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Schmidt, Thomas Mitchell

INTERACTIONS AMONG THE SULFUR, HYDROGEN, AND CARBON METABOLISMS OF BEGGIATOA ALBA AND A COMPARISON TO THIOTHRIX NIVEA

The Ohio State University Ph.D. 1985

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University Microfilms International INTERACTIONS AMONG THE SULFUR, HYDROGEN, AND CARBON METABOLISMS OF BEGGIATOA ALBA AND A COMPARISON TO THIOTHRIX NIVEA

A DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University

by

Thomas Mitchell Schmidt, B.S., M.S.

The Ohio State University

1985

Reading Committee: Approved by:

Dr. Patrick R. Dugan

Dr. Kathleen E. Kendrick Dr. William R. Strohl

Dr. Robert M. Pfister Adviser ACKNOWLEDGMENTS

Learning is a tremendous experience. Unfortunately, few people have the luxury of pursuing it full time. I would like to acknowledge some of the people and organizations that have given me such an opportunity.

Thanks are due to my adviser, Dr. William R. Strohl, for his encouragement and flexibility throughout my doctoral studies. I thank

The Department of Microbiology, especially its chairman, Dr. Robert M.

Pfister, and The Program of Environmental Biology for financial support throughout my years in graduate school. Research and study funds were also obtained from grants from The National Science Foundation, The

Marine Biological Laboratory in Woods Hole, Massachusetts, and The NASA

Microbial Ecology Program.

Parts of this work were performed in the laboratories of Dr. Yehud

Cohen and Dr. Etana Padan of the Hebrew University. Their work continues to provide me with an example of excellent research. Thanks are also due to Dr. Riccardo Guerrero for his help with the work on

Chromatium, and Mr. Jim Porter for his able assistance with the respirometry. I am grateful to Dr. Patrick R. Dugan, Dr. Kathleen E. Kendrick,

Dr. Nelson H. Lawry, and Dr. Robert M. Pfister for their critical review of this dissertation. Their questions and comments have surely improved the quality of this document.

Without my wife Susan, the completion of this dissertation would have been much less enjoyable; her patience and sense of humor were a welcome refuge during the past three years. Finally, I thank my parents, Phyllis and Eddie, for teaching me that anything is possible.

iii VITA

NAME: Thomas Mitchell Schmidt

SOCIAL SECURITY NUMBER: 278-46-6031

DATE AND PLACE OF BIRTH: January 16, 1956. Lorain, Ohio

EDUCATION:

M.S. Environmental Biology, 1981; The Ohio State University, Columbus, Ohio. B.S. Biology, 1978; The University of Michigan, Ann Arbor, Michigan.

AREAS OF SPECIAL INTEREST:

Microbial Physiology and Ecology Sulfur and Hydrogen Metabolism Bacterial Energetics

GRANTS AND AWARDS:

July 1984 - August 1984. Participant in NASA sponsored research program, "Microbes and the Global Sulfur Cycle," San Jose, California.

September 1983 - December 1983. Invited Researcher, The Hebrew University of Israel, Jerusalem, Israel.

June 1982 - August 1982. Participant in Woods Hole Microbial Ecology Course. Recipient of stipend for research, Marine Biological Laboratory, Woods Hole, Massachusetts.

June 1980 - June 1981. Recipient of Sigma Xi Grant for study on the effects of Ixtoc oil on the sea grass ecology of the Gulf of Mexico.

June 1980 - September 1980. Recipient of Explorers Club Grant for on-site research in the Gulf of Mexico.

iv INVITED SEMINARS, LECTURES, AND SYMPOSIA:

Metabolic Diversity in the Family Beggiatoaceae. The Ohio State University. Columbus, Ohio. October, 1984.

Sulfide oxidation by the colorless, filamentous, sulfur bacteria. The Hebrew University of Israel, Jerusalem. November, 1983.

Structures of Filamentous Sulfur Bacteria. NASA research program. San Jose, California. July, 1984.

PUBLICATIONS and CONTRIBUTIONS IN BOOKS:

Strohl, W. R., and Schmidt, T. M. 1984. Mixotrophy of the colorless, sulfide-oxidizing, gliding bacteria Beggiatoa and Thiothrix. In Microbial Chemoautotrophy, W. R. Strohl and 0. H. Tuovinen, (eds). The Ohio State University Press, Columbus, Ohio.

Baca, B. J.f Schmidt, T. M., and Tunnel, J. W. 1982. Ixtoc oil in seagrass beds surrounding Isla de Media. In Proceedings of the International Symposium on Ixtoc 1. In press.

Schmidt, T. M. 1981. The uptake of petroleum-derived hydrocarbons by the sea grass Thalassia testudinum (Hydrocharitacea). M.S. Thesis, The Ohio State University, Columbus, Ohio.

PUBLISHED ABSTRACTS:

Sprouse, M. L., Schmidt, T. M., and Strohl, W. R. 1984. Sulfide induced appearance of plasmids in Beggiatoa. Abstract; Annual Meeting of the American Society for Microbiology. H 53, p 101.

Vinci, V. A., Schmidt, T. M., and Strohl, W. R. 1983. Protein synthesis by Beggiatoa alba. Abstract; Annual Meeting of the American Society for Microbiology I< 191, p 208.

Schmidt, T. M., and Castenholz, R. W. 1982. The effects of sulfide on cyanobacterial photosynthesis in marine microbial mats. Biological Bulletin, October. ASSISTANTSHIPS:

June 1983 - present. Graduate Research Associate, Deptartment of Microbiology, Ohio State University.

January 1981 - June 1983. Graduate Teaching Associate, Department of Microbiology, Ohio State University.

September 1980 - December 1980. Research Associate, Program of Environmental Biology, Ohio State University.

September 1979 - June 1980. Graduate Teaching Associate, Program of Environmental Biology, Ohio State University.

June 1979 - September 1979. Research Associate at Stone Laboratory, Freshwater Biological Station on Lake Erie, Ohio State University. TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

VITA ...... iv

TABLE OF C O N T E N T S ...... vii

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

LIST OF ABBREVIATIONS...... xiii

CHAPTER

I. Introduction ...... 1

II. Sulfide and Acetate Oxidation by Beggiatoa alba B 1 8 L D ...... 12

Introduction ...... 12

Materials and Methods ...... 13

R e s u l t s ...... 18

Discussion ...... 32

III. A comparison of the sulfur metabolisms of Thiothrix nivea JP3 and Beggiatoa alba B18LD. . . 41

Introduction ...... 41

Materials and Methods ...... 42

R e s u l t s ...... 45

Discussion...... 53

vii Page

IV. Endogenous and hydrogen-stimulated reduction of sulfur by Beggiatoa alba under anoxic conditions. 56

Introduction ...... 56

Materials and Methods ...... 57

R e s u l t s ...... 62

Discussion...... 71

V. Protein synthesis by Beggiatoa alba under heterotrophic and sulfide-oxidizing conditions . 77

Introduction...... 77

Materials and Methods ...... 78

R e s u l t s ...... 83

Discussion...... 94

VI. SUMMARY ...... 97

APPENDICES

Appendix A. Trace elements solution containing sulfate salts ...... 101

Appendix B. Trace elements solution containing chloride salts ...... 102

Appendix C. SDS PAGE electrophoresis...... 103

LITERATURE CITED ...... 105

viii LIST OF TABLES

Table Page

1.1. Morphological and physiological comparison of Beggiatoa alba B18LD and Thiothrix nivea JP3 ...... 9

2.1. Growth of B. alba B18LD filaments in soft agar gradients containing combinations of sulfide, titanium citrate, acetate, and bicarbonate ...... 20

2.2. Electron transport inhibitors tested on cells of B. alba B 1 8 L D ...... 23

2.3. Effect of electron transport inhibitors on acetate oxidation in B. alba B 1 8 L D ...... 25

2.4. Solubility of 35s accumulated by cells of B. alba B18LD exposed to 35s_sul f i d e 2 9

2.5. Effect of electron transport inhibitors on sulfide oxidation in B. alba B 1 8 L D ...... 30

3.1. Solubility of 35s accumulated by filaments of T. nivea JP3 exposed to 35s_sulfide 4 7

4.1. Hydrogenase activities in strains of Beggiatoa and Vitreoscilla...... 68

4.2. Effect of FCCP dissolved in DMSO on methylviologen- dependent hydrogen evolution by whole cells of B. alba B 1 8 L D ...... 69

•3 5.1. Fraction of cell-bound H-leucine incorporated in trichloroacetic acid precipitable material from cells of B. alba B 1 8 L D ...... 88

6.1. Summary of metabolic rates in B.alba B18LD filaments . . 99

ix LIST OF FIGURES

Figure Page

1.1. Light micrograph of Beggiatoa alba B18LD trichomes. . . 3

2.1. Sulfide/oxygen gradient cultures of B. alba B18LD . . . 19

2.2. Effect of respiratory inhibitors on oxygen consumption by cells of B>^ alba strain B18LD...... 21

2.3. Proposed electron transport chain in alba strain B18LD and the classical sites of action by electron transport inhibitors ...... 24

2.4. Effect of 1 mM sodium sulfide on the rate of oxidation of [2- C]-acetate to 14C02 by cells of B. alba B18LD # # 2 6

2.5. alba B18LD cellular accumulation of from ^S-sulfide in the presence and absence of oxygen . . . 28

2.6. Endogenous and sulfide-stimulated oxygen consumption in whole cells of B_j_ alba B 1 8 L D ...... 31

2.7. Proposed model of sulfide oxidation by Beggiatoa. . . . 38

3.1. Cellular accumulation of from ^^S-sulfide by trichomes of B^ alba B18LD and nivea JP 3 . . . . 46

3.2. Sulfide- and thiosulfate-dependent oxygen consumption by filaments of nivea JP3...... 48

3.3. Endogenous respiration of filaments of T. nivea JP3 . • 49

x Figure

3.4. Release of fr0m cells of nivea JP3 containing radiolabeled sulfur inclusions ...... 51

3.5. Oxygen consumption and sulfate production by sulfur- containing filaments of nivea JP3 suspended in basal salts solution ...... 52

4.1. Microelectrode apparatus...... 58

4.2. Microelectrode studies of oxygen and sulfide gradients resulting from the endogenous metabolism of a tuft of cells of B. alba B 1 8 L D ...... 63

4.3. Effect of nitrogen and hydrogen on sulfide production by tufts of B^ alba B18LD filaments embedded in agar. . 64

4.4. Apparatus for measuring the anaerobic reduction of sulfur to sulfide ...... 65

4.5. Anaerobic reduction of intracellular sulfur to sulfide by cells of Chromatium vinosum and filaments of B. alba containing and lacking sul f u r...... 66

4.6. Hydrogen consumption by filaments of EL alba B18LD. . . 70

4.7. Model of sulfur metabolism in Beggiatoa ...... 75

5.1. Accumulation of ^ S after exposur to ^ S - s u l f i d e by heterotrophically grown cells of .B^ alba B18LD before and after a 2 hour incubation in the presence of 1 mM sulfide...... 84

5.2. Effect of chloramphenicol on sulfide oxidation by heterotrophic cells of EL_ alba B18LD ...... 85

5.3. Effect of chloramphenicol on sulfide oxidation by heterotrophic cells of EL alba B18LD incubated for 2 hours in 1 mM sulfide...... 86

5.4. Effect of 1 mM sulfide on the incorporation of [U- H]-leucine into cells of B. alba B 1 8 L D ...... 87

xi Figure Page

5.5. SDS-polyacrylamide gel of proteins precipitated from cells of alba B18LD grown in thepresence or absence of sulfide, and of the enriched sulfurinclusions . . . 90

5.6. Silver-stained polyacrylamide gel of the proteins precipitated from alba B18LD cells grown in the presence or absence of s u l f i d e ...... 91

5.7. Autoradiogram of sulfide-induced proteins in filaments of B. alba B 1 8 L D ...... 92

5.8. Composite of lanes 1, 5, and 6 from the silver—stained polyacrylamide gel and the autoradiogram of that gel. . 93

xii LIST OF ABBREVIATIONS

Abbreviation

AC Acetate medium ACS Acetate medium + 1 mM sodium sulfide BSS Basal salts solution CAP Chloramphenicol CPM Counts per minute CYT Cytochrome DBMIB Dibromothymoquinone DMSO Dimethylsulfoxide DPM Disintegrations per minute EDTA Disodium dihydrogen ethylene diaminetetraacetate dihydrate FCCP Carbonylcyanide-jD-trifluoromethoxyphenylhydrazone FP Flavoprotein HOQNO B-r^-heptyl-^-hydroxyquinoline-n-oxide 8-HQ 8-hydroxyquinoline KCM Potassium cyanide Relative molecular mass n a B Nicotinamide adenine dinucleotide NaN, Sodium azide PAGE Polyacrylamide gel electrophoresis PHB Poly-^-hydroxybutyric acid Phen 1,10-o-phenanthroline POPOP 1,4-bis-[2]-(5-phenyloxazolyl)benzene PPO 2-diphenyloxazole Q8 Ubiquinone 8 RuBP Ribulose-1,5-bisphosphate SDS Sodium dodecyl sulfate TCA Trichloroacetic acid TTFA Thenoyltrifluoroacetone CHAPTER I

INTRODUCTION

Gliding trichomes of the filamentous bacterium Beggiatoa were first

described by Vaucher (1803), and later named Beggiatoa by Trevisan

(1845). Winogradsky (1887, 1888) was among the first to experiment with the organism. He documented the formation of sulfur inclusions when the trichomes were exposed to hydrogen sulfide and observed the eventual disappearance of the sulfur inclusions, which he attributed to the oxidation of sulfur to sulfate. Based on these and other observations,

Winogradsky developed the concept of bacterial chemoautotrophy. He proposed that the oxidation of inorganic sulfur compounds served as the sole energy-generating pathway, while total cell carbon was derived from carbon dioxide. He later confirmed his theory of autotrophy with studies on organisms of the genus Nitrobacter (Winogradsky 1890).

In the years since Winogradsky’s experiments, research on organisms of the genus Beggiatoa has been sporadic. There has been a renewed interest in the physiology and ecology of these bacteria in the past ten years, rt.. ed in the number of papers recently published on

Beggiatoa. The original and recent research on Beggiatoa physiology and ecology is reviewed in several manuscripts including: Wiessner (1981),

Jdrgensen (1982), Nelson and Jannasch (1983), Larkin and Strohl (1983),

1 2

and Strohl and Schmidt (1984). An updated characterization of the genus

will appear in the ninth edition of Bergey's Manual of Determinative

Bacteriology in 1986. The following sections of this chapter provide an

introduction to the genera Beggiatoa and Thiothrix, and a review of the

literature that pertains to the results reported in this dissertation.

