin and John

Buchner, Lyla Lipscomb, and Dave Samuels have been great sounding boards and, at times, comic relief. I also cannot neglect to thank Wendy Dustman, who seems to be everything to everyone and has helped me in innumerous ways, large and small.

My ―outside‖ friends have been as much a part of this effort as my academic colleagues. I appreciate all of the reminders that a real-world exists outside the lab and not being told I am crazy more often than was absolutely necessary.

Finally, I have to thank my family, particularly my parents and brother, for all of their support and encouragement. Without them, this pursuit would have been much more difficult.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ...... 1

Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of

high salt, alkaline pH and elevated temperature approach a physico-

chemical boundary for life? ...... 1

Temperature and pH optima of extremely halophilic Archaea: a mini-review ...... 13

2 NATRONOLIMNOBIUS AEGYPTIACUS SP. NOV., AN AEROBIC

EXTREMELY HALOPHILIC ALKALITHERMOPHILIC ARCHAEON

ISOLATED FROM THE ATHALASSOHALINE WADI AN NATRUN,

EGYPT ...... 42

3 NATRANAEROBIUS JONESII SP. NOV. AND NATRANAEROBIUS GRANTII

SP. NOV., TWO ANAEROBIC HALOPHILIC ALKALITHERMOPHILES

ISOLATED FROM THE KENYAN-TANZANIAN RIFT ...... 54

4 ADAPTIVE MECHANISMS AND INTRACELLULAR PH REGULATION IN

NATRANAEROBIUS SPECIES, ANAEROBIC,

HALOALKALITHERMOPHILIC BACTERIA ...... 62

5 CONCLUSIONS...... 96

APPENDIX

A CALCULATION OF BIOENERGETIC PARAMETERS ...... 98

vii

LIST OF TABLES

Page

Table 1.1: Growth characteristics of extremophiles ...... 34

Table 1.2: [Na+], pH and temperature optima and ranges for extremely halophilic Bacteria ...... 35

Table 1.3: [Na+], pH and temperature optima and ranges for extremely halophilic Archaea ...... 38

Table 2.1: Differential characteristics of strain JW/NM-HA 15T and closely related strains...... 53

Table 3.1: Substrate utilization profile of strains JW/NM-KB 43T and JW/NM-KB 411T ...... 60

Table 3.2: Differential characteristics of strain JW/NM-KB 43T, JW/NM-KB 411T and

closely related strains ...... 61

Table 4.1: Changes in intracellular [K+] and [Na+] under varying conditions ...... 94

Table 4.2: Survival of Natranerobius thermophilus and Natronovirga wadinatrunensis after

UV radiation exposure...... 95

viii

LIST OF FIGURES

Page

Figure 1.1: Correlation of [Na+] optimum and pH or temperature optima of extreme

halophiles...... 28

Figure 1.2: Clustering of poly-extremophiles relative to other extreme halophiles ...... 30

Figure 1.3: Correlation of [Na+] optimum and pH or temperature optima of extremely

halophilic Archaea ...... 31

Figure 1.4: Correlation of [Na+] optimum and pH or temperature optima of extremely

halophilic Archaea ...... 33

Figure 2.1: Light microscopic image of Strain JW/NM-HA 15T ...... 51

Figure 2.2: Phylogenetic relationship of strains to closely related microorganisms ...... 52

Figure 3.1: Light microscopic images of strains JW/NM-KB 43T and JW/NM-KB 411T ...... 68

Figure 3.2: Phylogenetic relationship of strains JW/NM-KB 43T and JW/NM-KB 411T to

closely related microorganisms ...... 69

Figure 4.1: Bioenergetic properties of N. jonesii and N. grantii ...... 91

Figure 4.2: Effect of extracellular pH on membrane potential and ΔpH ...... 92

Figure 4.3: γ-radiation resistance of Natranaerobius thermophilus ...... 93

CHAPTER ONE

INTRODUCTION AND REVIEW OF THE LITERATURE

This introduction describes the diversity of extremely halophilic Bacteria and Archaea, according to pH and temperature optima and specifically discusses the physiological properties of poly-extremophiles, e.g., haloalkalithermophiles. Figures 1.1a and b and 1.3a and b illustrate the frequency of pH and temperature optima amongst extremely halophilic Bacteria and

Archaea, and show that most extremely halophilic microorganisms cluster at near-neutral pH and mesophilic temperature optima. Figures 1.2 and 1.4 show the relationship of poly-extremophilic

Bacteria and Archaea, respectively, with relation to other extremely halophilic microorganisms.

Throughout this discussion, the uniqueness of these poly-extremophiles is highlighted and sets the stage for the work discussed in Chapters 2-4. Finally, the main objectives of this work are outlined in the final section.

______Adapted from:

Bowers, K.J., Mesbah, N.M, & Wiegel, J. (2009). Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physic-chemical boundary for life? Saline Systems. 5, 9 (23 November 2009). Bowers, K.J., & Wiegel, J. To be submitted to Extremophiles.

BIODIVERSITY OF POLY-EXTREMOPHILIC BACTERIA: DOES COMBINING THE

EXTREMES OF HIGH SALT, ALKALINE PH AND ELEVATED TEMPERATURE

APPROACH A PHYSICO-CHEMICAL BOUNDARY FOR LIFE?

Extremely halophilic Bacteria

It is frequently asked: what are the physical and chemical boundaries for life? How extreme can conditions become and still support life? In respect to one extreme—haline conditions—the answer is simple; growth has been observed at saturated concentrations of sodium salts, mainly

NaCl. But what happens if one tries to increase the solubility of these salts by increasing the temperature? This overview deals with the diversity of bacteria able to grow under concomitant extreme growth conditions, namely high sodium salt concentration, alkaline pH and elevated temperature, and thus with the recently isolated anaerobic halophilic poly-extremophiles.

Different authors use different definitions for what constitutes a halophile; one definition identifies microorganisms which grow optimally at Na+ concentrations greater than 0.2 M as halophiles (Oren 1996). For this review, the authors wish to focus upon bacterial microorganisms that grow at the upper limits of the combined extremes, and in the case of halophiles, optimally at Na+ concentrations of 1.7 M, or the equivalent of 10% NaCl. These microorganisms are defined as extreme halophiles, in contrast to those microorganisms which merely tolerate such sodium concentrations or which grow optimally at marine salt concentrations of approximately 3.5% w/v (Table 1.1). In this review Na+ concentrations are given in mol/liter, rather than % NaCl, since some of the alkaliphilic halophiles require media with carbonates, usually provided as sodium carbonates, for pH control (Mesbah et al. 2007,

2008). Therefore the [Na+] comes both from the sodium carbonates and from the supplemented

NaCl. Many of the well-known extreme halophiles are Archaea (over one hundred species in the family Halobacteriacae alone); however, some extremely halophilic bacteria have been described (Table 1.2), and this review will focus exclusively on extremely halophilic Bacteria.

These bacteria have been isolated from various extreme environments such as solar thalassohaline salterns (i.e., originating from marine waters) (Mouné et al. 1993), athalassohaline

(e.g., Wadi An Natrun, Egypt) (Mesbah et al. 2007) and ancient thalassohaline (e.g., Great Salt

Lake, UT, USA) (Fendrich 1998) salt lakes, marine environments (Kim et al. 2007), and fermented fish sauces (Pakdeeto et al. 2007). Additionally, among the validly published taxa

(e.g., published in, or publication validated by, the International Journal of Systematic and

Evolutionary Microbiology), the extremely halophilic Bacteria are relatively equally distributed between aerobic and anaerobic species, with the addition of four facultative anaerobes, such as

Halomonas sinaiensis (Romano et al. 2007) and Thiohalorhabdus denitrificans (Sorokin et al.

2008). Examples of extremely halophilic bacteria—representing different types of extrema— include Halorhodospira halochloris (basonym Ectothiorhodospira) (Imhoff et al. 1996), which, at 4.62 M, has one of the highest [Na+] optima (Imhoff et al. 1977); Halomonas taeanensis, which is capable of growing over the unusually wide range of 0-5.13 M Na+ (Lee et al. 2005); and Natranaerobius grantii, which tolerates saturated NaCl concentrations in its growth medium at elevated temperature and alkaline pH (Bowers et al. 2008).

Bacterial extreme halophiles exhibit various physiological and nutritional properties

(Mesbah et al., 2008, Sorokin et al. 2006, Adkins et al. 1993, Liaw et al. 1992) and belong to different phylogenetic groups such as the order Actinomycetales from the phylum

Actinobacteria; the order Sphingobacteriales from the phylum Bacteroidetes; the orders

Bacillales, Halanaerobiales and Natranaeriobiales from the phylum Firmicutes; the orders

Rhizobiales and Rhodospirillales from the subphylum α-Proteobacteria; and the orders

Chromatiales, Oceanospirillales and Pseudomonadales from the subphylum γ-Proteobacteria.

Although many extreme halophiles are mesophilic or neutrophilic, moderately thermophilic extreme halophiles have been described, along with several alkaliphilic extreme halophiles.

Microorganisms which have two extreme growth optima are generally described using the two specific extrema, e.g., alkaliphilic halophiles. However, only a very few extreme halophiles able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated so far. This review will focus on the pH and temperature optima of extremely halophilic bacteria, with a focus on those with alkaline pH optima, above 8.5, and elevated temperature optima, above 50ºC. These microorganisms are considered extremophiles, and, if all three conditions are required for optimal growth, are termed by the authors ―poly-extremophiles‖.

They are of great interest, as their adaptive mechanisms give insight into the abilities of bacteria to survive in environments which were previously considered prohibitive to life, as well as to possible properties of early evolutionary and extraterrestrial life forms (Wagner et al. 2008). The purpose of this overview is to discover whether a correlation exists amongst the validly published bacterial taxa between the extents of halophily, alkaliphily and/or thermophily. In other words: is the extent of one extreme condition limiting the concomitant extent of other extreme growth condition (e.g., a higher temperature optimum requiring a less alkaline pH or a lower sodium salt concentration)?

Currently (September 2009), there are over sixty validly published species (i.e., published or validated in the International Journal of Systematic Bacteriology/Systematic and Evolutionary

Microbiology, as listed at www.bacterio.cict.fr) which are extremely halophilic, according to the description. Of these species, approximately thirty percent have [Na+] optima of less than 2.0 M

(equivalent to approximately 12% w/v NaCl), nineteen of which are published at 1.7 M

(equivalent to approximately 10% w/v NaCl). Approximately forty-five percent of the extremely halophilic species have published [Na+] optima equal to or greater than 2.0 M but less than 3.4

M, and only thirteen microorganisms (approximately 25%) have published [Na+] optima equal to or greater than 3.4 M (equivalent to approximately 20% (w/v) NaCl). Additionally, approximately thirty percent of the species tolerate [Na+] 5.0 M or greater (equivalent to approximately 29% w/v NaCl). Among these microorganisms, only three—Halorhodospira halochloris, Halanaerobium lacusrosei and the unpublished Natranaerobius grantii—have been described which grow in the presence of saturated NaCl (i.e., 5.5 to 6.5 M, since the saturation point is dependent upon media composition, growth pH and temperature) (Imhoff et al. 1977,

Bowers et al. 2008, Cayol et al. 1995). Clearly, as the [Na+] increases the number of known microorganisms with the adaptive mechanisms that enable them to thrive under these conditions decreases. However, while the number of microorganisms with [Na+] optima above 3.0 M is small, a significantly larger number of bacterial halophiles are able to tolerate 3.0M [Na+]; in fact, all of the extreme halophiles with a published [Na+] maximum are able to do so (Table 1.2).

pH optima and ranges of extreme halophiles

Interestingly, out of all the established extremely halophilic bacteria, only nineteen species have pH optima of 8.5 or greater (Table 1.2). Although many salt lakes and salterns from which these organisms were isolated have alkaline pH values. Of these, only ten species combine an elevated pH optimum with a [Na+] optimum of 2.0 M or greater. The distribution of the [Na+] and pH optima are shown in Figure 1.1a; clearly, the combinations of pH optimum 9 with a

[Na+] optimum of approximately 1.7 M is the most highly represented, followed by the

combinations of pH optima 7 and 8 with [Na+] optimum of approximately 1.7 M. Theoretically, if the adaptive resources of a microorganism are being utilized heavily to deal with one type of environmental stress (e.g., osmotic stress) there will be less available resources to deal with other types of environmental stressors: in this case, the elevated pH. Microorganisms with pH values for optimal growth above 8.5 carry with them the usual energetic problems of alkaliphiles, e.g., an inverted pH gradient and thus a suboptimal proton motive force (Mesbah et al. 2008). In the case of extremely halophilic alkaliphiles these problems are exacerbated by the need to keep the intracellular sodium concentration below toxic levels, which is frequently as low as a few mM

(Padan et al. 2000, Oren 2002). This complication may explain the more prevalent occurrence of microorganisms growing optimally in environments that are pH neutral or near neutral.

However, it could also be an artifact of the fact that researchers have investigated the alkaline halobiotic environments and the biodiversity of their microorganisms less than those of neutral or slightly acidic halobiotic environments. Mesbah et al. (2007) have shown that the biodiversity of the alkaline athalassohaline lakes of Wadi An Natrun (North Egypt) is relatively high. Similar observations were made by Ghozlan et. al. regarding saline habitats of Alexandria, Egypt (2006) and Duckworth et. al. regarding various alkaline soda lakes (2009). Obligately alkaliphilic halophilic bacteria from the lakes of the Wadi An Natrun include: Natranaerobius thermophilus

(Mesbah et al. 2007), Natranaerobius trueperi and Natronovirga wadinatrunensis (Mesbah et al.

2009). Each of these microorganisms has a pH55°C optimum of 9.5 or greater as well as a [Na+] optimum greater than 2.5 M. As previously recommended by Wiegel (1998), the pH values for

N. thermophilus, N. jonesii, N. trueperi, N. wadinatrunensis and N. grantii were measured at each microorganism’s optimum growth temperature, denoted with a superscript (i.e., pH55˚C).

The pH measurement of an alkaline, complex growth medium which is at an elevated

temperature (i.e., 55°C) with a pH probe calibrated at a much lower temperature (i.e., 25°C) will yield a pH measurement that can be upwards of one unit greater than that measured with a pH probe calibrated at the elevated temperature (Wiegel 1998). A number of other species have pH optima of 8.5-9 or greater, and of these, three species—Halorhodospira halochloris (Imhoff et al. 1996, Imhoff et al. 1977), Halorhodospira abdelmalekii (Imhoff et al. 1977, Imhoff et al.

1981) and Natroniella acetigena (Zhilina et al. 1996)—also have [Na+] optima of greater than

2.5 M, whereas the more-studied Salinibacter ruber, with a [Na+] optimum around 4 M, grows optimally at pH 8.0 and does not grow above pH 8.5 (Antón et al. 2002). Overall, the number of anaerobic and aerobic haloalkaliphiles is similar (there are nine anaerobic and eleven aerobic haloalkaliphiles); interestingly, however, of the group of organisms just discussed, which have a

[Na+] optimum greater than 2.5 M as well as a pH optima greater than 8.5, all are obligately anaerobic with a fermentative metabolism.

Elevated temperature optima and ranges of extreme halophiles

The other environmental stressor of interest is elevated temperature. Figure 1.1b contrasts, in similar fashion to Figure 1.1a, the correlation between temperature optima with the [Na+] optima of the halophiles under review. Elevated temperature optima—which is, for true thermophiles, above 50˚C—are even more infrequent amongst the published extremely halophilic eubacteria than are alkaline pH optima. An additional problem for thermophiles is that, at the elevated temperature, the cell membrane becomes more permeable to the diffusion of protons and to increased Na+ diffusion (Vossenberg et al. 1999)—especially in saline environments—making it more difficult for the cell to keep the intracellular [Na+] at a millimolar level against the molar extracellular concentration of Na+. The increased Na+ permeability is less pronounced than the

increased proton permeability, but is significant in extremely halophilic conditions. Of the extremely halophilic bacteria with determined temperature optima, only eight have temperature optima equal to or greater than 50ºC. Dichotomicrobium thermohalophilum and Halorhodospira halophila have published temperature optima of 50ºC (Hirsh et al. 1989, Raymond et al. 1969),

Halonatronum saccharophilum of 55˚C (Zhilina et al. 2001), Halothermothrix orenii of 60˚C

(Cayol et al. 1994), the unpublished Natranaerobius jonesii of 66ºC (Bowers et al. 2008),

Natranaerobius thermophilus of 53ºC (Mesbah et al. 2007), Natranaerobius trueperi of 52˚C

(Mesbah et al. 2009), and Natronivirga wadinatrunensis of 51˚C (Mesbah et al. 2009). Three others, Salinibacter ruber, Halorhodospira halochloris and the unpublished Natranaerobius grantii, are thermotolerant, and have temperature optima of 46-48˚C, just below the thermophilic designation (Table 1.2). Of these two groups of organisms only D. thermohalophilum and S. ruber are aerobes; the remaining organisms are all obligately anaerobic. While there are few true thermophilic extreme halophiles, many species—approximately forty percent of the validly published halophiles—are able to tolerate temperatures above 50˚C (Table 2). Most species

(60%) have temperature optima of 40˚C or below: twenty-five percent of these have temperature optima of 38-40˚C, and approximately fifty percent have temperature optima of 32-37˚C. The remaining twenty-five percent have temperature optima between 30 and 32˚C. It is important to note that not all species considered have published temperature optima. Interestingly, no strictly psychrophilic (Topt ≤15˚C and Tmax ≤ 20˚C) extreme halophiles have been described to date, though many psychrophiles and psychrotolerant species, such as Psychrobacter okhotskensis (Trange 5-35°C, Topt 25°C), tolerate up to 4 M Na+ (Yumoto et al. 2003); however the published [Na+] optima of these microorganisms are below 1.7 M.

