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1987 Regulation of Dextransucrase Secretion by Proton Motive Force in Leuconostoc Mesenteroides. David Roland Otts Louisiana State University and Agricultural & Mechanical College

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Order Number 8728211

Regulation of dextransucrase secretion by proton motive force in Leuconostoc mesenteroides

Otts, David Roland, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1987

U-M-I 300 N. Zeeb Rd. Ann Arbor, MI 48106

Regulation of Dextransucrase Secretion by Proton Motive Force

in

Leuconostoc mesenteroides

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Microbiology

by David Roland Otts B.S., University of Wisconsin, 1983 August, 1987 ACKNOWLEDGEMENTS

I would like to thank Dr. E. C. Achberger for reviewing this

manuscript and for providing stimulating discussion on all aspects of

microbiology during my tenure at Louisiana State University. I thank

Dr. D. F. Day for providing the impetus for this project as well as

providing the necessary lab space. I also thank Dr. E. S. Younathan,

Dr. V. R. Srinivasan, Dr. J. M. Larkin, and Dr. H. D. Braymer for

reviewing this manuscript. I would also like to thank my lab cohorts,

David Koenig and Gregory Siragusa, for listening to ray ideas even when

they didn't make much sense. Appreciation is extended to Dr. J. A.

Polack and the Audubon Sugar Institute for providing the funds and facilities for the entirety of this work. I dedicate this work to my

wife Holly and my children Rachel and Tyler. Their love, patience,

and understanding have made this work possible.

ii TABLE OF CONTENTS

Page ACKNOWLEDGEMENT ii

TABLE OF CONTENTS . iii

LIST OF FIGURES „ v

LIST OF TABLES vi

SYMBOLS AND ABBREVIATIONS vii

ABSTRACT viii

INTRODUCTION 1

LITERATURE REVIEW 3

I. Sucrose Metabolism in Leuconostoc mesenteroides 3

II. Chemiosmotic Theory 8

III. Generation of Proton Motive Force in Bacteria 13

IV. Ion transport and its relationship to Proton Motive Force 17

V. Utilization of Proton Motive Force by Bacteria 19

VI. Protein Export in Bacteria 21

VII. Energetic Requirements of Protein Secretion 25

MATERIALS AND METHODS 30

I. Organism 30

II. Preparation of packed cells 30

III. Determination of cellular dry weight 32

IV. Growth of cells in batch fermenters at constant pH.... 32

V. Extraction and assay of extracellular dextransucrase.. 33

VI. Determination of the pH stability of dextransucrase... 34

VII. Determination of extracellular polysaccharide 35

VIII. Preparation and use of ionophores 35

iii IX. Determination of cytoplasmic and extracellular water spaces 36

X. Determination of ApH 36

XI. Determination of A4* 37

XII. Calculation of the proton motive force (Ap) 38

XIII. Isolation and assay of the membrane H+-ATPase 39

XIV. Statistics 39

XV. Chemicals 40

RESULTS 41

I. Use of an aqueous-phase system to purify extracellular dextransucrase 41

II. Effect of ionophores on the growth of packed cells.... 41

III. Effect of ionophores on the secretion of dextransucrase by packed cells 44

IV. Determination of Ap in packed cells 52

V. Effect of cations on the generation of ApH 55

VI. Effect of cations on Af 55

VII. Effect of potassium on Ap 60

VIII. Activity of the membrane H+-ATPase 60

IX. Inhibition of dextransucrase secretion by sodium 64

X. Dextransucrase secretion by cells grown in batch fermenters at constant pH 64

XI. Determination of Ap in cells grown at constant pH 69

XII. Effect of methyltriphenylphosphonium bromide on Ap and dextransucrase secretion 69

XIII. Effect of nigericin on dextransucrase secretion 77

DISCUSSION 80

LITERATURE CITED 91

VITA 112

iv LIST OF FIGURES

PAGE Figure 1. Effect of gramicidin D on the growth of .L. mesenteroides 43

Figure 2. Medium pH of ionophore treated cultures 45

Figure 3. Effects of nigericin and valinomycin on the production of extracellular dextransucrase 47

Figure 4. Effect of methyltriphenylphosphonium bromide on dextransucrase secretion 50

Figure 5. Effect of gramicidin D on dextransucrase secretion.... 51

Figure 6. Effect of the addititon of ionophores after allowing dextransucrase export 53

Figure 7. The proton motive force of packed cells grown in LMK 100 medium 54

Figure 8. Regulation of internal pH by packed cells 56

Figure 9. Effect of cations on the generation of ApH 57

Figure 10. Effect of cations on the generation of A4* 59

Figure 11. Effect of K+ on the proton motive force 61

Figure 12. pH activity curve of membrane H+-ATPase 62

Figure 13. Effect of pH on the secretion of dextransucrase by cells grown in pH-controiled fermenters 67

Figure 14. Effect of pH on the stability of dextransucrase 68

Figure 15. Effect of the medium pH on the generation of proton motive force 70

Figure 16. Effect of medium pH on the internal pH 71

Figure 17. Dependence of methyltriphenylphosphonium mediated inhibition of dextransucrase secretion on the medium pH 74

Figure 18. Dependence of dextransucrase secretion on proton motive force 75

Figure 19. Dependence of dextransucrase secretion on the trans­ membrane pH gradient after treatment with methyltriphenylphosphonium bromide 76

v LIST OF TABLES

PAGE Table 1. Media composition 31

Table 2. Effect of ionophores on the production of extra­ cellular polysaccharide 46

Table 3. Effect of pH on valinomycin mediated inhibition of dextransucrase secretion 49

Table 4. Effect of cations on the cytoplasmic volume 58

Table 5. Effect of DCCD on the generation of proton motive force 63

Table 6. Cell growth as a function of medium pH 65

Table 7. Effect of methyltriphenylphosphonium bromide on the generation of proton motive force at pH 6.0 72

Table 8. Effect of nigericin on Ap and dextransucrase secretion 78

vi SYMBOLS AND ABBREVIATIONS

Symbol Definition

mV millivolt

Ap Proton motive force (mV)

A^ Membrane Potential (mV)

ApH Transmembrane pH gradient (pH units)

-ZApH Contribution of the pH gradient to Ap (mV)

AUu Electrochemical potential of protons IJ+ AG Gibbs Free energy change

F Faraday's Constant

R Gas Constant

Z 2.3 RT/zF where z = valency of the ion

T Temperature in degrees Kelvin

TPP+ Tetraphenylphosphonium (bromide)

MPTP+ Methyltriphenylphosphonium (bromide)

DCCD N,N'-dicyclohexylcarbodiimide

CCCP Carbonylcyanide-m-chlorophenylhydrazone

ATP Adenosine triphosphate

NAD+ Nicotinamide adenine dinucleotide (oxidized)

NADH Nicotinamide adenine dinucleotide (reduced)

MES 2[morpholino]ethanesulfonic acid

vii ABSTRACT

The relationship between proton motive force and the secretion of the enzyme dextransucrase (E.C. 2.4.1.5) in Leuconostoc mesenteroides was investigated. The transmembrane pH gradient was determined by measurement of the uptake of radiolabeled benzoate or methylamine while the membrane potential was determined by measurement of the uptake of radiolabeled tetraphenylphosphonium bromide. Leuconostoc mesenteroides was able to maintain a constant proton motive force of

-130 mV when grown in fermenters at constant pH while a value of

-140 mV was determined from concentrated cell suspensions. The contribution of the membrane potential and transmembrane pH gradient to the proton motive force varied depending on the cation concentration and pH of the medium. The differential rate of dextransucrase secretion was relatively constant at 1040 AmU/Amg dry weight when cells were grown at pH 6.0 to pH 6.7. At this pH range, the cell interior was alkaline with respect to the growth medium.

Above pH 7.0, dextransucrase secretion was severely inhibited. This inhibition was accompanied by an inversion of the pH gradient across the cytoplasmic membrane as the cell interior became acidic with respect to the growth medium. Treatment of cells with the lipophilic cation methyltriphenylphosphonium bromide had no effect on the rate of dextransucrase secretion at pH 5.5 but inhibited secretion by 95 % at pH 7.0. The inhibition of secretion by methyltriphenylphosphonium bromide was correlated to the dissipation of the proton motive force by this compound. Values of proton motive force greater than -90 mV were required for maximal rates of dextransucrase secretion. The

viii results of this study strongly indicate that dextransucrase secretion

Leuconostoc mesenteroides is dependent on the presence of a proton gradient across the cytoplasmic membrane. The results further suggest that dextransucrase secretion is coupled to proton influx into the cell.

ix INTRODUCTION

The production of dextran by the bacterium Leuconostoc mesenteroides has been a nuisance to the sugar cane industry for decades (23,54). Processing problems associated with dextran include increased juice viscosity, poor clarification, and crystal elongation.

The sugar refineries have recently instituted monetary penalties to the producers of raw sugar for the dextran content in their raw sugar.

This policy has made the control of dextran formation in the raw sugar factories a priority.

Dextran is produced by the action of dextransucrase (E.C.

2.4.1.5) on sucrose (124). This enzyme has been extensively studied over the past forty years (79,80,81,100,101,124,125) yet relatively little is known about the regulation of its production (90,149). The research that has been done on the regulation of dextransucrase production has centered on the optimization of dextran formation, rather than the control of it. A study on the regulation of dextransucrase production with a focus on the inhibition of its production is thus lacking.

Dextransucrase is an extracellular enzyme (100,101,149). The cell must have an efficient mechanism to transport this protein across the cytoplasmic membrane if it is going to act on its substrate, sucrose. Over the past seven years, the involvement of proton motive force in the process of protein secretion has been investigated in

Escherichia coli (5,19,25,26,162). Several proteins that are secreted into the periplasmic space of Escherichia coli were demonstrated to be dependent on the presence of the proton motive force. A study on the

1 2 energetic requirements of (3-lactamase secretion demonstrated that a specific level of proton motive force was required for optimal secretion (5). It was of interest, then, to ask whether proton motive force plays a role in the regulation of dextransucrase secretion in

Leuconostoc mesenteroides. LITERATURE REVIEW

I. Sucrose Metabolism in Leuconostoc mesenteroides

Leuconostoc mesenteroides produces several enzymes that act on

sucrose (64,81). In addition to the extracellular enzymes dextran-

sucrase and levansucrase (81,125), sucrose phosphorylase is also

produced (151,156). This enzyme catalyzes the hydrolysis of sucrose

by the addition of inorganic phosphate to the glucose moiety of the

sucrose molecule. The products of the reaction are fructose and

glucose-l-phosphate (156). Sucrose phosphorylase is a constituitive

enzyme and is thought to be located intracellularly (151).

In addition to fructose and glucose-l-phosphate, the metabolism

of sucrose entails the production of dextran and fructose by

dextransucrase (100) and the production of levan and glucose by levan­

sucrase (125). The organism lacks a phosphoenolpyruvate:hexose

phosphotransferase system (127) and instead phosphorylates glucose and

fructose via ATP-dependent enzymes (127,130). Glucose is fermented

by the phosphoketolase pathway, generating 1 ATP under anaerobic

conditions (28). The fermentation products of this pathway are

ethanol, lactic acid, and (X>2 in equal molar quantities (28). The

growth of several strains of mesenteroides has been demonstrated to

be improved in the presence of oxygen (62,96). During aerobic growth,

ethanol production is reduced while acetate becomes a major end-

product of glucose metabolism (163). In the pathway to ethanol formation, acetyl-phosphate is formed by the action of phosphoketolase on xyulose-5-phosphate (163). Acetate is then formed from acetyl-

phosphate by the action of acetate kinase. This enzymatic reaction

3 4

generates the production of 1 ATP (163). Thus, the ATP yield under aerobic conditions is twice that under anaerobic conditions for the catabolism of glucose.

When glucose is the sole carbon source under aerobic growth conditions, the production of acetate at the expense of ethanol

presents a redox problem for the cell as it needs to regenerate NAD+ for catabolic functions (57). It has been demonstrated that the regeneration of NAD+ is accomplished by NADH oxidase (96). This enzyme utilizes oxygen as an electron acceptor to oxidize NADH (96).

The production of this enzyme has been shown to be stimulated when _L. mesenteroides is grown in the presence of oxygen while alcohol dehydrogenase is repressed (57,96). Not all strains of L:. mesenteroides produce NADH oxidase (96). These strains do not produce acetate from acetyl-phosphate and thus do not generate an additional ATP from glucose fermentation.

In L. mesenteroides, sucrose fermentation differs from glucose utilization in that a major end product of sucrose fermentation is mannitol (151) Mannitol is produced by the reduction of fructose by mannitol dehydrogenase (151). This enzyme is intracellular and is

NADH-dependent (151). The mannitol that is produced is excreted by the cell (151). The production of mannitol allows the cell to maintain its redox balance and thus not all acetyl-phosphate is reduced to ethanol. Additional ATP is then available for the cell under both aerobic and anaerobic conditions when grown in sucrose medium. The reaction scheme for fructose utilization is:

3 Fructose ===> 1 lactic acid + 1 acetic acid + 1 CC^ + 2 mannitol 5

Although Ij. mesenteroides will utilize oxygen as an electron acceptor, it does not carry out oxidative phosphorylation as it lacks a cytochrome system (96). This characteristic is exploited in selective media for Leuconostoc. The addition of sodium azide to media will make them selective for Leuconostoc and other organisms that lack electron transport chains (62).