Beggiatoa

Organisms of the genus Beggiatoa are microaerophilic filamentous

bacteria that inhabit the zone adjacent to the oxic/anoxic interface in

freshwater and marine sediments. At this interface, where sulfide and

oxygen coexist (Jdrgensen and Revsbech 1983), trichomes of Beggiatoa

oxidize sulfide to sulfur. The sulfur is stored in periplasmic sulfur

inclusions (Maier and Murray 1965; Strohl et al. 1981a; Lawry et al.

1981). These inclusions are refractile when photographed through a

light microscope (Figure 1.1).

Sulfide oxidation is the energy source for the chemolitho-

autotrophic growth of at least one strain of Beggiatoa (Nelson and

Jannasch 1983) and may be a supplemental energy source for growth on

ac.etate (GDde et al. 1981). Most Beggiatoa strains thus far tested have

the capacity for heterotrophic growth on acetate in the presence of

oxygen (Pringsheim 1964; Strohl and Larkin 1978). However, members of

the genus Beggiatoa lack catalase (Burton and Morita 1964; Strohl and

Larkin 1978), and so growth under highly oxygenated conditions is

limited. Many researchers have noted the beneficial effects of a

lowered oxygen tension on the growth of Beggiatoa (Faust and Wolfe 1961;

Kowallik and Pringsheim 1966; Strohl and Larkin 1978; Nelson and Figure 1.1. Light micrograph of Beggiatoa alba B18LD trichomes. The trichomes, which are between 1.6 and 2.0 umeters wide were photographed while gliding on an agar surface. S°; periplasmic sulfur inclusions. 4

Castenholz 1981a). The oxidation of sulfide may eliminate the need for

catalase by detoxifying metabolically formed hydrogen peroxide (Burton

and Morita 1964). Alternatively, sulfide oxidation may simply alleviate the toxic effects of sulfide in the environment, while that anion provides an optimal redox potential for growth.

By creating a sulfide/oxygen gradient in the laboratory, Nelson and

Jannasch (1983) measured the chemoautotrophic growth of a marine strain of Beggiatoa. This study confirmed the previous controversial reports of autotrophic growth of Beggiatoa in cultures containing both sulfide and oxygen (Keil '1912; Kowallik and Pringsheim 1966). The microoxic environment created by sulfide is also required for expression of nitrogenase activity (Nelson et al. 1982).

Whatever the effect of sulfide and its oxidation might be, marked concentrations of Beggiatoa filaments in nature virtually always coincide with the presence of hydrogen sulfide (Wiessner 1981). In the environment studied by Jorgensen and Revsbech (1983). more than 99% of the sulfide oxidation in Beggiatoa mats was biological. Thus, the biological oxidation of sulfide competes successfully with chemical oxidation in these microoxic environments.

The position of the oxic/anoxic zone in the environment is dependent on the production and use of sulfide and oxygen by microorganisms, and the diffusion of these compounds through the sediments (Jorgensen 1977). In environments where cyanobacteria are present, oxygenic photosynthesis during daylight hours produces molecular oxygen that diffuses into the sediments, lowering the oxic/anoxic interface. The interface rises at night in these 5

environments due to decreased photoproduction of oxygen and the upward diffusion of sulfide produced by the sulfate-reducing bacteria

(Jorgensen 1977). In these environments, Beggiatoa trichomes migrate through the sediments on a diurnal cycle (Nelson and Castenholz 1982), presumably to remain at the oxic/anoxic interface. The gliding motility exhibited by these microorganisms may be due to oxygen or sulfide chemotaxis in combination with a photophobic response (Nelson and

Castenholz 1982a).

In addition to the general characteristics of these gradient habitats, the microenvironment of Beggiatoa has been investigated using microelectrodes (Jorgensen and Revsbech 1983). The microenvironment was found to be extremely dynamic, with filaments of Beggiatoa playing a major role in controlling the chemical variables of the environment, particularly the oxygen and sulfide profiles.

Filaments of Beggiatoa are most likely exposed to periods of anoxia in their microenvironment as oxygen gradients shift. Therefore, the organism must be able to obtain energy anaerobically if the organism is to produce sufficient maintenance energy, as well as the energy needed to glide to an oxic zone. Since nitrate apparently cannot be utilized by cells of Beggiatoa in dissimilatory nitrate reduction (Vargas 1984,

MS thesis, The Ohio State University), sulfur is an obvious alternative electron acceptor for anaerobic metabolism.

In preliminary experiments, Nelson and Castenholz (1981a) maintained sulfur-containing cells of Beggiatoa strain 75-2a for five days in the absence of oxygen. Sulfide was detected in these anoxic cultures, suggesting that sulfur was reduced to sulfide as the terminal 6

electron-accepting reaction (Nelson and Castenholz 1981a). If sulfur is

utilized as an anaerobic terminal electron acceptor, this may help to

explain why copious amounts of sulfur are stored by the cells. An organism capable of both aerobic and anaerobic metabolism would surely be favored in a dynamic environment such as that of Beggiatoa.

The use of sulfur as an anaerobic terminal electron acceptor occurs in several genera of bacteria that are distantly related based on 16S rRNA analysis (Fox et al. 1980). Both purple and green photosynthetic sulfur bacteria reduce sulfur to sulfide under dark, anoxic conditions.

Cells of Chromatium strain 6412 oxidize glycogen and form poly-B-hydoxybutyric acid (PHB) with excess reducing power being shuttled to sulfur reduction (van Gemerden 1968). This provides a means of substrate-level phosphorylation and explains the motility of

Chromatium cells in the dark. Triiper and Schlegel (1964) also observed the dark reduction of sulfur to sulfide by cells of Chromatium okenii.

The oxidation of hydrogen appears to be coupled to sulfur reduction in cells of Chromatium strain D (Hendley 1955), thus expanding the potential for dark anaerobic metabolism in the Thiorhodaceae.

Chlorobium cells also reduce sulfur anaerobically in both the presence and absence of light (Paschinger et al. 1974). Filaments of the halophilic cyanobacterium Oscillatoria limnetica respire in the dark, with sulfur being reduced in the terminal electron-accepting reaction

(Oren and Shilo 1979). The strictly anaerobic bacterium Desulfuromonas acetooxidans is the only organism that has been described with the capacity for anaerobic, chemotrophic growth utilizing sulfur as the sole terminal electron acceptor (Pfenning and Bieble 1976). 7

In all of these bacteria, sulfide oxidation and sulfur reduction

are catalyzed enzymatically. Flavocytochrome cj-552 isolated from

Chromatium vinosum possesses sulfideicytochrome jj oxido-reductase activity and also catalyzes the reduction of sulfur to sulfide (Fukumori and Yamanaka 1979). Cytochrome Cg isolated from Desulfovibrio desulfuricans possesses sulfur reductase activity in the presence of hydrogen, sulfur, and hydrogenase (Fauque et al. 1979). Although no attempt has been made to isolate any of these enzymes from Beggiatoa, a complete respiratory chain is present and may be involved in the oxidation of sulfide and the reduction of sulfur. Cells of Beggiatoa alba B18LD contain cytochromes c (Cannon et al. 1979), b., a, and o, and ubiquinone 8 (Strohl, Schmidt, and Larkin; manuscript in preparation).

That same study indicates that cytochromes b, c, a, and o are also present in several other strains of Beggiatoa.

When sulfide is oxidized by filaments of Beggiatoa, sulfur accumulates in periplasmic sulfur inclusions. These sulfur inclusions are enclosed by an electron-dense envelope between 3 and 5 nm thick

(Lawry et al. 1981; Strohl et al. 1981a). The inclusions are usually spherical and filled with sulfur (Lawry et al. 1981). Filaments of

Beggiatoa alba B15LD grown in the absence of sulfide contain what appear to be empty sulfur inclusion envelopes (Strohl et al. 1982). Because the sulfur inclusion are periplasmic, enzymes involved in the transitions of sulfur are presumably also in the periplasmic space.

These enzymes might be soluble, bound to the outside of the cytoplasmic membrane, or associated with the sulfur inclusion envelope.

There is indirect evidence that the sulfur stored in inclusions is 8

oxidized to sulfate in some strains of Beggiatoa. Nelson and Jannasch

(1983) noted that the pH of the medium in sulfide/oxygen gradients

decreased in the area surrounding Beggiatoa filaments. They attributed

this to sulfate produced from sulfur oxidation. Also, filaments of

Beggiatoa removed from enrichments or nature are occasionally devoid of

sulfur (Winogradsky 1888; Strohl and Larkin 1978). Because sulfide

oxidation is not required for growth of Beggiatoa cells, Minges et al.

(1983) suggested that enzymes involved in the deposition of sulfur might

be plasmid-encoded. The absence of plasmids in some filaments, could

also account for the absence of sulfur in those filaments. No

correlation, however, was found between sulfide oxidation and the

presence of any particular plasmid (Minges et al. 1983).

Thiothrix

Bacteria of the genus Thiothrix are colorless, filamentous,

sheathed, sulfide-oxidizing organisms that are morphologically similar

to filaments of the genus Beggiatoa (Winogradsky 1888; Bland and Staley

1978; Larkin 1980; Larkin and Shinabarger 1983). A comparison of

Beggiatoa and Thiothrix, adapted from Strohl and Schmidt (1984), is

presented in Table 1.1. The major differences between the genera are:

(i) the presence of a sheath around the trichomes of Thiothrix; (ii) the presence of a holdfast which attaches Thiothrix filaments to substrates;

(iii) the lack of gliding by Thiothrix trichomes; and (iv) the requirement of a reduced sulfur compound for growth. The only currently accepted species of Thiothrix is T. nivea (Larkin and Strohl 1983;

Larkin and Shinabarger 1983). Table 1.1. Morphological and Physiological Comparison of Beggiatoa alba B18LD and Thiothrix nivea JP3a.

Characteristic Beggiatoa Thiothrix

Trichome Formation + + Sheath - + Holdfast - + Rosette formation - + Hormogonia formation +/- + Gliding of intact trichomes + Gliding of hormogonia + + Sulfur inclusions from sulfide + + Sulfur inclusions from thiosulfate + + TMPD oxidation + + Cytochromes b,c,a,o c,(others ?) Quinones UQ 8 UQ 8 Acetate assimilation + + Acetate oxidation to CO^ + ? Reduced sulfur requirement - + Sulfide-dependent oxygen consumption + +

a Adapted from Strohl and Schmidt 1984 10

Filaments of Thiothrix are found attached to organic and inorganic objects in flowing, sulfide-containing waters, such as the effluent from sewage plants (Farquhar and Boyle 1971; Merkel 1975) or marsh streams, attached to rotting seaweed. Thus, even though Beggiatoa and Thiothrix filaments are morphologically and physiologically similar, their habitats are distinct. Filaments of Beggiatoa glide through the sediments in response to changing chemical and physical variables, whereas filaments of Thiothrix are sessile and thrive in flowing waters that provide an adequate supply of nutrients.

An understanding of the metabolic potential of Thiothrix has awaited isolation of pure cultures; these were obtained only recently

(Larkin 1980). Past attempts to isolate Thiothrix have been hindered, most likely by the difficulty in providing the appropriate balance between oxygen and sulfide, and carbon dioxide and organic carbon. The strain used in this study, JP3, was isolated by Larkin (1980) from enrichments inoculated with Thiothrix filaments found in John Pennekamp

State Park, Florida.

Objectives

The work reported in this dissertation is the result of experiments designed to delineate the metabolic capacity of Beggiatoa filaments in view of the organism's unique habitat. The studies are conducted to test the potential for autotrophic growth in the type strain of

Beggiatoa alba, strain B18LD (Mezzino et al. 1984; Skerman et al. 1980), and to determine at what point electrons released from the oxidation of sulfide might enter the electron transport chain. The potential for 11

dogenous, anaerobic metabolism and the anaerobic coupling of hydrogen oxidation to sulfur reduction in cells of strain B18LD is also investigated. The sulfur metabolism is compared to that in cells of

Thiothrix nivea strain JP3.

Proteins extracted from cells of strain B18LD grown in the presence and absence of sulfide are examined using polyacrylamide gel electrophoresis. The rate of protein synthesis, as well as the increased rate of synthesis of individual proteins, is measured after the addition of sulfide to a heterotrophic culture. A preliminary characterization is then made of the proteinaceous sulfur-inclusion envelope. CHAPTER II

SULFIDE AND ACETATE OXIDATION BY BEGGIATOA ALBA B18LD

Introduction

The potential for chemolithoautotrophic growth of a marine strain of Beggiatoa has recently been studied by Nelson and Jannasch (1983).

Under the microoxic conditions present in sulfide/oxygen gradient cultures, filaments of the marine Beggiatoa grew with sulfide as the energy source and carbon dioxide as the primary source of carbon for biosynthesis (Nelson and Jannasch 1983).

In the present study, sulfide/oxygen gradients are used to culture a freshwater strain of Beggiatoa alba, strain B18LD. Several gradient designs are tested to examine the roles of acetate, sulfide, and titanium citrate, a redox dye, in the growth of filaments of strain

B18LD.

The functioning of the respiratory electron transport chain in the presence and absence of sulfide is investigated with the use of electron transport inhibitors, and a model for sulfide oxidation is presented.

12 13

Materials and Methods

Growth conditions

Beggiatoa alba strain B18LD (ATCC 33555) was grown in the basal salts solution described below, containing either 500 mg/1 (6 mM) sodium acetate (AC medium) or 500 mg/1 sodium acetate and 240 mg/1 sodium sulfide (1 mM) added after autoclaving (ACS medium). The basal salts solution (BSS) contained per liter: 200 mg 10 mg

K2HP0|,t 10 mg MgSOj, 7H20; 75 mg CaCl2 2H20, and

5 ml of a microelement solution (Appendix A). All media were adjusted to pH 7.2 and sterilized by autoclaving.