Poly-extremophiles: extreme halophiles with elevated temperature optima and alkaline pH optima

Of the group of thermophilic extremely halophilic Bacteria, only the anaerobic microorganisms

Natranaerobius thermophilus, Natranaerobius jonesii, Natranaerobius trueperi and

Natronovirga wadinatrunensis demonstrate elevated pH optima (9.5-10.5) and [Na+] optima

(3.7-3.9 M), a group we term poly-extremophiles. Also of note are Halorhodospira halochloris which has a slightly lower temperature optimum of 48˚C, a pH optimum of 8.5 and a [Na+] optimum of 4.62 M and Natranaerobius grantii which has a temperature optimum of 46˚C, a pH45˚C optimum of 9.5 and [Na+] optimum of 4.3 M (Table 1.2). The uniqueness of these poly- extremophiles when compared to other known, extremely halophilic Bacteria is illustrated in

Figure 1.2. On all three axes, [Na+], pH and temperature, these bacteria fall much farther along the axis than do other extreme halophiles. The fact that these microorganisms can not only survive but thrive under these multiple extreme conditions has extended the known boundaries for life at a combination of multiple extrema. Microorganisms living in extreme environments utilize a number of adaptive mechanisms in order to enable them to proliferate, and this is true to an even greater extent of poly-extremophiles. Cytoplasmic acidification for pH adaptation under halophilic growth conditions using multiple monovalent cation/proton antiporters with various pH ranges (Padan et al. 2001), the combined use of organic compatible solutes (Roberts

2005) and intracellular accumulation of K+ (Oren 2002) for adaptation to osmotic pressure are three of the adaptive mechanisms employed by this group of microorganisms (Oren 2006,

Mesbah et al. 2008, Mesbah et al. 2009).

To date, the only true anaerobic poly-extremophiles have been isolated from sediment samples taken from one of two locations. Three of the four anaerobic extremely halophilic alkalithermophiles, N. thermophilus, N trueperi and N. wadinatrunensis, were isolated from the solar-heated, alkaline, hypersaline lakes of the Wadi An Natrun, Egypt (temperatures up to 60˚C measured in the salt brine) (Mesbah et al. 2007, 2009). The Wadi An Natrun is a series of eight lakes in northern Egypt noted for their salinity and alkaline pH. Halorhodospira halochloris, an anaerobic phototrophic purple bacterium, which is a thermotolerant, rather than thermophilic alkaliphilic halophile, was also isolated from the Wadi An Natrun (Imhoff et al. 1977). N. jonesii, and the thermotolerant N. grantii, were isolated from sediment samples from Lake

Magadi, in the Kenyan Rift Valley (Bowers et al. 2008). Lake Magadi, like the lakes of the

Wadi An Natrun, is noted for its salinity and alkalinity. In places, the temperature of the lake exceeds 45˚C; however, the lake is fed by saline hot springs in addition to being heated by sun rays.

The question remains: do the physiological and bioenergetic demands of dealing with one extreme condition (e.g., elevated temperature) limit an organisms’ ability to meet the demands of two additional extreme conditions (e.g., elevated pH and high sodium concentrations) or reduce the extent of the other extrema to lower levels? A first look at the distribution of the peaks in

Figure 1.2 suggests that yes, amongst the presented microorganisms, the more extreme the optimum is for one condition, the less extreme the optima for other conditions tend to be.

However, the recent isolation and cultivation of the anaerobic poly-extremophiles in the novel order Natranaerobiales, which grow with doubling times around 3-4 h (Mesbah et al. 2007,

Bowers et al. 2008, Mesbah et al. 2009) and which require elevated temperatures, alkaline pH

and high [Na+] in order to survive, demonstrate that microorganisms can thrive under combinations of multiple extrema. Furthermore, there is a notion that aerobic extremophiles, with higher ATP yields via electron transport phosphorylation should be better suited to extreme conditions than are anaerobic fermentative extremophiles, which have significantly lower ATP yields per substrate utilized, but so far there are no validly published aerobic Bacteria with similar combinations of growth conditions such as those of the anaerobic Natranaerobius species. The point needs to be stressed that with further investigation into alkaline halobiotic environments more, possibly many more, bacterial poly-extremophiles will be isolated and identified. As noted by Foti et al. (2008), few species identified from hypersaline habitats via culture independent methods are closely related to validly described species. Additionally, in the study performed by Foti et al. (2008) on Russian soda lakes, none of the validly described species were defined as haloalkaliphilic or haloalkalitolerant; however, other studies on Kenyan and Egyptian soda lakes have identified uncultured clones related to validly described haloalkaliphiles (Mesbah et al. 2007, Rees et al. 2004). While the utility of culture independent methods cannot be disputed, the presence of a microorganism in an environment does not necessarily imply that the particular environment under investigation represents the optimal environment for the growth of the microorganism. The only way to learn the true ranges and optimum growth conditions for a particular species is to characterize the cultured species, therefore limiting our discussion to validly published microorganisms. Although nearly every month novel halophiles are published in the International Journal of Systematic and

Evolutionary Microbiology the majority of them are Archaea, or grow only at slightly elevated salt concentration, and are not bacterial extreme halophiles. The authors predict that when investigators focus more on isolating extreme halophiles, and especially poly-extremophilic

halophiles, from extreme habitats the present list will be significantly extended, and the present limits of combined alkaline pH, elevated temperature and [Na+] could be pushed to more extreme values. Then the questions arise: what are the final boundaries? Does a super bacterium exist which can grow at the presently known limit of alkalinity, around pH 12 (or conversely, at acidity of around pH 1); at the present limit of temperature, around 121˚C (or conversely, at temperatures below -12˚C); as well as at saturated [Na+]? Thus far, no single aerobic or anaerobic bacterial or archaeal isolate has been found with optima even near these levels; however, this lack of knowledge certainly does not rule out the existence of such a microorganism. The authors hope that this overview will stimulate further investigation and isolations of these intriguing poly-extremophilic bacterial halophiles and elucidation of their unique physiological and biochemical properties for biotechnological applications.

TEMPERATURE AND PH OPTIMA OF EXTREMELY HALOPHILIC ARCHAEA

Extremely halophilic Archaea

Recently a review was conducted of halophilic bacteria which focused upon extremely halophilic bacteria, with Na+ concentration optima for growth of 1.7 M or greater (Bowers et al. 2009); the review identified sixty-one extremely halophilic bacteria. It is commonly known that archaea are inhabitants of some of the most extreme environments on the planet; validly published species include: Picophilus torridus and Picophilus oshimae, which grow at pH values as low as -0.2, and 60°C (Schleper et al. 1996); Methanopyrus kandleri, which grows optimally at 110°C, and maximally at 122°C (Kurr et al. 1991, Takai et al. 2008); and Thermococcus barophilus, which grows at 40 MPa (Marteinsson et al. 1999). The minimum Na+ optimum for growth of extreme halophiles is 1.7 M, or the equivalent of 10% NaCl (w/v), as previously defined (Oren et al.

2002; Bowers et al. 2009). There are numerous species which merely tolerate such sodium concentrations or which grow optimally at marine salt concentrations of approximately 3.5% w/v; however, the authors focus on microorganisms which grow optimally at the upper limits of combined extremes, this review will be limited to extremely halophilic Archaea. Using this criterion, one hundred seven extreme halophiles were identified (February 2010) amongst the validly published species of the domain Archaea (i.e., published or validated in the International

Journal of Systematic Bacteriology/Systematic and Evolutionary Microbiology, as listed at www.bacterio.cict.fr). In this review Na+ concentrations are given in mol/liter, rather than %

(w/v) NaCl, since some of the alkaliphilic halophiles require media with carbonates, usually provided as sodium carbonates, for pH control (Mesbah et al. 2007; Mesbah et al. 2008).

Therefore the [Na+] comes both from the sodium carbonates in the media buffer and from the supplemented NaCl. These archaea have been isolated from various and diverse environments

such as thalassohaline (i.e. originating from marine waters) solar salterns (Tomlinson et al.

1986); athalassohaline, e.g. Wadi an Natrun, Egypt, (Bowers et al. 2009a) and ancient thalassohaline salt lakes, e.g. Great Salt Lake, UT, USA (Paterek et al. 1988); saline soil (Ihara et al. 1997); and fermented food products (Roh et al. 2007). Unlike the extremely halophilic

Bacteria, which are relatively evenly distributed between aerobic and anaerobic species, the extremely halophilic Archaea are predominately obligate aerobes; out of the one hundred seven species identified (as of February 2010), only five species are obligately anaerobic, with the addition of fourteen facultative aerobes. The extremely halophilic Archaea encompass different types of extrema; for example, Halorhabdus utahensis, at 4.6 M, has the highest [Na+] optimum

(Waino et al. 2000); whereas Haloferax mediterranei is capable of growing over the unusually wide [Na+] range of 1.0-5.2 M (Torreblanca et al. 1986). Additionally, there are eight species, including Natrialba aegyptia and Halobiforma nitratireducens which tolerate Na+ saturation

(Hezayen et al. 2001; Hezayen et al. 2002) and Natronolimnobius ‘aegyptiacus’, which tolerates

Na+ saturation at the highest temperature and alkaline pH (Bowers et al. 2009a).

Many extreme halophiles are mesophilic or neutrophilic; only moderately thermophilic extreme halophiles have been described, with Topt of 50°C or above, along with several alkaliphilic extreme halophiles. Several of the extremely halophilic Archaea grow optimally at either alkaline pH ( ≥ 8.5) or at elevated temperatures ( ≥ 50°C); however, only a very few of the extreme halophiles are able to grow optimally under triply extreme conditions: high [Na+], alkaline pH, and elevated temperatures. These microorganisms are considered poly- extremophiles (Table 1.1), if all three conditions are required for optimal growth, as previously described by Bowers et al. (2009).

Of the one hundred seven validly published, extremely halophilic Archaea, no species has a [Na+] optimum of less than 2 M (approximately 12% w/v NaCl); a stark contrast to the thirty percent of validly published, extremely halophilic Bacteria (Bowers et al. 2009).

Approximately forty percent of the extremely halophilic species have published [Na+] optima equal to or greater than 2.0 M (approximately 12% w/v NaCl) but less than 3.4 M

(approximately 20% w/v NaCl), approximately thirty-six percent of the species have [Na+] optima greater than or equal to 3.4 M but less than 4.0 M (approximately 24% w/v NaCl), and approximately twenty-two percent (23 species) have published [Na+] optima of equal to or greater than 4 M. Additionally, approximately sixty percent of all the extremely halophilic

Archaea (63 species) tolerate greater than 5 M Na+ in their growth medium (approximately 29% w/v NaCl), including eight species which tolerate Na+ saturation, i.e., 5.5 to 6.5 M, since the saturation point is dependent upon media composition, growth pH and temperature (Table 1.3).

In contrast to the extremely halophilic Bacteria all of the extremely halophilic Archaea with published [Na+] maxima are able to tolerate greater than 4 M Na+, with the exception of

Methanohalophilus mahii which has a [Na+] maximum of 2.5 M (Paterek et al. 1988).

pH optima and ranges of extremely halophilic Archaea

Out of all the validly published extremely halophilic Archaea, only nineteen have pH optima of

8.5 or greater (Table 1.3), and therefore are haloalkaliphiles (Table 1.1), although many of the environments from which these microorganisms were isolated have alkaline pH values (Foti et al. 2008; Mesbah et al. 2007; Rees et al. 2004). All these species combine an alkaline pH optimum with a [Na+] optimum of 2 M or greater, and twelve of these species have a [Na+] optimum of 3.4 or greater, although only three: Halorubrum luteum; Natronolimnobius

‘aegyptiacus’, a recent isolate from the authors; and Halorubrum alkaliphilum combine an alkaline pH optimum (9.8, 9.5 and 9.0, respectively) with a [Na+] optimum of greater than 4 M

(4.2, 4.5 and 4.1, respectively) (Hu et al. 2008; Bowers et al. 2009; Feng et al. 2005). The distribution of the [Na+] and pH optima of the extremely halophilic archaea (Figure 1.3a), display a bifurcated distribution, i.e., with many pH optima near 7, and the others near pH 9.

The combinations of pH optimum 7.5 with a [Na+] optimum of approximately 3.4 M is the most highly represented, followed by the combinations of pH optima 7.3 and 9 with [Na+] optimum of approximately 3.4 M. This is opposite what was seen upon examination of the extremely halophilic Bacteria; in that case, the most highly represented combination was pH 9 and [Na+] optimum 1.7 (Bowers et al. 2009). Microorganisms with pH values for optimal growth above

8.5 carry with them the unique energetic problems of alkaliphiles, e.g., an inverted pH gradient and thus a suboptimal proton motive force (Bowers et al. 2009; Kevbrin et al. 2004; Krulwich

1998). In the case of extremely halophilic alkaliphiles these problems are exacerbated by the need to keep the intracellular sodium concentration below toxic levels, which is frequently as low as a few mM (Mesbah et al. 2009; Oren 2002; Padan 2000). This complication may explain the more prevalent occurrence of microorganisms growing optimally in environments that are pH neutral or near neutral. However, as discussed by Bowers et al. (2009), this could be due to the fact that researchers may simply have not tried to isolate microorganisms able to thrive under these combined stressors. The biodiversity of the highly alkaline athalassohaline lakes of Wadi

An Natrun (North Egypt) is relatively high (Mesbah et al. 2007), as are the saline habitats of

Alexandria, Egypt (Ghozlan et al. 2006) and the various alkaline soda lakes of Rift Valley,

Kenya (Duckworth et al. 2006). Currently, the only identified obligately alkaliphilic archaeal species from the lakes of the Wadi An Natrun is Natronolimnobius ‘aegyptiacus’, which has a

pH55°C optimum of 9.5, and grows optimally above 50°C (Bowers et al. 2009a). However, several obligately alkaliphilic archaeal species have been isolated from Lake Magadi and the surrounding Rift Valley, Kenya, including Natronobacterium gregoryi, Halorubrum vacuolatum,

Natrialba magadii, Natronomonas pharaonis, and Natronococcus amylolyticus; each of these microorganisms has a pH optimum of 9.0 or greater (Table 1.3). As previously recommended

(Wiegel 1998), the pH values for N. ‘aegyptiacus’ were measured at the microorganism’s optimum growth temperature, denoted with a superscript (i.e., pH55˚C). The pH measurement of an alkaline, complex growth medium which is at an elevated temperature (i.e., 55°C) with a pH probe calibrated at a much lower temperature (i.e., 25°C) will yield a pH measurement that can be upwards of one unit greater than that measured with a pH probe calibrated at the elevated temperature (Wiegel 1998); this should be considered when comparing the pH optima of alkaliphiles to the pH optima of alkalithermophiles. Three other species are alkalitolerant, and have pH optima of 8.0; of these, only one, Halopiger xanaduensis, has a [Na+] optimum of greater than 4 M (Gutiérrez et al. 2007). With the exception of Halobiforma nitratireducens, which is a facultatively aerobic species, all of the extremely halophilic, alkaliphilic Archaea are obligate anaerobes (Table 1.3). This again, is in contrast to the distribution of haloalkaliphilic

Bacteria, which are relatively evenly distributed between aerobic and anaerobic species.

Temperature optima and ranges of extremely halophilic Archaea

The third environmental stressor of interest is elevated temperature. Figure 1.3b contrasts, in similar fashion to Figure 1.3a, the correlation between temperature optima with the [Na+] optima of the halophiles under review. Elevated temperature optima—which is, for true thermophiles, above 50˚C (Table 1.3)—are even more infrequent amongst the published extremely halophilic

eubacteria than are alkaline pH optima. Only eleven of the extremely halophilic archaea have temperature optima of 50°C or greater, seven of which are equal to 50°C (Table 1.3).

Natronolimnobius ‘aegyptiacus’, at 55°C, has the highest temperature optimum of all the archaeal extreme halophiles; Haloferax elongans, Haloarcula quadrata, and Haloferax mediterranei have temperature optima of 51-53°C (Allen et al. 2008; Oren et al. 1999;

Torreblanca et al. 1986). As orginally discussed in Vossenberg et al. (1999) at elevated temperature, the cell membrane of thermophilic microorganisms become more permeable to the diffusion of protons and to increased Na+ diffusion —especially in saline environments—making it more difficult for the cell to keep the intracellular [Na+] at a millimolar level against the molar extracellular concentration of Na+, and is possibly a reason that, so far, no hyperthermophilic extreme halophiles have been isolated. The group of extremely halophilic, thermophilic archaea contains one anaerobe, Haloarcula quadrata (Torreblanca et al. 1986) and two facultative aerobes, Halorhabdus utahensis and Haloferax denitrificans (Waino et al. 2000; Tomlinson et al. 1986); all the other microorganisms are aerobic. Fourteen other microorganisms have temperature optima between 45 and 48°C, and are thermotolerant. This group contains two anaerobes, Methanohalobium evestigatum and Halorhabdus tiamatea (Zhilina et al. 1987;

Antunes et al. 2008), but no facultative aerobes. While there are relatively few truly thermophilic extreme halophiles, many species—approximately seventy-five percent of the extreme halophiles with published temperature maxima—are able to tolerate temperatures above

50˚C (Table 1.3). Just over half of the extremely halophilic archaeal species (63%) have temperature optima of 40˚C or below: sixty-three percent of these species exhibit temperature optima of 38-40˚C. The remaining species have temperature optima between 28 and 35˚C. It is important to note that not all extremely halophilic species considered have published temperature

optima. Interestingly, no strictly psychrophilic (Topt ≤15˚C and Tmax ≤ 20˚C) extreme halophiles have been described to date; however, Halorubrum lacusprofundi, despite its optimum growth temperature of 33°C, dominates the archaeal population of Deep Lake, Antarctica, which has a

[Na+] of 3.6-4.8 M and a temperature ranging from -18 to 12°C (Caviccioli 2006).