Dextransucrase (E.C. 2.4.1.5) is produced by species of

Leuconostoc. Streptococcus, and Lactobacillus (59,81). This enzyme polymerizes the glucosyl moiety of sucrose to form dextran, an a-(l->6)-linked glucan with various degrees of branching (79).

Dextransucrase has been purified from several Ij. mesenteroides strains

(79,81,100). The dextransucrase from strain NRRL B-512F has been extensively studied (100,101,108,124,125). This enzyme has been purified by a variety of techniques but probably the most novel was the use of aqueous-phase partitioning (116). The use of a polyethylene glycol-dextran phase partition system allowed the purification of dextransucrase to be achieved with high recovery and high specific activity (116). The phase system removes contaminating levansucrase from the enzyme preparation (116).

There has been some discrepancy on the molecular weight of

B-512F dextransucrase determined by various groups (81,101).

Molecular weights of 64-65,000 daltons as well as 177,000 daltons have been reported (81,100,101). The dextransucrase of this strain has been shown to aggregate (81). This may affect the size determinations depending on the conditions of purification (81,100) and on the amount of contaminating dextran present (81,100,116). The enzyme has a pi of 6

4.1 (100) and has been demonstrated to contain carbohydrate other than

dextran (81). Acid hydrolysis, followed by paper chromotography,

showed that the major carbohydrate was mannose (125). The pH optimum

of the enzyme is between pH 5.0 to pH 5.5 (81). The reported values

of the Km of the enzyme for sucrose range from 15 to 20 mM (81,125).

When carbohydrates other than sucrose are present in the reaction

mixture, the D-glucosyl group of sucrose is diverted from the

synthesis of dextran and is added to the carbohydrate (124).

When these carbohydrates, or acceptors, are of low molecular weight,

the products are also short, low molecular weight oligosaccharides

(124). When sucrose or dextran concentrations are low, a hydrolysis

reaction predominates (90).

There have been few studies on the regulation of

extracellular dextransucrase production in Leuconostoc mesenteroides.

Dextransucrase is an inducible enzyme that is only produced when

the organism is grown in the presence of sucrose (108). The

production of dextransucrase is not subject to catabolite repression as enzyme is produced in glucose medium with added sucrose.

Continuous culture studies also failed to demonstrate dextransucrase

production under conditions of glucose limited growth (90). The

production of dextransucrase by L^. mesenteroides differs from that of the oral streptococci as in the latter the enzyme is constitutive

(61,157).

Tsuchiya et al^. (149) examined the effect of various medium components on the production of extracellular dextransucrase. They found that the level of extracellular enzyme obtained was dependent on the sucrose concentration in the medium. Additionally, they found 7

that ammonium ions had a detrimental effect on enzyme production.

Enzyme production was found to be optimal at pH 6.7. In cultures where the pH of the medium was not controlled, enzyme production declined once the medium pH fell below 6.0 (149).

The dependence of dextransucrase production on the sucrose concentration in the growth medium has been taken advantage of by various workers to produce large quantities of dextransucrase. Use of a semicontinuous fermentor with sucrose as the batch feed allowed for the production of 8 U/ml of dextransucrase as compared to 2 U/ml with a normal batch fermentor (104).

Dextransucrase synthesis has been demonstrated to take place on membrane-bound and free ribosomes in L,. mesenteroides (83,84).

Intracellular, membrane-bound, and extracellular dextransucrase were isolated in this study (83). Addition of colchicine to cells inhibited the transport of dextransucrase and caused accumulation of the intracellular form (84).

Magnetism has also been demonstrated to affect dextransucrase secretion (92). When 70-120 mT of magnetic force was applied to L. mesenteroides, higher levels of dextransucrase were found in the culture medium. The pH of the cultures subjected to the magnetic force was higher than that of cultures without the force. Penicillin further enhanced the effect of magnetism (92).

The regulation of dextransucrase production in the oral streptococci has been more thoroughly studied than in ]±. mesenteroides. Membrane lipid composition has been postulated to effect glucosyltransferase secretion in Streptococcus salivarius. 8

Tween 80 markedly stimulated the production nf extracellular glucosyltransferase and also increased the --degree of unsaturation of the membrane lipid fatty acids (59,60). The productrsuon ©f glucosyl­ transferase has also been demonstrated to be stimulated by K+ in the growth medium (74,98). Increasing the K+ concentteation ibn the medium increased the degree of unsaturation of the memtaKve lipM fatty acids

(98).

The secretion of glucosyltransferase was 'dHiivanKtjrated to be inhibited by Na+ in Streptococcus sanguis '(74)„ TimMlbition of secretion could also be obtained by treating the cieMs with gramicidin (74). It was postulated that proton motii're tfoncss (Ap) was involved in the process of secretion, though no actual measurements of

Ap were made (74„158).

II. Chemiosmotic Theory.

According to the chemiosmotic theory (42.,102), the .cytoplasmic membrane forms topologically closed structures that are? essentially impermeable to Ions, specifically H+. Protasis that axe vectorally pumped outside the cell by various metabolic, fiatihways establish a gradient of protons across the membrane. The transport -:of charge associated with the proton across the membrane generates ana electrical potential across the membrane with the cytoplasm 'being aneegatiw-e. The transport of protons also generates a pH gradient across tihe membrane with the cytoplasm alkaline with respect to the exterior. Both gradients contribute to the electrochemical potential ®-f protons,

Au , which is the force that tends to pull protons 'back across the n+u membrane into the cell. The free energy change ;(MSi) as protons flow 9

down these two gradients is equal to:

[H+]in AG = Auu = FAf - 2.3RT(log ) (1) H+ [H ]out

In this equation, Ay„ is defined as the difference in electrochemical n+ potential of protons across the cytoplasmic membrane and has the unit

of kcal (102). The membrane potential is represented by the symbol

AV. The gas constant is represented by R while T is equal to the

temperature in degrees Kelvin. To convert this expression to units of

electrical potential, all terms in the equation are divided by

Faraday's constant (F). Equation 1 then becomes:

Au„ 2.3 RT [H+]in «+_ = Ap = A4* log (2) F F [H ]out

The logarithmic factor is equivalent to the pH difference across the

cell membrane, ApH = pHQUt - P^n* Commonly, 2.3RT/F is given the

abbreviation Z and at 25°C is equivalent to 59 mV. Making these

substitutions equation 2 becomes:

Ap = AV - 59ApH (3)

The proton motive force (Ap) has the unit of mV.

The study of proton motive force and how it affects cell

processes has been advanced with the use of ionophores. Ionophores

are compounds that increase the ionic permeability of membranes

(39,120). Two general classes of ionophores are mobile carriers and

channel formers. Mobile carriers are hydrophobic compounds that can

bind specific ions and actively transport them across cell membranes. 10

Valinomycin is a mobile carrier ionophore which catalyzes the transport of K+, Rb+, NH^+ and Cs+ (120). Valinomycin is an antibiotic produced by species of Streptomyces and consists of alternating hydroxy- and amino acids (120). Due to its structure, the + 4 + affinity of valinomycin for Na is approximately 10 less than for K

(120). Valinomycin is uncharged but it acquires the charge of the complexed ion (39). Valinomycin is commonly used to dissipate AT due to its transport of charge across the membrane (39,120). Cells treated with valinomycin have been demonstrated to increase ApH to partially compensate for the decrease in AW in a number of systems

(1,75,150).

A mobile carrier that is commonly utilized to dissipate ApH is nigericin. Like valinomycin, the active form of nigericin is a cyclic compound but differs in that nigericin loses a proton when it binds a cation (120). Nigericin forms a neutral complex with an ion and generally catalyzes the exchange of K+ for H+ (39). Other ionophores which catalyze similar electroneutral exchanges are monensin and dianemycin (39,120). The usual result of nigericin treatment of bacterial cells is the equilibration of K+ and H+ gradients across the cytoplasmic membrane (120).

Uncouplers are chemical agents that uncouple ATP synthesis from respiration (39,120). Examples include dinitrophenol, carbonylcyanide- m-chlorophenylhydrazone (CCCP), and salicylanilide (39,120). These compounds are acidic, lipid soluble substances that can pass through the lipid bilayer of the membrane when combined with protons. The transport of protons across the membrane thus dissipates Ap. When treated with uncouplers, cells increase their rate of respiration with 11

no ATP synthesis (120). Uncouplers are commonly used to deenergize

cells (25,26,33,63).

The accurate determination of cytoplasmic volume is crucial for

correct determinations of A1}1 and ApH (58,70,123). Most techniques

employ the use of membrane permeable and impermeable radiolabeled

probes to estimate the cytoplasmic volume and contaminating 3 14 extracellular fluid (58,70). Cells are incubated with 1^0 and a C-

probe that is generally a polymer such as dextran (58), inulin (51),

or polyethylene glycol (58). Alternatively, low molecular weight

nontransportable solutes, such as sucrose, are sometimes employed

(58). After sufficient incubation time to allow for equilibration,

cells are separated from the extracellular fluid by either centrifugation through silicone oils (8) or by membrane filtration

(1,63). Membrane filtration offers the advantage of being rapid and

the disadvantage of large volumes of contaminating extracellular fluid (58). Silicone oil centrifugation minimizes extracellular fluid contamination but can be disadvantageous for use with strictly aerobic

organisms due to poor diffusion of oxygen through the silicone oil

(58).

Determination of AH1 in bacteria relies on indirect rather than on

direct methods (58,70,123). The use of microelectrodes has been reported with giant IS. coli cells (70), but for practical purposes

bacterial cells are too small for this type of measurement at

physiological conditions. Valinomycin plus K+ has been utilized to determine the membrane potential (71). At equilibrium, the concentration gradient of K+ across the membrane is equal to AV. The 12

+ 4* AT can be calculated from the distribution of radiolabeled K or Rb ions through use of the Nernst equation (58,70). This method is valid only if bacteria are depleted of energy-yielding compounds (70). For example, K+ may be accumulated by ATP-dependent transport proteins in

E. coli (34).

The most commonly used probes for AT are radiolabeled lipophilic cations (70,123). These include DDA+ (N,N-dibenzyl N,N- dimethylammonium) (70), TPP+ (tetraphenylphosphonium) (58,70,123) and

TPMP+ (triphenylmethylphosphonium) (38,123). These cations are able to diffuse through phospholipid bilayers due to their dispersed positive charge (123). In theory, these cations should distribute themselves across cell membranes according to AT. Thus, at equilibrium, the potential of the ion should equal that of AT and the

Nernst equation can be utilized to calculate AT. This is applicable provided the cations are not actively transported or metabolized by the cell (123). In addition, the concentration of the probe selected should not be high enough to dissipate AT (70,123). Lipophilic cations have been demonstrated to equilibrate rapidly across biological membranes. Equilibration times range from less than 1 minute in mitochondria (123) to 5-10 minutes in Streptococcus (4,72).

The major setback to the use of these probes is the significant amount of binding to cellular components (94,123). Commonly, the amount of radiolabeled probe accumulated by cells after deenergization (51) or treatment with n-butanol (8,72) is taken to equal the amount of probe that is bound to cellular components. The difference between this value and that obtained from energized cells is then used to calculate

AT (72,94). 13

Determination of ApH is accomplished by measuring the

accumulation of weak acids such as benzoate (8,12) or weak bases such

as methylamine (12,70). Weak acids are utilized when the interior

compartment is alkaline with respect to the media while weak bases are

used when it is acidic compared to the medium. Care must be taken to

use low concentrations of these compounds (in the micromolar range) as

dissipatory effects are seen at higher concentrations. (129). As with

the AV probes, the weak acids or bases should not be actively

transported or metabolized by the cell (12,70). Another recently 31 developed technique is the use of P-NMR to measure intracellular pH

(147). The determination of ApH by these two techniques was shown to

to be in good agreement (70,147).

III. Generation of Proton Motive Force in Bacteria.

The generation of a proton motive force can be accomplished

through the expulsion of protons by an electron transport chain (102)

or by the action of ATPase (35,78). Under anaerobic conditions,

facultative anaerobes generate Ap essentially by the latter mechanism.

Consequently, the Ap generated under anaerobic conditions is less than

that generated under aerobic conditions (66,67). Staphylococcus

aureus maintains a Ap ranging from -250 mV to -160 mV under aerobic

conditions at pH 6.0 to pH 7.5 respectively (67). The same organism

grown under anaerobic conditions maintains a Ap 50 to 100 mV less

than under aerobic conditions (67). In general, fermentative bacteria

maintain a Ap less than -150 mV (70). Examples include -120 mV for

Streptococcus cremoris (144), -143 mV for Streptococcus lactis (72), 14

-120 inV for Clostridium thermoaceticum (8), -150 mV for Streptococcus

faecalis (4,78), and -106 mV for Clostridium acetobutylicum (51).

The central tenet to Mitchell's chemiosmotic theory was that

cells had a mechanism to convert the energy stored in the potential

difference of protons across a membrane to chemical energy. This link

has been shown to be the ATP synthase, or ATPase. This enzyme can either act to hydrolyze ATP or synthesize ATP, depending on the

direction of proton flow through the enzyme (35,103). The ATPase is also referred to as a H+-ATPase because of this coupling of proton flow to its action on ATP.