Strain B18LD was also grown in sulfide/oxygen gradients similar to those used by Nelson and Jannasch (1983). The gradients were formed in a soft agar solution in culture tubes (16 x 150 mm) containing 0.15? agar in BSS and supplemented with either 2 mM NaHCO^ or 6 mM sodium acetate. This soft agar was layered over a 2% agar plug containing BSS and 4 mM sodium sulfide at pH 7.2 or 4 mM titanium citrate. Titanium citrate was prepared by adding 5 ml of 15% titanium citrate to 50 ml of

0.2 M sodium citrate and adjusting the pH to 7.0 with sodium bicarbonate. The neutralized solution was stored in a 125 ml serum bottle under a nitrogen atmosphere, and sterilized by autoclaving.

Culture tubes in which sulfide and titanium citrate were omitted from the 2% agar plugs were used as controls. Cultures in autotrophic gradients were maintained by transfering 0.1 ml of soft agar containing

B18LD filaments to fresh gradients every two weeks. 1A

Acetate oxidation

Acetate-dependent oxygen consumption was measured with, the Warburg

respirometer as described by Umbreit et al. (1957). Cells were

harvested during exponential growth in AC media by centrifugation at

8,000 x g for 10 min, and washed once in sterile BSS. The resulting

cell pellet was resuspended to a density of ca. 0.3 mg protein/ml. Two

ml of the concentrated cell suspension were dispensed into Warburg

flasks. The center well of the flasks contained pleated filter paper (5

mm x 25 mm) saturated with 0.2 ml of 20% potassium hydroxide. The

sidearm of each flask contained 222 p i of 60 mM sodium acetate.

Inhibitors were added to the cell suspension at the concentrations designated in Table 2.3, then the flasks were attached to manometers.

Fifteen minutes after the addition of the inhibitors, sodium acetate was

added to the reaction mixture from the sidearm of the flask. Duplicate

flasks were prepared for each inhibitor tested. The temperature of the reaction flasks was maintained at 23 C, and manometric readings were taken every 15 min. Duplicate flasks containing distilled water were used in each experiment as thermobarometers to determine any fluctuations in the level of the manometer fluid due to changes in temperature or pressure.

Isotopic determination of the rate of acetate oxidation was performed in 25 ml Konte reaction flasks. Three ml aliquots of a concentrated cell suspension, prepared as described previously, were dispensed into the reaction flasks. A glass well, containing pleated filter paper saturated with hydroxylamine hydroxide, was suspended from the stopper in each flask to absorb any C02 emitted. The effect of 15

respiratory inhibitors on acetate oxidation was determined in duplicate samples by adding inhibitors to reaction flasks 15 min before the addition of 333 p i of [2-^^Cl-acetate (1 }jCi/30 umol/ml). The effect of sulfide on [2-^C]-acetate oxidation was determined by adding 2 mM sulfide (pH 7.2) to duplicate reaction flasks 15 min before the addition of 333 jul of [2-^i*C]-acetate. The final concentration of acetate in the reaction mixture was 3 mM - a concentration greater than that required for the maximal rate of acetate oxidation (Strohl et al. 1981b). Flasks were shaken at 120 strokes/second in a Dubnoff metabolic shaker at 23 C. The reaction was stopped and the dissolved

C02 was liberated from the medium by the addition of 0.5 ml of glacial acetic acid. After 30 min, the pleated filter paper was removed and placed in a toluene-based scintillation cocktail containing 5.0 g/1

2-diphenyloxazole (PPO) and 50 mg/1

1,4-bis-{2}-(5-phenyloxazolyl)benzene (P0P0P) (Patterson 1965). The samples were counted in the Beckman model LS 6800 scintillation counter that was calibrated with 3H and 14c standards.

Sulfide oxidation

Cells harvested from cultures in late exponential growth in AC medium were washed once and resuspended in basal salts. Ten ml aliquots of the concentrated cell suspension, containing approximately 0.2 mg protein/ml, were dispensed into 50 ml Erlenmeyer flasks, and the vessels were shaken at 120 strokes/min in a Dubnoff metabolic shaker at 23 C.

When anoxic conditions were required, oxygen was removed from the flask by flushing with nitrogen for 10 min, then carefully capping the flask 16

with a rubber stopper. Sodium [35s]_Sulfide (8.5 /jCi/20 /imol/ml) was added to each flask to a final concentration of 1 mM. Samples of

200 jul were removed at timed intervals and filtered through Whatman glass fiber filters. The filters were washed with BSS at pH 3 to remove any unbound label, and then dried at 60 C for 2 h. The dried filters were counted in the scintillation cocktail described previously.

Quenching was determined by using an internal standard of

(U-1 ^O-toluene. Standard calculations were used to convert disintegrations per minute (dpm) to moles of sulfide per mg protein.

The effect of respiratory inhibitors on sulfide oxidation was measured by adding inhibitors to the cell suspension 15 min before the addition of sulfide. Six mM sodium acetate was added to duplicate reaction flasks to test the effect of acetate oxidation on sulfide oxidation.

Sulfide-dependent oxygen consumption was measured in the Warburg apparatus by the method described for acetate oxidation, except that the sidearm of each flask contained 222 pi of 10 mM sodium sulfide in place of 60 mM sodium acetate. The sulfide was added to 2 ml of the cell suspension in the respirometer flask at the start of the experiment.

Duplicate measurements of the chemical oxidation of sulfide were made with respirometer flasks containing 2 ml BSS without cells. The oxidation of radiolabeled sulfide to labeled sulfur was measured concurrently with the same cell suspension.

Protein determinations

The concentration of total cell protein was estimated by the method of Lowry et al. (1951), after extraction of sulfur with 95% ethanol for 17

1 h, followed by heating the samples at 90 C in 1N NaOH for 10 min to digest the cells. Bovine serum albumin was used as a protein standard.

Chemicals

Isotopes were obtained from Amersham Corp. (Arlington Heights,

Illinois). Sodium [35s]-sulfide was dissolved in deoxygenated water containing unlabeled sulfide such that the final concentration of sulfide was 20 mM at pH 7.2. Unlabeled crystals of sodium sulfide were washed in distilled water and dried before being weighed. The sulfide solution was stored under a nitrogen atmosphere at 4 C. The nitrogen atmosphere was replenished after each use. 18

Results

Growth in Gradients

Filaments of Beggiatoa alba B18LD grew in a layer 1 nun thick in the soft agar cultures containing 2 mM NaHC03 and a 4 mM sulfide plug.

This band of cells formed 12 mm below the surface of the soft agar (Fig

2.1). Cultures have been maintained for three months in these autotrophic sulfide/oxygen gradients with transfers every two weeks.

When sulfide was omitted from the 2% agar plug in the gradient cultures, there was no observable growth (Fig 2.1). Titanium citrate (4 mM), which lowers the redox potential of media, did not replace the requirement for sulfide in these soft agar cultures (Table 2.1).

The addition of acetate to the upper layer of the gradient cultures resulted in a band of growth 2 mm thick that began 2 mm below the surface of the air/agar interface. The band of growth in the acetate-supplemented agar was near the surface when the bottom plug contained sulfide, titanium citrate, or unsupplemented BSS (Table 2.1).

Acetate Oxidation

Oxygen was consumed by cells of strain B18LD suspended in AC medium at a rate of 3.60 /jl 02/min/mg protein (Fig 2.2). This consumption is equivalent to a rate of 160 nmol Og/min/mg protein. These rates were determined after the appropriate corrections were made for fluctuations in temperature and pressure as determined with the 19

Figure 2.1. Sulfide/oxygen gradient cultures of B. alba B18LD. The bottom layer of tubes 1,2, and 3 contain four mM sodium sulfide in basal salts solution containing 2% agar. Zones of growth 1 mm wide are visible 12 mm from the air/agar interface in the first three tubes (arrows). Sulfide was omitted from tubes 4 and 5. The top layer of all tubes was supplemented with 2 mM sodium bicarbonate and 0.15% agar. 20

Table 2.1. Growth of B. alba B18LD filaments in soft-agar gradients containing combinations of sulfide, titanium citrate, acetate, and bicarbonate.

Contents of Contents of Description of growth hard agar plug soft agar overlay after 5 days3 (bottom) (top layer)

Salts*3 acetate0 Ring, 2 mm

Salts NaHC03 No growth

Salts + 4 mM acetate Ring, 2 mm titanium citrate

Salts + *1 mM NaHC03 No growth titanium citrate

Salts + 4 mM acetate Ring, 2 mm sodium sulfide

Salts + 4 mM NaHC03 Ring, 12mm sodium sulfide

a The position of the zone of growth was measured from the upper surface of the soft agar layer

b Basal salts solution (Chapter II)

c Acetate concentration, 6 mM; NaHC03 concentration, 2 mM. Figure 2.2 Effect of respiratory inhibitors on oxygen consumption by consumption oxygen on inhibitors respiratory of Effect 2.2 Figure

UL 02/ MG PROTEIN 200 250 300 100 150 350 400 450 0 - 50 0 cells of B. alba strain B18LD. No inhibitor (X ); (X inhibitor No B18LD.strain alba B.of cells 50 uM HOQNO (■ ); 1 (■ mM HOQNO 50uM 15 04 03 105 30 60 45 30 IE (MIN) TIME 8 H (-HQ ♦ 1);NaN^ mM (A >• 120 21 22

thermobarometer. The rate of release of 1i*C02 fr0m

[2— ]—acetate was 65 nmol C02/min/mg protein.

The inhibition of acetate oxidation was determined by measuring the

effect of respiratory inhibitors on acetate-dependent oxygen consumption

and the release of 11*C02 from [2_1i,C]-acetate. The

inhibitors tested and the solvent that they are dissolved in are listed

in Table 2.2. The sites of inhibition for these respiratory inhibitors are diagrammed in Figure 2.3. Figure 2.2 shows the rates of oxygen consumption for three of the inhibitors tested. The slope of the best fitting line for each series of measurements was calculated, using the equation for a linear regression, and is reported in Table 2.3. The inhibitors that decreased the rate of acetate-dependent oxygen consumption had a similar effect on the rate of release of ^ C 0 2 from [2-1l*C]-acetate (Table 2.3). The solvents used for the inhibitors, ethanol and dimethylsulfoxide (DMSO), did not significantly affect acetate-dependent oxygen consumption or the release of

11*C02 from [2-1l*c)“acetate. The rate of acetate oxidation, measured isotopically, was decreased by approximately 18% in the presence of 2 mM sulfide (Figure 2.U).

Sulfide Oxidation

The rate of sulfide oxidation was determined by measuring the accumulation of 35$° When the cells were exposed to

[3^S]-sulfide at pH 7.2. The initial rate of oxidation was approximately 65 nmol/min/mg protein in the presence of oxygen; in the absence of oxygen there was no measurable uptake after the first 10 min 23

Table 2.2. Electron transport inhibitors tested on cells of B. alba B18LD.

Inhibitor Abbreviation Solvent

Dibromothymoquinone DBMIB DMSOa

2-n-heptyl-JJ-hydroxy- HOQNO Ethanol quinoline-n-oxide

8-hydroxyquinoline 8HQ Ethanol

Potassium cyanide KCN Water o-phenanthroline Phen Ethanol

Sodium Azide NaN3 Water

Thenoyltriflouro- TTFA Ethanol acetate

Dinitrophenol DNP Ethanol

a DMSO: dimethylsulfoxide 24

Figure 2.3 . Proposed electron transport chain in alba strain B18LD and the classical sites of action of electron transport inhibitors. See Table 2.2 for an explanation of abbreviations not listed below, and for the solvent used for each inhibitor.

8-HQ KCN TTFA DBMIB phen HOQNO NaN \J^ vl' ^ NAD ^ FP --- ^ Q8 ---- > cyt b ---- ^ cyt c ----> cyt o 'I' cyt a

Abbreviations: NAD - nicotinamide adenine dinucleotide; FP - flavoprotein; Q8 - ubiquinone 8 ; cyt - cytochrome. 25

Table 2.3. Effect of electron transport inhibitors on acetate oxidation in B. alba B18LD.

Percent of Percent of n D Inhibitor3 Concentration u2 consumption CO2 evolution0

None - - - 100 100

Ethanol 1 % 100 98

TTFA 1.0 mM NPd 3

DBMIB 20 uM 11 NP

8-HQ 1.0 mM 12 22

Phen 2.0 mM 10 6

HOQNO 50 uM 73 91

KCN 1.0 mM 2 3

NaN3 1.0 mM 5 49

DNP 0.5 mM 1 2

a See Table 2.2 for a list of inhibitor abbreviations, and the solvent used for each inhibitor b Control rate of acetate-dependent oxygen consumption: 162 nmol/min/mg protein c Control rate of 12*C0_ release from [2- 1i,C]-acetate: 72 nmol/min/mg protein d experiment not performed Figure 2.M. Effect of 1 mM sodium sulfide on the oxidation of oxidation the on sulfide 1 sodium mM of Effect 2.M.Figure

UMOLES C 0 2 / MG PROTEIN .5 2 0 samples. of acetate. Each point is the average of duplicate duplicate of average isthe point Each acetate. of 15 minbefore added (x) .the was saltsin addition 1 basal (■)sulfide ormM salts basal Either B18LD. 2 1 - to ^C-acetate 10 1 i

(Fig 2.5). Approximately 90% of the labeled elemental sulfur

accumulated in the presence of oxygen could be dissolved in acetone,

benzene, or 95% ethanol (Table 2.4).

The relative effects of several electron transport inhibitors on

sulfide oxidation are listed in Table 2.5. All of the inhibitors of

acetate oxidation suppressed sulfide oxidation except for

dibromothymoquinone (DBMIB). DBMIB at concentrations five times the

amount necessary to inhibit acetate oxidation had no effect on sulfide

oxidation. Acetate at a final concentration of 6 mM inhibited sulfide

oxidation by 50%..

The rate of sulfide-dependent oxygen consumption was determined

concurrently with the measurement of 35s_Sulfide oxidation. In this

experiment (Fig 2.6), the rate of sulfur accumulation was 38 nmol/min/mg

protein. This rate was the same for cells grown in AC or ACS media.