Poly-extremophiles: extremely halophilic Archaea with concomitant elevated temperature optima and alkaline pH optima

Of all the extremely halophilic Archaea, only two species are extremely halophilic, obligately alkaliphilic and thermophilic, and are thus termed poly-extremophiles: Natrialba hulunbeirensis, which has a [Na+] optimum of 3.4 M, a pH optimum of 9.0, and a temperature optimum of 50°C

(Xu et al. 2001); and Natronolimnobius ‘aegyptiacus’, which has a [Na+] optimum of 4.5, a pH optimum of 9.5 and a temperature optimum of 55°C (Bowers et al. 2009a). Also of note are

Natronorubrum tibetense, with a [Na+] optimum of 3.4, a pH optimum of 9, and a thermotolerant temperature optimum of 45°C (Xu et al. 1999) and Natronorubrum bangense, with a [Na+] optimum of 3.8, a pH optimum of 9.5 and a thermotolerant temperature optimum of 45°C (Xu et al. 1999). All four of these noteworthy species are aerobic (Table 1.3). These poly- extremophiles cluster uniquely when compared to other known, extremely halophilic Archea

+ (Fig. 1.4). This is further demonstrated when extremely halophilic species (Na opt ≥ 3.4 M) which are also either thermophilic (Topt ≥ 50°C) or obligately alkaliphilic (pHopt ≥ 8.5), along with the two identified poly-extremophiles are plotted (Fig. 2). Natronorubrum bangense, while nearly unique amongst archaeal species in its poly-extremophily, is neither extraordinarily halophilic nor alkaliphilic. However, on all three axes, [Na+], pH and temperature,

Natronolimnbius ‘aegyptiacus’ falls much farther along the axis than do the other extremophiles.

The fact that this archaeal microorganism can not only survive but thrive under these multiple extreme conditions has helped, similarly to the poly-extremophilic Bacteria of the order

Natranaerobiales to extend the known boundaries for life at a combination of multiple extrema.

Microorganisms living in extreme environments utilize a number of adaptive mechanisms in order to enable them to proliferate, and this is true to an even greater extent of poly- extremophiles. Cytoplasmic acidification for pH adaptation under halophilic growth conditions using multiple monovalent cation/proton antiporters with various pH ranges (Mesbah et al. 2009;

Slonczewski et al. 2009), the combined use of organic compatible solutes (da Costa et al. 1998;

Roberts 2005) and intracellular accumulation of K+ (Mesbah et al. 2008; Oren 2002) for adaptation to osmotic pressure are three of the adaptive mechanisms employed by this group of microorganisms (Oren 2006).

To date, the only true anaerobic poly-extremophilic Archaea have been isolated from sediment samples taken from one of two locations: solar-heated, alkaline, hypersaline lakes of the Wadi An Natrun, Egypt, with temperatures up to 60˚C measured in the salt brine (Bowers et al. 2009), and an unnamed soda lake in the Hulunbeir Prefecture in China (Xu et al. 2001).

With further investigation into alkaline halobiotic environments more, archaeal poly- extremophiles will likely be isolated and identified. As noted both by Foti et al., (2008) and

Mesbah et al. (2007), few species identified from hypersaline habitats via culture-independent methods are closely related to validly described species. Additionally, in the study performed by

Foti et al., (2008) on Russian soda lakes, none of the validly described species were defined as haloalkaliphilic or haloalkalitolerant; however, other studies on Kenyan and Egyptian soda lakes have identified uncultured clones related to validly described haloalkaliphiles (Mesbah et al.,

2007; Rees et al. 2004). While the utility of culture independent methods cannot be disputed, the

presence of a microorganism in an environment does not necessarily imply that the particular environment under investigation represents the optimal environment for the growth of the microorganism. The only way to learn the true ranges and optimum growth conditions for a particular species is to characterize the cultured species, therefore limiting our discussion to validly published microorganisms. Although nearly every month novel halophiles are published in the International Journal of Systematic and Evolutionary Microbiology, the majority of them

Archaea, many grow only at slightly elevated salt concentration, and are not extreme halophiles.

The authors hope that this mini-review will encourage other scientist to investigate and characterize other novel poly-extremophiles and further expand the knowledge base of their habitats, limitations and adaptive mechanisms.

Acknowledgements

This work was financially supported by grant MCB 060224 from the National Science

Foundation and grant AFOSR 033835-01 from the Air Force Office of Scientific Research.

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Figure 1.1 Correlation of [Na+] optimum and pH or temperature optima of extreme halophiles.

Figure 1.1a. Correlation between [Na+] optimum and pH optimum. [Na+] optimum (M) is plotted against pH optimum; number of microorganisms at each locus is plotted on z-axis as indicated by color coding; no representation (zero) is indicated in the darkest shade.

Figure 1.1b. Correlation between [Na+] vs. temperature optima. [Na+] optimum (M) is plotted against temperature optimum (˚C); number of microorganisms at each locus is plotted on z-axis as indicated by color coding; no representation (zero) is indicated in the darkest shade.

Figure 1.2. Clustering of poly-extremophiles relative to other extreme halophiles.

Representation of extremely halophilic Bacteria for which both additionally-considered growth conditions (pH and temperature) approach or exceed thermophilic and alkaliphilic levels. The optima of the discussed recently isolated poly-extremophiles cluster in the upper range for each criterion, well-separated from other representative microorganisms. The z-axis color coding depicts the temperature optimum of each bacterium. Species represented: 1. Natranaerobius jonesii, 2. Natronovirga wadinatrunensis, 3. Natranaerobius thermophilus, 4. Natranaerobius grantii, 5. Natranaerobius truperi, 6. Halorhodospira halochloris, 7. Dichotomicrobium thermohalophilum, 8. Halanaerobacter salinarius, 9. Salinibacter ruber

Figure 1.3 Correlation of [Na+] optimum and pH or temperature optimum of extremely halophilic Archaea

Figure 1.3a. Correlation between [Na+] optimum and pH optimum. [Na+] optimum (M) is plotted against pH optimum; number of microorganisms at each locus is plotted on z-axis as indicated by color coding; no representation (zero) is indicated in the darkest shade.

Figure 1.3b. Correlation between [Na+] vs. temperature optima. [Na+] optimum (M) is plotted against temperature optimum (˚C); number of microorganisms at each locus is plotted on z-axis as indicated by color coding; no representation (zero) is indicated in the darkest shade.

Figure 1.4. Clustering of poly-extremophilic Archaea relative to other extremely halophilic

Archaea. Representation of extremely halophilic Archaea for which one or both additionally- considered growth conditions (pH and temperature) exceed thermophilic and alkaliphilic levels.

The peaks representative of the discussed recently isolated poly-extremophiles are labeled numerically, and are delineated below. The z-axis color coding depicts the temperature optimum of each bacterium. Species represented: 1. Natronolimnobius ‘aegyptiacus’, 2. Natrialba hulunbeirensis; also represented: Halalkalicoccus tibetensis, Haloarcula quadrata, Halobiforma nitratireducens, Halorhabdus utahensis, Halorubrum alkaliphilum, Halorubrum luteum,

Halorubrum vacuolatum, Haloterrigena hispanica, Natrialba magadii, Natrialba hulunbeirensis,

Natronolimnobius baerhuensis, Natronomonas pharaonis, Natronorubrum bangense, and

Natronorubrum tibetense; all of which meet two out of three criteria for poly-extremophily.

Table 1.1 Growth Characteristics of Extremophiles

Growth Characteristic Minimum Optimum Maximum Halotolerant - [Na+] < 0.2 M [Na+] > 0.2 M Halophilic [Na+] ≥ 0.2 M 0.2 M < [Na+] < 1.7 M - Extreme [Na+] ≥ 0.2 M [Na+] ≥ 1.7 M - Alkalitolerant pH ≥ 6.0 pH < 8.5 pH > 9.0 Alkaliphilic Facultative pH < 7.5 pH ≥ 8.5 - Obligate pH ≥ 7.5 pH ≥ 8.5 pH ≥ 10.0 Thermotolerant - T < 50˚C T > 50˚C Thermophilic - T ≥ 50˚C T > 55˚C

apH should be measured at optimum growth temperature (Wiegel 1998) b10% added NaCl equals 1.7 M Na+ c – indicates no boundary at this parameter

Table 1.2. [Na+], pH and Temperature Optima and Ranges for Bacterial Extreme Halophiles

Na+opt. Na+ range Temp. Temp. Genus Species (M) (M) pH opt. pH range opt. range Isolated from Aerobic

Actinopolyspora halophila 3.42 1.71- 5.13 37 10- 43 dairy salt Alkalibacillus halophilus 2.55 0.85-5.10 7.5 6.0- 9.0 37 25-45 hypersaline soil Xin-Jiang, China Arhodomonas aquaeolei 2.57 1.03-3.42 7.0 6.0-8.0 37 20-45 petroleum reservoir production fluid Bacillus persepolensis 1.71 0.85-3.42 8.3 7.0-10.0 40 25-45 Howz-Soltan lake, Iran Dichotomicrobium thermohalophilum 2.43 1.37-3.80 8.5 5.8-9.5 50 20-65 solar lake near Elat (Sinai) Filobacillus milosensis 2.39 0.34-3.94 7.8 6.5-8.9 38 -42 Palaeochori Bay, Milos, Greece Gracilibacillus saliphilus 2.13 0.17-3.74 7.0 6.0-8.0 33 4-45 Ebinur Lake, China Haloglycomyces albus 1.71 0.51-3.06 7.25 5.0-9.0 37 15-40 Xinjiang Province, China Halomonas aquamarina 1.71 0-3.42 9.0 7.5-10 37 5-50 Pacific Ocean Halomonas campaniensis 1.71 0-2.7 9.0 7-10 37 10-43 Malvizza, Campania, Italy Halomonas campisalis 1.5 0.2-4.5 9.5 6.0-11.0 30 4-50 Alkali Lake, Washington, USA Halomonas cupida 1.5 0.2-4.5 9.5 6.0-12.0 30 4-50 marine habitats Halomonas denitrificans 1.71 0.34-3.42 8.0 5.0-10.0 35 5-50 seawater, Anmyeondo, Korea Halomonas gomseomensis 2.05 0.17-3.42 8.0 6.0-10.0 30 5-45 solar saltern, Anmyeondo, Korea Halomonas gudaonensis 2.57 0.17-3.42 8.0 8.0-9.0 30 10-42 oil contaminated saline soil, China Halomonas ilicicola 1.71 0.34-2.98 6.5 6.0-9.0 37 25-42 Santa Pola, Spain Halomonas janggokensis 2.57 0.17-3.42 8.0 6.0-10.0 30 5-45 Janggok saltern, Anmyeondo, Korea Halomonas maura 2.57 0.17-3.42 7.5 6.0-10.0 30 5-45 solar saltern, Asilah, Morocco Halomonas salaria 3.42 0-4.28 8.0 5.0-10.0 30 10-45 seawater, Anmyeondo, Korea Halomonas shengliensis 3.42 0-4.28 7.5 5.0-10.0 30 10-45 Shengli oilfield, China Halomonas taeanensis 2.57 0-4.28 8.0 7.0-10.0 35 10-45 solar saltern, Taean, Korea Halomonas variabilis 1.6 1.2-4.9 7.5 6.5-8.4 33 15-37 Great Salt Lake, Utah Kushneria indalinina 1.71 0.51-4.28 7.2 5.0-9.0 35 10-42 solar saltern, Almaria, Spain Nocardiopsis kunsanenesis 1.71 0.51-3.42 9.0 37 saltern, Kunsan, Korea Nocardiopsis lucentensis 1.71 0.51-4.28 9.0 5.0 - 37 salt marsh, Alicante, Spain Oceanobacillus kapialis 1.71 0.09-4.08 8.0 6.0-9.0 37 8-43 fermented shrimp paste, Thailand Rhodospirillum sodomense 2.05 1.03-3.42 7.0 40 25-47 Dead Sea Saccharopolyspora halophila 2.13 0.51-3.42 7.5 6.0- 8.5 33 10-45 Xinjiang Province, China Saccharopolyspora qijiaojingensis 2.13 1.02-3.74 7.0 5.0-8.0 28 20-40 Xinjiang Province, China Salinibacter ruber 4.00 2.57- 8.0 6.0-8.5 47 -52 saltern crystallizer ponds, Spain

Salinicoccus albus 1.71 0.17-5.10 8.5 6.0-10.0 25 5-40 Yunnan, China Salinicoccus alkaliphilus 1.71 0-4.28 9.0 6.5-11.5 32 10-46 Baer Soda Lake, China Salinicoccus iranensis 1.71 0.17-4.28 7.5 6.5-10 35 5-45 textile wastewater, Qom, Iran Salinicoccus luteus 1.71 0.17-4.28 9.0 7.0-11.0 30 4-45 desert soil sample, Egypt Salinicoccus roseus 1.71 0.15-4.28 8.0 6.0-9.0 Salinicoccus siamensis 1.71 0.26-4.28 8.5 6.0-9.0 37 15- 45 fermented shrimp paste

Na+ Na+ range Temp. Temp. Genus Species opt. (M) (M) pH opt. pH range opt. range Isolated from Anaerobic

Actinopolyspora iraqiensis 2.56 0.86- 4.27 7.5 35 16- 40 saline soil, Iraq Halanaerobacter chitinivorans 3.0 0.5-5.0 7.0 45 23-50 Solar saltern, California Halanaerobacter salinarius 2.57 0.86-5.13 7.8 5.5- 8.5 45 10-50 Salin-de-Giraud, France

Halanaerobaculum tuisiense 3.57 2.38- 5.10 7.3 5.9- 8.4 42 30- 50 El -Djerid Chott, Tunisia Halanaerobium kushneri 2.05 1.54-3.08 7.5 6.0-8.0 40 20-45 reservoir production fluid, Oklahoma Halanaerobium lacusrosei 3.42 1.03-5.82 7.0 40 20-50 Retba Lake, Senegal Halanaerobium praevalens 2.14 0.34-5.13 7.2 6.0- 9.0 37 5-50 Great Salt Lake, Utah Halanaerobium saccharolyticum 1.7 0.51-5.1 7.5 6.0-8.0 40 15-47 Lake Civash subsp. saccharolyticum Halanaerobium saccharolyticum 2.1 0.8- 4.3 7.0 6.3- 8.7 40 20- 47 Retba Lake, Senegal subsp. senegalense Halocella cellulosilytica 2.57 0.86- 3.42 7.0 5.5- 8.5 39 20- 50 Lake Sivash, France Halonatronum saccharophilum 2.05 0.51-2.91 8.5 7.7-10.3 55 18-60 Lake Magadi, Kenya Halorhodospira abdelmalekii 3.08 0.86-5.13 9.2 44 Halorhodospira halochloris 4.62 1.71-5.81 8.5 8.1- 9.1 48 33- 50 Wadi An Natrun, Egypt Halorhodospira halophila 3.76 1.54-5.14 7.8 50

Halothermothrix orenii 1.71 0.68-3.42 7.0 5.5-8.2 60 45-68 Chott El Guettar (lake), Tunisia Natranaerobius grantii 4.3 2.9-sat. 9.5 7.5-10 46 31-52 Lake Magdi, Kenya Natranaerobius jonesii 3.9 3.1-5.0 10.5 8.5-11.5 66 47-71 Lake Magdi, Kenya Natranaerobius thermophilus 3.9 3.1-4.9 9.5 8.3-10.6 53 35-56 Wadi An Natrun, Egypt Natranaerobius trueperi 3.7 3.1-5.4 9.5 7.8-11.0 52 26-55 Wadi An Natrun, Egypt Natroniella acetigena 2.57 1.71-3.42 9.7 8.1-10.7 37 -42 Lake Magdi, Kenya Natronovirga wadinatrunensis 3.9 3.1-5.3 9.9 8.5-11.5 51 24-58 Wadi An Natrun, Egypt

Facultatively anaerobic

Halomonas sinaiensis 2.57 0.86- 5.13 7.0 6.0- 9.0 35 25- 50 Ras Muhammad Park, Egypt Halospina denitrificans 3.0 2.0-5.0 6.7-8.5 Halovibrio denitrificans 2.25 2.0-5 6.7-8.5 hypersaline lake, Mongolia Salinivibrio siamensis 1.71 0.17-3.74 8.0 5.0-9.0 37 10- 47 fermented fish, Thailand Thiohalorhabdus denitrificans 3.0 2.0-5.0 7.8 6.5-7.2 35 hypersaline lake, Siberia, Russia ablank cells indicate data not found bShaded cells indicate data meeting or exceeding one of the following criteria: + [Na ]opt ≥ 2.0 M, pHopt ≥ 8.5, Topt ≥ 50˚C c For reasons of brevity, citations for the description of the species can be found in the List of Prokaryotic Names with Standing in Nomenclature, www.bacterio.cict.fr (updated monthly)

Table 1.3 [Na+], pH and Temperature Optima and Ranges for Extremely Halophilic Archaea

Na+ pH Genus Species Na+ opt range pH opt range T opt T range O2 Isolated from Natrinema altunense 3.7 1.7- 7.4 6.9-8.0 - - facultative salt lake, China Natrinema gari 3 1.7- 6.3 5.5-8.5 39 20-60 facultative fermented fish sauce Natrinema versiforme 3.9 1.5- 6.8 6.0-8.0 42 20-53 facultative Aibi salt lake, China Haloterrigena saccharevitans 3.2 1.71-? 7.5 6.5-8.5 44 24-58 facultative Aibi salt lake, China Halorhabdus utahensis 4.6 1.5-5.1 6.9 5.5-8.5 50 17-55 facultative Great Salt Lake, Utah Halomicrobium mukohataei 3.3 2.5-4.5 - 6.2-8.0 43 - facultative salt flats, Argentina Halomicrobium mukohataei 3.3 2.5-4.5 - - 40 - facultative Argentina Halogeometricum boriquense 3.9 1.36-? - - 40 - facultative Cabo Rojo, Puerto Rico Haloferax denitrificans 2.5 1.5-4.5 6.7 6.0-8.0 50 30-55 facultative solar salterns, S.F. Bay, CA (T) Haloferax sulfurifontis 2.55 1.02-sat 6.6 4.5-9.0 35 18-50 facultative Zodletone Spring, OK Halobiforma lacisalsi 3.5 1.7- 7.5 6.5-9.0 44 24-57 facultative salt lake, China Halobiforma nitratireducens 3.5 2.5-sat 8.5 8.0-10.5 39 26-44 facultative Chahannao soda lake, China Haloarcula hispanica 3.9 2.5-5.2 7 6.0-8.0 38 25-50 facultative marine salterns, Spain (T) Haloarcula vallismortis 4.3 2.5- 7.5 5.5-8.5 40 20-45 facultative salt pools, Death Valley Methanohalophilus mahii 2 .5-2.5 7.5 - 35 -45 anaerobic Great Salt Lake, Utah Methanohalobium evestigatum 4.3 2.6-5.1 7.3 6.0-8.3 48 35-60 anaerobic saline lagoons, Sivash Halorhabdus tiamatea 4.6 1.71-5.1 6.8 5.5-8.5 45 15-55 anaerobic Shaban Deep, Red Sea Haloarcula quadrata 3.9 2.7-4.3 6.8 5.9-8.0 52 -55 anaerobic brine pool, Sinai, Egypt Natronorubrum aibiense 2.9 2.0-4.3 8 6.5-9.5 45 20-50 aerobic Aibi salt lake, China Natronorubrum bangense 3.8 2.0-4.3 9.5 8.0-11.0 45 22-55 aerobic soda lake, Tibet Natronorubrum sulfidifaciens 3.1 2.1-4.8 9 8.0-10.0 46 20-55 aerobic Aiding Salt Lake, China Natronorubrum tibetense 3.4 2.0-5.1 9 8.5-11.0 45 22-55 aerobic soda lake, Tibet Natronomonas pharaonis 3.5 2.0-5.2 9 7.0-10.0 37 25-50 aerobic Lake Magadi, Kenya Natronolimnobius aegyptiacus' 4.5 2.8-sat 9.5 7.3-10.8 55 31-59 aerobic Wadi An Natrun, Egypt Natronolimnobius baerhuensis 3.4 2.6- 9 7.0-10.0 37 30-46 aerobic soda lakes, China Natronolimnobius innermongolicus 3.1 1.7- 9.5 7.5-10.0 45 19-54 aerobic soda lakes, China Natronococcus amylolyticus 3.1 146-5.1 9 8.0-10.0 43 22-50 aerobic Lake Magadi, Kenya