The H+-ATPase is a complex that consists of two main portions, and FQ. The catalytic portion of the complex is F^ which is an extrinsic membrane protein and consists of five different subunits

(35,103). The FQ portion is a transmembrane complex that acts as the

"proton pore" through the membrane (35,103).

Several studies have linked the H+-ATPase to Ap. These have included work with chloroplasts (160), chromatophores (91), and submitochondrial particles (145). Purification of the H+-ATPase complex from PS3, a thermophilic bacterium, and incorporation into proteoliposomes demonstrated the link between Ap and ATP synthesis

(138). ATP was synthesized only when a ApH was applied to the liposomes in the presence of K+ and valinomycin (138) or when an electric field was applied in a voltage-pulse experiment (126).

Proton motive force dependent ATP synthesis was also achieved in

Streptococcus lactis, even though under normal physiological conditions the organism is unable to do so (97). This was achieved through the imposition of a proton motive force across the membranes 15

of vesicles prepared from the organism (97).

The H+/ATP stoichiometry, (i.e. the number of protons necessary

to synthesize 1 molecule of ATP), can be calculated from a comparison

of Ap and the phosphorylation potential (42,102). Reported values

include 2 or 3 for mitochondria (102), 3 for chloroplasts (103), 3 for

Paracoccus denitrificans (42), and 3 for JE. coli (68).

Mutations within the genes coding for the H+-ATPase have been

classified as unc mutations (35). Cells with these mutations are

uncoupled and cannot synthesize ATP via a proton gradient. EL coli strains that carry these mutations are unaMe to grow on organic acids

such as lactate and succinate, but will grow fermentatively on glucose

(13).

Although the major purpose of the H+-ATPase in respiratory

organisms is the synthesis of ATP, it has recently been proposed that

the major function of the enzyme in fermentative bacteria is to maintain the intracellular pH by the active extrusion of protons

(12,78). Kobayashi et al. (78) has demonstrated that the activity of

H+-ATPase, as well as its synthesis, is stimulated under conditions that acidify the cytoplasm.

The compound N,N'-dicyclohexylcarbodiimide (DCCD) has been used extensively to inhibit the H+-ATPase in the study of proton motive force (18,19,51). This compound binds to the hydrophobic part of the

H+-ATPase and blocks the transmembrane movement of protons, thus inhibiting its catalytic function (107).

Another mechanism has been proposed for the generation of proton motive force by bacteria grown under fermentative conditions (99). 16

The energy-recycling model postulates that metabolic end-products can be excreted with protons out of the cell. As a result of the translocation of protons, a proton motive force is generated. Using the fermentative bacterium Streptococcus cremoris as the model system

(134), the fate of lactate translocation out of the cell has been examined. The metabolic scheme of this organism was simplified to:

Glucose + 2ADP + 2Pi + 2(n-l) H+. <==> 2 lactate" +2 ATP ' in out + 2 n H+ + 2 H,0 out 2

As a consequence of this equation, if n=l there will be no generation of membrane potential as a result of lactate efflux. However, if n=2, the efflux process results in the translocation of 4 protons and thus the- translocation of 2 positive charges per 2 molecules of lactate excreted. As a result of this, both AV and ApH can be generated by lactate excretion and less ATP has to be hydrolyzed to generate Ap.

Lactate efflux has been demonstrated to drive the uptake of leucine in deenergized cells (112). The uptake was shown to be sensitive to protonophores but not DCCD, an inhibitor of the ATPase. This indicated that ATP was not the driving force for leucine transport

(112). Additionally, uptake of tetraphenylphosphonium demonstrated that lactate efflux increased A4* by 51 mV, indicative that lactate is translocated across the membrane with more than one proton (112).

Lactate translocation has also been studied in membrane vesicles from anaerobically grown EL coli (143). Alteration of the components of Ap through variation of the external pH in the presence of valinomycin or nigericin demonstrated that at pH 5.5 lactate uptake was driven by ApH while at pH 8.0 A4* was the main driving force. 17

Thus, at alkaline pH lactate transport is electrogenic while at pH 5.5

it is electroneutral (143). These results indicate a variable

H+/stoichiometry. This has also been demonstrated in Streptococcus faecalis where the ratio of proton influx to lactate influx increased from about 1 at pH 6.5 to about 2 at pH 7.5 (133,144). It has been

postulated that the pH effect on stoichiometry is caused by a change in the dissociation state of the lactate carrier (144).

IV. Ion transport and its relationship to Proton Motive Force.

Virtually all bacteria expend energy to expel intracellular sodium. The reasons for this are unclear, though it is believed that 4* "1" Na competes with K , the major cytoplasmic cation (4,30), for use in various cell processes (43). Aerobic bacteria, such as 12. coli, •I* *4" utilize Na /H antiporters which are dependent on Ap, not ATP, for their driving force (9,128). West and Mitchell (159) found that sodium increased the proton permeability of 1J. coli cytoplasmic + + membranes and first postulated the presence of a bacterial Na /H antiporter. Work in various laboratories has demonstrated this antiporter activity through the use of inverted (106) and right-side out (9) membrane vesicles. The energetics of sodium efflux has also been carried out in vivo in IS. coli (14). This study confirmed the results obtained with vesicles; sodium efflux was dependent on the electrochemical proton gradient rather than ATP (14,15,128).

Na+ extrusion by Streptococcus faecalis differs from that in _E. coli due to the use of an ATP-driven pump by the former (47). In initial studies, net Na+ movement and ^Na+/Na+ exchange were observed 18

only in cells capable of generating ATP (47). Cells were demonstrated

to extrude Na+ against a 100-fold concentration gradient in the

presence of dissipators of both ApH and A4* (47). The production of

inverted membrane vesicles has allowed for the isolation of a sodium-

stimulated ATPase that is distinct from F^Fg-ATPase as it was not

inhibited by DCCD (65). It was demonstrated that elevated activities

of both Na+ extrusion and Na+-stimulated ATPase could be detected when

cells were grown in the absence of Ap (65). Further work has + + demonstrated that the Na -ATPase is actually a secondary K transport

system named Ktrll (65,77). Mutants that lack the Na+-ATPase also

lack Ktrll. Ktrll and the Na+-ATPase are induced in parallel when cells are grown in high sodium media, particularly under conditions

that limit the generation of Ap (65). The uptake of K+ in exchange for Na+ is thought to be an electroneutral process (43,65).

The effects of K+ on Ap in bacteria are well documented (4,6,71).

In K+-depleted cells of Streptococcus faecalis, the addition of K+ ions depolarized the membrane by 60 mV (6). This decrease in AT was compensated by an increase in ApH leaving Ap unchanged (6). Bakker and Harold (4) showed that K+ accumulation only occurred in metabolizing cells. The accumulation of K+ could be prevented by reagents that short circuited the proton circulation. It was noted that the gradient of accumulation was too steep (i.e. 50,000 fold) to

be sufficiently accounted for by AT (4). They concluded that K+ accumulation in j5. faecalis involves both ATP and AT and is regulated + + by Ap, or by a membrane carrier that translocates K inward with H

(4). K+/H+ antiporters have been identified in both IS. coli (34) and + / + _S. faecalis (73). The K /H antiporter in IS. coli has been implicated 19

S. faecalis (73). The K+/H+ antiporter in E. coli has been implicated in the mechanism for internal pH regulation in alkaline environments

(12,69).

The pH of the growth medium also greatly affects the relative contribution of At and ApH to Ap. In growing j5. lactis cells, At decreased from -108 mV to -83 mV during a pH change from 6.8 to 5.1 while -ZApH increased from -25 mV to -60 mV over the same pH range

(72). The Ap was kept relatively constant over this pH range. The interconversion of At and -ZApH has also been demonstrated in a variety of other fermentative and respiring bacteria (1,8,51,75,132).

The decrease in At is thought to occur via the electrogenic import of cations, in particular K+, to buffer Ap due to the increased export of protons at lower external pH values (4,6,30).

V. Utilization of Proton Motive Force by Bacteria.

Proton motive force has been demonstrated to affect the transport of lactose and proline by altering the redox state of dithiols in the respective carrier proteins (32). Oxidation of a dithiol to a disulfide increased the Km for both carriers. The addition of dithiothreitol restored the full activity of the carriers (85).

Evidence for the involvement of dithiols located at the cytoplasmic side of the membrane was obtained from studies with inside-out membrane vesicles (32). The imposition of a proton motive force protected the L-proline carrier against inactivation by glutathione hexane maleimide (32). The results indicated that two redox- sensitive dithiol groups are involved in the transport of proline.

The establishment of a proton motive force by whole cells ensures the 20

dithiols located on the cytoplasmic side of the membrane, thus making

that side a low affinity form of the protein.

Proton motive force has been suggested to be critical in DNA

transformation of Bacillus subtilis (150). In this study, the effects

of ionophores, weak acids, and lipophilic cations on the components of

the proton motive force and on the entry of transforming DNA into the

cells were investigated. DNA entry into the cells was demonstrated to

be severely inhibited under conditions where Ap was small and also

under conditions where ApH was small and A 4* was high. This suggested

that Ap, in particular ApH, functions as the driving force for DNA

uptake in transformation (150).

One of the better studied systems that has been examined for

proton motive force involvement is bacterial motility (49,75,132).

Translational swimming of starved 15. subtilis cells was induced by an artificially created Ap (75). Similar results were also obtained with a motile Streptococcus sp. (141) and Rhodospirillum rubrum (141).

Two groups have examined the amount of Ap required for motility in 13. subtilis (75,132). Titration of A4* with valinomycin plus K+ demonstrated that at least -30 mV of Ap was required for flagellar rotation (75). At approximately -100 mV, no further increase in rotation rate could be demonstrated. This indicated that saturation of the system had been achieved. The alkalophilic bacterium, Bacillus strain YN-1, has been demonstrated to utilize Na+ rather than protons to drive its flagellar motors (49).

Although the generation of a proton motive force is vital for various cell processes (30,43), it has been suggested that it is not 21 obligatory for cell growth (44,76). Treatment of Streptococcus faecalis with gramicidin D dissipated Ap but still allowed for cell growth to occur (44). Growth was dependent on the presence of a complex medium supplemented with a high concentration of K+. Growth was also pH dependent as alkaline pH values (ca. pH 8.0) supported better growth than lower pH values (44). Treatment of IS. coli with the protonophore CCCP dissipated both AT and ApH, however cell growth still occurred at pH 7.5 in the presence of glucose (76).

Substitution of succinate or lactate for glucose in the growth medium inhibited the growth of 12. coli in the presence of CCCP. This indicated that oxidative phosphorylation was inhibited by CCCP treatment (76).

VI. Protein Export in Bacteria.

The export of proteins is an important aspect of bacterial physiology. Extracellular enzymes facilitate the utilization of macromolecular nutrients by degrading polymers to low-molecular weight products that can be assimilated by the bacterial cell. Examples of enzymes that act in this manner include the pectinase and cellulase of

Erwinia chrysanthemi (2). Conversely, extracellular enzymes are also utilized by the bacterial cell to synthesize polymers. Examples include the dextransucrases of Leuconostoc (81) and the oral

Streptococci (61) and the levansucrase of Bacillus subtilis (36).

Other functions of extracellular proteins in bacteria include those that provide defense mechanisms such as the beta-lactamase of

Bacillus licheniformis (46) and E. coli (5). Cell to cell communication is also achieved through the use of extracellular 22

proteins. The sex pheromone produced by Streptococcus faecalis (121)

is secreted by donor cells to facilitate conjugation to recipient

cells (121).

In bacteria, most exported proteins are synthesized as precursors

containing an amino-terminal sequence which is not found in the mature

protein (154). These sequences have been detected by comparing the

native protein with the products of iji vitro translation under

conditions where processing is prevented (154). They have also been

determined from comparing the amino-terminal end of the mature protein

with the nucleotide sequence of the structural gene (154). Many of

these amino-terminal sequences, or signal peptides, have been compared

and although there is not strict sequence homology, there are some

general features that are shared by most. The signal peptide is

anywhere from 20 to 40 amino acid residues in length. The amino-

terminus of the peptide contains 1-3 positively charged amino acid

residues, usually consisting of either arginine or lysine. The amino-

terminus is followed by a stretch of 14-20 primarily hydrophobic amino

acids. It has been postulated that the function of the positively

charged residues is to make the initial contact with the membrane via

interaction with negative charges, possibly those on membrane

phospholipids (111). The hydrophobic core is postulated to form the first membrane transversing portion of the protein. Alteration of the

hydrophobic portion of the signal peptide has inhibited the export of

proteins in a variety of bacterial systems (22,46,119). Accumulation

of precursors in the cytoplasmic membrane have been shown via these alterations. 23

The processing of signal peptides in .E. coli is accomplished by the action of two enzymes; leader peptidase, or signal peptidase I, and lipoprotein leader peptidase, signal peptidase II (24,29). Signal peptidase I has been purified to homogeneity and consists of a single polypeptide chain of 36,000 daltons (161). Its 323 amino acids span the membrane with the carboxy-terminus exposed to the periplasm.