The oxygen consumption due to chemical sulfide oxidation was 0.35 pl/min/flask. The endogenous rate of oxygen consumption for

sulfur—containing cells was 0.25 pi (^/min/mg protein and for cells

lacking sulfur the rate was 0.37 pi Og/min/mg protein. Accounting

for the oxygen consumption due to chemical sulfide oxidation and

endogenous oxygen consumption, the rate of sulfide-dependent biological

oxygen consumption was 0.45 pi 02/min/mg protein. This is

equivalent to a rate of oxygen consumption of 20 nmol/min/mg protein. Figure 2.5. B. alba B18LD cellular accumulation of 35g from sodium 35gfrom of accumulation cellular B18LD alba B. 2.5. Figure

UMOLES S ACCUMULATED / MG PROTEIN 0.2 0-4 0.6 0-8 1.6 - ^]sliei h rsne « ad bec (■) («) absence and presence thein T^S]-sulfide foye. ahpit steaeaeo ulct samples. duplicate of average isthe point Each oxygen. of TIME TIME (MIN) 28 Table 2.4. Solubility of 35g accumulated by filaments of B. alba B18LD exposed to 35s_suifide. Samples of the culture were filtered through glass fiber filters and washed with three ml of solvent.

Solvent CPM / Filter % of Control

Basal salts, pH=3 77,921 100

Ethanol 5,526 7

Acetone 6,789 8

Benzene 7,371 9

10% Trichloroacetic 98,416 126 acid 30

Table 2.5. Effect of electron transport inhibitors on sulfide oxidation in B. alba B18LD.

Inhibitor3 Concentration % of control*3

Ethanol 1.0 % 99

TTFA 0.5 mM 24

8-HQ 2.0 mM 34

Phen 1.0 mM 24

HOQNO 50 uM 85

DBMIB 20 uM 85 60 uM 99 100 uM 90

KCN 1.0 mM 6

NaN3 1.0 mM 13

a See Table 2.2 for a list of inhibitor abbreviations, and the sovent used for each inhibitor. b Control rate for accumulation of 35g from Na235s: 53 nmol/min/mg protein. All values are the average of duplicate samples. 0 IS 30 45 60 75 90 10S TIME (MIN)

Figure 2.6. Endogenous and sulfide-stimulated oxygen consumption in whole cells of EL alba B18LD. Endogenous respiration was measured in cells containing ( X,) or lacking (▼ ) sulfur inclusions. The effect of sulfide on oxygen consumption was measured by adding 1 mM sodium sulfide at time 0 to cells containing (♦ ) or lacking ( ■ ) sulfur inclusions. The chemical oxidation of sulfide was measured by adding 1 mM sulfide to basal salts solution ( • ). 32

Discussion

Growth in Gradients

Many researchers have been unable to culture Beggiatoa autotrophically (Faust and Wolfe 1961; Scotten and Stokes 1962; Strohl and Larkin 1978; Nelson and Castenholz 1981a). Recently, Nelson and

Jannasch (1983) expanded on the efforts of Keil (1912) and Kowallik and

Pringsheim (1966) to duplicate the natural habitat of Beggiatoa. In so doing, they succeeded in culturing a marine strain of Beggiatoa autotrophically.

The adaptation of their technique for freshwater strains has resulted in the maintenance of cultures of strain B18LD for three months in autotrophic sulfide/oxygen gradients. Sulfide is apparently the sole energy source under these conditions, since elimination of sulfide prevents growth. Sulfide could not be replaced by titanium citrate, a redox dye that lowers redox potential, suggesting that sulfide does more than simply provide an optimal redox potential for growth of Beggiatoa filaments under these conditions.

The addition of acetate to the upper layer of the soft-agar cultures always resulted in a band of growth 2 mm from the air/agar interface, regardless of the contents of the lawer layer. This is most likely due to the decreased oxygen tension in the agar resulting from acetate-stimulated oxygen consumption. 33

Acetate Oxidation

The majority of Beggiatoa isolates are capable of heterotrophic

growth utilizing acetate as the sole source of carbon and energy

(Scotten and Stokes 1962; Pringsheim 1964; Strohl and Larkin 1978).

Some strains require additional nutrients, such as yeast extract, for

growth (Pringsheim 1964; Burton et al. 1966). Nevertheless, all

Beggiatoa strains tested to date are capable of heterotrophic growth,

including the chemolithoautotrophic strain isolated by Nelson and

Jannasch (1983).

A complete tricarboxylic acid cycle and glyoxylate pathway have

been detected in strain B18LD (Strohl; manuscript in preparation) and

are probably present in strain 75-2a (Nelson and Castenholz 1981b).

Additionally, an electron transport chain consisting of NADH

dehydrogenase, ubiquinone 8 , and cytochromes a, b, c, and £ have been

detected in strain B18LD (Strohl, Schmidt, and Larkin; manuscript in

preparation). Because all the components of a typical aerobic, heterotrophic bacterial metabolism are present, the following stoichiometry for the complete oxidation of acetate is proposed:

ch3COOH + 2 02 ------> 2 C02 + 2 H20

The rate of acetate-dependent oxygen consumption in cells of B18LD was 160 nmol/min/mg protein. The rate of release of 1i*C02 fr0m

[2-^^C]-acetate was 65 nmol/min/mg protein, which is in close agreement with the Vmgx value of 72 nmol/min/mg protein determined by Strohl et al. (1981b) for the release of 1i,C0 2 from 34

[2-^C ]-acetate. Because only one of the carbon atoms of acetate

was labelled in these experiments, the recovery of labelled CO^

should have been one-half of the amount of oxygen consumed, assuming

that both carbons are oxidized at a similar rate. The rate of oxygen

consumption was 2.4 fold greater than the rate of ^ C 0 2

generation. These values suggest that the initial oxidation of acetate

proceeds according to the proposed stoichiometry.

A variety of respiratory inhibitors are known that inhibit the oxidation of substrates by interfering with the action of a component of the electron transport chain (Table 2.2). The classical sites of

inhibition for these electron transport inhibitors in bacteria are shown in Figure 2.1. The results of the inhibitor experiments show that most of the inhibitors reduce the rate of acetate oxidation in cells of strain B18LD, as measured by either the rate of acetate-dependent oxygen consumption or the release of ^ C 02 from [2-^C]-acetate.

These levels of inhibition of respiration at the concentrations used are typical for aerobic chemotrophic bacteria (Heinen 1971).

Heterotrophic growth of cells of strain B18LD utilizing acetate as the sole carbon and energy source results in excessive deposition of poly-B-hydroxybutyric acid (PHB), accounting for up to 50% of the dry weight of cells grown heterotrophically on acetate (Strohl et al.

1981b). Deposition of PHB is usually an indication of unbalanced growth

(Shively 1974). Addition of sulfide to the growth medium alleviates the excessive deposition of PHB (Gifde et al. 1981). This effect may be the result of sulfide (i) acting as a supplemental energy source (Gude et al. 1981), (ii) providing an optimal redox potential for growth, (iii) 35

detoxifying metabolically formed hydrogen peroxide (Burton and Morita

1964; Nelson and Castenholz 1981a), or (iv) some combination of these effects.

Sulfide Oxidation

Because strain B18LD is capable of growth in autotrophic media in the presence of sulfide, but not in its absence, sulfide oxidation can apparently provide the energy required for growth. Since oxygen is required for the oxidation of sulfide, I propose that there is a transfer of electrons from sulfide to oxygen. When the rate of sulfur accumulation from sulfide and the rate of sulfide-dependent oxygen consumption were determined simultaneously, oxygen consumption was calculated to be 20 nmol/min/mg protein and sulfur accumulation was calculated at 38 nmol/min/mg protein. These values indicate the following stoichiometry for sulfide oxidation:

2 h2S + 02 ^ 2S° + 2 H2o

The rate of sulfide oxidation calculated from the amount of 35§ accumulated from Na2^ S varies between 38 and 380 nmol/min/mg protein. The rate is dependent upon the density of cells used in the experiment, the volume of medium in which the cells are resuspended relative to the flask size, and the rate of shaking. All of these factors are related to the amount of oxygen available to the cells.

Additionally, sulfide reacts chemically with oxygen, so that any sulfide added above the Vmax concentration would have the effect of lowering 36

the rate of sulfide oxidation by decreasing the amount of available

oxygen. There is also a reaction between Nag^s ancj iron that

is present in the basal salts solution. The resulting complex, FeS, is

black and can bind to extracellular polysaccharides, resulting in

artificially high measurements for the rate of sulfide oxidation.

The transfer of electrons from sulfide to oxygen may be coupled to

proton translocation across the cytoplasmic membrane via an electron

transport system as described in the chemiosmotic theory (Mitchell

1966). To test the possible involvement of specific electron transport

components, the inhibitors tested with acetate oxidation were employed to determine at what point electrons released from sulfide oxidation may enter the electron transport chain. The quinone analog DBMIB acts as a specific antagonist of ubiquinone function, inhibiting the

NADH-dependent reduction of cytochrome b (Poole et al. 1975; sun and

Crane 1976). Because DBMIB inhibited the oxidation of acetate, but not the oxidation of sulfide, ubiquinone 8 is probably not involved in the transfer of electrons from sulfide oxidation. In contrast to these results, quinones in Oscillatoria limnetica are an essential part of the electron transfer chain involved in sulfide oxidation (Oren and Padan

1977).

The other compounds tested are inhibitors of either metallo-proteins or flavins. These inhibitors suppressed the oxidation of sulfide in cells of strain B18LD, suggesting that flavin-linked metallo proteins (Bartsch et al. 1968) are involved in the oxidation of sulfide. A pathway in which electrons from sulfide oxidation enter at cytochrome c via a flavocytochrome, as found in Chromatium (Truper and 37

Fischer 1982; Gray and Knaff 1982), is a likely possibility for

Beggiatoa.

Alternatively, the electrons from sulfide oxidation may never cross the cytoplasmic membrane. Since cytochromes c and o are mostly soluble in cells of strain B18LD (Strohl, Schmidt, and Larkin; manuscript in preparation), the coupling of sulfide oxidation to oxygen reduction may be periplasmic. If this is the situation, a pH gradient could still be generated across the cytoplasmic membrane since two protons may be released in the periplasmic space during the oxidation of hydrogen sulfide to sulfur. In a similar situation, a pH gradient resulting from the periplasmic oxidation of ammonium is apparently coupled to energy production in cells of Nitrosomonas (Olson and Hooper 1983; Hooper

1984).

These two possible pathways for electrons released from sulfide oxidation are diagrammed in Figure 2.7. This model includes a representation of the periplasmic sulfur inclusions bounded by a proteinaceous envelope.

Mixotrophy

Mixotrophy refers to a type of metabolism in which simultaneous use is made of organic and inorganic sources for energy or carbon or both

(Kelly 1971). Since sulfide oxidation appears to support autotrophic growth of strain B18LD, it is possible that sulfide is a supplemental energy source for cells growing on acetate, as suggested by Gu’de et al.

(1981). In addition to the possibility for mixotrophic energy generation, there may also be mixotrophic carbon utilization with carbon 38

BEG GIATOA FP

,2H+

cell wall

HS'

Figure 2.7. Proposed model of sulfide oxidation by Beggiatoa FP - flavoprotein; Q8 - ubiquinone 8; cyt - cytochrome S° - sulfur contained within sulfur inclusion envelope. Electrons released from the oxidation of hydrogen sulfide might enter either the membrane-associated electron transport chain (i), or be coupled to the periplasmic reduction of oxygen (ii). In both cases, protons are released into the periplasm. 39

dioxide and acetate both providing a source for cellular carbon. A

mixotrophic metabolism may allow the organism, living in an nutrient

limited environment, to capitalize on any organic material that may be

present.

Strohl et al. (1981b) noted that sulfide inhibited the oxidation of

acetate, changing both the Vmax and Km of acetate oxidation.

This was offered as evidence for mixotrophic energy generation since the

addition of an alternative energy source, sulfide, was expected to

lessen the requirement for energy generated from acetate oxidation.

Sulfide inhibition of acetate oxidation was also detected in this study

(Fig 2.1U. However, it was also noted that acetate inhibited the

oxidation of sulfide (Fig 2.7). Since both acetate oxidation and

sulfide oxidation are linked to oxygen consumption, it is possible that

with cells concentrated approximately ten-fold, oxygen limited both

reactions. Oxygen limitation, as well as some sort of metabolic

regulation under mixotrophic conditions, could explain the differences

in the Vmgx an(j ^ Df acetate oxidation noted by Strohl et al.

(1981).

It is only under the microoxic conditions present in

sulfide/oxygen gradients that the autotrophic potential of Beggiatoa has

been observed (Nelson and Jannasch 1983). Likewise, the potential for

nitrogen fixation was observed only under gradient conditions (Nelson et

al. 1982). The oxidation of sulfur to sulfate has also been suggested based on growth in sulfide/oxygen gradients (Nelson and Jannasch 1983)

and by the observation that some filaments removed from their low redox

environment or enrichment cultures contained no sulfur (V/inogradsky 40

1888). The unique environment created in the sulfide/oxygen gradient is required for the expression of several physiological traits and may be required to delineate the physiological capacity of the organism, including its potential for mixotrophy. CHAPTER III

A COMPARISON OF SULFUR METABOLISMS IN BEGGIATOA ALBA B18LD

AND THIOTHRIX NIVEA JP3

Introduction

The filamentous bacterium, Thiothrix nivea, accumulates sulfur in sulfur inclusions when the cells are exposed to either sulfide or thiosulfate (Strohl and Schmidt 1984). The sulfur inclusions are membrane-bounded in filaments of Thiothrix (Bland and Staley 1978) as are the sulfur inclusions in cells of Beggiatoa alba B18LD.

Unlike 13^ alba B18LD, filaments of T^ nivea JP3 do not grow in the absence of reduced sulfur compounds (Strohl and Schmidt 1984), suggesting an obligately lithotrophic means of energy generation.

Strain JP3 is easily cultured in the laboratory in the ACS medium used for strain B18LD (Chapter II). These two strains of filamentous sulfur bacteria are used in this study to compare the rates of accumulation of sulfur from sulfide and thiosulfate, and the oxidation of sulfur to sulfate.

41 42

Materials and Methods

Strains and Growth conditions

Thiothrix nivea strain JP3 (courtesy of John Larkin, Louisiana

State University) and Beggiatoa alba strain B18LD were grown in ACS medium as described in chapter II.

Oxidation of sulfide and thiosulfate

Cells of strain B18LD or strain JP3 were harvested from mid-exponential phase cultures by centrifugation at 8,000 x g for 10 min. The resulting cell pellets were washed once in BSS (Chapter II) and resuspended to a density of approximately 0.2 mg protein/ml in BSS.