Na+ pH Genus Species Na+ opt range pH opt range T opt T range O2 Isolated from Natronococcus jeotgali 4.1 1.3-5.1 7.5 7.0-9.5 41 21-50 aerobic fermented shrimp, Korea Natronococcus occultus 2.8 1.4-5.2 9.8 8.0- 38 25-50 aerobic Natronobacterium gregoryi 3 2.5-5.2 9.8 8.0- 37 25-40 aerobic Rift Valley, Kenya Natrinema ejinorense 3.4 1.8- 7 6.0-8.5 37 25-50 aerobic Lake Ejinor, China Natrinema pallidum 3.9 1.7- 7.4 6.0-8.4 39 - aerobic salted cod Natrinema pellirubrum 3.9 2.0- 7.6 6.0-8.6 - 20-45 aerobic salted hide Natrialba aegyptia 2.75 1.6-sat 7.5 6.0-9.0 40 -60 aerobic Aswan, Egypt Natrialba asiatica 4 2.0-sat. 6.8 6.0-8.0 35 -50 aerobic beach sand, japan (T) Natrialba chahannaoensis 2.6 1.7-5.1 9 8.5-10.5 45 20-55 aerobic soda lake, China Natrialba hulunbeirensis 3.4 2.04-5.1 9 8.5-10.5 50 20-55 aerobic soda lake, China Natrialba magadii 3.5 2.0-5.2 9.5 8.5-11.0 39 20-50 aerobic Lake Magadi, Kenya Natrialba taiwanensis 3.5 2.0-sat. 7.7 5.0-10.0 38 -55 aerobic solar salts, Taiwan Halovivax asiaticus 3.4 2.5-4.3 7.3 6.0-9.0 37 25-45 aerobic Lake Ejinor, China Halovivax ruber 3.4 2.5- 7.3 6.0-9.0 37 25-45 aerobic Lake Xilinhot, China Haloterrigena hispanica 3.4 2.2-4.0 7 6.5-8.5 50 37-60 aerobic Fuenta de Piedra Lake, Spain Haloterrigena jeotgali 3.1 1.7-5.1 7.3 6.5-8.5 41 17-50 aerobic shrimp jeotgal Haloterrigena limicola 3.1 1.7-5.1 7 6.5-9.0 48 30-61 aerobic Aibi salt lake, Xin-Jiang, China Haloterrigena longa 3.1 1.7-5.1 7.3 6.5-9.0 43 30-56 aerobic Aibi salt lake, Xin-Jiang, China Haloterrigena salina 3.4 2.5-5.0 7.5 6.0-9.0 37 25-50 aerobic Lake Xilinhot, China saline crystallizer pond, Puerto Haloterrigena thermotolerans 3.3 2.0-? 7.3 6.5-8.2 50 -60 aerobic Rico Haloterrigena turkmenica 3.4 2.0- - - 45 - aerobic saline soil, Turkmen Halostagnicola larsenii 3.4 2.5-5.0 7.5 6.0-9.0 37 25-50 aerobic Lake Xilinhot, China Halosimplex carlsbadensee 4.3 3.4-5.1 - 7.0-8.0 38 22-50 aerobic Carlsbad, New Mexico Halosarcina pallida 3.1 1.7-4.3 6.5 6.0-8.5 30 25-45 aerobic Zodletone Spring, OK Halorubrum aidingense 2.6 1.7-4.3 7.5 7.0-9.0 41 25-52 aerobic Aiding Salt Lake, China Halorubrum alkaliphilum 4.1 1.8-5.2 9 8.0-10.5 38 20-44 aerobic soda lake, Xin-Jiang, China Halorubrum arcis 4.2 2.2-5.2 7.5 6.0-8.5 42 25-55 aerobic Qinghai-Tibet Plateau Halorubrum californiense 4 2.5-5.0 7.3 6.5-8.5 37 25-42 aerobic Cargill Solar Plant, California

Na+ pH Genus Species Na+ opt range pH opt range T opt T range O2 Isolated from Halorubrum chaoviator 4.3 2.0-5.0 7.4 7.0-8.5 37 28-50 aerobic Baja California, Mexico Halorubrum coriense 2.5 2.0-5.2 - - 50 30-56 aerobic marine salterns (T) Halorubrum distributum 2.9 1.7-5.2 - - 41 26-50 aerobic Halorubrum ejinorense 3.4 2.5-5.0 7.5 6.0-10.0 37 25-50 aerobic Lake Ejinor, China Halorubrum ezzemoulense 3.4 2.5-4.3 7.3 6.5-9.0 39 22-50 aerobic Ezzemoul Sabkha, Algeria Halorubrum kocurii 3.4 2.5- 7.5 6.0-9.0 37 25-55 aerobic Lake Bagaejinor, China Halorubrum lacusprofundi 3 1.5-5.2 - - 33 1-40 aerobic Deep Lake, Antarctica Halorubrum lipolyticum 2.6 1.7-4.8 7.5 7.0-9.0 47 25-58 aerobic Aibi salt lake, Xin-Jiang, China Halorubrum litoreum 3.4 2.0-5.1 7.3 6.0-9.5 40 20-55 aerobic Fuqing solar saltern, China Halorubrum luteum 4.2 2.5-5.2 9.8 7.5-10.5 35 17-41 aerobic Lake Chagannor, China Halorubrum orientale 3.4 2.5-5.0 8 6.0-10.0 40 25-50 aerobic Lake Ejinor, China Halorubrum saccharovorum 4 1.5-5.2 - - 50 30-56 aerobic Halorubrum sodomense 2.1 0.5- - - 40 - aerobic Dead Sea Halorubrum tebenquichense 4.25 2.5-5.2 - 7.0-10.0 40 35-50 aerobic Lake Tebenquiche, Chile Halorubrum terrestre 4.25 2.6-5.1 7.5 5.0-9.0 41 28-50 aerobic saline soil Halorubrum tibetense 3.2 1.7-5.2 9.3 8.0-10.5 39 22-45 aerobic Lake Zabuye, Tibet Halorubrum trapanicum - 2.5-5.2 - - 37 - aerobic Trapani salt, Norway Halorubrum vacuolatum 3.5 2.6-5.1 9.5 8.5-10.5 38 20-50 aerobic Lake Magadi, Kenya Halorubrum xinjiangense 3.3 2.0-5.2 7.3 6.0-10.0 40 10-54 aerobic Xiao-Er-Kule Lake, China 2.38- Haloquadratum walsbyi 3.06 6.12 - 6.0-8.5 - 25-45 aerobic solar saltern ponds Haloplanus natans 3 2.6-4.3 7 6.5-8.0 40 37-52 aerobic Dead Sea water mixture Halopiger xanaduensis 4.3 2.5-5.0 8 - 37 -45 aerobic Lake Shangmatala, China Halomicrobium katseii 4.3 3.4-5.2 7.3 6.5-10.0 39 35-50 aerobic Lake Tebenquiche, Chile Haloferax alexandrinum 4.25 1.71-sat 7.2 5.5-7.5 37 20-55 aerobic solar saltern, Egypt Haloferax elongans 3 1.7-5.1 7.4 7.0-9.0 53 30-55 aerobic Shark Bay, Australia (T) Haloferax gibbonsii 3.5 1.5-5.2 6.8 5.0-8.0 38 15-55 aerobic marine salterns, Spain (T) Haloferax larsenii 2.8 1.0-4.8 6.8 6.0-8.5 44 25-55 aerobic solar saltern, China Haloferax lucentensis 4.3 1.7-5.1 7.5 5.0-9.0 37 10-45 aerobic Alicante, Spain

Na+ pH Genus Species Na+ opt range pH opt range T opt T range O2 Isolated from Haloferax mediterranei 2.5 1.0-5.2 6.5 - 51 20-55 aerobic marine salterns, Spain (T) Haloferax mucosum 3 1.7-5.1 7.4 6.0-10.0 47 23-55 aerobic Shark Bay, Australia (T) Haloferax prahovense 3.5 2.5-5.2 7.3 6.0-8.5 43 23-51 aerobic Telega Lake, Romania Haloferax volcanii 2 1.5-4.5 7 5.0-8.0 45 30-55 aerobic Dead Sea Halococcus dombrowskii 3.9 2.55-? 7 5.8-8.0 39 - aerobic salt mine, Bad Ischl, Austria Halococcus hamelinensis 2.55 2.13-5.1 - 4.0-9.0 37 - aerobic Shark Bay, Australia (T) Halococcus morrhuae 4 2.5- 7.2 5.5-7.9 35 28-50 aerobic various saline waters (T) Halococcus qingdaonensis 3.06 1.71-? 6 4.0-9.0 38 - aerobic Qingdao, China Halococcus saccharolyticus 4 2.5- 7.2 6.0-8.0 37 - aerobic marine salterns (T) Halococcus salifodinae 3.9 2.13-? 7 6.8-9.5 40 - aerobic salt mine, Austria Halococcus thailandensis 4.2 2.13-? 7.4 6.0-10.0 37 15-45 aerobic fish sauce, Thailand Halobiforma haloterrestris 3.4 2.2-sat. 7.5 6.0-9.2 42 - aerobic salt soil, Aswan, Egypt Halobaculum gomorrense 2 1.0- 5.5 5.5-8.0 40 -50 aerobic Dead Sea Halobacterium jilantaiense 3.3 2.7-5.2 7.3 5.5-8.5 40 22-55 aerobic Jilantai salt lake, China Halobacterium noricense 2.7 2.1- - 5.2-7.0 41 - aerobic salt mine, Altausee, Australia Halobacterium piscisalsi 3.9 2.6-5.1 7.3 5.0-8.0 39 20-60 aerobic Haloarcula amylolytica 3.1 2.0-5.1 7.3 6.5-9.0 41 20-52 aerobic Aibi salt lake, China Haloarcula argentinensis 2.8 2.0-4.5 - - 40 - aerobic saltern soil, Argentina Haloarcula japonica 3.5 2.5-5.0 7.3 6.0-8.0 44 24-45 aerobic saltern soil, Japan Haloarcula marismortui 3.7 1.7-5.1 - - 45 - aerobic Dead Sea Halalkalicocus jeotgali 2.55 1.7-5.1 7 6.5-9.0 41 21-50 aerobic jeotgal Halalkalicocus tibetensis 3.4 1.4-5.2 9.8 8.0-9.5 40 23-47 aerobic Lake Zabuye, Tibet Haladaptatus paucihalophilus 2.9 0.8-5.1 6.3 5.0-7.5 28 25-45 aerobic Zodletone Spring, OK a - indicates data not found bShaded cells indicate data meeting or exceeding one of the following criteria: + [Na ]opt ≥ 3.4 M, pHopt ≥ 8.5, Topt ≥ 50˚C c (T) indicates a known thalassohaline environment d For reasons of brevity, citations for the description of the species can be found in the List of Prokaryotic Names with Standing in Nomenclature, www.bacterio.cict.fr (updated monthly)

CHAPTER TWO

NATRONOLIMNOBIUS AEGYPTIACUS SP. NOV., AN AEROBIC, EXTREMELY

HALOPHILIC ARCHAEON ISOLATED FROM THE ATHALASSOHALINE WADI AN

NATRUN, EGYPT

______Bowers, K.J., Sarmiento B., F., Mesbah, N.M, & Wiegel, J. To be submitted to the International Journal of Systematic and Evolutionary Microbiology.

Abstract

Aerobic haloalkalithermophilic archaeal strains were isolated from the sediments of Wadi

An Natrun, a depression in the Sahara Desert (Egypt) containing seven alkaline, hypersaline lakes. Strains were isolated from four lakes. Cells were rod-shaped and non-motile. Optimal growth occurred at pH55°C 9.25-9.75, with no growth at or above pH 10.9 and at or below pH 7.1; at 54-57°C, with no growth at or above 65°C and at or below 29°C; and at 4.4-4.5 M Na+, with growth at Na+ saturation and no growth at or below 2.6 M Na+. The strains were obligately aerobic and chemoorganotrophic. Strain JW/NM-HA 15T was selected for further characterization. The mol% G+C content of genomic DNA was 60.2. Phylogenetic analysis showed that strain JW/NM-HA 15T and the related strains belonged to the family

Halobacteriaceae within the order Halobacteriales. Based upon genotypic and phenotypic characteristics, it is proposed that the strains represent a novel species, Natronolimnobius aegyptiacus sp. nov., with type strain JW/NM-HA 15T

Introduction

The Wadi An Natrun is a depression in the Sahara Desert which contains seven sun- heated and highly alkaline lakes, which range in salinity from 1.5 to 5 M and pH 9-11 and exhibit temporal and local temperatures of up to 60°C (Mesbah et al. 2007a; Taher 1999). The characteristics of the region make it an ideal place for the isolation of poly-extremophiles, or microorganisms which live optimally at high [Na+], alkaline pH, and elevated temperatures. The microbial communities of the Wadi An Natrun are exposed not only to a combination of high salt, alkaline pH values, and elevated temperature, but to intense solar irradiation and periods of desiccation. Several bacterial microorganisms have previously been isolated from this region, including Halorhodospira halochloris (Imhoff et al. 1977), a haloalkaliphile, Natranaerobius thermophilus (Mesbah et al. 2007b), and Natronovirga wadinatrunensis (Mesbah et al. 2009), both of which are haloalkalithermophiles. To our knowledge, this is the first report of a poly- extremophilic archaeon isolated from the Wadi An Natrun region.

Isolation and cultivation

Mixed water and sediment samples were collected from lakes Hamra, UmRisha, Abu Dawood and Baida of the Wadi An Natrun during May 2005. The pH values and salinity of the water at the time of collection are described in Mesbah et al. (2007). The aerobic, carbonate-buffered medium used for enrichment and initial cultivation contained (l-1): 200 g NaCl, 0.1 g

. MgSO4 7H2O, 1.0 g KCl, 0.3 g KH2PO4, 6.8 g Na2CO3, 3.8 g NaHCO3, 0.5 g yeast extract

(Difco), 0.5 g Bactopeptone (Difco), 0.5 g Casamino acids (Difco), 0.8 g glucose and 0.4 g pyruvate. The pH55°C was adjusted to 9.0 with 5 M HCl. The enrichment cultures became turbid after 60 hours incubation at 55°C in a shaking incubator. Culture vessels were 150 mL serum

bottles containing 50 mL media. Pure cultures were obtained by repeated pour-plating in carbonate-buffered media and 1.5% (w/v) agar. Purity of the cultures was confirmed by 16S rRNA sequencing. The isolates were maintained in carbonate-buffered liquid medium at pH55°C

9.5 and 55°C under aerobic conditions, and subcultured for maintenance after three to four weeks of storage.

Colony and cell morphology

Colonies were observed in agar pour plates, but not on agar streak plates, after 2-3 days of incubation and were 1.0-1.5 mm in diameter, ovoid, and yellow-white in color. Colonies turned pink upon extended stationary phase exposure to light. Cell morphology was observed via light microscopy (Olympus VANOX phase-contrast microscope). Exponentially growing cells in liquid culture of strain JW/NM-HA 15T were straight rods of 0.5-0.75 μM in diameter and 1.5-

2.0 μM in length (Fig. 2.1). Cells were found singly and in chains, particularly upon entry to stationary phase. No active motility was observed using phase-contrast microscopy. Cells stained Gram-negative.

Cultural and physiological characteristics

The optimal conditions for growth of strain JW/NM-HA 15T were tested in carbonate-buffered medium with 0.05% (w/v) each of yeast extract, Bactopeptone and Casamino acids, 64 mM

Na2CO3 and 32 mM NaHCO3 (before pH adjustment). Complex media components are required; equal parts can be substituted (e.g. Bactopeptone for yeast extract), but at the expense of doubling times and culture density. The temperature range for growth at pH55°C 9.0 and 3.4 M

Na+ was determined using a shaking temperature-gradient incubator (Scientific Industries, Inc.).

Strain JW/NM-HA 15T was thermophilic, with a growth temperature optimum of 55°C and a

growth range of 31-59°C. No growth was observed at 29°C and below or at 61°C or above during incubations up to 340h.