Signal peptidase I lacks a cleavable leader sequence (24). The purified enzyme in the presence of nonionic detergents can efficiently process precursors of M13 coat protein, leucine-binding protein, OmpA and OmpF proteins, as well as the LamB protein (155).

This enzyme has been demonstrated to cleave precursors of eukaryotic as well as prokaryotic origin (117). Signal peptidase I has been demonstrated to act on precursor protein molecules after translocation into the membrane has commenced (24). Dalbey et^ al. (24) constructed an 12. coli strain in which the synthesis of signal peptidase I was under the control of the arabinose B promoter. Under conditions where the leader synthesis was repressed, precursors to maltose binding protein as well as OmpA protein accumulated in the cytoplasmic membrane. The precursors were demonstrated to be anchored at the outer surface of the membrane by their suceptibility to digestion with proteinase K (24). The results suggested that signal peptidase I acts on precursors on the periplasmic side of the IS. coli cytoplasmic membrane.

Signal peptidase II action is restricted to precursors that contain a cysteine residue after the cleavage site that is covalently modified with a glyceride thioether group. A consensus sequence has been compiled for the target site of signal peptidase II (121). It 24 been compiled for the target site of signal peptidase II (121). It consists of Leu-Leu-Ala-Gly-Cys, with cleavage occuring between the glycine and cysteine residues. Modification of the cysteine residue is absolutely required for correct processing to occur (46,52,53).

Proteins processed by this enzyme include the penicillinase of J3. licheniformis and the Braun lipoprotein in J3. coli. Signal peptidase

II is inhibited by the antibiotic globomycin, which contains a structural analog of the cleavage site on the target signal peptide

(29). This antibiotic has been utilized to demonstrate accumulation of precursors in the above described systems (55). The purified enzyme consists of a single polypeptide chain of 18,000 daltons (148).

In gram-negative cells, the enzyme is located exclusively in the inner membrane (148).

Although a proper signal sequence has been found to be mandatory for efficient export, evidence has accumulated that export-specific information exists within the mature protein (24,86,87). Through the use of stepwise deletions, Dalbey and Wickner (24) demonstrated that a region carboxy-terminal to the membrane-spanning domain of signal peptidase I was necessary for efficient translocation. Similar studies have been done with chain-terminating mutants in the (3- lactamase gene (86,87) and in the gene coding for arginine-binding protein (16).

Protein export in bacteria can occur both cotranslationally and posttranslationally (122,136). Using membrane impermeable labeling agents, polypeptide chains of exported proteins have been labeled as they cross the cytoplasmic membrane (134,135,136). Randall (122) has demonstrated that certain secreted proteins are translocated 25

posttranslationally. Translocation of the maltose binding protein occurred only after 80% of the protein had been synthesized while ribose-binding protein was translocated only after its synthesis was complete. The data suggested that entire domains of polypeptides are

transferred after their synthesis (122).

There is accumulating evidence for other protein complexes involved in bacterial protein export. Genetic studies have been utilized in E. coli to identify these components. Temperature- sensitive mutants in two genes, prlA (secY) and secA, have been isolated (3,121). These mutations result in the accumulation of unsecreted cytoplasmic precursors (3,121). The prlA (secY) gene encodes a 49,000 dalton hydrophobic protein that is in the cytoplasmic membrane (3). This protein has been demonstrated to act post­ translationally to enhance the translocation of pro-OmpA across the cytoplasmic membrane (3).

VII. Energetic Requirements of Protein Secretion.

The first demonstration of a requirement for proton motive force during export of protein by bacteria was done with the insertion of phage M13 coat protein into the cytoplasmic membrane of £. coli.

(26). Treatment of cells with arsenate to deplete cellular ATP levels had no effect on the conversion of the precursor to the mature trans­ membrane form. Treatment of cells with uncouplers such as carbonylcyanide m-chlorophenylhydrazone (CCCP) caused the precursor to be bound to the cytoplasmic side of the membrane (26). This indicated a role for proton motive force in the integration of the coat protein into the membrane. 26

Uncouplers have been used to examine the role of proton motive force in the secretion of a variety of proteins in IS. coli. Daniels et al. (25) demonstrated inhibition of processing of leucine-specific binding protein by CCCP as well as with valinomycin. They postulated a specific requirement for membrane potential in export. The use of a uncA mutant that was deficient in membrane H+-ATPase demonstrated that the export of the OmpF and LamB proteins was dependent on proton motive force and not due to depletion of cellular ATP (33).

The first examination of the actual levels of proton motive force necessary for protein export was done on the 3-lactamase in IS. coli

(5). In this study, the magnitude of the membrane potential was manipulated by varying the concentrations of K+ in the presence of valinomycin in the medium. The pH gradient was manipulated by varying the external pH of the experiment. The export of @-lactamase was inhibited over a wide range of values for either AV or -ZApH.

However, the level of total proton motive force where half-maximal inhibition was observed was constant at approximately -150 mV.

Similar experiments using a mutant strain defective in the membrane

H+-ATPase demonstrated that cellular ATP was not depleted as a result of dissipation of the proton motive force.

By using both in vitro and in vivo assays, it has been shown that conditions which prevent the generation of proton motive force across the mitochondrial membrane block the uptake of mitochondrial precursors (41,110,118). Valinomycin plus K+ prevented the import of the ADP/ATP translocator as well as ATPase subunit precursors into

Neurospora crassa mitochondria (131,142). The addition of nigericin 27

mitochondria (131). These results suggested that membrane potential alone is required for protein import. This is supported by evidence showing that import of the ADP/ATP translocator protein could be driven by a diffusion potential generated by valinomycin plus K+, even under conditions where the membrane was made permeable to protons through the use of CCCP (118).

The energetic requirements of export in gram-positive bacteria have not been as extensively studied as in .E. coli. The secretion of an alpha-amylase by Bacillus amyloliquefaciens is dependent on proton motive force (104a). This -was demonstrated through the use of uncouplers or valinomycin plus K+. When treated with these compounds, a precursor form of the enzyme accumulated in the cytoplasmic membrane. No actual measurements of proton motive force were made

(104a).

Unlike translocation across bacterial membranes, proton motive force has been shown to have no involvement in export in yeast microsomal membranes (153). Through the use of an in vitro, cell-free translocation system, it was shown that translocation was totally dependent on the availability of ATP (153). Ionophores such as valinomycin and monensin, as well as the uncoupler CCCP, had no effect on translocation.

The development of an _in vitro protein translocation system in jE. coli (17) has allowed for a closer examination of the energy requirements of protein export. Alkaline phosphatase and OmpA protein were demonstrated to require ATP rather than a proton motive force for efficient translocation (18). ATP could support translocation, though 28

less efficiently, in the presence of uncouplers or with membranes

prepared from mutants defective in the membrane H+-ATPase (19).

Further work demonstrated that the H+-ATPase is not involved in ATP-

dependent translocation (19,20). Strain CK1801 membrane vesicles,

devoid of H+-ATPase, could use ATP for protein translocation in the

presence of magnesium almost as efficiently as membrane vesicles with

a functioning H+-ATPase. The addition of D-lactate, which generates

Ap via oxidative respiration (13), stimulated the ATP-dependent

translocation of OmpA at low ATP concentrations. These results

suggested that Ap acts only in a contributory fashion in translocation

(18,19). Examination of the effects of various nucleotide analogs

demonstrated the requirement for ATP involved the specific recognition

of both the adenosine and phosphate moieties of ATP (20).

One drawback to the above described studies was the absence of

determinations of Ap under the various experimental conditions

utilized. In a separate study, measurement of A^ was done under

different experimental conditions through the use of fluorescence

quenching (37). Using membrane vesicles devoid of H+-ATPase, ATP or

NADH could only support 20% of the translocation of OmpA observed when

both were present. The data indicated that both A 4* and ATP were necessary for optimal pro-OmpA assembly into membrane vesicles (37).

Stripping membranes of the F1 subunit of the H+-ATPase rendered membranes incapable of both generating Af and translocating OmpA (37).

A study on the translocation of a chimeric ompF-lpp protein has

provided additional evidence for proton motive force involvement in protein export (162). The precursor protein is translocated post- translationally in an in_ vitro translocation system. Concurring with 29

other studies (18,19,20), translocation was equally efficient with

membrane vesicles from a strain that had its genes for the H+-ATPase

deleted or with wildtype cells (17). The uncoupler CCCP severely

inhibited the translocation of the chimeric protein. This inhibition

was not suppressed by the addition of ATP to the in vitro system

(162). Similar results were obtained with the ionophores nigericin

and valinomycin. ATP was shown, however, to have a significant role

in translocation as the addition of glucose and hexokinase, an ATP-

consuming system, almost totally inhibited the translocation of the

chimeric protein.

The studies described above demonstrate there is no universal

mechanism for driving protein export in all organisms. Prokaryotes

have been demonstrated to rely on proton motive force and/or ATP to

drive protein export. Unfortunately, studies on the energetic requirements of protein export in bacteria have centered on only two

organisms, 13. coli and .B. amyloliquefaciens. Both of these organisms rely on respiratory pathways to generate an electrochemical potential and synthesize ATP (69,70,75,132). Thus, the energetic requirements of protein export in organisms with other modes of metabolism remain to be elucidated. MATERIALS AND METHODS

I. Organism.

Leuconostoc mesenteroides ATCC 10830 was purchased from the

American Type Culture Collection, Rockville, MD. It was transferred daily in All Purpose Tween Broth (APT) (Difco Co., Detroit, MI) and biweekly on APT agar plates.

II. Preparation of packed cells.

Leuconostoc mesenteroides ATCC 10830 was maintained on APT agar and broth. To prepare packed cell suspensions, cells were grown overnight in standing culture at 30°C in APT broth and then transferred (5% vol/vol) into 100 ml of fresh APT broth. This culture was incubated, without shaking, at 30°C until the turbidity at 660 nm

(A&6o) reached approximately 0.5. The cells were harvested by centrifugation and washed twice with the medium of interest (Table 1).

The buffer MES (2[N-morpholino]ethanesulfonic acid) was included at a concentration of 200 mM to prevent rapid acidification of the growth medium by the packed cell suspension. The cells were resuspended to

9 ml in 10/9 strength medium in a 25 ml screw capped tube (Corex).

The cells were allowed to equilibrate in a 30°C water bath for 5 minutes after which 1 ml of a 50 % (wt/vol) sucrose solution was added to initiate growth and dextransucrase synthesis. The cell concentration at the start of each experiment gave an A^Q between 4.5 to 5.0.

30 Table 1. Media composition, (a)

BASAL MEDIUM (LM)

COMPONENT GRAMS/L

Tryptone 10.0"

Yeast Extract 7.0

Sucrose (b) 50.0

Tween 80 1.0 ml

MES (c) 19.5

MEDIUM COMPONENTS

LMK100 LM + 50 mM K2HPO4

LMK250 LM + 50 mM K HP0. + 150 mM KC1 2o 4

LMNalOO LM + 50 mM Na2HP04

LMNa250 LM + 50 mM Na HP0. + 150 mM NaCl 2o 4

(a) The pH of the media varied according to the experimental conditions.

(b) Autoclaved separately as a 50 % (wt/vol) solution.

(c) 2[N-morpholino]ethanesulfonic acid was included only in media for experiments with packed cells. 32

III. Determination of cellular dry weight.

Cellular dry weight was determined by the measurement of the

of appropriate culture dilutions and comparison to the values obtained

from a standard curve. To generate the standard curve, cells were

grown in LMK250 medium and harvested by centrifugation. The cell

pellets were washed twice with distilled water and resuspended to

various densities in 10 ml volumes. Aliquots were transferred to

aluminum weigh boats that had been heated at 110°C for 24 hours. The

turbidity of these aliquots was determined at 660 nm. The weigh

boats containing the cell suspensions were heated at 110°C for 24

hours. After this time, the weigh boats were removed from the oven

and transferred to dessicators where they were allowed to cool. The

weigh boats were weighed on an analytical balance and then returned to

the oven. The samples were again heated for 24 hours and reweighed.

The' cellular dry weight was determined from the difference of the

weight of the weigh boat with the dried cell paste and the weigh boat.

The turbidity values were then plotted against the corresponding

weights determined by this method. An A^Q of 5.0 was equal to 1.85

mg/ml dry weight cells.

IV. Growth of cells in batch fermenters at constant pH.

Leuconostoc mesenteroides ATCC 10830 was maintained on APT agar

and broth (Difco Laboratories, Detroit Mich.). For all experiments, cells were grown overnight for 16 hr in APT broth. A 5% (vol/vol) inoculum from this culture was transferred to a 500 ml fermenter

(Model C30 Bioflo, New Brunswick scientific Co., Edison, New Jersey)

that contained LMK100 medium lacking MES buffer (see table 1). 33

Sucrose was prepared as a 50 % stock solution in water that was adjusted to pH 7.0 and autoclaved separately from the medium. Sterile sucrose (50 ml) was added to 450 ml of LMK100 medium that was prepared in 10/9 strength. The medium pH was maintained by the addition of 5 N

K0H by a Model pH-40 Automatic pH Controller and Pump Module (New

Brunswick Scientific Co., Edison, New Jersey). All fermentation experiments were carried out at 30°C with an agitation rate of 100 rpm and no aeration. Cell concentrations were determined as dry weights from measurements of the turbidity at 660 nm from appropriate culture dilutions.