Ten ml of the concentrated cell suspension were dispensed into 50 ml

Ehrlenmeyer flasks. The oxidation of sulfide to sulfur was measured after adding [35s]-sulfide (2 ^iCi/5 umol) to a final concentration of 2 mM, and removing 200 jul aliquots at timed intervals. The aliquots were filtered through Gelman GA-3 glass fiber filters (Gelman Sciences,

Inc. Ann Arbor, MI) and washed with 2 ml of either BSS (pH 3), 95% ethanol, benzene, acetone, or trichloroacetic acid (TCA).

Sulfide- or thiosulfate-dependent oxygen consumption was measured with the Warburg respirometer at 30 C. The center well of the respirometer flasks contained pleated filter paper saturated with 250 ;ul of 20% potassium hydroxide. Three ml of a washed and concentrated cell suspension, prepared as described above, were dispensed into the flasks 43

and 333 >ul of reduced sulfur compound (10 mM) were added to the sidearms of specified flasks. The flasks were attached to manometers; after a 15 min incubation at 30 C, the contents of the sidearm were added to the cell suspension. Manometric readings were recorded every 15 min.

Flasks containing 3 ml of twice distilled water and 333 ^Jl of distilled water in the sidearm were treated the same as flasks containing cells, these flasks were used as thermobarometers.

Oxidation of cellular sulfur

Filaments of strain B18LD or strain JP3 were harvested from exponential phase cultures in ACS medium and washed once in sterile BSS.

In an attempt to deplete the cellular reserves of sulfur, these cells were resuspended in BSS to the volume from which they were harvested, and incubated at 23 C on a rotary shaker at 150 rpm. After twelve hours, the cells were examined microscopically for the presence of sulfur inclusions and prepared for respirometry as previously described.

Cells that were harvested from ACS media and not starved for sulfur were also prepared for respirometry.

The production of sulfate by sulfur-containing filaments of strain

JP3 was measured during one of the respirometry experiments. At 30 min intervals, two Warburg flasks were removed from the respirometer and the contents were assayed for the presence of sulfate by the barium chloride turbidometric method (Faber et al. 1955). The concentration of sulfate determined by this method in the presence of large quantities of organic material is not absolute and should be considered as a relative measurement (Faber et al. 1955). 44

The oxidation of cell-bound sulfur was also measured isotopically after growing cultures of strain B18LD and strain JP3 in ACS medium containing 1 mM [35s]_sulfide (2 pCi/ 5 umol). Cells harvested from the radioactive medium were washed twice in basal salts and resuspended to their original denisty in oneof the following: BSS containing sulfate salts (Appendix 1), BSS containing chloride salts instead of sulfate salts (Appendix 2), or BSS (- sulfate) + 1 mM sodium sulfide. All solutions were adjusted to pH 7.2. Four 200 ul samples were taken from each flask during a 22-hour period. Two of the samples were filtered through Gelman glass fiber filters and washed with 2 ml of

BSS at pH 3.0. The radioactivity on the filters was counted as described in Chapter II. The other two samples were centrifuged for 2 min in a microfuge, then 100 ul of the supernatant were added to a toluene-based scintillation cocktail containing 5 g/1 PP0, and 50 mg/1

P0P0P in 33% Triton X-100 in toluene (Patterson 1965) and counted as described in Chapter II. 45

Results

Microscopic observation of filaments of Thiothrix nivea JP3 grown

in ACS medium revealed the presence of numerous refractile granules, similar to the sulfur inclusions in filaments of Beggiatoa alba B18LD.

These inclusions were depleted from filaments of strain JP3, but not from filaments of strain B18LD, after being harvested from ACS media, washed, and incubated in basal salts for twelve hours. Sulfur inclusions in filaments of strain B18LD were visible 48 hours after the cells were removed from sulfide, at approximately the same concentration as was initially present.

Upon exposure to [35s]-sulfide, Thiothrix filaments depleted of sulfur inclusions accumulated radioactivity at a rate similar to cells of B. alba B18LD (Fig 3.1). This radioactivity was readily soluble in

95? ethanol, acetone, carbon disulfide, or benzene, but not 10% TCA or

BSS at pH 3 (Table 3.1). The oxidation of both sulfide and thiosulfate by sulfur-starved trichomes of strain JP3 resulted in constant rates of oxygen consumption (Fig 3.2).

Sulfur-containing filaments of nivea JP3 consumed oxygen at a constant rate when resuspended in BSS, whereas the endogenous respiration of sulfur-starved filaments of strain JP3 was negligible

(Fig 3.3). The oxidation of intracellular sulfur was confirmed by isotopic measurement of the release of 35g from radiolabeled sulfur inclusions. Filaments of strain JP3 or strain B18LD were suspended in

BSS and the change in the concentration of radioactivity in the cells Figure 3.1. Cellular accumulation of 35s from 35s_suifi

JJMOL S INCORPORATED / MG PROTEIN pH = 3. Each pont is the average of duplicate samples. duplicate of average isthe pont Each 3.= pH T. nivea JP3 (° ). Cells were collected on glass on collected Cellswere ). (° JP3 nivea T. ie fles n ahd3 ih2m fbsl salts,basalof 2 ml 3xwith andwashed filters ( fiber B18LD alba ■ ) and of trichomes by IE (MIN) TIME 47

Table 3.1. Solubility of 35$ accumulated by filaments of T. nivea JP3 exposed to 35s sulfide. Culture samples were filtered through glass fiber filters and washed with 3 ml of solvent.

Solvent cpm/filter % of control

Basal salts (pH 3) 78,513 100

Ethanol 9,148 12

Acetone 7,806 10

Benzene 10,676 14

Carbon disulfide 8,217 1°

Trichloroacetic 134,178 171 acid Figure 3.2. Sulfide- and thiosulfate-dependant oxygen consumption by consumption oxygen thiosulfate-dependant and Sulfide- 3.2. Figure JUL 02 CONSUMED / MG PROTEIN 100 120 0 esrdfrflmns upne i aa salts inbasal suspended filaments for measured fe h diino 1 Msdu ufd ( sulfide 1sodium mM ■of addition ) the or after filaments of nivea JP3. Oxygen consumption was consumption Oxygen JP3. nivea of filaments m oimtislae ) t 45min. (at thiosulfate sodium 1*) mM 53 45 30 15 TIME (MIN) TIME 60 75 SO 105 120 (♦), 48 Figure 3.3. Endogenous respiration of filaments of filaments Endogenousrespirationof T. JP3.nivea 3.3.Figure

JLIL 02 CONSUMED / FLRSK 200 2S0 300 350 100 ISO 400 50 - f10Ketuis (■)200 units or100 Klett units Klett of Filaments lacking sulfur (♦ ) (♦ control.asasulfurused lacking were Filaments Sulfur-containing filaments were suspended toa suspended density filamentswere Sulfur-containing 30 60 IE (MIN) TIME 30 120 150 (A). 180 49 50

and in the medium was monitored. Cells of strain JP3, but not strain

B18LD, released 35g into the medium from the radioactive sulfur inclusions (Fig 3.4). The release of radioacitivity was independent of the presence of 1 mM sulfide or sulfate in the basal salts solution (Fig

3.4). None of the conditions tested resulted in the release of 35s from filaments of alba B18LD.

At least one of the products of sulfur oxidation in strainJP3 is sulfate. Chemical measurments of sulfate detected a steady increase in sulfate in the medium over a four hour time period (Fig 3.5). Figure 3.4. Release of 35s from cells of 1\ nivea JP3 nivea 1\ of cells from 35s Releaseof 3.4.Figure

CPM (X10 ) / 0.2 ML SAMPLE solution at pH 3.pH at solution filtered (■) were sulfide + BSS (▼),and sulfate - BSS ngasfbrflesadwse ihbsl salts basal with andwashed filters fiber glasson (x),sulfate+ BSS from cells of Samples (sulfide ■). uft (▼, rbsl at otiig uft n 1 mM and sulfate containing salts (basal or sulfate ▼), sulfate containing salts intervals timed at removed were themedium and cells the of Samples inclusions. sulfur radiolabeled containing fe el f ie eerssedd inbasal resuspended were nivea of aftercells ells C TIME (HOURS) TIME Medium ( ) x ( aa saltswithout basal 51 0 80 60 80 120 180 180 TIME (MIN)

Figure 3. Oxygen consumption (* ) and sulfate production ( ■ ) by sulfur-containing filaments of T . nivea JP3 suspended in basal salts solution. 53

Discussion

Descriptions of filaments of Thiothrix removed from their natural

environment or from laboratory enrichments have always included a

description of refractile inclusions recognized as sulfur globules

(Farquar and Boyle 1971; Merkel 1975; Larkin and Shinabarger 1983). In

this laboratory, axenic cultures of Thiothrix nivea strain JP3 were maintained on AC medium supplemented with either sodium sulfide or

sodium thiosulfate. When grown in the presence of either sulfide or thiosulfate, filaments of JP3 contained refractile inclusions similar to the sulfur inclusions in filaments of Beggiatoa alba. The inclusions

in strain JP3 have been identified as sulfur globules based on the solubility of the inclusions in ethanol, acetone, benzene, and carbon disulfide. Elemental sulfur is soluble in each of these solvents

(Windolz 1976). The rate of oxidation of sulfide to sulfur is similar in filaments of Thiothrix JP3 and Beggiatoa B18LD.

Sulfur inclusions in filaments of Thiothrix were not visible 12 hours after the removal of the cells from sulfide-containing medium, suggesting that the sulfur is further metabolized. The accumulated sulfur in strain JP3 filaments is evidently oxidized to sulfate, regardless of the presence of 13.5 mM sulfate in the medium or the presence of 1 mM sulfide in the medium. Since the assay for sulfate is influenced by the presence of organic carbon, a stoichiometry between sulfur oxidation, sulfate production, and oxygen consumption could not be determined. 54

The sulfur inclusions in alba B18LD filaments are visible for a minimum of 72 hours after removal from sulfide-containing medium. The lack of sulfur oxidation in cells of strain B18LD is also supported by the rates of oxygen consumption measured for strain B18LD filaments containing or lacking sulfur inclusions. Whereas the rate of oxygen consumption by cells of T^ nivea JP3 was dramatically increased when the cell contained sulfur inclusions, the presence of sulfur inclusions in strain B18LD filaments did not affect the rate of oxygen consumption.

Filaments of B.alba B18LD containing 35g sulfur inclusions released an undectetable amount of radioactivity in the experiments reported herein; in a separate experiment only 3% of the label from radiolabeled sulfur inclusions was released during a three day incubation in heterotrophic medium (W.R. Strohl; unpublished results). The lack of sulfur oxidation by strain B18LD filaments under the conditions tested is presumably due to the lack of required enzyme activity that may result from; (i) enzyme inhibition; (ii) repression of enzyme synthesis; or (iii) lack of genes encoding one or more of the enzymes. The inability of strain B18LD cells to oxidize sulfur leaves a potential source of electrons untouched. The stored sulfur can, however, be used as a terminal electron acceptor under anoxic conditions (Chapter II); this anaerobic capacity may be the reason that sulfur is stored and not readily oxidized to sulfate in strain B18LD filaments.

The cellular oxidations of sulfide, thiosulfate, and sulfur are all linked with oxygen consumption in T^ nivea JP3. These reactions apparently supply energy for the growth of cultures of strain JP3. All attempts to culture Thiothrix in the absence of a reduced sulfur source 55 have been unsuccessful (Strohl and Schmidt 1984; Larkin and Shinabarger

1983). This lack of growth indicates the obligatory operation of a lithotrophic energy generating system. Autotrophic growth of Thiothrix filaments, however, has not been observed. As with Beggiatoa, the potential for chemolithoautotrophic growth may only be expressed under stringently defined environmental conditions. CHAPTER IV

ENDOGENOUS AMD HYDROGEN-STIMULATED REDUCTION OF SULFUR

BY BEGGIAOTA ALBA B18LD UNDER ANOXIC CONDITIONS

Introduction

The dynamic microenvironment of Beggiatoa demands that the organism

is metabolically versatile. One of the possible requirements for growth

in an environment of changing sulfide and oxygen gradients is the

ability to conserve energy in both the presence and absence of oxygen.

The growth of Beggiatoa in the presence of oxygen is well documented

(Chapter I). There is little evidence, however, to suggest that

Beggiatoa can grow in the absence of oxygen. If Beggiatoa filaments

must survive periods of anoxia, or glide actively back to an oxic zone,

energy must be conserved anaerobically. Since neither nitrate nor

nitrite can serve as an anaerobic electron acceptor (Vargas 1984, MS

thesis, Ohio State Univ.), another terminal electron acceptor must be used.

The coupling of the oxidation of NADH or hydrogen to the reduction of sulfur to sulfide is a thermodynamically feasible reaction (Pfennig

and Bieble 1976) that may provide Beggiatoa filaments with energy under

anoxic conditions. This study tests the anaerobic potential of cells of

B. alba B18LD.

56 57

Materials and Methods

Strains and Growth Conditions

Beggiatoa strains B18LD, B25RD, and B15LD (Mezzino et al. 1984),

75-2a (Nelson and Castenholz 1981a), and SM-1 (gift from Dr. Siegfried

Maier, Ohio University), and Vitreoscilla strains B23SS (Minges et al.

1983) and ATCC 15551 were grown in AC or ACS medium as described in

Chapter II. Chromatium vinosum (gift from Dr. Riccardo Guerrero) was grown under conditions in which sulfur accumulated in the cells

(Guerrero et al. 1984).

Microelectrode Studies

A 5% inoculum of strain B18LD was introduced into ACS media. An inoculum of this size results in the formation of visible tufts during early exponential growth. These tufts were removed from the medium, rinsed gently in BSS (Chapter II), and embedded in a 2% agar cube measuring approximately 3 mm per side. The cube was pierced by thin glass rods that were anchored to the bottom of a mixing bowl (Fig 4.1).

The suspended agar cube containing the Beggiatoa tuft was perfused with basal salts. Air, nitrogen, or hydrogen was bubbled through the salts solution at room temperature, 23 C.

Microelectrodes were positioned with a micromanipulator to measure sulfide and oxygen gradients around and through a tuft of strain B18LD cells embedded in agar, while the reaction vessel was bubbled with air.

The electrodes were then relocated at the surface of the tuft of cells 58

MICRO- ELECTRODES

BSS CELLS

Figure 4.1. Microelectrode apparatus. Air, nitrogen, or hydrogen was bubbled through the basal salts solution (BSS) in which the embedded cell pellet was immersed. A micromanipulator was used to position the microelectrodes. 59

in the agar cube and either nitrogen or hydrogen was bubbled through the

reaction vessel.