The pH range and optimum for the growth of strain JW/NM-HA 15 T was determined at 55°C in the presence of 3.4 M Na+ in carbonate-buffered medium. All pH measurements were performed as described previously (Wiegel, 1998; Mesbah & Wiegel, 2006) with a microelectrode

(Accumet combination microelectrode with calomel reference; Cole-Parmer) calibrated at the optimum growth temperature with pH standards preheated to the same temperature. The pH of the medium was adjusted by the addition of sterile HCl (5 N) or NaOH (3 M). The optimum pH55°C for the growth of strain JW/NM-HA 15T was 9.5, and the range for growth was 7.25-

10.75. No growth was observed at pH55°C 7.1 or below or at pH55°C 10.9 or above.

The salinity range for growth of strain JW/NM-HA 15T was determined in carbonate-buffered

55°C medium at pH 9.5 by adding NaCl. Taking into account the 0.16 M Na2CO3/NaHCO3 present in the medium as a buffer, the total Na+ concentration range for growth of strain JW/NM-

HA 15 T was 2.9 M to saturation (5.5 M). The optimal Na+ concentration is at 4.5 M, or the equivalent of approximately 27% w/v NaCl. No growth occurred at 2.8 M Na+ or below; the upper limit was given by solubility of sodium salts in the alkaline growth medium. The shortest measured doubling time for strain JW/NM-HA 15T at optimal conditions, i.e., 55°C, pH55°C 9.5 and 4.5 M Na+, was 4.2 h.

For substrate-utilization tests, cultures were incubated for up to seven days in the previously described medium prepared without glucose and pyruvate and growth was judged strong if, in the third successive transfer, the OD600 of the culture was twice that of a control culture incubated with 0.05% (w/v) each of yeast extract, Bactopeptone and Casamino acids but no

supplemented test substrate. Growth was judged moderate if the OD600 of the culture was between 1.5-2 times that of the control culture. Strain JW/NM-HA 15T exhibited a moderately broad substrate spectrum (see below). Strain JW/NM-HA 15T was negative for catalase and oxidase activities, gelatin liquefaction and casein degradation. The strain was obligately aerobic and failed to grow in anaerobic media containing nitrate or DMSO.

Biochemical characteristics were determined under aerobic conditions using the API ZYM and

API ZYM 20E systems (bioMérieux). Strain JW/NM-HA 15T yielded positive results for alkaline phosphatase, esterase (C 4), esterase lipase (C 8), lipase (C 14), naphthol-AS-bi- phosphohydrolase and β-glucuronidase, and negative results for valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, α-galactosidase, α- and β-glucosidase,

N-acetyl-β-glucosaminidase, α-fucosidase, citrate utilization, H2S production, tryptophan deaminase, and indole production.

Cells were not viable after heat treatment (5 min at 100°C, 15 min at 85°C). Strain JW/NM-HA

15T, the only tested strain, did not remain viable after 24 hours desiccation when incubated in a desiccator above CaCl2, with a relative humidity of 0.2 g H2O/ g.

For short-term preservation, cultures of the strain were stored at room temperature and transferred every four weeks. For long-term preservation, cells in late exponential growth phase were harvested, resuspended in fresh, carbonate-buffered medium mixed glycerol (40-50% v/v) and stored at -80°C. Viability is more than 12 months.

Phylogenetic analysis

The nearly complete 16S rRNA gene sequence for strain JW/NM-HA 15T (1,279 bp) was determined by Macrogen, Inc. (Seoul, Korea) and compared with all GenBank entries by BLAST

search (http://www.ncbi.nlm.nih.gov/BLAST). Multiple sequence alingnments were created with the CLUSTAL_X Program (http://clustal.org/download/current). Trees were constructed by using the MEGA software package (http://www.megasoftware.net). Each tree was a consensus of 1000 replicate trees. The sequences from the isolated strains were aligned with those of representatives of the family Halobacteriaceae. The nearly complete 16S rRNA gene sequence of the strains, including the type strain JW/NM-HA 15T, were located in a phylogenetic cluster within the order Halobacteriales and genus Natronolimnobius. Strain JW/NM-HA 15T showed a 94% sequence similarity to Natronolimnobius baerhuensisT and a 95% sequence similarity to Natronolimnobius innermongolicus.

Taxonomic conclusions

Strain JW/NM-HA 15T belongs to the order Halobacteriales and is within the confines of the family Halobacteriaceae (Fig. 2.2). Strain JW/NM-HA 15T differs from Natronolimnobius baerhuensis in several traits (Table 2.1). Aside from the 16S rRNA sequence, the main phenotypic differences are in growth conditions and substrate utilization. Strain JW/NM-HA 15T has a significantly higher temperature optimum, slightly higher pH and [Na+] optima for growth, wider temperature and pH ranges, and a longer doubling time. On the basis of phenotypic and phylogenetic data, we propose that strain JW/NM-HA 15T represents the type strain of a novel species, Natronolimnobius aegyptiacus.

Description of Natronolimnobius aegyptiacus sp. nov.

Natronolimnobius aegyptiacus (a.e.gyp.ti.a'cus. L. masc. adj. aegyptiacus, pertaining to Egypt,

Egyptian).

Cells are rods 0.5-0.75 μM in diameter and 1.5-2.0 μM in length, non-motile and catalase- and oxidase-negative. Halophilic: optimal growth occurs at 4.5 M Na+; no growth occurs at Na+ concentrations at or below 2.8 M, and growth occurs up to Na+ saturation. Obligately alkaliphilic: pH55°C range 7.25-10.75, with an optimum at pH55°C 9.5; no growth occurs at pH55°C

7.1 or below or below or at pH55°C 10.9 or above. Thermophilic: temperature range for growth is

31-59°C, with an optimum at 55°C. No growth at or below 29°C or at or above 61°C.

Obligately aerobic. Chemoorganotrophic. In the presence of 0.05% (w/v) each of yeast extract,

Bactopeptone and Casamino acids, crotonate, fructose, galactose, glucose, glucouronic acid, inulin, maltose, mannose, pyruvate, starch and trehalose were used as carbon and energy sources

(0.12% w/v, pH55°C 9.5). Arabinose, cellobiose, ethanol, inositol, lactose, maltose, mannitol, methanol, ribose, sodium formate, sorbitol, sucrose, xylan, xylitol and xylose were not utilized.

Nitrate is reduced to nitrite. Produces arginine dihydrolase and β-galactosidase in the presence of 0.5% (w/v) each of yeast extract, Bactopeptone and Casamino acids, 0.08% (w/v) glucose and

0.04% (w/v) pyruvate; under the same conditions does not utilize citrate or produce urease, indole or acetoin (API ZYM, API ZYM 20E). The DNA G + C content of the genomic DNA is

60.2 % mol% (HPLC; Mesbah et al. 1989).

The type strain JW/NM-HA 15T was isolated from sediment of Lake Fazda, Wadi An Natrun,

Egypt.

Acknowledgments

We would like to thank M. Mesbah for help with sample collection at the Wadi An Natrun and J.

Gilleland for laboratory assistance. This work was supported by AFOSR 033835-01 and NSF

10-21-RR182-359 to J. Wiegel.

References

Imhoff, J.F., Trüper, H.G. (1977). Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch Micro 114:114-121.

Itoh, T., Yamaguchi, T., Zhou, P. & Takashina, T. (2005). Natronolimnobius baerhuensis gen. nov., sp. nov. and Natronolimnobius innermongolicus sp. nov., novel haloalkaliphilic archaea isolated from soda lakes in Inner Mongolia, China. Extremophiles, 9, 111-116.

Mesbah, N. M. & Wiegel, J. (2006). Isolation, cultivation and characterization of alkalithermophiles. In Methods in Microbiology volume 35: Extremophilic Microorganisms, pp. 451-468. Edited by F. A. Rainey and A. Oren, London: Elsevier.

Mesbah, N. M., Abou-El-Ela, S. H. & Wiegel, J. (2007a). Novel and unexpected prokaryotic diversity in water and sediments of the alkaline, hypersaline lakes of the Wadi An Natrun, Egypt. Microb Ecol 54, 598-617.

Mesbah, N. M., Hedrick, D. B., Peacock, A. D., Rohde, M. & Wiegel, J. (2007b). Natranaerobius thermophilus gen. nov. sp. nov., a halophilic, alkalithermophilic bacterium from soda lakes of the Wadi An Natrun, Egypt, and proposal of Natranaerobiaceae fam. nov. and Natranaerobiales ord. nov. Int. J. Syst. Evol. Microbiol. 57, 2507-2512.

Mesbah, N.M., Wiegel, J. (2009). Natronovirga wadinatrunensis gen. nov., sp. nov. and Natranaerobius trueperi sp. nov., two halophilic, alkalithermophilic microorganisms from soda lakes of the Wadi An Natrun, Egypt. Int J Syst Evol Microbiol 59:2042-2048.

Taher, A. G. (1999). Inland saline lakes of Wadi El Natrun depression, Egypt. Int J Salt Lake Res 8, 149-170.

Wiegel, J. (1998). Anaerobic alkalithermophiles, a novel group of extremophiles. Extremophiles 2:257-267.

Figure 2.1 Light microscopic image of strain JW/NM-HA 15T

Bar, 2.0 µm.

Figure 2.2. Phylogenetic relationship of strains to closely related microorganisms

Neighbour-joining tree based on 16S rRNA gene sequences, showing the position of strain

JW/NM-HA 15T to its closest relatives within the family Halobacteriaceae. All members of the genus Natronlolimnobius are shown. Genbank accession numbers for the sequences are shown in parentheses. Numbers at nodes are bootstrap values based on 1000 replicates; only values greater than 50% are shown. Bar, one nucleotide substitution per 100 nt.

Table 2.1. Differential characteristics of strain JW/NM-HA 15T and closely related species

1, JW/NM-HA 15T; 2, Natronolimnobius baerhuensisT; 3. Natronolimnobius innermongolicusT.

+, Positive; –, Negative.

Characteristic 1 2 3 Length of rods (µm) 1.5-2.0 3-6 3-5 Na+ range (optimum) (M) 2.8-sat. (4.5) 2.6-ND (3.4) 1.7-ND (2.6- pH range (optimum) 7.25-10.75 7-10 (9) 7.5-10 (9.5 Temp. range (optimum) 31-59 (55) 30-46 (37) 19-543.4) (38 -43) Utilization of: (9.5) Acetate(°C) – + – Arabinose – + + Fructose + – – Lactose – + – Maltose + + – Mannose + + – Sensitive to (50µg/mL): Anisomycin – + + Erythromycin – + + Data for N. baerhuensisT and N. innermongolicusT from Itoh, et al. 2005

All above species utilized galactose, glucose and pyruvate; all above species were sensitive to bacitracin, novobiocin and rifampicin.

CHAPTER THREE

NATRANAEROBIUS JONESII SP. NOV. AND NATRANEROBIUS GRANTII SP. NOV., TWO

ANAEROBIC HALOALKALITHERMOPHILES ISOLATED FROM THE KENYAN-

TANZANIAN RIFT

______Bowers, K.J., Mesbah, N.M, & Wiegel, J. To be submitted to the International Journal of Systematic and Evolutionary Microbiology.

Abstract

Two novel strains of haloalkalithermophilic bacteria were isolated from the sediments of

Lake Magadi, Kenya, which is an alkaline, saline lake in the Kenya Rift Valley. Strains

JW/NM-KB 43 T and JW/NM-KB 411T were non-motile, Gram-stain positive rods. Spore formation was not observed. Growth of strain JW/NM-KB 43 T occurred at pH55°C 8.5-11.4, with no growth observed at or above pH55°C 11.6 or at or below pH55°C 8.3; 44-71°C, with no growth observed at or above 73°C or at or below 42°C; and 3.1-5.0 M Na+ (2.3 M added NaCl), with no growth at or above 5.3 M Na+ or at or below 2.9 M Na+. Growth of strain JW/NM-KB 411 T occurred at pH55°C 7.8-10.6, with no growth at or above pH55°C 10.8 or at or below pH55°C 7.6;

32-53°C, with no growth at or above 55°C, or at or below 30°C; and 2.6 M-saturation Na+ (2.7

M added NaCl) with no growth at or below 2.6 M Na+. An upper Na+ limit to growth could not be determined due to the solubility of sodium salts in the alkaline growth medium. Optimal growth of strain JW/NM-KB 43T occurred at 60-66°C, pH55°C 10.5 and 3.9 M Na+ (2.3 M added

NaCl). Optimal growth of strain JW/NM-KB 411T, a thermotolerant to moderately thermophilic microorganism, occurred at 46-48°C, pH55°C 9.5 and 4.3 M Na+ (2.3 M added NaCl). Both strains were obligately anaerobic and chemoorganotrophic, and produced formate and acetate as major fermentation products from sucrose. The G+C content of genomic DNA was 41.5 and

40.9 mol%, respectively. Phylogenetic analysis showed that both strains belonged to the family

Natranaerobiaceae within the order Natranaerobiales of the phylum Firmicutes. Based upon genotypic and phenotypic characteristics, it is proposed that strain JW/NM-KB 43T represents the type strain of a novel species, Natranaerobius jonesii sp. nov., and that strain JW/NM-KB

411T represents the type strain of a novel species, Natranaerobius grantii sp. nov.

The Rift Valley is a volcanically active region that contains six highly alkaline lakes which range in salinity from 1 to 5 M (Grant, 1999) and pH 8.5-11.5 and above (Rees et al.,

2003). Lake Magadi is the southernmost lake in the Kenya Rift Valley, and is characterized by large deposits of trona, or sesquicarbonate of soda (Na2CO3/NaHCO3) (Owenson 1997). The characteristics of the lake make it an ideal place for the isolation of extremophiles; a previously published halophilic microorganism, Halonatronum saccharophilum, was also isolated from this lake (Zhilina et al. 2001).

Recently, Mesbah et al. described several halophilic, alkalithermophilic microorganisms from the athalassohaline lakes of the Wadi An Natrun, Egypt (2007; 2009). These microorganisms display a unique combination of adaptations to multiple extreme conditions in that they grow optimally above 50°C, pH55°C 9.5 as well as above 3.4 M Na+. These microorganisms, Natranaerobius thermophilus, Natranerobius truperi and Natronovirga wadinatrunensis are members of the order Natranaerobiales and represent the only currently identified bacterial poly-extremophiles (Bowers et al. 2009). On the basis of the physiological and phylogenetic evidence presented here, we propose that strains JW/NM-KB 43T and JW/NM-

KB 411T are novel members of the genus Natranaerobius from this order. Strain JW/NM-KB

43T also represents a novel poly-extremophile and is adapted to combinations of multiple extremes.

Isolation and cultivation of strains JW/NM-KB 43T and JW/NM-KB 411T

Mixed water and sediment samples were collected from Lake Magadi, in Rift Valley, Kenya.

Strain JW/NM-KB 43 T and strain JW/NM-KB 411 T were isolated from core samples taken from beneath the trona bed of the lake. The temperature of the cores at the time of sampling was

30°C, and the included water had a pH of 11 (Owenson 1997). Strain JW/NM-KB 43T was isolated directly from enrichment samples provided by Brian Jones, whereas Strain JW/NM-KB

411T was re-isolated from a co-culture provided by William Grant. The anaerobic, carbonate-

-1 buffered medium used for enrichment and initial cultivation contained (l ): 0.2 g KH2PO4, 0.1 g

MgCl2, 0.5 g NH4Cl, 0.2 g KCl, 100 g NaCl, 68 g Na2CO3, 38 g NaHCO3, 0.7 g cysteine.HCl, 5 g yeast extract, 5 g tryptone, 5 g glucose, 1 ml of trace element solution (Kevbrin & Zavarzin

1992) and 10 mL of vitamin solution (Wolin et al. 1963). The pH55°C was adjusted to 9.5 with anaerobic 5 M HCl. The enrichment cultures became turbid after 48 hours incubation at 55°C.

Pure cultures were obtained in repeated dilution in anaerobic carbonate-buffered media

(Ljungdahl & Wiegel 1986). To ensure that the cultures were axenic, isolates were purified by four successive rounds of 1:10 dilution rows to extinction. The isolates were maintained in the

55C carbonate-buffered medium at pH 9.5 and 55°C under anaerobic conditions (100% N2) by using a modified Hungate technique (Ljungdahl & Wiegel, 1986). The isolates were also judged to be axenic based on the cleanness of the 16S rRNA sequencing results.

Cell morphology

Colonies were not formed on agar plates or in agar shake-roll tubes. Cell morphology was observed via light microscopy (Olympus VANOX phase-contrast microscope). Exponentially growing cells in liquid culture of strain JW/NM-KB 43T were long, straight to curved rods, 0.25-

0.5 µm in diameter and 2-3 µm in lenth (Fig. 3.1a). Exponentially growing cells in liquid culture of strain JW/NM-KB 411T were long, straight rods, 0.25-0.5 µm in diameter and 1-2 µm in

length (Fig. 3.1b). Cells of both strains were found singly and in chains and stained Gram- positive in all growth phases (Doetsch 1981). Motility was not observed in either strain using phase-contrast microscopy.

Endospores were not observed via light microscopy after heat treatment (5 min at 100°C or 15 min at 85°C). Cultures of both strains failed to retain viability after heat treatment (5 min at 100°C, 15 min at 85°C). Cultures of neither strain remained viable after 24 hours desiccation

(incubated above CaCl2). Cells of both strains stained Gram-positive both in the early exponential and the stationary growth phases (Doetsch 1981).

Cultural and physiological characteristics

The optimal conditions for growth of strains JW/NM-KB T 43 and JW/NM-KB 411 T were tested in carbonate-buffered medium with 0.2% (w/v) yeast extract and tryptone, 640 mM Na2CO3 and

+ 320 mM NaHCO3 (before pH adjustment, yielding a base Na concentration of 1.6 M). The temperature ranges for growth were determined using a temperature-gradient incubator

(Scientific Industries, Inc.), the temperature range for growth (at pH55°C 9.5). Strain JW/NM-KB

43T was thermophilic, with a growth temperature optimum between 60 and 66°C and a growth range of 44-71°C. No growth was observed at 42°C and below or at 73°C or above. Strain

JW/NM-KB 411T was thermotolerant, with a growth temperature optimum between 46 and

48°C, and a growth range of 32-53°C. No growth was observed at 30°C or below or at 55°C or above.