V. Extraction and assay of extracellular dextransucrase.

Dextransucrase was separated from interfering enzymes through the use of an aqueous two-phase partition technique (116). For each sample, 0.35 ml of culture supernatant was added to a 1.5 ml microfuge tube containing 0.35 ml of a 10% Dextran T-500 solution. The tube's contents were mixed and then 0.7 ml of a 20% polyethylene glycol (PEG) solution (m.w. 3350) were added. The contents were again mixed and then centrifuged in a microfuge for 3 minutes. The upper phase, which consisted primarily of PEG, was removed with a pasteur pipette and discarded while the lower dextran-rich phase was brought to a volume of 0.5 ml with distilled water. Then, 0.5 ml of the PEG solution was added, mixed, and centrifuged. This was repeated twice with the final dextran phase diluted to 0.5 ml with 0.1 M sodium acetate pH 5.0.

Dextransucrase activity was measured by following the rate of production of reducing sugars using either the Nelson-Somogyi

(109,137) or the 3,5-dinitrosalicylic acid method (139). Fructose was 34

used as the standard. Final concentrations in the assay were: sucrose, 150 mM; CaC^, 1 mM; sodium acetate pH 5.0, 100 mM; and dextran T-40, 1 mg/ml. One unit is defined as the amount of enzyme that catalyzes the formation of 1 ymole of fructose per minute at 30°C and pH 5.0. Protein was determined by the method of Lowry at al.

(95). Levansucrase activity was measured by following the rate of reducing sugar production from raffinose. The buffer for the assay of dextransucrase was used, with the substitution of 150 mM raffinose for sucrose and the exemption of dextran T-40.

The differential rate of dextransucrase secretion was determined from the slope of an extracellular dextransucrase activity vs cellular dry weight graph. The unit for the specific production of dextransucrase was expressed as the increase of extracellular dextransucrase per increase of cellular dry weight (AmU/ Amg dry weight).

Kinetic constants were determined from the method of Lineweaver and Burk (93) using enzyme that had been purified by the aqueous two- phase partition technique described above.

VI. Determination of the pH stability of dextransucrase.

To determine the pH stability of dextransucrase, culture supernatant of an 11 hour, LMK100 grown, culture was separated into 50 ml fractions and the pH was adjusted to various values between pH 5.0 to pH 8.0 with K0H or HC1. Immediately after adjusting the pH of the fractions, dextransucrase was purified by the aqueous two-phase partition technique described above and assayed for dextrnasucrase 35

activity. To mimic the conditions in the fermenter, the 50 ml

fractions were held at 30°C for 7 hours. At this point,

dextransucrase was purified and assayed as described above.

VII. Determination of extracellular polysaccharide.

Total extracellular polysaccharide was defined as the total

carbohydrate precipitated from culture supernatant by 67% (vol/vol)

ethanol. To 0.5 ml of culture supernatant, 1.0 ml of absolute ethanol

was added. Precipitated polysaccharide was recovered by

centrifugation for 2 minutes in a microfuge. This precipitate was

washed three times with 67% (vol/vol) ethanol, dried, and then

resuspended to 0.5 ml with distilled water. Total carbohydrate was

determined with the phenol-sulfuric assay (31) using glucose as the

standard.

VIII. Preparation and use of ionophores.

With the exception of valinomycin, stock ionophore solutions were

prepared in ethanol at the following concentrations: nigericin 5

mg/ml, gramicidin D 2 mg/ml and methyltriphenylphosphonium bromide

(MPTP+) 200 mM. Valinomycin was prepared in acetone at a

concentration of 5 mg/ml. For all experiments, ionophores were added

such that the final concentration of ethanol, or acetone, did not

exceed 1.0%. A control culture was included in each experiment that

had appropriate amounts of ethanol or acetone added. In experiments

utilizing packed cells, ionophores were added at 45 minutes after

sucrose induction unless otherwise specified. In studies employing

cultures grown at a constant pH in batch fermenters, ionophores were

added at 6 hours after inoculation unless otherwise specified. 36

IX. Determination of cytoplasmic and extracellular water spaces.

Cytoplasmic and extracellular water spaces were determined by 2. A 3 incubating cells in the presence of [ C]inuiin and 1^0 (66). One ml

of growing cells was transferred to a 15 x 100 mm capped culture tube,

the radiolabeled probes were added, and the tube was incubated in a

30°C water bath for 5 minutes. Then, 0.4 ml of this mixture was

centrifuged through 0.4 ml of silicone oil ((79-82%) Dimethyl-(18-

21%)-diphenylsiloxane copolymer) for 1 minute at 10,000 rpm in an

Eppendorf microfuge. The upper aqueous layer and the silicone oil

were removed by vacuum with a Pasteur pipette. The cellular pellets

were resuspended and quantitatively transferred to 20 ml scintillation

vials and dissolved with 10 ml of scintillation fluid. All samples 14 3 were counted for C and H on a Packard TRI-Carb scintillation counter and corrected for quench via a quench curve. The counts obtained for each radiolabeled probe were divided by its specific activity. This value was taken to equal the amount of probe present in the sample.

X. Determination of ApH.

The ApH values were calculated from the uptake of [^C]benzoate

(6.4 pM, 43 Ci/mol) or [^C]methylamine (2.1 uM, 49 Ci/mol) (12,66).

One ml of growing cells was transferred to a 15 x 100 mm capped culture tube and the ApH probe was added. After 5 minutes of incubation at 30°C, 0.4 ml of this suspension was centrifuged through silicone oil as described in the determination of cytoplasmic volumes.

After centrifugation, 25 yl of the upper aqueous layer was removed and transferred to a scintillation vial. This sample represented the 37

concentration of the probe in the extracellular medium (i.e.

[benzoate]Qut). The remainder of the upper aqueous layer and the

silicone oil were removed by vacuum with a Pasteur pipette. The cell

pellet was resuspended with distilled water and quantitatively

transferred to a scintillation vial. This sample represented the

concentration of the probe inside the cells (i.e. [benzoate].^) as

well as the contribution of the probe from the contaminating

extracellular fluid. The equations used to determine ApH were:

Ka Hout pKa pHoUt 1. ApH = LogfBenzoatel (1 + l0P " P ) - 10 " [Benzoate]Q"t

2. ApH = LogTmethylamine1^ (1 + lC)P^out p^a) - iop^out [methylamine] t

Equation 1 was used to calculate ApH when benzoic acid was the ApH

probe while equation 2. was used when methylamine was the probe. The

pH gradient was converted to millivolts by multiplying ApH by 2.3 RT/F

where R is equal to the gas constant, F is Faraday's constant, and T

is the temperature of the assay in degrees Kelvin. This

multiplication factor, which commonly is represented by Z, is equal to

60 at 30°C.

XI. Determination of A^.

The membrane potential was determined by measuring the uptake of 3 + [ H]tetraphenylphosphonium bromide (TPP ) (54,66). One ml of growing

cells was transferred to a 15 x 100 mm culture tube, TPP+ was added,

and the suspension was incubated at 30°C for 5 minutes. After this

time, 0.4 ml was centrifuged as described above. The extracellular 38 concentration of TPP+ was determined by counting 25 yl of the upper aqueous layer after centrifugation. Non-specific binding of the probe to cellular components was corrected for by treating a separate tube with 5% (vol/vol) n-butanol for 30 minutes prior to addition of the isotope. The value obtained from liquid scintillation counting for the butanol-treated cells was subtracted from that of the untreated culture. The . membrane potential was calculated according to the Nernst equation (123) as follows:

-RT 2.3Log[TPP+]. AV = in zF [TPP ] „ L Jout where R is the gas constant, F is Faraday's constant, and T is the temperature in degrees Kelvin. The letter z represents the valency of the probe. Substitution of the values of the respective terms in this expression reduces this equation to:

+ AV = 60.05 Log[TPP ]in

[TPP+] _ L Jout

XII. Calculation of the proton motive force.

The proton motive force was calculated according to the following equation: Ap = AT - ZApH where AT is the membrane potential, ApH is the transmembrane pH gradient, and Ap is the proton motive force which has the unit of millivolts (mV). 39

XIII. Isolation and assay of membrane ATPase.

L. mesenteroides ATCC 10830 protoplasts were prepared as previously described (113). Briefly, cells were cultivated in APT

Broth overnight at 30°C. Then, 500 ml of fresh APT Broth was inoculated with 50 ml of this culture and incubation was continued at

30°C until the A^Q reached approximately 0.5. The cells were harvested, washed with protoplast buffer, and resuspended to 80 ml with the same buffer. Lysozyme was added at a concentration of 2 mg/ml and incubation was carried out at 37°C for 1 hour. Protoplasts were lysed with the addition of NaCl to give a final concentration of

1.5 M. The suspension was incubated at room temperature for 10 minutes and then centrifuged at 3000 x g for 10 minutes to remove unlysed cells. The resulting supernatant was then centrifuged at

30,000 x g for 30 minutes. The pellet was washed twice with 50 mM

Tris, 10 mM MgSO^ pH 7.5 and was stored at -20°C until assayed.

Membrane ATPase was assayed by following the production of inorganic phosphate from ATP hydrolysis (11). Assay buffer consisted of 5 mM

ATP, 5 mM MgC^, and 100 mM Tris-HCl for pH above 7.5.

XIV. Statistics.

All determinations were done in triplicate unless otherwise indicated. The sampling size is represented by the letter n in these circumstances. The values are reported as the sampling mean and where indicated are ± the standard deviation. 40

XV. Chemicals.

All radioactive compounds were obtained from New England Nuclear,

Medford, Mass. Silicone oil was obtained from Petrarch Systems,

Bristol, PA. All other chemicals were of reagent grade and are available commercially. RESULTS

I. Use of an aqueous-phase system to purify dextransucrase.

The study of dextransucrase regulation in L. mesenteroides

required a method that would accurately measure the levels of

extracellular dextransucrase under various conditions. One assay that

is commonly used to measure dextransucrase activity, the measurement

of reducing sugar production, is not feasible under normal growth

conditions due to the large background of reducing sugar found in the

medium. Additionally, the organism also produces extracellular

levansucrase which will also produce reducing sugar from sucrose.

These problems were circumvented by the use of an aqueous two-phase

partition technique (116). The procedure outlined in the Materials

and Methods section recovered dextransucrase at 95 % yield with a

specific activity of 30 U/mg protein. The preparation lacked

levansucrase activity as judged by its inability to cleave raffinose,

a substrate for the levansucrase but not the dextransucrase from this

organism (125). The Km of the enzyme for sucrose was determined to be

20 mM from a Lineweaver-Burke plot. This is within the range of Km reported in the literature for this enzyme (80,100). The Vmax was calculated to be 3.31 ymol/min/mg protein under the assay conditions

utilized.

II. Effect of ionophores on the growth of packed cells.

Ionophores are a valuable tool in examining the energetic requirements of various systems (39,120). In order to determine

41 42

whether or not proton motive force was involved in the regulation of

dextransucrase secretion, the effects of various ionophores were

examined using packed cells. Extracellular, dextransucrase could be

determined after short periods of growth using this method.

Leuconostoc mesenteroides ATCC 10830 was grown in various complex

media that was supplemented with 200 mM MES to prevent rapid

acidification of the media. The organism grew equally well in

LMK100, LMK250, and LMNalOO media (see Materials and Methods). The

mean generation time in each medium was 80 ± 5 minutes (n=5). The

cells lowered the culture pH from 6.7 to 5.8 in approximately three

hours, regardless of medium composition. The addition of valinomycin

to LMK250 medium at a concentration of 10 Ug/ml at pH 6.6 essentially

doubled the mean generation time (154 ± 5 minutes, (n=3). When added

to cultures at pH values below 6.0, there was no effect on cell

growth. Methyltriphenylphosphonium bromide (MPTP+), a lipophilic

cation that is also a dissipator of membrane potential, affected

growth in a manner similar to valinomycin when added at a pH of 6.6.

The mean generation time of such treated cells was 158 ± 6 minutes

(n=3). Cells treated with gramicidin D, a channel forming ionophore

that equilibrates ions and protons across cytoplasmic membranes (120),

required K+ in the growth medium to sustain growth (Figure 1). The

mean generation times in LMK250 and LMK100 media were essentially the

same as values of 100 ± 5 minutes (n=3) and 102 ± 6 minutes (n=3) + + were obtained respectively. Substitution of Na for K as the

predominant cation in the growth medium prevented growth of ATCC 10830

in the presence of gramicidin D. 43

90 120

TIME (min)

Figure 1. Effect of gramicidin D on the growth of L. mesenteroides ATCC 10830. Gramicidin D was added at a concentration of 10 ^g/ml at t =>45 min to cultures in either LMK250 or LMNalOO media. Symbols:

©, LMK250;0 LMK250 + gramcidin D; A , LMNalOO; ^ , LMNalOO + gramcidin D. 44

Interestingly, cells treated with gramicidin D were able to lower

the culture pH as effectively as control cultures (Figure 2). This

is in contrast to the results obtained with monensin and

nigericin, both equilibrators of protons across membranes. In the

presence of either of these ionophores, growth and acid production

were severely inhibited once the medium pH fell below 6.2 (Data not

shown). This indicated that these ionophores were having different

mechanistic effects on the cells as compared to gramidicin D.