Measurements of sulfide production

Anaerobic sulfide production by cells of strain B18LD and

Chromatium vinosum was measured using the bubbling device diagrammed in

Figure 4.2. This apparatus permitted the continual removal of sulfide

so that the accumulation of sulfide would not interfere with the cell metabolism.

To measure sulfur reduction to sulfide, cultures of strain B18LD

grown in either AC or ACS medium were harvested from mid-exponential

phase growth, washed in BSS, and resuspended to their original density

in BSS. Nitrogen was flushed through the system, and the outflowing gas was bubbled through two test tubes each containing 10 ml of 2% zinc acetate to bind any sulfide produced. The zinc acetate tubes were changed every hour during the course of the experiment, and the concentration of sulfide bound by zinc acetate was determined by the methylene blue method of Kline (1969).

Cultures of Cj_ vinosum grown in Pfennig's media (Pfennig and Triiper

1981) to mid-exponential phase were transferred directly to the bubbling device, and residual sulfide was removed by flushing the system with nitrogen. The reaction vessel was illuminated with a 100 watt incandescent bulb positioned 30 cm from the reaction vessel. Once sulfide was no longer detected in the zinc acetate solution, as determined by the methylene blue assay, the culture vessel was wrapped with aluminum foil and the dark, anaerobic production of sulfide was 60

measured in the manner previously described for EL alba B18LD.

Hydrogen evolution and uptake assays

To measure hydrogen evolution, exponentially growing filaments of

Beggiatoa and Vitreoscilla were harvested by centrifugation, washed in

sterile basal salts, and concentrated approximately ten-fold in basal

salts containing 25 mM HEPES buffer. The concentrated cell suspension

contained from 0.1 to 0.3 mg protein/ml. Protein concentration was

determined by the method of Lowry et al. (1951), after extraction of sulfur with 95% ethanol for 1 hour, and after heating the samples at 90

C in 1N NaOH for 10 min to digest the cells.

Two ml of the concentrated cell suspension were dispensed into 9 ml serum bottles (Wheaton Scientific, Millville, NJ) and sealed with rubber stoppers and aluminum caps. Forty jul of 100 mM methylviologen in 10 mM phosphate buffer, pH 8.0, were added to each flask. Where designated, carbonylcyanide-£-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, was added to the concentrations shown in Table 4.2. The bottles were flushed with nitrogen for 15 min and the hydrogen evolution assay was initiated by the addition of 0.1 ml of 100 mM sodium dithionite (in distilled water) to a final concentration 10 mM. The bottles were shaken at 120 strokes per minute at 23 C in a metabolic shaker.

Headspace gas samples of 100 ul were withdrawn from the bottles every 30 min and analyzed by injecting into a Varian Aerograph 3700 gas chromatograph equipped with a 3 m stainless steel column packed with molecular sieve 5 X (30-40 mesh). The following temperatures were used: injector, 100 C; column, 30 C; thermal conductivity detector, 150 C; 61

and filament, 300 C. Nitrogen, at a flow rate of 30 cc per min, served as the carrier gas. The output from the gas chromatograph was recorded on a Model 252A strip chart recorder (Linear Instruments Corp., Costa

Mesa, CA). Peak heights were measured and compared to a standard curve prepared from the heights measured for hydrogen standards in nitrogen.

Hydrogen uptake was measured by dispensing 1.5 ml of the concentrated cell solution into 9 ml serum bottles. After flushing with nitrogen for 15 min, of the headspace was replaced with 1 atm hydrogen. The headspace was sampled at 60 min intervals and analyzed by gas chromatography as previously described. 62

Results

Microelectrode studies

While air was bubbled around a tuft of Beggiatoa alba strain B18LD embedded in an agar cube (Fig 4.1), the endogenous metabolism lowered the concentration of dissolved oxygen to an undetectable level at the surface of the cell pellet. At the point where the oxygen concentration neared zero, sulfide was detected and reached a concentration of 10 uM at a distance of 50 urn inside the cell pellet (Fig 4.2).

When nitrogen, instead of air, was bubbled through the microelectrode apparatus, a constant rate of sulfide production was measured at the surface of the cell pellet (Fig 4.3). When the nitrogen was replaced with hydrogen an increased rate of sulfide production was observed. This increased rate was detected approximately four minutes after the addition of hydrogen (Fig 4.3).

Endogenous anaerobic respiration

In the bubbling apparatus, designed to provide short term anoxic conditions with the continual removal of sulfide (Fig 4.4), sulfide was produced at a rate of 6.7 nmol/min/mg protein by sulfur-containing trichomes of strain B18LD (Fig 4.5). Trichomes that lacked sulfur inclusions did not produce any detectable sulfide over a period of four hours (Fig 4.5). Sulfide was produced at a rate of 5.0 nmol/min/mg protein by cells of Chromatium vinosum, a purple photosynthetic iue .. ireetoesuiso xgn * adslie («) (*)sulfide and oxygen of studies Microelectrode 4.2. Figure

SULFlDE:OXYGEN U iM I 30 hs aust h ih ersn pstosi h agar.the in positions represent right the to values those zero indicate a position inside the cell pellet, while while cellpellet, the inside aposition indicate zero inurn Distances which tuftofthe aremeasured edge outer the from B18LD. alba Beggiatoa of cells of tufta of metabolism endogenous the from resulting gradients s eintd as is designated DISTANCE

100

150 20 25 30 35 40 63 Figure 4.3. Effect of nitrogen (♦ ) and hydrogen (* ,■) on sulfide onsulfide ,■) (* hydrogen ) (♦ and nitrogen of Effect 4.3. Figure SULFIDE (UM) 10 12 14 8 0 the time indicated by the arrows. the by indicated time the the apparatus. Nitrogen was replaced with hydrogen at hydrogen with replaced was Nitrogen apparatus. the surface of the tuft while nitrogen was bubbled through bubbled was nitrogen while tuft theat the of positioned surface was electrode sulfide The agar. in embedded B18LD strain of cells of tufts by production 2 6 IE (MIN) TIME 8 10 12 14 618 16 64 65

C 3 zn-acetate

Figure 4.4. Apparatus for measuring the anaerobic reduction of sulfur to sulfide. The arrow indicates the direction of gas flow. Figure 4.5. Anaerobic reduction of intracellular sulfur to sulfide to sulfur intracellular of reduction Anaerobic 4.5. Figure

UMOLES S s PRODUCED / MG PROTEIN 1.2 1.4 1.6 1.8 .2 .4 .6 .8 0 1 0 . lacnann () n lcig * sulfur. (*) lacking and (♦) containing alba B. ( vinosum ■Chromatium ofof cellsby )filaments and 60 IE (MIN) TIME 2 180 120 240 66 67

bacterium (Fig 4.5).

Anaerobic Hydrogenase Activity

A survey of several strains of Beggiatoa revealed the presence of hydrogenase activity in all strains tested (Table 4.1). Two strains of the closely related genus, Vitreoscilla, had similar hydrogenase acitivities. FCCP at concentrations up to 5 x 10“5 m did not reduce the level of hydrogen production by strain B18LD filaments (Table

4.2). Hydrogen production was detected only in the presence of both methylviologen and dithionite. Cells of strain B18LD exposed to sulfide alone, or in combination with either methylviologen or dithionite, produced no hydrogen.

Anaerobic hydrogen consumption by strain B18LD was measured directly by gas chromatography (Fig 4.6). Trichomes lacking sulfur inclusions showed no consumption of hydrogen. Sulfur-containing trichomes consumed hydrogen at a constant rate of 7.91 nmoles/min/mg protein. The uptake of hydrogen by boiled cells was undetectable. 68

Table 4.1. Hydrogenase activities in strains of Beggiatoa and Vitreoscilla. Activity was calculated from the in vitro assay of methylviologen-dependent hydrogen production.

Hydrogen produced Genus Strain (nmol/hr/mg protein)

Beggiatoa B18LD 151

B25RD 365

B15LD 292

75-2a 400

SM-1 131

Vitreoscilla ATCC 15551 137

B23SS 434 69

Table U.2. Effect of FCCP dissolved in DMSO on methylviologen-dependent hydrogen evolution by whole cells of B. alba B18LD.

Concentration of FCCPa Hydrogen evolution ( M ) (nmol/hr/mg protein)

1 x 10-6 189

1 x 10"5 179

5 x 10“5 200

DMSQb alone 188

a FCCP: Carbonylcyanide-p-trifluoromethoxyphenylhydrazone b DMSO: Dimethylsulfoxide 0 60 120 160 240 300 TIME (MIN)

Figure 4.6. Hydrogen consumption by filaments of B. alba B18LD. Boiled cells that contained ( ► ) or lacked (♦ ) sulfur were used as a chemical control of hydrogen uptake. Measurements of hydrogen consumption by sulfur-containing cells (•* ) and cells lacking sulfur ( ■ ) began 60 min after the addition of hydrogen to allow for the equilabration of hydrogen between the headspace and the medium. 71

Discussion

Sulfide is a lithotrophic energy source for at least one marine strain of Beggiatoa (Nelson and Jannasch 1983). The freshwater strain,

B18LD, also appears to demonstrate sulfide-dependent autotrophic growth, indicating that sulfide is a lithotrophic energy source (Chapter II).

Sulfur accumulates in periplasmic inclusions when Beggiatoa filaments are exposed to sulfide (Strohl et al. 1981, Strohl et al.

1982). Whereas the stored sulfur might be oxidized to sulfate, this additional step has not been observed in cells of strain B18LD (Chapters

II and III). It is possible that the oxidation of sulfur by filaments of strain B18LD requires specific environmental conditions such as the microoxic conditions present in sulfide/oxygen gradients.

In the absence of oxygen, the sulfur stored by strain B18LD filaments is mobilized by the cells and reduced to sulfide. Filaments lacking sulfur inclusions produced no sulfide, discounting the possibility that the sulfide is a product of sulfate reduction or the degradation of sulfur-containing proteins. Anaerobic sulfide production by Beggiatoa was first observed in strain 75-2a by Nelson and Castenholz

(1981a) and was thought to be the means by which filaments of that strain survived a period of several days in the absence of oxygen. It is now evident that anaerobic pathways for the reduction of sulfur to sulfide do exist in cells of B^_ alba B18LD. It remains unknown, however, whether the anaerobic pathways are linked to energy conservation. 72

Both the aerobic and anaerobic metabolisms of Beggiatoa can be

studied with microelectrodes. Microelecrode studies on tufts of

Beggiatoa B18LD embedded in agar and flushed with air show that the

cells consumed oxygen. This was apparently due to the endogenous

oxidation of stored carbon, since no other substrate was readily

available to the cells in these experiments. A constant rate of

endogenous oxygen consumption has been measured for cells of strain

B18LB (Chapter II). Carr et al. (1967) also noted a high rate of

endogenous oxygen consumption in a Pringsheim strain of Beggiatoa. At the point where the oxygen concentration was lowered to undetectable

levels near the embedded tuft of B18LD filaments, sulfide was detected.

Sulfur had evidently replaced oxygen as the terminal electron acceptor.

The rate of anaerobic sulfur reduction in strain B18LD is similar to the dark anaerobic rate measured for the purple sulfur bacterium,

Chromatium vinosum. Cells of Chromatium remain motile during periods of darkness (van Gemerden 1968); it has been proposed that the catabolism of stored polysaccharides (glycogen) to pyruvate via the

Embden-Meyerhoff pathway provides the ATP necessary for cell maintenance and motility. The reduction of pyruvate to PHB would then occur via acetyl Co-A (van Gemerden 1968). One fourth of the NADH produced in the breakdown of glycogen was consumed in the formation of PHB, with the remainder used in the reduction of sulfur to sulfide (van Gemerden

1968). This metabolic route is one of a number of possible pathways that might be utilized for the anaerobic reduction of sulfur in strain

B18LD.

With microelectrodes positioned at the surface of a tuft of B. alba 73

B18LD, a constant rate of sulfide production was detected when nitrogen

was bubbled through the medium. Because the rate of sulfide production

was stimulated by substituting hydrogen for nitrogen, the coupling of

hydrogen oxidation and sulfur reduction was investigated. Hydrogen

consumption was detected only when strain B18LD filaments contained

sulfur, suggesting that the consumption of hydrogen was linked to sulfur

reduction. The coupling of hydrogen oxidation to sulfur reduction has

been observed in cells of Desulfovibrio (Fauque 1979) and is also

evident in cells of Chlorobium (Paschinger et al. 1974).

Hydrogenase activity was observed in several strains of Beggiatoa

as well as in two strains of the closely related genus Vitreoscilla.

This activity was detected when the artificial electron donor,

methylviologen, was present in combination with dithionite. Hydrogen

production can be due to the enzyme nitrogenase as well as hydrogenase.

However, cells of strain B18LD possessed hydrogenase activity after they were grown in the presence of 5 mM N H^d „hich inhibits nitrogenase

in Beggiatoa (Nelson et al. 1982). Hydrogen production in strain B18LD was resistant to treatment with the uncoupler FCCP. Since hydrogen

production by nitrogenase is energy-dependent, FCCP should eliminate the

production of hydrogen by nitrogenase by inhibiting energy conservation.

For these reasons, methylviologen-dependent hydrogen production has been

attributed to the enzyme hydrogenase and not nitrogenase.

There was no production of hydrogen by cells of B^ alba in the

absence of both methylviologen and dithionite. Neither of these

compounds, alone or in combination with sulfide, led to the production

of hydrogen. Unlike the condition existing in cells of Oscillatoria 74

limnetica (Belkin and Padan 1978), addition of sulfide to the cell

suspension did not result in hydrogen production. Because no hydrogen

was produced in the presence of sulfide it is likely that the enzyme in

B. alba B18LD is an uptake hydrogenase.

The hydrogenase of strain B18LD appears to be coupled to the

reduction of intracellular sulfur to sulfide. In preliminary

experiments, it was also shown that the hydrogen consumption can be coupled to the reduction of oxygen. It is not known if hydrogen is available in the natural habitat of Beggiatoa. Since several strains of

Beggiatoa, including strain B18LD, contain nitrogenase (Nelson et al.

1982), the hydrogenase may function in recycling hydrogen produced by the side reaction of nitrogenase. The relationship between hydrogenase and nitrogenase in Beggiatoa may be of economic as well as academic importance. Hollis (1979) recognized the potential for Beggiatoa spp. to provide fixed nitrogen in the rhizosphere of rice plants, an ideal habitat for Beggiatoa (Joshi and Hollis 1977; Hollis 1979).