The pH ranges and optima for the growth of strains JW/NM-KB 43 T and JW/NM-KB

411T were determined at 65 and 48°C, respectively, in carbonate-buffered medium. All pH measurements were performed as described previously (Wiegel, 1998; Mesbah & Wiegel, 2006),

with a microelectrode (Accumet combination microelectrode with calomel reference; Cole-

Parmer) calibrated at the optimum growth temperature with pH standards preheated to the same temperature. The pH of the medium was adjusted by the addition of sterile, anaerobic HCl (5 N)

65°C T or Na2CO3 (3 M). The optimum pH for the growth of strain JW/NM-KB 43 was 10.5, and the range for growth was 8.5-11.4. No growth was observed at pH65°C 8.3 or below, or at pH65°C

11.6 or above. Strain JW/NM-KB 411T grew optimally at pH48°C 9.5, and between pH48°C 7.8 and 10.6. No growth was observed at pH48°C 7.6 or below or at pH48°C 10.8 or above.

The salinity range for growth of strain JW/NM-KB 43T was determined in carbonate-buffered

65°C medium at pH 10.5 by adding NaCl. Taking into account the 1.6 M Na2CO3/NaHCO3 present in the medium as a buffer, the total Na+ concentration range for growth of strain JW/NM-

KB 43 T was 3.1-5.0 (with no growth at or below 2.9 M or at or above 5.3 M Na+) and at an optimum of 3.9 M, or the equivalent of approximately 23% NaCl (w/v). Strain JW/NM-KB

411T grew over total Na+ ion concentration range of 2.9 M to saturation (approximately 5.5 M), with an optimum at 4.3 M, or the equivalent of approximately 25% NaCl (w/v). No growth occurred at 2.6 M Na+ or below; the upper limit to growth exceeded the solubility of sodium salts in the alkaline growth medium. The shortest measured doubling time for strain JW/NM-KB

411T at optimal conditions, i.e., 48°C, pH48 °C 10.5 and 4.3 M Na+, was 4.0 h. The doubling time for strain JW/NM-KB 43T at optimal conditions, i.e., 63°C, pH65°C 10.5 and 3.9 M Na+, was

4.5 h.

For substrate-utilization tests, cultures were incubated for up to five days and growth was judged strong if, in the third successive transfer, the OD600 of the culture was twice that of a control culture incubated with only 0.2% (w/v) yeast extract and tryptone. Growth was judged

moderate if the OD600 of the culture was between 1.5 and two times that of the control. Both strains exhibited a moderately wide substrate spectrum (Table 3.1). The substrate utilization patterns are presented in the species descriptions below. Both strain JW/NM-KB 43T and strain

JW/NM-KB 411T were negative for catalase and oxidase activities, gelatin liquefaction and casein degradation. Both strains were obligately anaerobic, since no growth was observed in the presence of air or 0.02% (v/v) oxygen.

Use of electron acceptors was determined by measuring growth (increase in OD600, for all acceptors) as well as production of sulfide, ammonium and nitrate, and colour change. In the presence of 0.2% (w/v) each yeast extract and tryptone, strain JW/NM-KB 43T utilized the

2- 2- - following as electron acceptors: S2O3 (20 mM), SO4 (20 mM), NO3 (20 mM). None of the

2- following electron acceptors were utilized: SO3 (20 mM), MnO2 (10 mM), iron (III) citrate

[20 mM, determined by A562 of Fe(II)-ferrozine complex (Stookey, 1970)]. Under the same

T 2- - conditions, JW/NM-KB 411 utilized S2O3 (20 mM), NO3 (20 mM), and MnO2 (10 mM) as

2- electron acceptors, and did not utilize any of the following electron acceptors: SO4 (20 mM),

2- SO3 (20 mM), iron (III) citrate (20 mM). The main organic fermentation products from 20 mM sucrose for both strains were acetate and formate. No fermentation products could be detected in the presence of 0.5% (w/v) yeast extract and 0.5% (w/v) tryptone alone, suggesting that they cannot serve as sole carbon and energy sources, but serve as a source of growth factors.

Biochemical characteristics were determined under anaerobic conditions using the API ZYM and

API ZYM 20E systems (bioMérieux). Both strains yielded positive results for arginine dihydrolase and β-galactosidase and negative results for valine arylamidase, cystine arylamidase,

trypsin, α-chymotrypsin, acid phosphatase, α-galactosidase, α- and β-glucosidase, N-acetyl-β- glucosaminidase, α-fucosidase, citrate utilization, H2S production, tryptophan deaminase, and indole production.

For short-term preservation, cultures of both strains were stored at room temperature and transferred every three weeks. For long-term preservations, cells in late exponential growth phase were harvested anaerobically, resuspended in fresh, pre-reduced medium mixed with anaerobic glycerol (1:1 ratio) and stored at -80°C. To avoid prolonged lag phases and loss of viability, strict anaerobic techniques had to be employed for both strains.

Chemotaxonomic characteristics

Attempts to purify peptidoglycan from cells of strains JW/NM-KB 43T and JW/NM-KB 411T were unsuccessful, and no isomer of diaminopimelic acid was detected in either strain. It was concluded that the amount of peptidoglycan in the strains are below detectable amounts (Peter

Schumann, personal communication).

The DNA G+C content of strains JW/NM-KB 43T and JW/NM-KB 411T were determined by HPLC as described by Mesbah et al. (1989) with the modifications of Lee et al.

(2005), using S1 nuclease and 0.3 M sodium acetate (pH 5.0). The G+C content of genomic

DNA of strain JW/NM-KB 43T was 41.5 mol%; the G+C content of genomic DNA of strain

JW/NM-KB 411T was 40.9 mol% (mean of five replicate analyses).

Phylogenetic analysis

The nearly complete 16S rRNA gene sequences for strains JW/NM-KB 43T and JW/NM-KB

411T (1389 and 1405 bp, respectively) were determined by Macrogen, Inc. (Seoul, Korea), and compared with all GenBank entries by BLAST search (http://www.ncbi.nlm.nih.gov/BLAST).

Multiple sequence alingnments were created with the CLUSTAL_X Program

(http://clustal.org/download/current). Trees were constructed by using the MEGA software packates (http://www.megasoftware.net). Distances were calculated by using the Jukes-Cantor algorithm and branching order was determined via the neighbor-joining algorithm. Each tree was a consensus of 1000 replicate trees. The sequences from the isolated strains were aligned with those of representatives of the order Natranaerobiales. The nearly complete 16S rRNA gene sequence of strains JW/NM-KB 43T and JW/NM-KB 411T were located in a phylogenetic cluster within the order Natranaerobiales and genus Natranaerobius. Strain JW/NM-KB 43T and strain JW/NM-KB 411T each showed a 96% sequence similarity to Natranaerobius thermophilus, a 94% sequence similarity to Natronovirga wadinatrunensis and a 95% sequence similarity to each other indicating that they constitute two different species of the genus

Natranaerobius.

Taxonomic conclusions

Phylogenetically, strains JW/NM-KB 43T and JW/NM-KB 411T belong to the order

Natranaerobiales and are within the confines of the family Natranaerobiaceae (Fig. 3.2). Strain

JW/NM-KB 43T differs from Natranaerobius thermophilus in several traits (Table 3.2). Beside the differences in the 16S rRNA sequence, the main phenotypic differences are in growth conditions and substrate utilization. Strain JW/NM-KB 43T has higher temperature optimum by more than 10°C, a more alkaline pH optimum and a higher [Na+] optimum for growth, as well as wider temperature and pH ranges, and a longer doubling time. On the basis of phenotypic and phylogenetic data, we propose that strain JW/NM-KB 43T represents the type strain of a novel species, Natranaerobius jonesii.

Beside the differences in the 16S rRNA sequence, strain JW/NM-KB 411T could be distinguished from both Natranaerobius thermophilus and strain JW/NM-KB 43T by having a significantly lower temperature optimum (about 10 and 20°C, respectively) and range, and a higher [Na+] optimum, and the ability to grow at saturated NaCl concentrations. Additonally, strain JW/NM-KB 411T has a slightly different cellular morphology and a doubling time longer than that of Natranaerobius thermophilus and shorter than that of strain JW/NM-KB 43T (Table

3.2). Based on the 16S rRNA sequence and physiological properties, we propose that strain

JW/NM-KB 411T represents the type strain of a novel species, Natranaerobius grantii.

Description of Natranaerobius jonesii sp. nov.

Natranaerobius jonesii (jones´i.i. N.L. gen masc. n. jonesii of Jones, referring to Brian Jones, in honor of his contributions to the field of the diversity and the physiology of extremophiles).

Cells are narrow rods (0.25-5 µm by 2-3 µm), non-motile and catalase- and oxidase-negative.

Cells stain Gram positive at all growth phases. Formation of endospores was not observed.

Halophilic: optimal growth occurs at 3.9 M Na+ (1.6 M from sodium carbonates, 2.3 M NaCl); no growth occurs at Na+ concentrations at or below 2.9 M or at or above 5.3 M. Obligately alkaliphilic: pH65°C range 8.5-11.4, with an optimum at pH65°C 10.5; no growth occurs at pH65°C

8.3 or below or below or at pH65°C 11.6 or above. Thermophilic: temperature range for growth is

26-71°C, with an optimum at 66°C. No growth at or below 42°C or at or above 73°C.

Obligately anaerobic. Chemoorganotrophic. In the presence of 0.2% (w/v) yeast extract and tryptone, glucose, mannose, sucrose, ribose, glucouronic acid, pyruvate, and casamino acids

were used as carbon and energy sources (0.5% w/v, pH65°C 9.5). Arabinose, cellobiose, trahalose, lactose, starch, mannitol, sorbiotl, xyitol, ethanol (0.5% w/v) were not utilized.

2- 2- Organic fermentation products from sucrose are acetate and formate. S2O3 , SO4 and nitrate

2- were utilized as electron acceptors. SO3 , MnO2, iron (III) citrate were not reduced. Produces arginine dihydrolase and β-galactosidase in the presence of 0.2% yeast extract/tryptone and 0.5% glucose. Does not utilize citrate or produce urease, indole or acetoin. The DNA G + C content of the genomic DNA is 41.5% mol% (HPLC).

The type strain JW/NM-KB 43T was isolated from sediment of Lake Magadi, Rift Valley,

Kenya.

Description of Natranaerobius grantii sp. nov.

Natranaerobius grantii (grant´i.i. N.L. gen masc. n. grantii of Grant, referring to William Grant, in honor of his contributions to the fields of alkaliphiles and of halophiles).

Cells are narrow rods (0.25-0.5 µm by 1-2 µm), non-motile and catalase- and oxidase-negative.

Cells stain Gram-positive. Formation of endospores were not observed. Extremely halophilic: optimal growth occurs at 4.3 M Na+ (1.6 M from sodium carbonates, 2.7 M NaCl); no growth occurs at Na+ concentrations at or below 2.6 M and grows in the presence of NaCl saturation at pH55°C 9.5 and 48°C. Obligately alkaliphilic: pH45°C range 7.8-10.6, with an optimum at pH45°C

9.5; no growth occurs at pH45°C 7.6 or below or below or at pH45°C 10.8 or above.

Thermotolerant: temperature range for growth is 32-53°C, with an optimum at 48°C. No growth

at or below 30°C or at or above 55°C. Obligately anaerobic. Chemoorganotrophic. In the presence of 0.2% (w/v) yeast extract and tryptone, glucose, fructose, arabinose, trehalose, sucrose, lactose, pyruvate and casamino acids were used as carbon and energy sources (0.5% w/v, pH65°C 9.5). Mannose, cellobiose, starch, glucouronic acid, sorbitol, xylitol and ethanol

(0.5% w/v) were not utilized. Organic fermentation products from sucrose are acetate and

2- 2- 2- formate. S2O3 , MnO2 and nitrate were utilized as electron acceptors. SO4 , SO3 , iron (III) citrate were not reduced. Produces arginine dihydrolase and β-galactosidase in the presence of

0.2% yeast extract/tryptone and 0.5% glucose. Does not utilize citrate or produce urease, indole or acetoin. The DNA G + C content of the genomic DNA is 40.9% mol% (HPLC).

The type strain JW/NM-KB 411T was isolated from sediment of Lake Magadi, Rift Valley,

Kenya.

Acknowledgements

We would like to thank B. Jones and W. Grant for enrichment cultures, and J. Gilleland and L.

Varghese for laboratory assistance. This work was supported by AFOSR 033835-01 and NSF

10-21-RR182-359 to J. Wiegel.

References

Doetsch, R.N. (1981). Determinative methods of light microscopy. In Manual of Methods for General Bacteriology, pp. 21-33. Edited by P. Gerhardt, R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg & G.B. Philips, Washington, DC: American Society for Microbiology.

Grant, S., Grant, W. D., Jones, B. E., Kato, C. & Li, L. (1999). Novel archaeal phylotypes from an East African alkaline saltern. Extremophiles 3, 139-145.

Kevbrin, V. V. & Zavarzin, G. A. (1992). The effect of sulfur compounds on the growth of the halophilic homoacetic bacterium Acetohalobium arabaticum. Microbiologia 61, 812-817.

Lee, Y. J., Wagner, I. D., Brice, M. E., Kevbrin, V. V., Mills, G. L., Romanek, C. S. & Wiegel, J. (2005). Thermosediminibacter oceani gen. nov. sp. nov. and Thermosediminibacter litoriperuensis sp. nov., new anaerobic thermophilic bacteria isolated from Peru margin. Extremophiles 9, 375-383.

Ljungdahl, L. & Wiegel, J. (1986). Working with anaerobic bacteria. In Manual of Industrial Microbiology and Biotechnology, pp. 84-96. Edited by A. L. Demain and N. A. Solomon, Washington D.C.: American Society for Microbiology.

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159-167.

Mesbah, N.M., & Wiegel, J. (2009). Natronovirga wadinatrunensis gen. nov., sp. nov. and Natranaerobius trueperi sp. nov., two halophilic, alkalithermophilic microorganisms from soda lakes of the Wadi An Natrun, Egypt. Int J Syst Evol Microbiol 59:2042-2048.

Owenson, G. G. (1997). Obligately anaerobic alkaliphiles from Kenya soda lake sediments. Ph.D thesis, University of Leicester, UK

Rees, H.C., Grant, W.D., Jones, B.E.,& Heaphy, S. (2004). Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods. Extremophiles 8:63-71.

Stookey, L. L. (1970). Ferrozine-A new spectrophotometric method for iron. Anal Chem 42, 779-781. Wiegel, J. (1998a). Anaerobic alkalithermophiles, a novel group of extremophiles. Extremophiles 2, 257-267.

Wolin, E. A., Wolin, M. J. & Wolfe, & R. S. (1963). Formation of methane by bacterial extracts. J Biol Chem 238, 2882-2886.

Zhilina, T.N., Zavarzin, G.A., Detkova, E.N., & Rainey, F.A. (1996). Natroniella acetigena gen. nov. sp. nov., an extremely haloalkaliphilic, homoacetic bacterium: a new member of Haloanaerobiales. Curr Microbiol 32:320-326.

Figure 3.1. Light microscopic images of strains JW/NM-KB 43T and JW/NM-KB 411T

Figure 3.1a. Strain JW/NM-KB 43T

Bar, 2.0 µm.

Figure 3.1b. Strain JW/NM-KB 411T

Bar, 2.0 µm.

Figure 3.2. Phylogenetic relationship of strains JW/NM-KB 43T and JW/NM-KB 411T to closely related microorganisms

Neighbour-joining tree based on 16S rRNA gene sequences, showing the position of strains

JW/NM-KB 43T and JW/NM-KB 411T to their closest relatives within the order

Natranaerobiales. All members of the order are shown. The tree was rooted with

Halanaerobium praevalens DSM 2228 as the outgroup. Genbank accession numbers for the sequences are shown in parentheses. Numbers at nodes are bootstrap values based on 1000 replicates; only values greater than 50% are shown. Bar, two nucleotide substitutions per 100 nt.

Table 3.1. Substrate utilization profile of strains JW/NM-KB 43T and JW/NM-KB 411T.

Substrate JW/NM-KB 43T JW/NM-KB 411T Arabinose – + Casamino acids + + Cellobiose – – Ethanol – – Fructose + + Glucose + + Glucouronic acid + – Lactose + + Mannitol – – Mannose + – Methanol – – Pyruvate + + Ribose + + Sorbitol – – Starch – – Sucrose + + Trehalose + + Xyitol – – Xylose – – +, Positive; –, Negative.

Table 3.2. Differential characteristics of strain JW/NM-KB 43T, JW/NM-KB 411T and closely related strains

Strains: 1, JW/NM-KB 43T; 2, JW/NM-KB 411T; 3, Natranaerobius thermophilusT; 4,

Natranaerobius truperi; 5, Natronovirga wadinatrunensis. +, Positive; –, Negative.

Characteristic 1 2 3 4 5 Length of rods (µm) 5-7 2-4 3-5 2-3 4-5 Na+ range (optimum) (M) 3.1-5.0 (3.9) 2.9-sat. 3.1-4.8 3.1-5.4 3.3-5.3 pH55°C range (optimum) 8.5-11.4 7.8-10.6 8.3-10.6 8.0-10.8 8.5-11.5 Temp. range (optimum) 44-71 (63) 32-(4.3)53 (47) 35-(3.6)56 (53) 26-(3.8)55 (52) 26-(3.9)56 (51) Doubling time (h) (10.5)4.5 (9.5)4.0 (9.5)3.5 (9.5)3.0 (9.9)2.0 Utilization(°C) of: Cellobiose - - + + - Fructose + + + - + Glucose + + - + - Mannose + - - - + Pyruvate + + + - + Ribose + + + + - Trehalose + + + - + API ZYM tests: Arginine dihydrolase + + - + - Citrate utilization - - - - + β-galactosidase + + - + - Ornithine - - - - + DNA G+C content 41.5 40.9 40.4 41.0 41.7 decarboxylase (mol%) Data for N. thermophilusT from Mesbah et al 2007, data for N. truperiT and N. wadinatrunensisT from Mesbah et al 2009.