III. Effect of ionophores on the secretion of dextransucrase.

The effects of ionophores on extracellular polysaccharide

production by IJ. mesenteroides ATCC 10830 are shown in Table 2. The

addition of nigericin and valinomycin, which should dissipate both AW

and ApH, inhibited extracellular polysaccharide production. The

addition of dissipators of ApH inhibited polysaccharide production

over five fold in K+ and Na+ media.

The effects of the addition of nigericin, valinomycin, or a

combination of the two ionophores on the export of dextransucrase are

shown in Figure 3. Initially, at a culture pH of 6.33, the amount of

extracellular dextransucrase obtained from valinomycin treated cells

was depressed as compared to nigericin or control treated cells. As

the pH of the medium decreased, valinomycin treated cells exported

dextransucrase at a higher rate. Conversely, nigericin treated cells

were inhibited in their ability to export dextransucrase as the culture pH decreased. Cells treated with monensin in Na+ containing

medium (LMNalOO) gave results essentially identical to those obtained with nigericin treated cells in potassium medium (data not shown). 45

6.4

6.0

5.6

5.2

60 120 130 240 300

TIME {min )

Figure 2. Culture pH of cells treated with gramicidin D or nigericin. Gramicidin D or nigericin were added to cultures in LMK250 or LMNalOO media at t • 45 min. Symbols: ©, LMK250 and LMNalOO;O, LMK250 + 10

Ag/ml gramicidin D; O , LMNalOO + HMg/ml gramicidin D;A, LMK250 + 10 /(g/ml nigericin. 46

Table 2. Effect of ionophores on the production of extracellular polysaccharide.(a)

Medium Ionophore Polysaccharide'3

LMK250 None 1050 Valinomycin (10 Ug/ml) 750 Gramicidin D (10 ug/ml) 320 Nigericin (1 yM) 180 Valinomycin (10 Ug/ml) + 0 Nigericin (1 yM)

LMNalOO None 660 Monensin (5 yg/ml) 115 Gramicidin D (10 ug/ml) 0

(a) Ionophores were added to cultures at a pH of 6.6. Cells were

allowed to incubate for 150 minutes and then extracellular

polysaccharide was determined as described in Materials and

Methods.

(b) Extracellular polysaccharide = Ug/mg dry weight cells. 47

120 180 240

TIME (min ]

Figure 3. Effects of nigericin and valinomycin on the production of extracellular dextransucrase. Ionophores were added to cultures at t b 45 min at the following concentrations; control, (• ); nigericin,

10 jug/ml ( B ); valinomycin, 10 /fg/ml (A); valinomycin, 10/«g/ml and nigericin, 10 ifg/ml (O). Dextransucrase was purified and assayed as described in Materials and Methods. 48

Cells treated with a combination of valinomycin and nigericin were unable to produce extracellular dextransucrase. The effect of

valinomycin on dextransucrase export was dependent on the medium pH

(Table 3). As the medium pH decreased, the ability of valinomycin to inhibit dextransucrase export also decreased. Valinomycin showed virtually no effect on dextransucrase export when it was added to cells at a pH of 5.5, suggesting that AT was not critical for efficient dextransucrase secretion at this pH.

In order to determine whether valinomycin was inhibiting dextransucrase export by dissipating AT or by dissipating a K+ gradient, export in the presence of MPTP+ was examined. When MPTP+ was added at a concentration of 1 mM to cells at pH 6.6, the effect on dextransucrase export was essentially identical to that of valinomycin (Figure 4). This suggested that AT was specifically affected. Cultures treated with gramicidin D were deficient in the export of dextransucrase in LMK250 medium (Figure 5). This inhibition + also paralleled the effects of valinomycin and MPTP . This indicates that gramicidin D does not act as a dissipator of Ap in this system but may only be dissipating AT.

To determine whether the ionophores altered dextransucrase activity, they were added at the same concentrations used in the above described experiments to a dextransucrase preparation that had been aqueous-phase partitioned from an exponentially growing culture.

There was no significant effect of the ionophores on dextransucrase activity (data not shown). To eliminate the possiblity that the ionophore effects on extracellular dextransucrase levels were due to the result of growth inhibition rather than secretion, cells were 49

Table 3. Effect of pH on valinomycin mediated inhibition of dextransucrase secretion, (a)

Medium pH % Inhibition

6.1 ' 70 5.9 55 5.7 45 5.5 0 5.3 0

(a) Cells were incubated in LMK250 medium containing 5% sucrose with

valinomycin added at a concentration of 10 yg/ml at the

appropriate culture pH. The percentage inhibition was determined

as the % decrease of dextransucrase secreted (U/mg dry weight) as

as compared to a control culture with no valinomycin added. 50

250

200

150

100

TIME {min)

Figure 4. Effect of methyltriphenylphosphonium bromide (MPTP+) on dextransucrase secretion. MPTP* was added to a concentration of 1 mM at t - 45 min (* ). Control, (©); valinomycin, 10 *g/ml ( D ). 51

S

60 _

SO 120

TIME (min)

Figure 5. Effect of gramicidin D on dextransucrase export in LMK250 medium. Gramicidin D was added at a concentration of 10ug/ml at t = 45 min. Symbols: LMK250, (©); LMK250 + gramicidin D, (B ). 52 incubated for two hours in the presence of sucrose prior to the addition of ionophores. Sufficient dextransucrase would then have been exported to provide ample fructose for cell growth. The results of this experiment are shown in Figure 6. When ionophores were added at culture pH values of 6.17, only nigericin significantly affected the growth of L. mesenteroides. The addition of MPTP+ produced a slight lag followed by normal growth whereas valinomycin and gramicidin had no affect on cell growth. The export of dextransucrase, however, was severely inhibited by all the ionophores examined. This suggests that the secretion of dextransucrase, not the production of the enzyme, was specifically affected.

These results suggested that proton motive force was involved in dextransucrase secretion. In order to further examine this hypothesis, it was necessary to measure the components of the proton motive force under various conditions. Packed cells were again chosen for these studies.

IV. Determination of Ap in packed cells.

The Ap of growing cells of Leuconostoc mesenteroides is shown in

Figure 7. Equilibration of all the probes utilized was rapid and complete within five minutes. The cytoplasmic volume was calculated to be 1.64 ± 0.10 yl/mg dry weight cells (n=30). The organism was able to increase -ZApH from -10 mV to -50 mV as the external pH fell from 6.6 to 5.0. This increase came at the expense of the membrane potential as AT decreased from -130 mV to --90 mV over this pH range.

This gave a relatively constant value of Ap of -140 mV over a wide range of external pH. As the pH of the medium was raised above 7, Ap 53

•" "—

Q O ^ I g 2

E

1 1 1 1 1 1

250 Ui

p £ 1 £ 150 g£

Q§.! 50 / , 150 210 91 150 210

TIME (min)

Figure 6.. Effect of the addition of ionophores after allowing dextransucrase export. Cells were allowed to incubate in the presence of 5% sucrose in LMK250 medium for 120 minutes to allow abundant dextransucrase production. At this point, ionophores were added at the following concentrations: valinomycin, 10 ug/ml (0); MPTP , 100 yM (A); gramicidin D, 10 ug/ml (A); nigericin, 10 Ug/ml (•). A separate culture containing no ionophores (©) acted as the control. 54

Figure .7. Proton motive force of L. mesenteroides ATCC

10830 in LMK100 medium. Cells were grown and assayed as

described in Materials and Methods. The data presented are

the averages calculated from three separate experiments.

Symbols: ( A ) AP. ( ® ) AT. ( • ) -Z ApH. 55

steadily decreased due to the increased positivity of -ZApH. IJ.

mesenteroides was unable to maintain a constant internal pH (Figure

8). Only over a narrow range of external pH from 6.6 to 7.0, was the

internal pH held constant. Although the contribution of ApH to Ap

increased with decreasing external pH, A^ was always the predominant

component. The addition of MES buffer to the culture medium had no

effect on the generation of ApH (Data not shown). This buffer has

been demonstrated to dissipate ApH in other fermentative bacteria (8).

V. Effect of cations on the generation of ApH.

The effect of Na+ or K+ on ApH is shown in Figure 9. The

stimulation of ApH generation by the presence of K+ increased as the

external pH decreased, but was prevalent over a wide range of external

pH. Increasing the Na+ content in the growth medium significantly

inhibited the ability of the cells to generate ApH. This was true

over a wide range of external pH values. Substi'tution of 250 mM Na+

for 250 mM K+ decreased -ZApH by approximately 20 mV. The

concentrations of cations utilized had no effect on the cytoplasmic

volume indicating that the effect on ApH was not due to changes in the

internal volume of the cell (table 4).

VI. Effect of cations on A1}*.

The effect of Na or K on the generation of Af is shown in

Figure 10. The decrease in A^ as a function of increasing K+ was constant over a wide range of external pH as increasing the added K+ from 100 to 250 mM decreased A'l7 by approximately 20 mV. Substitution of Na+ for K+ as the primary medium cation had no significant effect 56

Figure 8. Effect of external pH on internal pH in L. mesenteroides. The intracellular pH was determined by measuring the accumulation of [^C] benzoic acid or [14C] methylamine as described in Materials and Methods. The data represents averages obtained from three experiments. 57

-50

-10_

10 _

5.0 10 7.0

Figure 9. Effect of K+ or Na+ on the generation of ApH by

L^. mesenteroides. The data represent averages from three experiments. Symbols: ( 9 ) LMKIOO, ( H ) LMK250, ( O )

LMNalOO, ( • ) LMNa250. 4* (3) Table 4. Effect of K or Na on the cytoplasmic volume

MEDIUM VOLUME^)

LM 1.63 ± 0.10

LMK100 1.64 ± 0.10

LMK250 1.58 ± 0.08

LMNalOO 1.59 ± 0.09

LMNa250 1.70 ± 0.10

(a) The data represent the mean of three determinations.

(b) Volume = ul/mg dry vreight cells ( ± standard deviation) 59

J

P» {out)

Figure 10. Effect of K+ or Na+ on the generation of by

Ii- mesenteroides. The data represent averages obtained from three experiments. Symbols: ( © ) LMK100, (B) LMK250,

(O) LMNalOO, (•) LMNa250. 60 on A4\ These results suggest that internal Na+ is extruded from IJ. mesenteroides by electroneutral exchange with external protons.

VII. Effect of potassium on Ap.

The effect of increasing extracellular [K+] on the generation of

Ap is shown in Figure 11. At an external pH of 5.5, an increase the extracellular [K+] increased -ZApH at the expense of AV. An increase in the external K+ concentration by 500 mM increased -ZApH by 24 mV.

The membrane potential decreased by 18 mV over this range of K+.

Thus, Ap slightly increased from -135 mV to -141 over the range of external K+ examined. The organism, thus, can maintain a relatively constant Ap over a wide range of external K+.

VIII. Activity of the membrane ATPase.

The pH activity curve of H+-ATPase in isolated membranes from 1^. mesenteroides is shown in Figure 12. The pH optimum was between pH

5.0 to 5.5. Below pH 5.0, ATPase activity sharply declined as no activity could be observed at pH 4.5. The H+-ATPase activity was depressed at alkaline pH. To examine the role of the H+-ATPase in the generation of Ap in L. mesenteroides, packed cells were treated with DCCD, a known inhibitor of membrane ATPases in bacteria (107)

(Table 5). The addition of 250 uM DCCD to growing cells totally inhibited the generation of Ap. This suggests that the membrane H+-

ATPase is solely involved in generating Ap at the pH examined. 61

£6 -too

100 200 300 400 K*(mM)

Figure 11. Effect of K+ on Ap at pH 5.5. Cells were grown

in media containing different K+ concentrations and were

allowed to metabolize until the medium pH was lowered to pH

5.5. They were then assayed as described in Materials and

Methods. Symbols: (A) Ap. ( 0 ) AH1, ( © ) -Z A pH. 62

u

4

PH

Figure 12. The pH activity curve of the H+-ATPase activity

from isolated membranes of L. mesenteroides. Table 5. Effect of DCCD on the generation of Ap.

Treatment -ZApH AV Ap Doubling Time

Control -37 -109 -146 56

DCCD 13 3 16 548 64

IX. Inhibition of dextransucrase secretion by sodium.

It was noted that the level of dextran determined from supernatant of cells grown in Na+ medium was lower than that in K+ medium (Table 2). This occurred despite the lack of growth effects between the different media (Figure 1). Dextransucrase was isolated from culture supernatants of cells grown in LMK250 and LMNalOO media.

Dextransucrase from LMK250 grown cells reached 47 mU/mg dry wt at 110 minutes as compared to 28.1 mU/mg dry wt from LMNalOO grown cells.

This represented a 40 % inhibition of dextransucrase secretion. Thus, the presence of sodium in the growth medium inhibits the production of extracellular dextransucrase as well as dissipates the transmembrane pH gradient.