The potential for anaerobic sulfur metabolism shown in this chapter and the aerobic accumulation of sulfur discussed in Chapter II have led to the development of the model for sulfur metabolism presented in

Figure 4.7. Sulfur, which accumulates in the protein-bounded sulfur inclusions (Chapter V), can be mobilized and used as an electron acceptor for the anaerobic oxidation of endogenous carbon reserves and for the consumption of hydrogen.

This metabolic flexibility may be essential to an organism that exists in a changing environment such as that of Beggiatoa. While the organism appears to grow best in the presence of low concentrations of 75

BEGGIATOA

NADH

NAD ,cyt 2H+ cyt c ,cyt o

call membrane cell wall

Figure 4.7. Model of sulfur metabolism in Beggiatoa alba. NAD - nicotine adenine dinucleotide; for other abbreviations see Figure 2.8. The oxidation of hydrogen and of NADH are shown coupled to the reduction of sulfur. 76

oxygen, it is capable of survivng at least short periods of anoxia. In nature this may be the means by which filaments produce maintenance energy as well as the energy required to return to the oxic/anoxic interface. 77

CHAPTER V

PROTEIN SYNTHESIS BY BEGGIATOA ALBA B18LD IN THE PRESENCE

AND ABSENCE OF SULFIDE

Introduction

The filamentous gliding bacteria of the genus Beggiatoa are capable

of prototrophic growth on acetate (Strohl et al. 1981a), but are

characterized by the ability to oxidize hydrogen sulfide to sulfur. The

sulfur accumulates in cellular inclusions that are external to the cyto­

plasmic membrane, but internal to the complex cell wall (Lawry et al.

1981; Strohl et al. 1981a). Electron microscopy of B. alba B18LD

filaments has revealed that these sulfur inclusions are enclosed by an

electron-dense envelope approximately 3 nm thick (Lawry et al. 1981).

Noting that sulfide oxidation is not required for the growth of

Beggiatoa, Minges et al. (1983) set out to determine whether the genes

coding for proteins involved in sulfide oxidation are plasmid-encoded.

They were unable to demonstrate a correlation between the presence of

any plasmid and the ability to oxidize sulfide, but Sprouse et al.

(1984) reported the sulfide-induced amplification of two plasmids in

B18LD filaments.

In the present study, the rates of sulfide oxidation and protein

synthesis were measured in cells growing in the presence and absence of sulfide. Total cell protein, sulfur inclusion envelope protein, and sulfide-induced proteins were analyzed by gel electrophoresis. 78

Materials and Methods

Organism and growth conditions

Beggiatoa alba strain B18LD was used in all phases of this study.

It was cultured in the presence of sulfide (ACS medium) or its absence

(AC medium). The media and the basal salts solution (BSS) used for these experiments are described in Chapter II.

Sulfide oxidation

Heterotrophically grown cells of strain B18LD were harvested from exponential growth in 1 1 of AC media by centrifugation at 8,000 x g for

10 min. The cells were resuspended in 1 1 of BSS and two 500 ml volumes of this culture were placed into two 1 1 flasks. To test the effect of a 2 h sulfide incubation on the cells1 ability to oxidize sulfide, 2 mM sodium sulfide (pH 7.2) was added to one of the flasks and the cultures were incubated at 25 C for 2 h. The cells were harvested from the BSS cultures at 8,000 x g for 10 min, and the resulting cell pellets were resuspended to a density of approximately 0.2 mg protein/ml in BSS.

Fifteen ml of the concentrated cell suspensions from both cultures were added to 50 ml Ehrlenmeyer flasks. Sodium[35s]_ sulfide (8.25 jjCi/20 pmol/ml; pH 7.4) was added to a final concentration of 2 mM to each flask. Samples of 200 ul were removed at timed intervals and filtered through Gelman glass fiber filters. The filters were washed with 3 ml of ESS at pH 3, dried at 60 C overnight and counted as 79 previously described (Chapter II).

Six ml samples of the sulfide-induced and -uninduced concentrated cell pellets were added to 50 ml Ehrlenmeyer flasks to test the effect of chloramphenicol (CAP) on sulfide oxidation. CAP was added to a final concentration of 1 /Jg/ml 15 min before the addition of labeled sulfide to the cultures. Aliquots of 200 jul were removed every 10 min, then filtered and counted as previously described.

Incorporation of [l)3H]-leucine into proteins

The rate of protein synthesis in whole cells of strain B18LD was estimated by measuring the incorporation of [U-3H]-leucine into

TCA-precipitable macromolecules. Strain B18LD trichomes were harvested from either AC or ACS medium during late exponential phase, washed, and resuspended in 10 ml of either AC media or BSS to a density of approximately 0.3 mg protein/ml. Ten ml of the concentrated cell suspensions were dispensed into 50 ml Ehrlenmeyer flasks in a Dubnoff metabolic shaker at 23 C. Twenty-five ;j1 of [U-3H]-leucine (200 pCi/ 4 ^imol/ml) were added to each flask to begin the experiment. The effect of sulfide on the rate of incorporation of leucine into

TCA-precipitable material was determined by adding freshly prepared and neutralized sodium sulfide to a final concentration of 1 mM, 15 min prior to the addition of the labeled leucine. When included, additions of chloramphenicol were made 10 min prior to the addition of the labeled leucine. At timed intervals, 100 jul samples were removed from the reaction mixture to an equal volume of 10?. trichloroacetic acid (TCA) and kept on ice for 2 h. These samples were filtered through Gelman 80

GA-3 glass fiber filters (Gelman Science, Inc. Ann Arbor, MI) and were

washed sequentially with 2 ml of: (i) 5% ice cold TCA, (ii) 70% ice cold

ethanol, (iii) 70% ethanol (45 C), (iv) diethyl etherjethanol (1:1; 45

C) as described by Hanson and Phillips (1981). Assimilation of labeled

leucine into whole cells was measured by filtering 100 pi samples through glass fiber filters and washing with 2 ml of BSS at pH 3. In both cases, the filters were placed into scintillation vials, dried at

60 C overnight, and counted in a toluene-based scintillation cocktail

(Chapter II).

SDS-PAGE of total cellular protein and enriched sulfur inclusions

Enrichments for the sulfur inclusion envelope from strain B18LD were obtained from cells grown in ACS medium. Trichomes were harvested from 5 1 of a late log phase culture and resuspended in 10 ml of 100 mM phosphate buffer (pH 7.2) containing 2 mM EDTA. The cells were broken by sonication (3 x 30 sec bursts) with a Fischer Model 300 Sonic

Dismembrator, then incubated for 20 min at 37 C in the presence of 2.0 mg/1 lysozyme, 1.0 mg/ml phospholipase C, and 0.5 mg/ml DNAse. The sulfur inclusions were sedimented from this mixture by centrifugation at

5,000 x g for 10 min. The sulfur inclusions were resuspended in 100 mM phosphate buffer (pH 7.2) , followed by a 5-sec sonication, and then centrifuged at 5,000 x g for 5 min. The sulfur inclusion enrichments were washed four additional times with BSS. The pellet of sulfur inclusions was boiled for 2 min in 2% SDS containing 2-mercaptoethanol and glycerol.

Total cell protein and sulfur inclusion envelopes were analyzed on 81

a 15% SDS polyacrylamide gel prepared as described in Appendix C. The

gel was run for 6 h at a constant voltage of 130 V. The gel was stained

for 3 h with 0.1% Coomassie brilliant blue R250 dissolved in 7.5% acetic

acid in 50% methanol, then destained overnight in 7.5% acetic acid in

20% ethanol in water.

Effect of sulfide on protein synthesis

The effect of sulfide on the rate of synthesis of individual

proteins was determined by pulse-labeling proteins after the addition of

sulfide. Two 250 ml volumes were removed ‘from an exponentially growing culture in AC medium. One volume was used to inoculate 250 ml of fresh

AC medium, and one volume was used to inoculate 250 ml of fresh ACS medium (AC medium + 1 mM sodium sulfide). At intervals of 30 min, 1 h,

2 h, 3 h, and 4 h, 2.5 ml aliquots were removed from both the AC and ACS cultures and placed into 20 ml glass vials containing 10 /ul of

D,L-[U1I*C]-leucine (100 juCi/3.33 AJmol/ml). The vials were shen at

120 strokes per minute in a Dubnoff metabolic shaker at 23 C. The amount of labeled leucine incorporated into proteins during 30 min was measured as follows. Duplicate 100 ;ul samples were removed from the reaction mixtures and 2.3 ml of cold 10% TCA were added to the remaining

2.3 ml of cells. The two 100 pi samples were each added to 100 >ul of

10% TCA, and kept on ice for two hours before being filtered through glass fiber filters. The filters were washed with 2 ml of BSS (pH 3) and 2 ml of 95% ethanol, then dried and counted as described in Chapter

II. The radioactivity on the filters provided an estimate of the rate of protein synthesis. 82

The specific proteins synthesized were characterized by SDS PAGE.

The proteins precipitated from the 2.3-tnl aliquot were collected by centrifugation at 5,000 x g for 10 min. The pellet was washed once in

95? ethanol, then resuspended in 80 pi of SDS PAGE buffer. Twenty pi of

2M Trizma base were added to the protein suspension to neutralize any residual acid. The samples were boiled for 2 min, then 30 pi aliquots

(all of which contained approximately 25,000 cpm) were loaded onto a 15% polyacrylamide gel, prepared as previously described. The gel was run at a constant voltage of 130 V for six hours. The gel was fixed overnight in 50% methanol, then silver-stained in 0.25% silver nitrate and developed in 3% sodium carbonate containing 0.05% formalin. The staining was stopped after approximately 10 min by the addition of 2.3 M citric acid. This procedure was derived from that described by

Morrissey (1981). The gel was photographed, then destained by the method of Wray et al. (1981) and soaked in Enlightning (New England

Nuclear) for 30 min. The gel was then dried and an autoradiogram of the gel was made with Kodak X-Omat AR X-ray film exposed at -70 C for 10 days. 83

Results

Sulfide oxidation

Heterotrophically grown cells of strain alba B18LD oxidize

sulfide to sulfur within 30 sec after exposure to sulfide (Fig 5.1).

Preincubation in 2 mM sulfide for two hours had no effect on the amount

of sulfur accumulated over a period of 100 minutes, although the rate of

accumulation was slightly slower (Fig 5.1). Chloramphenicol had a

slight inhibitory effect on the rate of sulfide oxidation by both

heterotrophic cells of strain B18LD (Fig 5.2) and cells preincubated in

sulfide for 2 hours (Fig 5.3).

Uptake and incorporation of leucine

Figure 5.4 shows the uptake of [U-^Hj-leucine by cells of

Beggiatoa alba B18LD. Cells suspended in basal salts supplemented with

6 mM acetate as a carbon and energy source accumulated labeled leucine

at a rate of approximately 0.26 nmol/min/mg protein. The addition of

sulfide to the heterotrophically growing cells did not alter the rate of

protein synthesis (Fig 5.4). Chloramphenicol at a concentration of 5 pg/ml effectively inhibited the incorporation of leucine into

TCA-precipitated material (Fig 5.4). In a separate experiment, it was

found that approximately 45 % of the total leucine assimilated by cells

of strain E18LD was incorporated into TCA-precipitable macromolecules

(Table 5.1). Figure 5.1. Accumulation of 35S after exposure to 35s_sulfide 35s_sulfide to exposure after 35S of Accumulation 5.1. Figure jUMOLES SULFUR / MG PROTEIN 0 h rsneo 1sulfide. mM of presence albathe B18LD ( ) after and (* ■ before ) a of cells grown heterotrophically by IS 80 45 TIME (MIN) TIME 60 2 hu icbto in incubation hour 73 90 10S Figure 5.2. Effect of chloramphenicol on sulfide oxidation sulfide on chloramphenicol of Effect 5.2. Figure

UMOLES SULFUR / MG PROTEIN - 4 0 by heterotrophic cells of alba B18LD. alba chloramphenicol No of cells heterotrophic by 10 20 TIME (MIN) TIME (■); 90 1 ug/ml chloramphenicol chloramphenicol 1 ug/ml 40 90 60 (*). 85 Figure 5.3. Effect of chloramphenicol on sulfide oxidation by oxidation sulfide on chloramphenicol of Effect 5.3. Figure

UMOLES S ACCUMULATED / MG PROTEIN 12 8 - 4 - - eeorpi el icbtd o or n 1 mM in for2hours incubated cells heterotrophic hoapeio ( chloramphenicol A ). chloramphenicol No sulfide. (■); 1 ug/ml 86 Figure 5.4 Effect of 1 mM sulfide on the incorporation of incorporation the on 1 sulfide mM of Effect 5.4 Figure NMOLES LEUCINE / MG PROTEIN 10 12 14 0 2 4 6 8 0 One mM sulfide was added either 30 min before the before min 30 either added was sulfide mM One of leucine in the presence of 5 ug/ml chloramphenicol chloramphenicol 5ug/ml of presence the in leucine of of incorporation the to compared are rates These ( (leucine of time same addition ♦ )the at or■ ). is also shown Calso is ▼). sulfide of absence inthe leucine [U-3H]-leucine into cells of alba« B18LLD. alba« of cells into [U-3H]-leucine 10 20 TIME TIME

Table 5.1. Fraction of cell-bound 3H_].eucjine incorporated into trichloroacetic acid precipitable material from cells of alba B18LD. Samples were taken for 50 minutes after the addition of 3n-leucine to cells suspended in basal salts or basal salts + 0.05% acetate.

time (min) basal salts basal salts + acetate

10 0.49 0.46

20 0.45 0.45

30 0.35 0.40

40 0.40 0.44

50 0.40 0.49 89

Sulfur inclusion envelopes

The sulfur inclusions from cells of strain B18LD are surrounded by

a single, electron-dense layer approximately 4 nm wide (Lawry et al.

1581; Strohl et al. 1981a). Enrichment for the sulfur inclusions,

which depends on their sedimentation at very low velocity and resistance

to solubilization by lysozyme, phospholipase C, and DNase, resulted in

the enrichment of a protein with a relative molecular mass of 15,000.