CHAPTER FOUR

ADAPTIVE MECHANISMS AND INTRACELLULAR PH REGULATION IN

NATRANAEROBIALES SPECIES, ANAEROBIC HALOALKALITHERMOPHILIC

BACTERIA

______Bowers, K.J., Blamey, J. & Wiegel, J. To be submitted to Extremophiles.

Abstract

Natranaerobius jonesii and Natranaerobius grantii are thermophilic and thermotolerant microorganisms, respectively, and are both obligately halophilic and obligately alkaliphilic. This work sought to discover some of the adaptive mechanisms used by these microoroganisms in order to enable them not only to survive but to thrive under these multiple extreme conditions.

Growth of both N. jonesii and N. grantii were inhibited by the protonophore carbonyl cynanide m-chlorophenylhyrazone and the ionophores monensin, valinomycin and nigericin, suggesting that both proton and sodium motive forces exist across the membrane. Measurements of intracellular Na+ and K+ found total concentrations of less than 300 mM, which are not great enough to osmotically balance the cellular environments. 1H nuclear magnetic resonance data revealed the presence of several common compatible solutes, including glycine betaine and β- glutamate. When the extracellular pH55°C was increased, both N. jonesii and N. grantii maintained a pH gradient of approximately one unit across the cell membrane (alkaline out).

This corresponded with increases in both membrane potential and proton motive force. This is similar to results obtained in previous experiment with the type species of the genus,

Natranaerobius thermophilus. Additionally, N. jonesii, N. grantii and N. thermophilus all displayed a requirement for chloride in their growth medium. No Natranaerobius species displayed either desiccation or γ-radiation resistance. However, when tested, both N. thermophilus and N. wadinatrunensis displayed high levels of UV-radiation resistance.

Introduction

The species of the genus Natranaerobius are non-spore forming anaerobic, haloalkalithermophilic or thermotolerant bacteria which, depending upon the species, grow optimally at 48-60°C, pH55°C 9.5-10.5 (Wiegel 1998) and Na+ concentrations of 3.3-4.3 M

(Mesbah et al. 2007; 2009, Bowers et al. 2008). These species were isolated from solar or geothermally heated althalassohaline soda lakes, and are the only group of haloalkalithermophilic bacteria isolated this far (Bowers et al. 2009). Other bacterial microorganisms of note are Natronovirga wadinatrunensis, a closely related species

(Natranaerobiaceae) which is also haloalkalithermophilic (Mesbah et al. 2009a), and

Halorhodospira halochloris, which is a haloalkalithermotolerant bacterium (Imhoff et al. 1977).

The number of haloalkalithermophilic Archaea is similarly small; only two species, the aerobic

Natronolimnobius aegyptiacus and Natrialba hulunbeirensis (Bowers et al. 2009a, Xu et al.

2001), have currently been identified as haloalkalithermophiles. All of the currently validly described haloalkalithermophilic or thermotolerant Bacteria are anaerobic, as well as halophilic, alkaliphilic and thermophilic, mean that their growth requirements are extremely complex. All the Natranaerobius species are chemoorganotrophic, and so mainly generate energy by substrate level phosphorylation.

The electrochemical ion gradient across the cytoplasmic membrane of alkaliphiles is usually generated by a H+- or Na+ coupled ATPase (Dimroth & Cook, 2004). The anaerobic alkalithermophiles studied to date have been shown to use Na+ as a coupling ion for energy transducing processes (Ferguson et al., 2006, Prowe et al., 1996). However, thermophiles, including the Natranaerobius species, are faced with the challenge of passive permeation of both

H+ and, although to a lesser degree, Na+ through its cytoplasmic membrane. Membrane

permeability to both H+ and Na+ increases with temperature (Vossenberg et al., 1995), which poses a problem for alkaliphiles which must acidify their cytoplasm. Other alkalithermophiles account for increased membrane permeability to protons by modifying the stoichiometry of their

ATP synthase, maintaining a large inward sodium motive force, and by coupling transport to Na+ ions, and using Na+ as a coupling ion for energy transducing processes (Olsson et al. 2003,

Peddie et al. 1999, Ferguson et al. 2006), which is advantageous because membranes are less permeable to Na+ than to H+ (Konings et al. 2002). However, because Na+ permeability increases as NaCl concentration increases (Vossenberg et al. 1999), this presents a difficulty for organisms which are also halophilic.

This then presents interesting problems for organisms which live not only at one of these extreme conditions, but at multiple extreme conditions. Mesbah et al. (2009) showed that

Natranaerobius thermophilus has the ability to grow and regulate its intracellular pH at external pH values ranging from 8.0 to 10.5, exhibiting a constant ΔpH of approximately one pH unit. In this work, we show that two other related microorganisms, the extremely thermophilic

Natranaerobius jonesii and the thermotolerant Natranaerobius grantii, exhibit the same unusual property of maintenance of a constant ΔpH over their pH range for growth. Furthermore, the extent of other mechanisms for adapting to environmental extremes is discussed along with an observed requirement for chloride for growth of Natranaerobius species.

Materials and methods

Abbreviations, chemicals and radiochemicals.

Δp, proton motive force; ΔpNa+, sodium motive force; Δψ, membrane potential; ΔpH, pH gradient; ZΔpH, transmembrane proton gradient; CCCP, carbonyl cyanide m-

chlorophenylhydrazone. CCCP, monensin, nigericin and valinomycin were obtained from

Sigma-Aldrich. The radiochemicals were obtained from: [14C]methyltriphenylphosphonium

(TPP+), and [3H]water, American Radiolabeled Chemicals, Inc.; [3H]methylamine hydrochloride,

MP Biomedical, [3H]polyethylene glycol, PerkinElmer.

Media and culture conditions

Natranaerobius jonesii strain JW/NM-KB 43 and Natranaerobius grantii strain JW/NM-KB 411 were routinely grown anaerobically under a nitrogen gas phase in carbonate buffered medium as previously described but with 0.5% (wt/vol) sucrose (in lieu of glucose) as a carbon source

(Chapter 3). The pH of the medium was adjusted to the pH value being studied at 55ºC, as indicated by the superscript, using sterile, anaerobic 5 N HCl. Unless otherwise stated, pH values of all media and buffers were measured at 55ºC, as previously described (Wiegel 1998). Cellular protein was determined by the Biuret protein assay using bovine serum albumin as standard

(Gornall et al. 1949). The relationship between optical density and cellular protein was 320 mg of protein • liter-1 • 1 OD unit-1.

Determination of bioenergetic parameters.

For determination of ΔpH, Δψ and Δp, Natranaerobius jonesii and Natranaerobius grantii were grown in batch culture at 60ºC, 3.9 M Na+ and 48°C, 4.3 M Na+ respectively and at the pH55°C value being studied. Cells were harvested during mid-exponential phase by centrifugation (6000 x g, 30 min, 24 ºC) and washed three times in sterile anaerobic carbonate-buffered medium, pH55°C 9.5. All washing steps were carried out anaerobically inside a Coy anaerobic chamber

(Coy Laboratory Products Inc, Grass Lake, Michigan). Cells were resuspended to a final OD600 of 1.0 in anaerobic carbonate-buffered medium adjusted to the pH being studied. Cell suspensions were energized and incubated as described by Mesbah et al. (2009) and Cook et al.

(1996) with one of the following isotopes: [14C]methylamine (5.4 μM) or [3H]TPP+ (1 μM).

Supernatant and cell pellets were recovered as previously described by Mesbah et al. (2009) and

Cook et al. (1996); the supernatant and cell pellets were dissolved in scintillation fluid, and radioactivity (cpm) was determined with a Beckman LS 6500 Multi-purpose Scintillation

Counter.

Intracellular volume (5.8 ± 0.42 μL/ mg protein for N. jonesii and 5.7 ± 0.33 42 μL/ mg protein for N. grantii) was determined from the difference in partitioning of [3H]water (1 mM) and [14C]polyethylene glycol. Polyethylene glycol is not metabolized by N. jonesii or N. grantii

(data not shown). The Δψ across the membrane was calculated from the uptake of [3H]TPP+ according to the Nernst relationship. Non-specific TPP+ binding was estimated from valinomycin- and nigericin-treated cells (10 μM each).

Measurement of intracellular sodium and potassium ion concentrations.

Cells were harvested from pH55°C-controlled batch culture, and resuspended in isotonic anaerobic

+ medium adjusted to the pH, temperature, or Na value being studied to an OD600 of 1.0. This cell suspension was then centrifuged at 13,000 x g for 5 min at 24ºC. Five hundred microliters of supernatant were removed, and cell pellets were digested with 3N HNO3 for 24 hours at room temperature. Sodium and potassium concentrations in cell digests and supernatants were analyzed by inductively coupled plasma-optical emission spectrometry. Corrections were made for extracellular contamination of the cell pellet by Na+ and K+. The sodium motive force,

+ + + ΔpNa , was calculated from the equation 59 x log ([Na ]in/[Na ]out), where in and out refer to the concentration of Na+ inside and outside the cell, respectively.

Determination of Cl- requirement

Cells were grown in batch culture at the respective optimum conditions; NaCl was replaced with

- a combination of Na2CO3 and NaHCO3. Cl was added to the growth medium as NaCl, and 3M

+ Na2CO3 was used to achieve the necessary concentration of Na . The growth medium was

55°C adjusted to optimum pH with H2SO4. Cultures were incubated up to seven days and growth was assessed after the third successive transfer, in order to minimize the effect of Cl- transferred with the original inoculum. A parallel control was conducted in medium which contained no

- + added Cl and utilized a combination of NaCO3 and NaHCO3 to achieve the optimum Na concentration.

Investigation of desiccation resistance

To investigate resistance to desiccation, cells were grown in batch culture at optimum conditions; exponentially growing cells were harvested anaerobically by slow centrifugation (5,000 x g, 3 mins., 24°C). Decanting of supernatant was performed inside a Coy anaerobic chamber (Coy

Laboratory Products Inc, Grass Lake, Michigan), and pellets were dried anaerobically over

CaCl2 for 12, 24, 72, 120, and 168 h. After the desired desiccation period, pellets were rehydrated with sterile, anaerobic, carbonate-buffered growth medium and incubated at optimum conditions for up to fourteen days. Growth was judged positive or negative in comparison to an undesiccated control from the same batch of harvested cells.

Investigation of γ-radiation resistance

In order to determine survival of exposure to ionizing radiation, cells were grown in liquid medium under optimum conditions, harvested anaerobically using a Coy anaerobic chamber

(Coy Laboratory Products Inc, Grass Lake, Michigan) and the modified Hungate technique

(Ljungdahl and Wiegel, 1986) via centrifugation and resuspended in fresh carbonate–buffered

medium. 5 mL aliquots of cells in stationary phase were exposed at room temperature to a 60Co source. After exposure to 1, 3, 6 and 9 kGy in triplicate, cultures were inoculated in duplicate into fresh carbonate-buffered medium with 0.5% each yeast extract, tryptone, and appropriate carbon source, and dilution rows of ten 1:10 dilutions were performed. After fourteen days incubation, growth was gauged positive or negative in comparison to non-irradiated cultures from the same batch of harvested cells.

Investigation of UV radiation resistance

Resistance to UV radiation was tested using UVA (385 nm), UVB (312 nM) and UVC (254 nm) resulting in irradiance levels of 0.35 mW cm-2 (UVA) 0.38 mW cm-2 (UVB) and 0.645 mW cm-2

(UVC). 2 mL aliquots of culture were irradiated anaerobically for 0-34 h. Escherichia coli was used as a control culture. At the end of each irradiation interval, 0.1 mL of each cell suspension was removed and stained using LIVE/DEAD BacLight (Invitrogen). Stained cultures were viewed and survival percentage was calculated using fluorescence microscopy.

Results and Discussion

Effect of external pH and metabolic inhibitors on the growth of Natranaerobius jonesii and

Natranaerobius grantii in batch culture.

Natranaerobius jonesii is able to grow in the pH55°C range of 8.5 – 11.4, with no growth at pH55°C 8.3 or below or at pH55°C 11.6 or above (Figure 1a) (Bowers et al. 2008, Chapter 3) The main organic fermentation products from sucrose at pH55°C 10.5, 60ºC and 3.9 M Na+ were acetate and formate. Natranaerobius grantii is able to grow in the pH55°C range of 7.8 – 10.6, with no growth at pH55°C 7.6 or below or at pH55°C 10.8 or above (Figure 1a) (Bowers et al. 2008,

Chapter 3). The main organic fermentation products from sucrose at pH55°C 10.5, 60ºC and 3.9

M Na+ were acetate and formate, and small amounts of lactate. These products indicate fermentation via the Embden-Meyerhof pathway in both microorganisms. The addition of CCCP

(50 μM), monensin (30 μM), valinomycin (10 μM) and nigericin (10 μM) to exponentially growing cells of each species completely arrested growth, indicating that proton, sodium and potassium ion gradients exist and are important for growth under these conditions.

Intracellular pH in Natranaerobius jonesii and Natranaerobius grantii.

To study intracellular pH homeostasis and Δp generation by N. jonesii and N. grantii, the internal pH and membrane potential were determined in energized cell suspensions with radioactive probes: [14C]methylamine and [3H]TPP+, over the growth range, e.g. external pH 9.0 – 11.0 for

N. jonesii and 7.5-11.0 for N. grantii. To correct for non-specific binding, separate cultures treated with valinomycin or nigericin were incubated with [3H]TPP+ over the growth range.

These inhibitors cause complete growth arrest when added to cultures of N. jonesii and N. grantii during the exponential growth phase, thus are membrane active with these bacteria (data not shown). The ΔpH was determined from the distribution of [14C]methylamine with the

Henderson-Hasselbach equation, and ZΔpH was calculated by 59 mV multiplied by the ΔpH.

The energized cell suspensions were prepared from cultures in mid-exponential phase (grown at optimum pH55°C, optimum temperature, and the pH value being studied) and resuspended in carbonate-buffered medium adjusted to the experimental pH being tested. Both N. jonesii and N. grantii showed the unusual feature of maintaining a ΔpH homeostasis, with a value of ~ 1 unit; this feature was previously identified in the type species of the genus, N. thermophilus. As the external pH of N. jonesii was increased from pH55°C 9.0 to 11.0, the intracellular pH increased from 7.9 to 10.2 (Figure 1b). The membrane potential (ΔΨ) increased from -93 mV at pH55°C 9.0 to -134 mV at pH55°C 11.0 (Figure 2a). The Δp increased from -46 mV at pH55°C 9.0 to -91 mV at

pH55°C 11.0 (Figure 2b). Similar results were obtained in a parallel experiment with N. grantii; as the external pH was increased from pH55°C 7.5 to 10.0, the intracellular pH increased from 6.9 to

9.2 (Figure 1b). The membrane potential (ΔΨ) increased from -85 mV at pH55°C 8.0 to -132 mV at pH55°C 10.0 (Figure 2a). The Δp increased from -38 mV at pH55°C 75 to -80 mV at pH55°C 10.0

(Figure 2b). The increase in ∆p occurred mainly due to an increase in the membrane potential; the value for the ZΔpH only fluctuated by ~ 20 mV over the external pH range studied (data not shown).

The ΔpH values for N. jonesii and N. grantii (0.6-1.1 pH unit) were less than those reported for the anaerobic alkalithermophile Clostridium paradoxum (ΔpH maximum of 1.3 units). In C. paradoxum, the ΔpH reached a maximum value at the optimal extracellular pH for growth and decreased at both higher and lower extracellular pH values until it collapsed almost completely at pH 7.0 and 10.8 (Cook et al., 1996). However, the extent of ΔpH in N. jonesii and

N. grantii was consistent with those reported for Natranaerobius thermophilus (Mesbah et al.,

2009). As previously seen in N. thermophilus, the ΔpH remained constant over the entire pH range tested and did not collapse even near the upper and lower pH boundaries for growth.

The Δp is a sum of the ΔpH across the membrane and the membrane potential. The increase in the Δp at values above optimal external pH55 °C values correlated with a decrease in growth rate and cell densities, supporting the contradiction demonstrated by similar studies in N. thermophilus of the current hypotheses that optimal growth occurs at the largest value for the Δp

(Mesbah et al. 2009). The ΔpH across the membrane remained nearly constant over the entire tested pH range, whereas the membrane potential increased (Figure 2b). The increase in the value of the proton motive force mirrors that of the increase in ΔΨ. Again, these results are similar to those seen in previous experiments in N. thermophilus.

For N. thermophilus, it was postulated that the growth inhibition of the microorganism at values below the optimum external pH55°C (i.e., above 9.5) was not due to collapse of ΔpH, as has been reported for Clostridium paradoxum, but rather due to the inability of the intracellular machinery to adapt to alkaline conditions (Mesbah, 2008). This work lends further support to this hypothesis, as similar inhibition of growth rate and culture cell density were observed at pH values below the optimum extracellular pH55°C, with a concomitant maintenance of ∆pH.

Intracellular concentrations of sodium and potassium in Natranaerobius jonesii and

Natranaerobius grantii.

Intracellular Na+ and K+ levels were determined from exponentially growing cultures.

Exponentially growing cells of N. jonesii, grown under optimum conditions, had an intracellular

K+ concentration of 225 mM (extracellular K+ concentration was 8.4 mM) and an intracellular

Na+ concentration of 6 mM (extracellular Na+ concentration was 3.9 M). Exponentially growing cells of N. grantii, grown under optimum conditions, had an intracellular K+ concentration of

262 mM (extracellular K+ was 8.4 mM) and an intracellular Na+ concentration of 10 mM

(extracellular Na+ concentration was 4.3 M). Intracellular concentrations of K+ did not fluctuate significantly with increased or decreased extracellular concentrations of K+ or Na+ in either species, nor did it fluctuate with increased or decreased growth temperatures (Table 1). The intracellular K+ concentration of N. jonesii in energized cell suspensions did, however, increase from 179 mM at the below-optimum pH55°C 9.5 to 281 mM at the above -optimum pH55°C 11.0.

Similarly, the intracellular K+ concentration of N. grantii increased from 207 mM at pH55°C 8.0 to 324 mM at elevated pH55°C 10.0. The intracellular Na+ concentration of both species under these conditions remained below 8 mM. Furthermore, these data are consistent with that previously obtained for N. thermophilus (Mesbah et al. 2009), and suggest that the various

species of this genus, independent of individual extent of thermophily, halophily and alkaliphly, utilize the same mechanisms to deal with environmental stress conditions.