X. Dextransucrase secretion by cells grown in batch fermenters at constant pH.

The use of packed cells presented a disadvantage in that the medium pH continually decreased, presumably due to organic acid production by the organism. Thus, manipulation of the pH gradient, vital to the study of its involvement in dextransucrase secretion, was impossible. This problem was overcome by studying the secretion of dextransucrase in cultures grown at constant pH in batch fermenters.

The pH gradient could be manipulated by simply varying the medium pH at which the fermentation was run while A4* could be altered by an ionophore such as valinomycin or methyltriphenylphosphonium bromide.

The optimum pH for growth was between 6.0-7.0 (table 6). Within this pH range the mean doubling time was 31 ± 2 min (n=4). The mean doubling time increased when the medium pH was above or below this pH 65

Table 6. Cell growth as a function of medium pH.

Medium pH Mean Doubling Time Cell yield (a)

5.0 60 0.548 5.5 50 0.858 6.0 32 1.547 6.3 34 1.448 6.7 29 1.500 7.0 31 1.360 7.5 54 0.991 8.0 67 0.531

(a) Mg/ml dry weight at t = 6 hours 66

range. At pH 8.0, the mean doubling time increased to 67 min while at

pH 5.0 it increased to 60 min. Leuconostoc mesenteroides was able to

secrete dextransucrase over a wide pH range (Figure 13). An optimal

rate of 1240 AmU/Amg dry wt. was determined at pH 7.0. Decreasing the

medium pH had little effect on the rate of dextransucrase secretion.

Above pH 7.0, the rate of dextransucrase secretion was severely

inhibited. At pH 8.0, no dextransucrase could be detected in culture

supernatants. To determine whether the depressed levels of

dextransucrase were a result of pH inactivation, the pH stability of

dextransucrase was determined (Figure 14). Dextransucrase activity

decreased 40 % when incubated at pH 7.5 for 7 hours whereas it was

totally inactivated at pH 8.0. Thus, at pH 7.5 the low levels of

dextransucrase observed cannot be totally accounted for by the

inactivation of the enzyme. To further examine this, a culture grown

at pH 6.7 for 6 hours was shifted to pH 7.5 and extracellular

dextransucrase was determined at 10 minute intervals for 1 hour. This

shift decreased dextransucrase secretion to 107 AmU/Amg dry wt. and

had no effect on cell growth. After 1 hour at pH 7.5, the pH of the

medium was lowered back to 6.7. Dextransucrase secretion increased to

1130 AmU/Amg dry wt. This increase in the rate of dextransucrase

secretion was detected 10 minutes after the shift in culture pH to pH

6.7. These results indicated that the low levels of dextransucrase found in cultures at pH 7.5 were due to inhibition of secretion rather

than pH inactivation of the enzyme. 67

Medium pH

Figure 13. Extracellular dextransucrase production as a function of the medium pH. Culture supernatants were extracted and assayed for dextransucrase as described in

Materials and Methods between t=6 and t=7 hours. 68

s 6

§ i

Q£

e.o 10 s.o

pH

Figure 14. Effect of pH on the stability of dextransucrase.

Dextransucrase was pH adjusted to various pH values and held at 30 °C for 7 hours. Symbols: ( B) immediately after pH adjustment, (•) held at 30 ° C for 7 hours. 69

XI. Determination of Ap in cells grown at a constant pH.

The components of the proton motive force were determined at

various pH (Figure 15). Cells maintained a constant proton motive

force of -130 mV from pH 7.0 to pH 5.8. Below pH 5.8 Ap decreased and

reached a level of -112 mV at pH 5.3. The contribution of -ZApH to the

proton motive force increased from 0 mV at pH 7.0 to -50 mV at pH 5.3.

The membrane potential decreased from -130 mV to -62 mV over this

pH range. The organism was able to maintain a relatively constant

internal pH of 7.0 when grown within a pH range of 6.6 to 7.5 (Figure

16). Thus, above pH 7.0 ApH inverted (i.e. the interior was acidic with respect to the medium). The inverted ApH increased with increasing external pH until at a culture pH of 7.9 the internal pH was 7.18. This inversion of ApH caused a decrease in Ap even though

AT increased to -140 mV. As demonstrated previously through the use of packed cells (figure 8), the organism was unable to maintain a constant internal pH when the external pH was lowered below pH 6.6.

This decrease in internal pH, did not allow the organism to compensate for the decrease in AV and thus caused a decrease in Ap at lower pH values.

XII. Effect of methyltriphenylphosphonium bromide on Ap and

dextransucrase secretion.

The lipophilic cation methyltriphenylphosphonium (MPTP+) bromide was used to manipulate the membrane potential of growing cells while the transmembrane pH gradient was manipulated by varying the pH of the medium. The titration of Ap with MPTP+ is shown in Table 7. The effects of MPTP+ were not specific for AV as ApH was slightly "100

10

0

6.0 10 8.0

Medium pH

Figure 15. Effect of medium pH on the generation of Ap by L. mesenteroides. Cells were grown at different pH values for 6 hours and were assayed for ApH and AT as described in Materials and Methods. Symbols: (a) Ap,

( m ) AT . ( ® ) -Z A pH. 71

§

i 7 e

External pH

Figure 16. Effect of medium pH on the internal pH of L. mesenteroides. The internal pH was determined from 14 measurements of the accumulation of [ C] benzoic acid or

[1<4C] methylamine as described in Materials and Methods. 72

Table 7. Effect of MPTP+ on the generation of Ap at pH 6.0.

[MPTP+] (mM) -ZApH AY Ap

0 -35 -95 -130 0.1 -31 -85 -116 0.5 -31 -61 -92 1.0 -27 -54 -81 73 dissipated. Between pH 5.5 to 6.0, the addition of 1 mM MPTP+ inhibited ApH by 15%. Addition of MPTP+ at a concentration of 1 mM decreased the membrane potential by approximately 40 % regardless of the medium pH (data not shown). The addition of MPTP+ to the cells inhibited dextransucrase secretion in a pH dependent manner (Figure

17). At a culture pH of 5.5, MPTP+ had no effect on the rate of dextransucrase secretion. Dextransucrase secretion decreased as a function on increasing culture pH until at pH 7.0 it was inhibited by

95 % as compared to cells not treated with the lipophilic cation. When added to cells at pH 5.5, MPTP+ increased the doubling time of the cells from 96 to 146 minutes. At pH 7.0, MPTP+ increased the doubling time of the cells from 113 to 134 minutes. Thus, the pH dependent effects of MPTP+ on dextransucrase secretion were not the result of growth effects. The rate of dextransucrase secretion was demonstrated to be dependent on the proton motive force (Figure 18).

Dextransucrase secretion was severely inhibited at values of Ap less than -80 mV. Between -80 to -90 mV, there was a rapid rise in the rate of extracellular dextransucrase production. Above -90 mV there was no significant increase in the rate of dextransucrase export indicating that a saturation level had been reached. The ability of 1^. mesenteroides to secrete dextransucrase after treatment with MPTP+ was directly related to the presence of a pH gradient (inside alkaline) across the cell membrane (Figure 19). These results suggest that proton influx is required for efficient dextransucrase secretion. 74

tf 1200 •d bfi

900

600

300

5 6

Medium pH

Figure 17. Dependence of MPTP+ mediated inhibition of

dextransucrase secretion on medium pH. Symbols: ( O )

cultures treated with 1 mM MPTP+, ( H ) untreated control. 75

£

•o &c S

1000

-75 "100 -125

Ap (mV)

Figure 18. Dependence of extracellular dextransucrase

production on A p. Cells were treated with 1 mM MPTP+ at

different pH values between 5.5-7.0 and the rate of

dextransucrase secretion and Ap was determined as

described in Materials and Methods. Symbols: ( • ) culture

treated with 1 mM MPTP+, ( B ) untreated cultures. 76

800 _

600 _

400 _

200 __

mV

Figure 19. Dependence of dextransucrase secretion on the transmembrane pH gradient after treatment with 1 mM MPTP+.

Procedures are as described in the legend to figure 5. 77

XIII. Effect of nigericin on dextransucrase secretion.

At pH 7.5 extracellular dextransucrase production was severely inhibited (Figure 13). The proton motive force of L,. mesenteroides at this pH was higher than the level of Ap required for optimal dextransucrase secretion as determined by MPTP+ treatment (Figure 18).

At a culture pH of 7.5, ApH was equal to 0.48 (interior acidic).

Cellular processes dependent on proton influx would not be expected to function well at pH 7.5 due to the large amount of energy required to bring protons into the cell against the concentration gradient. To examine whether dextransucrase secretion was affected in this manner, nigericin was added to cells grown at pH 7.5 (Table 8). This ionophore is commonly used to dissipate pH gradients across biological membranes (39). Addition of nigericin to cells at pH 7.5 stimulated dextransucrase secretion four-fold, from 124 to 494 AmU/Amg dry wt. The pH gradient (interior acidic) was partially collapsed from 29 to 24 mV and Ap increased from -110 to -119 mV. Thus, partial dissipation of the inverted pH gradient stimulated the production of extracellular dextransucrase. When nigericin was added to cells at pH 6.7, the generation of ApH was severely, but not totally, inhibited with a corresponding increase in A^. This increase in AV kept Ap relatively constant. The doubling time of the cells increased from

116 to 254 minutes but there was no effect on the specific production of extracellular dextransucrase. This provides evidence that dextransucrase secretion is independent of general growth effects caused by the ionophore. Addition of nigericin at pH 6.3 totally dissipated ApH and increased A4*. However, growth and dextransucrase secretion were severely inhibited indicating the importance of the 78

Table 8. Effect of nigericin on Ap and dextransucrase secretion.

Q k Treatment pH -ZApH A^ Ap Growth Dextransucrase

Control 6.3 -34 -99 -133 116 1038 Nigericin 1 -129 -128 553 328

Control 6.7 -26 -115 -141 116 1038 Nigericin -6 -135 -141 254 1078

Control 7.5 29 -140 -111 107 124 Nigericin 24 -143 -119 105 494

(a) Doubling time between t= 6-7 hrs.

(b) AmU/Amg dry wt. 79 transmembrane pH gradient at pH 6.3. DISCUSSION

Most of the studies on the energetic requirements of protein

secretion in bacteria have utilized JS. coli as the model system

(5,19,21,37). Although the use of this organism offers the

investigator the advantage of a very well defined system, it offers

the disadvantage of too narrow a focus. What is true in 12. coli is

not true for all organisms. Thus, any theory that is espoused for all

bacteria from work done in only one organism must be tested in others.

The study of protein secretion by Leuconostoc mesenteroides offered

several advantages. This organism is very proficient at secreting

dextransucrase, as well as other enzymes. Additionally, this organism

lacks electron transport chains. It uses a strictly fermentative

metabolism and thus offers an energetic system distinct from that

found in JS. coli. Dextransucrase production by L. mesenteroides is

also very costly to the sugar industry due to the processing problems

caused by dextran (23,54). This project, then, was of interest as

both a basic and applied research problem.

The dependence of dextransucrase secretion on the protonic

potential has been demonstrated by manipulation of the components of

Ap with ionophores. The results shown in figure 18 demonstrate that,

between pH 5.5 to pH 7.0, dextransucrase export is dependent on proton

motive force. Optimal rates of dextransucrase secretion require Ap to

be greater than -90 mV. Above this level of Ap, there was no

significant increase in dextransucrase secretion. This "saturation"

of the dextransucrase secretion system by Ap is similar to the findings of Ap involvement in involvement in flagellar motility in 13.

80 81

subtilis (75,132). The maximal rate of flagellar rotation was achieved at values of Ap greater than -100 mV (132).

Interestingly, the proton motive force of JL. mesenteroides when grown above pH 7.0 was above the level of Ap required for optimal dextransucrase secretion but there was no enzyme detectable in culture supernatants. From experiments measuring the stability of dextran­ sucrase as a function of pH (Figure 14), it was clear that significant inactivation of enzyme occurs at pH 8.0. However, at pH 7.5, the turnover of dextransucrase due to pH was only 40 %. Extracellular enzyme levels were depressed by greater than 90 % as compared to optimum levels obtained at 7.0. The proton motive force at pH 7.5 was -112 mV, which was also above the saturation level of Ap required for optimal dextransucrase secretion. The proton gradient, however, was inverted (i.e. the interior of the cell was acidic with respect to the exterior)'as compared to cells grown below pH 7.0. Treatment of cells with nigericin at pH 7.5 partially dissipated the inverted gradient (Table 8) and stimulated dextransucrase by approximately four-fold. The result of shifting the medium pH from 6.7 to 7.5 and then back to 6.7 demonstrated that secretion of dextransucrase, and not cell growth, was specifically affected. The internal pH remained relatively constant at pH 7.0 when the external pH was varied from 6.7 to 7.5 (figure 16). This ruled out internal pH as a control factor in dextransucrase secretion. Thus, dextransucrase is dependent on the presence of a proton gradient that is oriented in the proper direction

(i.e. where the interior is alkaline with respect to the exterior).