This protein is considerably more abundant in cells grown in the

presence of sulfide than in its absence (Fig 5.5)

Effect of sulfide on protein synthesis

Although the overall rate of protein synthesis was unaffected by

the addition of sulfide, the rate of synthesis of specific proteins was

increased. TCA-precipitated proteins from whole cells grown in the presence or absence of sulfide were analyzed by SDS polyacrylamide gel electrophoresis. The pattern of proteins was similar in cells grown in the presence or absence of sulfide (Fig 5.6). However, there was

increased synthesis of several of these proteins upon the addition of

sulfide (Fig 5.7). One of these proteins (Fig. 5.7, arrow) has the same

electrophoretic mobility as the protein enriched from sulfur inclusions.

This can be seen in Figure 5.8, where lanes from the silver stained gel and the autoradiogram of that gel are aligned next to each other. 90

S 1 2 3 4 5 S' S

66.2

45.0

31.0

21.5

14.4

Figure 5.5 SDS-polyacrylamide gel of proteins precipitated from cells of alba B18LD grown in the presence (lane 2) or absence of sulfide (lane 1), and of the enriched sulfur inclusions (lanes 3,1*, and 5) after one, two, and three washes, respectively, in basal salts solution. Molecular weight markers are in lanes marked S and S , and labelled in Kd. Figure 5.6. Silver-stained polyacrylamide gel of the proteins precipitated from Bj_ alba B18LD cells grown in the presence or absence of sulfide. Lanes marked S and S' contain molecular weight markers (Kd). Lane 1: whole cell protein before the addition of sulfide; lanes 2 ,4,6 ,8 , and 10: proteins from cells transferred to AC medium 30 min, 1 hour, 2 hours, 3 hours, and 4 hours after the transfer (respectively); lanes 3 ,5 ,7 ,9 , and 11: proteins from cells transferred to AC medium containing 1 mM sulfide. These protein samples were taken 30 min, 1 hour, 2 hours, 3 hours, and 4 hours after the addition of sulfide. The protein band marked with the arrow corresponds to the 15,000 Mr 92

1 2 3 4 5 6 7 8 9 10 11

Figure 5.7. Autoradiogram of sulfide-induced proteins in filaments of IB^ alba B18LD. An autoradiogram of the gel pictured in Figure 5.8 shows the synthesis of several sulfide-induced proteins. Lanes 2,4,6,8 , and 10 contain proteins from cells transferred to AC medium 30 min, 1 hour, 2 hours, 3 hours, and 4 hours after the transfer (respectively). Lanes 3»5,7,9, and 11 contain proteins from cells transferred to AC medium containing 1 mM sulfide. The protein band marked with the arrow corresponds to the 15,000 Mr protein present in enrichments of the sulfur inclusion envelope (Figure 5.6). 93

1 2 3

66.0

45.0

14.2

Figure 5.8. Composite of lanes 1,5,and 6 from the silver stained polyacrylamide gel and the autoradiogram of that gel. The arrow indicates the protein associated with the sulfur inclusion envelope, that is synthesized upon the addition of sulfide. Lanes 1 and 2 correspond to the sample taken at time 0 , lanes 3 and 4 correspond to the sample taken 3 0 .min after the addition of sulfide, lanes 5 and 6 correspond to the sample taken 2 hours after the addition of sulfide, and lanes 7 and 8 correspond to samples 2 hours after the transfer to AC medium (lacking sulfide). Lane 9 contains molecular weight markers (Kd). 94

Discussion

Leucine is incorporated primarily into protein pools in bacteria,

and so the incorporation of radiolabeled leucine into TCA-precipitable macromolecules can be used to estimate the rate of protein synthesis.

■3 [ H]-leucine is taken up by whole cells of EL_ alba B18LD; about 45% of this label is precipitated by TCA. The incorporation of leucine into macromolecules is effectively inhibited by chloramphenicol (Vinci, VA et al. 1983; Abstract K191, ASM).

In Oscillatoria limnetica a two hour induction period in the presence of sulfide is required before sulfide is oxidized by the filaments (Oren and Padan 1978). Protein synthesis, which can be blocked by CAP, is required during this induction period in cells of 0. limnetica (Oren and Padan 1978). Incubation in sulfide for two hours did not significantly affect the rate of sulfide oxidation to sulfur in filaments of strain B18LD. Additionally, chloramphenicol had little effect on the oxidation of sulfide to sulfur in B18LD filaments. These observations indicate that enzymes involved in sulfide oxidation are normally present in the cells of strain B18LD, and no de novo synthesis is required for the initial oxidation of sulfide.

The sulfur resulting from sulfide oxidation accumulates in the periplasmic space of filaments of strain B18LD in inclusions that are bounded by a single unit membrane (Lawry et al. 1981; Strohl et al.

1981a). Due to the high density of sulfur, these inclusions can easily be separated from the majority of cell material by differential 95

centrifugation. Enrichments of sulfur inclusions from alba B18LD

analyzed by SDS polyacrylamide electrophoresis are enriched in a protein

with a relative molecular mass of approximately 15,000. This protein is

thought to be a structural protein that surrounds the sulfur globule,

with a function similar to the structural protein isolated from the

sulfur inclusions from the purple sulfur bacterium, Chromatium.

In cells of Chromatium, the proteinaceous envelope surrounding the

sulfur inclusion is thought to act primarily as a barrier, to separate

the sulfur from the interior of the cell and perhaps to provide binding

sites for the enzymes responsible for sulfur metabolism (Schmidt et al.

1971). The sulfur inclusion envelope purified from Chromatium vinosum

is composed of a single monomeric protein with a relative molecular mass

of 13,500 (Schmidt et al. 1971).

The 15,000 protein associated with the sulfur inclusion in

cells of J3j_ alba B18LD is abundant when the cells are grown in the

presence of sulfide, and is present in lesser quantities in cells grown

in the absence of sulfide. This finding is consistent with the

observation by Strohl et al. (1982) of rudimentary sulfur inclusion

envelopes in filaments of B^ alba B15LD grown in the absence of sulfide.

The enzymes for sulfide oxidation and the structural protein that

encloses the sulfur inclusions appear to be present in heterotrophically growing filaments of B^ alba B18LD, such that sulfur accumulates in the

cells immediately upon contact with sulfide. The continued oxidation of sulfide, however, may require the continued synthesis of the putative structural protein that is a major component of the sulfur inclusion envelope. Continued synthesis of the sulfur inclusion envelope may be 96

necessary to accomodate the increasing amount of stored sulfur.

Although the presence of sulfide does not change the overall rate

of protein synthesis in cells of strain B18LD, sulfide does increase the

rate of synthesis of certain proteins, including the 15,000 M r protein associated with the sulfur inclusions. This induction is evident within thirty minutes after the addition of sulfide, as would be expected if the sulfur inclusion envelope is continually synthesized when sulfide is being oxidized.

The accumulation of sulfur by Beggiatoa filaments may increase the viability of the organism, particularly if the filaments are able to use the stored sulfur as a terminal electron acceptor under anoxic conditions, as suggested in Chapter IV. CHAPTER VI

Summary

The presence of sulfur inclusions in filaments of Beggiatoa and

Thiothrix distinguishes these genera of bacteria from other genera of filamentous bacteria. Upon exposure to sulfide, sulfur accumulates in trichomes of Beggiatoa alba B18LD and Thiothrix nivea JP3 at similar

^es. The oxidation of sulfide is linked to the reduction of oxygen, and appears to serve as an energy source for the chemolithotrophic growth of filaments of strain B18LD and strain JP3. The growth of trichomes of strain B18LD in autotrophic sulfide/oxygen gradients suggests the operation of an autotrophic carbon dioxide assimilating pathway, but it is not known if such a pathway exists in cells of strain

B18LD. The few attempts to culture strain JP3 autotrophically have been unsuccessful. A summary of the metabolic rates in cells of B^ alba

B18LD is given in Table 6.1.

When strain B18LD filaments are grown heterotrophically on acetate, classic respiratory inhibitors effectively reduce the rate of acetate oxidation as measured by either acetate-dependent oxygen consumption or the release of from [2-^C]-acetate. These same inhibitors, except for the quinone analog dibromothymoquinone (DBMIB), reduce the rate of sulfide oxidation. Therefore, if the electrons from sulfide oxidation enter the membrane-associated electron transport chain it must be at a carrier after ubiquinone 8 . The movement of electrons 97 98

through the electron transport chain may result in the translocation of

protons across the cytoplasmic membrane, creating a pH gradient.

Alternatively, sulfide oxidation may be linked to oxygen consumption in

the periplasmic space. This may also result in a pH gradient across the

cell membrane, due to the protons released in the periplasm. In both

cases, the energy from sulfide oxidation might be conserved by the cells

as described by the chemiosmotic theory.

The capacity of strain B18LD cells to oxidize sulfide immediately

is not dependent upon previous exposure to sulfide. Thus, any proteins

involved in the initial oxidation of sulfide are constantly present in the cell. When sulfide is oxidized to sulfur, the sulfur accumulates in periplasmic sulfur inclusions that are enclosed by an electron-dense envelope 3-5 nm wide. Enriched sulfur inclusion preparations contain a monomeric protein with a relative molecular mass of aprroximately

15,000. This protein is not present at high concentrations in the cell unless the filaments are grown in the presence of hydrogen sulfide.

Although the overall rate of protein synthesis does not change upon exposure to hydrogen sulfide, the rate of synthesis of several proteins, including the protein with a Mr 15,000, is increased. The 15,000

protein is associated with the sulfur inclusion, and may be required for the continued oxidation of sulfide.

The stored sulfur in cells of strain B18LD is not oxidized to sulfate under the conditions tested, whereas sulfur oxidation to sulfate by cells of strain JP3 is readily detectable and is linked to oxygen consumption. The sulfur stored in the inclusions in strain B18LD, however, can be used by the cells as the terminal electron acceptor for Table 6.1. Summary of metabolic rates in Beggiatoa alba B18LD filaments.

Sulfide-dependent Sulfide oxidation oxygen consumption

38 - 380 nmol/min/mg protein 20 nmol/min/mg protein

Acetate-dependent Acetate oxidation oxygen consumtion

65 nmol/min/mg protein 160 nmol/min/mg protein

Endogenous sulfur reduction

7 nmol/min/mg protein

Hydrogen consumption

8 nmol/min/mg protein

Leucine incorporation

0.26 nmol/min/mg protein 100 endogenous metabolism under anoxic conditions. The rate of anaerobic reduction of sulfur to sulfide is stimulated by hydrogen; apperently due to an uptake hydrogenase coupled to the reduction of sulfur. Both the endogenous and the hydrogen-linked anaerobic reduction of sulfur may be vital to the organism, since Beggiatoa filaments are most likely exposed to periods of anoxia in their natural environment. The uptake hydrogenase may also be vital in recycling hydrogen produced by a side reaction of nitrogenase.

Although sulfide oxidation is not required for the growth of B. alba B18LD filaments in the laboratory, the oxidation of sulfide and the reduction of stored sulfur expand the metabolic capacity of the organism and may confer an advantage on the organism in its dynamic environment.

Our understanding of Beggiatoa physiology would surely benefit from a study of the organism's response to various environments that could be created in a chemostat. The potential for mixotrophic carbon utilization and mixotrophic energy generation could be explored by varying the concentrations of acetate and carbon dioxide, and sulfide and oxygen, in a steady state culture. During chemostat studies, the organisms response to redox potentials must also be examined, since the addition of sulfide to a culture not only supplies a potential energy source, but also decreases the redox potential of the culture.

As we dig deeper into the complexities of Beggiatoa in its gradient habitat, we are likely to find a metabolically versatile organism that interacts with the environment by regulating its metabolism in response to changes in the environment, or by moving to a more favorable position in the environment. 101

APPENDICES

Appendix A. Trace elements solution containing sulfate salts (Kowallik and Pringsheim 1966)

Compound mg / 100 ml

Na2EDTA 20.00

FeSOjj. 7h 2o 70.00

ZnSO^. 7H2o 1.00

MnSO^ 2!h 2 0 0.20

CuSO^. 5h 2o 0.0005

1.00 H3B03 c o (n o 3 )2 0.10

NagMoOy 0.10 102

Appendix B. Trace elements solution containing chloride salts.

Coumpound mg / 100 ml

Na2EDTA 20.00

FeCl2* 6H20 67.00

ZnCl2 0.48

MnCl2 «4H20 0.22

CuClg"* 2H20 0.0004

H3BO3 1.00

C0(N03 )2 0.10

Na2« Mo0/| 0.10 103

Appendix C. SDS-Page electrophoresis

The following solutions were made with ultrapure reagents.

Resevoir buffer (10x)

Tris-base 30.2 g Glycine 144.0 g

Add water to 1 1; filter. Before use, dilute 10 fold and add SDS to 0.12.

Stacking gel buffer (4x)

Tris-base 61.0 g

Dissolve Tris in 500 ml water and adjust pH to 6.8 with HC1. Add water to 1 1 and filter.

Separating gel buffer (4x)

Tris-base 182.0 g

Dissolve Tris in 500 ml water and adjust pH to 8.8 with HC1. Add water to 1 1 and filter.

Sample buffer

Stacking gel buffer (4x) 0.125 ml 102 SDS 0.30 ml 1002 glycerol 0.10 ml Mercaptoethanol 0.05 ml Water 4.25 ml Final volume 1.00 ml

Other solutions

(i) Acrylamide 302 w/v Bis-acrylamide 0.82 w/v

Filter and store in dark at 4 C.

(ii) Glycerol 502 v/v in water

Filter 1 0 A

(iii) SDS 10? w/v in water

Filter

(iv) Ammonium persulfate 10% w/v in water

Make fresh for each experiment. Ammonium persulfate should effervesce when added to water.

(v) Bromphenol Blue 0.2% w/v in water

(vi) TEMED

Must be stored at 4 C.

Preparing polyacrylamide gel

Prepare the separating gel first. Allow approximately 90 min for polymerization. Prepare and pour the stacking gel over the separating gel. The stacking gel should polymerize within 15 min.

15% separating gel Stacking gel

Water 9.3 ml 11.6 ml Separating-gel 10.0 ml 0 buffer Stacking-gel 0 5.0 ml Acrylamide:bis- 20.0 ml 3.8 ml acrylamide

Mix the solutions gently; avoiding excess aeration of the mixture. Degas for 15 min, then add:

10% SDS 0.4 ml 0.2 ml TEMED 10 ul 16 ul Ammonium persulfate 0.3 ml 0.28 ml

After loading the sample, run the gel at 130 V for 6 hours. 105

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