Cytoplasmic accumulation of K+ is common in halophilic microorganisms (Oren 2002,

Oren et al., 2002, Ventosa et al., 1998) However, even at its highest levels—281 mM in N. jonesii (at pH55°C 11.0) and 324 mM in N. grantii (at pH55°C 10.0) intracellular K+ is not sufficient to osmotically balance the extracellular Na+ concentrations. There are currently two known methods which are utilized by halophilic microorganisms in order to adapt to high osmotic pressure: the ―salt-in‖ strategy, in which the cells accumulate intracellular K+ and Cl- ions in order to maintain osmotic balance, and the ―salt-out‖ strategy, in which the cells exclude ionic salts from the cytoplasm to the greatest extent possible and accumulate organic compatible solutes, which provide osmotic balance (Oren, 2002). Among the studied halophiles, both aerobic and anaerobic, only four aerobic members of the archaeal order Halobacteriales have been reported to use a combination of the ―salt-in‖ and ―salt-out‖ strategies. The extremely halophilic members of the bacterial order Halanaerobiales, along with many extremely halophilic Archaea, use the ―salt-in‖ strategy for osmotic adaptation and accumulate molar concentrations of intracellular K+ (Oren, 2008), while N. thermophilus, the type species of the genus Natranaerobius, utilizes a combination of the ―salt-in‖ and ―salt-out‖ strategies (Mesbah et al. 2009). 1H NMR spectra of the compatible solute extracts of N. thermophilus show a pattern of mixed compounds which include glycine betaine, β-glutamate, and small amounts of the amino acids proline and valine, among other unidentified compounds (data not shown).

Like the moderate thermophile N. thermophilus, the extremely thermophilic N. jonesii and the thermotolerant N. grantii compensate for their reversed ΔpH by maintaining a large membrane potential which is positive outside the cell and relatively negative inside the cell, and

which increases as the extracellular pH increases (Figure 1d). Accumulation of large concentrations of K+ inside the cytoplasm would decrease that membrane potential. From this and preceding data, it has been concluded that the three Natranaerobius species analyzed thus far must use a combination of both ―salt-in‖ and ―salt-out‖ strategies.

Requirement for Cl- in Natranaerobius thermophilus, Natranaerobius jonesii and

Natranerobius grantii

All Natranaerobius species tested, N. thermophilus, N. jonesii and N. grantii, demonstrated a requirement for varying amounts of Cl- in their growth medium. The microorganism with the lowest temperature optimum, N. grantii, also required the least amount of added Cl- (0.2 M) for growth; the microorganism with the highest temperature optimum, N. jonesii, required the greatest amount of Cl- in its growth medium (1.4 M). N. grantii required 0.2 M Cl- for growth, and showed no growth at 0.1 M Cl- and below. N. jonesii required 1.4 M Cl- for growth, but showed no growth at 1.2 M Cl- or below. The type species of the genus, N. thermophilus, a moderate thermophile, required the intermediate concentration of 1.2 M Cl- for growth, but showed no growth at or below 1.0 M Cl-.

A requirement for chloride has previously been identified in several microorganisms, such as Halobacillus halophilus, Salinibacter ruber, and Halanaerobium praevalens (Roeβler and Müller 1998; Müller and Oren, 2003). Some microorganisms, such as H. praevalens and other members of the Halanaerobiales, use Cl- and other inorganic ions to remain iso-osmotic with their environments (―salt-in‖). In H. praevalens, intracellular Cl- concentrations (2.24 M) have been measured which are roughly equivalent to that of their extracellular environments (2.3

M) (Oren 1986). Similar results were obtained in experiments on S. ruber (Oren et al. 2002).

Other microorganisms use Cl- as a signaling molecule. Flagellin protein synthesis, and, to a

lesser degree, fliC gene expression, in H. halophilus is strongly dependent upon the presence of

Cl- (Roeβler and Müller, 2002), as is organic compatible solute transport and synthesis (―salt- out‖) (Roeβler and Müller, 2001); chloride has also been identified as a signal molecule for organic compatible solute modification in environments of changing salt concentration (Saum et al. 2008).

As previously discussed, Natranaerobius thermophilus has been shown to utilize both the

―salt-in‖ and the ―salt-out‖ strategies; therefore, it is difficult to postulate what purpose of the cell is served by the presence of Cl- ions. While its function is yet unknown, and such analysis is outside the scope of this investigation, what is certain is that all of the tested Natranaerobius species require some concentration of Cl- in order to thrive.

Desiccation and resistance of Natranaerobius thermophilus, Natranaerobius jonesii,

Natranaerobius grantii and Natronovirga wadinatrunensis

All of the currently known Natranaerobius and Natronovirga species are exposed to periods of desiccation in their natural habitat due to seasonal precipitation fluctuations (Owenson 1997,

Taher 1999). For this reason, it was expected that these species have the ability to survive desiccation, at least for short periods of time. Cells were desiccated as described above, and, after rehydration, were incubated for up to fourteen days; growth was judged positive or negative compared to an unexposed control culture. None of the four species displayed desiccation resistance at or beyond twelve hours (the shortest interval tested), and thus were extremely sensitive to desiccation.

γ-radiation resistance of Natranaerobius thermophilus

Cells of cultures exposed to 1 kGy γ-radiation had a survival rate of 0.01%; cells of cultures exposed to 3 kGy γ-radiation were no longer viable (Fig. 2). The resistance of N. thermophilus

to γ-radiation is slightly higher than to that of E. coli, which is not considered to be resistant to ionizing radiation (Daly 2009). Resistance to ionizing radiation exposure has been observed in a number of environmental isolates, most notably Deionococcus radiodurans, and seems to be linked to desiccation resistance (Mattimore and Battista, 1996). Other γ-radiation resistant organisms of note include Kineococcus radiotolerans (Phillips et al. 2002), and other

Deionococcus species, e.g., Deionococcus geothermalis (Ferreira et al. 1997). D. radiodurans, the most ionizing radiation resistant organism isolated to date, is capable of withstanding up to 6 kGy of γ-irradiation with less than a one log reduction in number, and up to 17 kGy of γ- irradiation (Daly 2009). K. radiotolerans is able to withstand at least 4 kGy with no log reduction in number, and up to 11 kGy γ-irradiation (Bagwell et al. 2008). N. thermophilus, does not display nearly such resistance to ionizing radiation. Cells of cultures exposed to 1 kGy

γ-radiation had a survival rate of 0.01%; cells of cultures exposed to 3 kGy γ-radiation were no longer viable (Fig. 2). The resistance of N. thermophilus to γ-radiation is slightly higher than to that of E. coli, which is not considered to be resistant to ionizing radiation (Daly 2009).

UV resistance of Natranaerobius thermophilus and Natronovirga wadinatrunensis

N. thermophilus and N. wadinatrunensis were exposed to three wavelengths of UV radiation:

385 nm, 312 nm and 254 nm. Results are shown in Table 4.2 At all wavelengths, N. thermophilus displayed greater resistance (as evidenced by a greater number of viable cells remaining after exposure). However, at 312 and 254 nm, N. wadinatrunensis displayed greater resistance at intermediate time points (data not shown). The reason for this variance is unknown, but may be due to differing resistance mechanisms (e.g. DNA repair mechanisms) or other unknown factors.

In conclusion, the known adaptive mechanisms of these unique bacteria are many and varied, and the number and variety of the unknown mechanisms may be even greater. The environments in which these bacteria thrive require specialized adaptations in order to maintain intracellular conditions compatible with growth not required of microorganisms living in less extreme conditions. This work only begins to elucidate those unique qualities, and the authors hope that the suite of adaptive mechanisms employed by these and other extremophiles will be further investigated in the future.

Acknowledgments

We would like to thank J. Gilleland for laboratory assistance. This work was supported by

AFOSR 033835-01 and NSF 10-21-RR182-359 to J. Wiegel.

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Figure 4.1. Bioenergetic properties of Natranaerobius jonesii and Natranaerobius grantii N. jonesii is represented by the solid line, N. grantii is represented by the broken line.

Figure 4.1a. pH range of N. jonesii and N. grantii

Figure 4.1b. Extracellular vs. intracellular pH of N. jonesii (upper panel) and N. grantii (lower panel)

Figure 4.2. Effect of extracellular pH on membrane potential and proton motive force N. jonesii is represented by the solid line, N. grantii is represented by the broken line.

Figure 4.2a. Effect of extracellular pH on membrane potential

Figure 4.2b. Effect of external pH on proton motive force

Figure 4.3. γ-radiation resistance of Natranaerobius thermophilus

Survival following exposure to γ-irradiation. Escherichia coli, Natranerobius thermophilus, Kineococcus radiotolerans, Deionococcus radiodurans

Table 4.1. Intracellular [K+] and [Na+] under varying conditions

N. thermophilus N. jonesii N. grantii K+ Na+ K+ Na+ K+ Na+ optimum 247 ±11 8 ±2 225 ±20 6 ±1.5 262 ±21 10 ±3 elevated pH 293 ±15 7 ±1.5 281 ±29 7 ±2 324 ±27 8 ±3 depressed pH 211 ±13 8 ±1 179 ±21 5 ±2 207 ±19 8 ±2 elevated [Na+] 240 ±15 7 ±3 232 ±19 6 ±1 255 ±21 11 ±2 depressed [Na+] 257 ±19 9 ±2 229 ±23 8 ±2 263 ±23 9 ±1.5 elevated temperature 252 ±16 8 ±2 230 ±17 5 ±2 257 ±19 9 ±3 depressed temperature 245 ±17 7 ±2 219 ±23 6 ±3 271 ±20 10 ±2

- all values in mM - N. thermophilus values from (Mesbah 2008).

Table 4.2. Survival of Natranerobius thermophilus and Natronovirga wadinatrunensis after UV radiation exposure

N. thermophilus N. wadinatrunensis E. coli 385 nm (34 j•cm-2) 75% at 30 h 60% at 30 h 0% at 2h 312 nm (46 j•cm-2) 80% at 28 h 70% at 28 h 0% at 2h 254 nm (60 j•cm-2) 50% at 28 h 45% at 28 h 0% at 2h

CHAPTER FIVE

CONCLUSIONS

This thesis has 1) provided the first known comparison of growth properties of extremely halophilic bacteria and archaea, elucidating and describing the characteristics of poly- extremophiles within this group, 2) resulted in the isolation and characterization of two bacterial members of a unique and novel group of poly-extremophiles, the anaerobic halophilic alkalithermophiles, 2) resulted in the isolation and characterization of an aerobic, haloalkalithermophilic archaeon, and 3) described bioenergetic aspects and adaptive mechanisms of several members of the group of anaerobic halophilic alkalithermophiles.

The body of work encompassing not only the isolation and characterization of poly- extremophiles as well as their unique adaptive mechanisms is small when compared to that of extremophiles which are adapted only to one extreme. This work attempts to contribute significant new details and insights to this field, specifically in the area of microorganisms that are adapted to survive and thrive under conditions of high salt concentration (≥ 1.7 M), alkaline pH values (≥ 8.5) and high temperature (≥ 50°C). Chapter One describes, in detail, the characteristics of poly-extremophilic bacteria and archaea, and enumerates currently known poly-extremophilic species.

The three novel poly-extremophiles isolated and characterized in the duration of this work were isolated from two separate habitats (Chapters Two and Three). The aerobic haloalkalithermophile Natronolimnobius aegyptiacus was isolated from the lakes of the Wadi An

Natrun, Egypt, which are sun-heated athalassohaline lakes characterized by NaCl concentrations which approach saturation (5.6 M), alkaline pH (up to 11.0), and elevated temperature (up to

60°C). The anaerobic, haloalkalithermophilic Natranaerobius jonesii and the anaerobic, haloalkalithermotolerant Natranaerobius grantii were isolated from Lake Magadi, in the Kenya

Rift Valley. Lake Magadi is a solar- and geothermally-heated athalassohaline lake characterized by extremely high Na+ concentrations, which is contributed both by NaCl and large amounts of

Na2CO3 and NaHCO3, elevated pH (10-12) and high temperatures (up to 60°C near hot springs).

The investigations of bioenergetic parameters in this thesis build upon prior work and expands our knowledge of the unique bioenergetic properties of anaerobic haloalkalithermophilic bacteria, and further demonstrates the need for greater understanding of the bioenergetic mechanisms of this unique group of microorganisms (Chapter Four). Finally, this work begins an investigation of the adaptive mechanisms of the anaerobic haloalkalithermophilic bacteria, including intracellular accumulation of monovalent cations, requirements for chloride, and resistance to desiccation and UV- and γ-irradiation (Chapter Four).

It is hoped that the work presented in this thesis not only expands our knowledge of this unique and novel group of microorganisms, but demonstrates the need for further exploration and study of poly-extremophiles. These studies will provide a deeper understanding not only of survival, but of microbial growth and proliferation under multiple extreme conditions.

APPENDIX A

CALCULATION OF BIOENERGETIC PARAMETERS

Calculation of Intracellular pH

The intracellular pH values of N. jonesii and N. grantii were calculated from the uptake of

[14C]methylamine into the cell. Methylamine permeates the cell membrane in unprotonated form, once in the cell, it becomes protonated and cannot diffuse out. The intracellular pH can be calculated from the equation:

pHin = pHout – log10 ([B]in/[BH+]in)

As described in Chapter 4, the intracellular pH of 0.9 mL of cells (OD600 1.0) was calculated.

The concentration of [14C]methylamine in the cell pellet was determined by liquid scintillation counting. The total amount of base in the cell pellet = amount of base in intracellular aqueous volume + amount of base in the extracellular aqueous pellet volume

The steps involved in this calculation are outlined below:

(1) Determine concentration of undissociated methylamine in the extracellular space [B]ex

(2) Determine intracellular volume (Vin) of the cell pellet: from the difference in partitioning of

3 14 H2O and [ C]polyethylene glycol

(3) Determine amount of methylamine in the intracellular space: Bin = Bp – Bex

Where Bin is the amount of methylamine in the intracellular space, Bp is the amount of

methylamine in the pellet, and Bex is the amount of methylamine in the extracellular space

(4) Determine intracellular pH:

pHin = pHout – log10 ([B]in/[BH+]in)

(1) Determination of the concentration of undissociated methylamine [B]ex

pHout = pKa methylamine +log ([B]ex/[BH+]ex)

The pHout is the extracellular pH at which the experiment is performed;

pKa of methylamine = 10.64

[BH+]ex = amount of methylamine in supernatant, in counts per minute (cpm)

Rearranging for [Bex]:

pHex – pKa pHex – pKa [Bex] = (10 / 1+ 10 ) X [BH+]ex

(2) Determination of the intracellular volume

Intracellular volume = total pellet volume – extracellular volume

Vin = Vp - Vex

3 2 a. Vp: measured from the amount of H O in the cell pellet

3 2 3 2 Vp = ( H Op / H Os) X volume of supernatant sample (μL)

3 H2Op = amount of H2O in pellet (cpm)

3 H2Os = amount of H2O in supernatant (cpm)

14 b. Vex: measured from accumulation of [ C]polyethylene glycol in the cell pellet;

polyethylene glycol is not taken into the cell, thus remains in the extracellular space

Vex = (PEGp / PEGs) X volume of supernatant sample (μL)

14 PEGp = amount of [ C]PEG in pellet (cpm)

PEGs = amount of [14C]PEG in supernatant (cpm)

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(3) Determination of the amount of methylamine in intracellular space METin

METin = METp – METex

METp= amount of methylamine in the pellet (cpm)

METex = (METs / volume of supernatant) X Vex

METex = amount of methylamine in extracellular space

METs = amount of methylamine in supernatant (cpm)

(4) Determination of intracellular pH

pHin = pHout – log10 ([Bin] / [BH+]in)

The total amount of methylamine inside the cell includes both the protonated and

unprotonated forms, thus:

METin = Bin + BH+in

At equilibrium, Bin = Bex, thus

Bin = (Bex / volume of supernatant sample) X Vin

BH+in = (BH+in + Bin) – Bin

Since Metin = BH+in, + Bin

then: BH+in = Metin - Bin

Metin has been calculated in step 3.

The intracellular pH is calculated from the equation:

pHin = pHout – log10 ([B]in / [BH+]in

The ΔpH is calculated from:

ΔpH = pHex - pHin

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Calculation of Electrochemical Membrane Potential (ΔΨ)

The Δψ across the membrane was calculated from the uptake of [14C]TPP+ according to the Nernst relationship. The Nernst equation relates the electrochemical equilibrium distribution of an ion to the membrane potential. The Δψ is positive outside the cell, negative inside.

Diffusion of [14C]TPP+ into the cell proceeds until electrochemical equilibrium is reached.

To correct for non-specific binding of [14C]TPP+ to cells and the amount of [14C]TPP+ in the extracellular space, [14C]TPP+ ―uptake‖ in cells that have been treated with 10 μM of both nigericin and valinomycin was measured. Valinomycin and nigericin both transport K+ across the membrane until electrochemical equilibrium is reached, thus abolishing the Δψ. As a result, there is no driving force for TPP+ uptake.

The Nernst equation:

Δψ = (2.3RT / mF) X Log10 (TPP+in / TPP+out)

R: the gas constant 8.314 J mol-1 K-1

T: temperature (Kelvin)

m: valency of ion

F: Faraday constant, 96.485 KC mol-1

Total amount of TPP+ accumulated in the pellet TPP+pf:

TPP+pf = TPP+p – TPP+p n/v

TPP+p = amount of TPP+ accumulated in cell pellet

TPP+p n/v = amount of TPP+ accumulated in cell pellet treated with nigericin/

valinomycin

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TPP+in = (TPP+pf / Vin)

TPP+out = (TPP+s / volume of supernatant)

TPP+s = amount of TPP+ in the supernatant (cpm)

The value for (2.3RT / mF) is 59.

Calculation of Proton Motive Force

pmf = ZΔpH + Δψ

Z = (2.3RT / mF) = 59

The ΔpH is multiplied by the Z factor to convert it to units of charge: mV.

Calculations modified from Mesbah 2008

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