The idea of regluation of dextransucrase secretion by the proton gradient is supported by the results obtained with cells grown in 82

sodium containing media. Virtually all bacteria expend energy to extrude internal sodium. In most bacteria, this task is entrusted to

Na+/H+ antiporters that are dependent on the protonic potential for their driving force. The extrusion of sodium in the fermentative bacterium Streptococcus faecalis is accomplished by the action of a

Na+-K+-ATPase (43,47). The results of this study suggest that L^. mesenteroides utilizes the protonic potential to drive the expulsion of sodium from the cell. The dissipation of ApH but not A4* suggests that Na+ is exchanged for H+ in an electroneutral manner. The dissipation of ApH by Na+ correlated with a decrease in the production of extracellular dextran and dextransucrase by J±. mesenteroides (Table 2). This further supports the hypothesis that dextransucrase secretion requires a protonic potential for efficient secretion. The inhibition of glucosyltransferase secretion by Na+ has also been noted in the oral streptococci (74,140). However, Na+ has been demonstrated to decrease A4* in that system (73). Thus, the oral streptococci utilize an electrogenic mechanism for the expulsion of

+ internal Na rather than a electroneutral one demonstrated in IJ. mesenteroides.

Apparently a value of proton motive force for IJ. mesenteroides is not equivalent at acidic and alkaline pH. The total dissipation of Mf and ApH by DCCD suggests that the H+-translocating ATPase is solely responsible for the generation of Ap in IJ. mesenteroides (Table 5).

It has been proposed that lactate efflux contributes to the generation of potential energy in fermentative bacteria (99,143). The results of this study suggest that this process does not contribute to the 83 generation of Ap in IJ. mesenteroides under the experimental conditions examined. Because of the large inverted pH gradient, the large membrane potential above pH 7.0 must be generated by additional path­ ways other than simply proton efflux via membrane H+-ATPases. Thus, the term "proton motive force" may not be applicable to the membrane energetics of L^. mesenteroides above pH 7.0. The organism may utilize other ions to drive solute transport systems such as is the case with alkalophilic bacteria (30,40). These organisms utilize Na+ to drive many transport systems (30) as well as flagellar movement (49).

Conversely, it is also possible that protons are held in a

"localized" fashion that allow proton driven solute transport possible even under conditions where the bulk phase to phase proton gradient is not directed in the correct orientation (82,105). It has been postulated that alkalophilic bacteria accomplish ATP synthesis by localized movement of protons across the cell membrane (40). This localized movement would not be detected by the methods currently used to estimate ApH. Apparently, dextransucrase is not driven in this manner. The secretion of dextransucrase at alkaline pH is suppressed as compared to pH values at or below pH 7.0. This does not rule out the possibility that localized proton flow could drive other transport systems in .L. mesenteroides.

ATP cannot be ruled out as an energy source for dextransucrase secretion as no measurements of the intracellular ATP levels of cells treated with various ionophores were made in this study. However, measurements of the bulk intracellular ATP concentration do not reflect the involvement of ATP in secretion. 12. coli mutants defective in the proton translocating ATPase are still able to utilize 84

ATP to drive protein export (19,20,21). Additionally, MPTP+ has been

documented in Bacillus subtilis to actually increase the intracellular

ATP concentration (63). Lipophilic cations induced cell lysis of 13. subtilis at the concentrations used in this study (63,164). Although

the growth of L. mesenteroides was slowed by the addition of MPTP+,

the effects were not as severe as were noted in 15. subtilis.

Lipophilic cations have been demonstrated to bind to cellular components (94,123). Thus, any biological effects caused by these compounds must be carefully examined. Unfortunately, radiolabeled

MPTP+ was not available for this research. However, the pH dependency of MPTP -mediated inhibition of dextransucrase secretion ((figure 17) was very similar to the action of valinomycin on packed cells (table

3). This indicated that the two compounds were exerting their effects on dextransucrase secretion by the same mechanism, namely the dissipation of the membrane potential.

The energetic requirements of protein export in bacteria have been examined predominantly in Escherichia coli (5,18,19,20,26,37).

This organism has been demonstrated to use both ATP (18^,19,20) and proton motive force to drive protein export (5,37,162). The proton motive force in E. coli is typically -175 to -200 mV (1,66). It has been demonstrated that a decrease in the proton motive force to -150 mV decreased the export of 8-lactamase by 50 % (5). This value of proton motive force is larger than that obtained by J^. mesenteroides under optimal growth conditions. The level of proton motive force where dextransucrase export was inhibited by 50 % can be calculated to be approximately -85 mV. This represents a decrease in Ap by 35 % as 85 compared to approximately 25 % for JS. coli (5). The results of this study suggest that a certain percentage of the proton motive force is required for efficient protein secretion. Thus, a fermentative organism such as L.. mesenteroides requires a smaller absolute value of

Ap for protein secretion than IS. coli, but the relative percentage of the normal Ap required is fairly similar for both organisms.

Certainly, more systems need to be examined in order to determine if this relationship holds true for all bacteria.

Protein export in several gram-positive bacteria has been suggested to be dependent on Ap. The export of a-amylase in Bacillus amyloliquefaciens was demonstrated to be inhibited by CCCP or valinomycin plus K+ (104a) while gramicidin was shown to inhibit the export of a glucosyltransferase in Streptococcus sanguis (74).

Neither of these studies addressed the question of whether protein export was driven solely by AT, ApH, or by Ap. This study, therefore, represents the first examination of the actual relationship between

Ap and protein secretion in gram-positive bacteria.

It is not yet clear what the exact role of proton motive force is in the process of protein secretion in bacteria. The most simplistic model would be the direct coupling of proton movement to protein translocation through the membrane. It is difficult to conceive how this would occur. This model would require a proteinaceous membrane complex that would act as both a proton pore and as a secretory apparatus. Alternatively, this protein export complex might require proton motive force for the maintenance of a proper conformation within the membrane as described by Bakker and Randall (5). This type of model has also been ascribed to the role of proton motive force in 86

peptidoglycan and teichoic acid synthesis in Bacillus subtilis (45).

Interestingly, Lancaster elt al. (89) have postulated two different

types of extracellular proteins in Bacillus licheniformis.

Penicillinase was secreted maximally during phosphate limited growth

and was unaffected by the presence of Na+ in the growth medium while

the secretion of a-amylase was severely inhibited under both of the

growth conditions. During phosphate limited growth, B. licheniformis

produces teichuronic rather than teichoic acids as the major cell wall

anionic polymer (89). Thus, the secretion of a-amylase was correlated

to the presence of teichoic acids in the cell wall. Inhibition of Ap

in 13. subtilis also inhibits teichoic acid synthesis (45). It would

be interesting to speculate that the secretion of dextransucrase is

dependent on teichoic acid synthesis. There is evidence that the

extracellular glucosyltransferase of S^. mutans is associated with

teichoic and lipoteichoic acids (88). It is possible that the

dependence of dextransucrase secretion in mesenteroides on Ap is

actually due to the dependence of cell wall polymer synthesis on Ap.

No measurements of this type were made in this study, however, so

proof of this hypothesis is still lacking.

Membrane lipid composition has been postulated to play an

important role in glucosyltransferase secretion in Streptococcus

(60,98). An increase in the K+ concentration of the growth medium

stimulated glucosyltransferase secretion and an increase in the amount of unsaturated membrane lipid fatty acids (98). Recently, Hope and

Cullis (50) demonstrated that the presence of a pH gradient across large unilamellar vesicle membranes affected the transbilayer 87

distribution of the membrane lipids. Oleic and stearic acid were

localized to the inner side of the membrane when the vesicle interior

was basic. When the vesicle interior was acidic, stearylamine and

sphingosine localized to the inner side. It is possible that the

direction of the pH gradient could affect the localization of membrane

lipids in L. mesenteroides and thus affect dextransucrase secretion.

Studies on the effect of Ap on'the membrane lipid composition would be

useful in the further elucidation of the role of Ap in dextransucrase secretion.

L^. mesenteroides maintains a level of proton motive force that is typical for organisms that utilize a strictly fermentative metabolism

(8,72,78). Like other fermentative organisms, the contribution of the membrane potential and the pH gradient to the proton motive force varies according to the external pH. Generally, as the external pH decreases the membrane potential also decreases. The decrease in the membrane potential is compensated by an increase in the contribution of the pH gradient to the proton motive force. This is also common in other bacteria (5,8,72,78). This interconversion is thought to pre­ vent the large buildup of charge across the membrane caused by the expulsion of protons from the cell (30,42). If too large a charge ratio is generated across the membrane, then the expulsion of protons from the cell would not be thermodynamically feasible. In most bacteria, the decrease in membrane potential is usually accomplished through the uptake of cations, in particular K+ (6,71). The results of this study suggest that JL. mesenteroides also utilizes cation transport to buffer Ap. An increase in the K+ concentration of the medium decreased A^ with a corresponding increase in -ZApH (Figure 88

11). The results suggest that L. mesenteroides may utilize K+ as the buffering cation for Ap.

Evidence has accumulated that a major role of the H+- translocating ATPase in fermentative bacteria is the regulation of internal pH (12,69,78). The data presented in figures 8 and 16 show that L. mesenteroides can only regulate its internal pH over a narrow range of external pH. Cells grown in pH-controlled fermenters were able to maintain an internal pH of 7.0 over an external pH range of

6.6 to 7.5. Conversely, packed cells inverted its pH gradient at pH

6.7 (figure 8). The discrepancy in internal pH regulation between the two growth conditions is not immediately obvious. In both packed cells and pH-controlled grown cells, the internal pH decreased as a function of decreasing external pH once the external pH dropped below approximately 6.5. The cells were able to increase ApH, but not enougth to compensate for the decrease in external pH. The response of the H+-ATPase to a change in pH demonstrates the integral role of the enzyme in the extrusion of protons from the cell (figure 12). A change in pH from 7.0 to 6.0 resulted in a 50% increase in ATPase activity. Thus, the ATPase can help to regulate the cell's internal pH. This type of regulation system is common among fermentative bacteria (12,78). The activity of the H+-ATPase greatly decreased over pH 7.0. These results indicate that as the cell's internal pH is raised the activity of the H+-ATPase is inhibited. This inhibition would then keep more protons in the cytoplasm and an inverted pH gradient could be established. Obviously, other factors are involved in the generation of the inverted pH gradient due to its occurance in 89

a pH range where the internal pH is kept constant. These other

factors are not apparent from the results obtained in this study.

The inhibitory action of gramicidin D on the growth of L.

mesenteroides in Na+-containing media is consistent with the findings

of Harold and Van Brunt in Streptococcus faecalis (44).

Interestingly, cells grown in medium containing high concentrations of

K+ were able to lower the medium pH as effectively as control cultures without the ionophore, even to acidic pH values (figure 2).

Gramicidin D is a channel forming ionophore that equilibrates ions and

protons across biological membranes (120) and thus should dissipate

both Af and ApH. Treatment of cells with nigericin (figure 2, table 7) suggests that growth is inhibited once the pH falls below

6.3. This suggests that gramicidin D is not acting as an equilibrator of protons in this system. If it was, then the growth effects seen should be similar to those obtained with nigericin. The inhibition of dextransucrase secretion by gramicidin D follows the same pattern as that of valinomycin and MPTP+ (figures 4,5) and thus may be acting as a dissipator of A^ but not ApH in this system.

The production of dextran by IJ. mesenteroides represents a major loss of profits for the sugar cane industry. The results of this study indicate two major avenues for the control of dextran formation by this organism. The factor that first comes to mind is the control of pH. The results of this study demonstrate the inability of the organism to secrete dextransucrase above pH 7.0. Although the enzyme was found to be relatively stable at pH 7.5, all activity was lost at pH 8.0. The pH of raw sugar cane juice is generally between 5.0-5.5, ideal for the production of dextransucrase and dextran. In the sugar 90

factory, the addition of lime in the liming tanks raises the pH sufficiently high enough to inactivate the enzyme and inhibit the production of dextransucrase by the organism. However, this is under ideal conditions. The product flow in a sugar factory is quite rapid and times do exist in the liming tank where the pH is not sufficiently high enough to inhibit production of dextran. The possiblity of washing the cane as it comes in on the conveyor belts with a basic solution could be explored. It is interesting to note that Texas does not have the dextran problems that Louisiana has. One of the major differences between the two states is the high degree of potash in the soil of Texas as compared to Louisiana. It would be attractive to speculate that the low dextran content in Texan sugar is due to the high cation concentration of the soil.

Many of the preservatives that are presently on the food market are weak acids that inhibit .bacterial growth by acidification of the cytoplasm (129). Since dextransucrase secretion is highly dependent on the presence of a proton gradient, these types of compounds would be highly effective in inhibiting extracellular dextransucrase production as well as growth of the organism. Examples of these weak acids include benzoate and sorbate (129). LITERATURE CITED

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David Roland Otts was born in Rockford, Illinois on October 13,

1961. After graduating from Beaver Dam Senior High School, Beaver Dam

Wisconsin, he attended the University of Wisconsin-Madison where he obtained his Bachelor of Science degree in bacteriology in 1983. In

August 1983, he began his graduate training at the Louisiana State

University in Baton Rouge.

112 DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: David R. Otts

Major Field: Microbiology

Title of Dissertation: Regulation of Dextransucrase Secretion by Proton Motive Force in Leuconostoc mesenteroides.

Approved:

c C-<- Major Professor and Chairman

'I\)kA^ ' V.rvA_^ Dean of the Graduate School

EXAMINING COMMITTEE:

tA'/ylu"vs

Date of Examination:

Monday, June 29 T 1987