Strategies of Nutrient Transport by Ruminal Bacteria JAMES B. RUSSELL Agricultural Research Service. USDA Comell University
Ithaca, NY 14853 HERBERT J. STROBEL
Department of Animal Science Comell University Ithaca, NY 14853 SCOTT A. MARTIN Departments of Animal and Dairy Science and Miaobiology University of Georgia Athens 30602 ABSTRACT
The survival of bacteria in natural environments like the romen depends on the ability of the bacteria to scavenge nutrients. It is now evident that mminal bacteria use a variety of transport mechanisms. Hydrophobic substances, such as ammonia and acetate, are permeable to the lipid bilayers of cell membranes and can be taken up by passive diffusion. Hydrophilic compounds (e.g., sugars, amino acids, peptides) do not easily pass through lipid bilayers and must be transported across cell membranes on carrier proteins. Facilitated diffusion can display saturable kinetics but does not result in accumulation of solute. Active transport can establish extremely high concentration gradients, and this work may be driven by the hydrolysis of chemical bonds (e.g., ATP) or ion gradients, which are coupled to solute symport. Many solute symports involve protons, but sodium systems also are common in mminal bacteria. The phosphotransferase system chemically modifies sugars as they pass across the cell membrane, and several ruminal bacteria have this method of group translocation. Many feed additives have either a direct or indirect effect on rumen bacterial transport. For instance,
Received September 15, 1989. Accepted January 19, 1990. 1990 J Dairy Sci 73:2996-3012
ionophores can inhibit transport by destroying (sometimes even reversing) ion gradients, lowering intracellular pH, or causing excessive ATP hydrolysis. (Key words: mminal bacteria, transport, ionophores) INTRODUCTION
It is thought that life began more than 4 billion yr ago by chemical reactions that gave rise to a variety of organic compounds. Although the exact mechanisms of biochemical evolution are still controversial (96, 99), it generally is accepted that the emergence of cells provided order and the possibility of even more complex processes. As cells proliferated, many of the substrates necessary for growth were depleted from the environment, and there was a need to scavenge nutrients. Many present-day bacteria are able to grow when energy sources and other nutrients are available at micromolar or even submicromolar concentrations. The rumen usually contains an abundance of feed, but this material is primarily composed of large, relatively insoluble, and sometimes complex polymers. These polymers must be degraded to low molecular weight substances (sugars, oligosaccharides, amino acids, peptides, etc.) by extracellular enzymes before they can be utilized by the bacteria. Because cell densities in the rumen are very high, and polymer degradation often is slow, the success of individual species and the composition of fermentation end-products is at least partially related to the affinity, specificity, and regulation of bacterial transport systems. 2996
SYMPOSIUM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
lipopolY'8ccheride
Outer
membrane
Protein
[ .-u-"'-Llt"Y"~
Porin
Periplumic :spl!lce
[
Cv to plesmic
• .....fi-I1iJ)...._i!lIII
Bindinq Protein li Poprotein PeptidoqJ'jc.n
[
membnne
Figure 1. Schematic representation of a gram-negative bacterial cell envelope.
2997
lipid membranes is low, other structures are needed to maintain cellular integrity. Most bacteria have a rigid layer of N-substituted glucosamine and muramic acid linked together by ~ 1,4 bonds. These polysaccharide chains are joined by short peptides, which can include diaminopimelic acid. The peptidoglycan of gram-positive bacteria is approximately 30 om thick, and this rigid matrix can account for 40 to 90% of the cell wall (88). In gram-negative species, the peptidoglycan accounts for only 5 to 10% of the wall. Outer Membrane
BACTERIAL CELL ENVELOPES
Cell Membranes
Cells are defined by a bilayer of phospholipids that contains varying amounts of protein (Figure 1). The cell membranes of all living organisms are morphologically similar, but the observation that fatty acid differences can be used as a tool in bacterial classification illustrates the potential variation (63). Environmental conditions can also influence lipid composition. According to the model of Singer and Nicholson (86), membrane lipids must be maintained in a fluid mosaic. H membranes are to be fluid over a range of temperatures, membrane composition must change. In Escherichia coli, fatty acid synthesis is regulated by a special dehydratase that replaces saturated fatty acids with unsaturated ones as temperature decreases (26). Since unsaturated fatty acids are biohydrogenated by ruminal bacteria, membrane fluidity is controlled by the proportion of branched-ehain fatty acids (44). Membrane proteins may constitute as much as 75% of cell membrane weight (69). Peripheral proteins can be removed easily from the cell membrane by chelating agents, but some membrane proteins are embedded in the lipid bilayer via hydrophobic domains. Proteins that traverse the cell membrane (e.g., transport proteins) often have several hydrophobic regions. Because the membrane is fluid, integral pr0teins can theoretically move in a lateral fashion (7). Whether membrane proteins are spatially organized is still a matter of speculation. Peptidoglycan
Because intracellular solute concentrations are usually very high and the tensile strength of
The outer membrane of gram-negative bacteria serves as a protective barrier that excludes high molecular weight substances and is chemically different from the cell membrane (37). The inner surface consists of phospholipids, but the outer surface is coated with irregular lipopolysaccharide chains, which are stabilized by divalent cations. The outer membrane is joined to the underlying peptidoglycan by lipoproteins. Porins traverse the outer membrane and act as channels for low molecular weight compounds. In E. coli, porins are peptide trimers with a molecular weight cut off of approximately 600 daltons (29). The outer membrane also contains transport proteins for specific high molecular weight substances (maltodextrins, cobalamin, etc.). The region between the cell membrane and the outer membrane is known as the periplasm. In E. coli, the periplasm contains more than 50 proteins at a density which resembles a gel rather than an aqueous solution (7). Periplasmic binding proteins have a high affinity for substrates and donate specific solutes to transport proteins in the cell membrane (3). PASSIVE DIFFUSION
Ammonia
Ammonia has been recognized as a substrate for rumen microbial growth since the 1950s (5), but there was considerable disagreement concerning the optimal concentration. Ammonia can pass across cell membranes by passive diffusion, but at neutral or acidic pH, most of the ruminal ammonia would be present as ammonium ion, a polar species that should not be Journal of Dairy Science Vol. 73, No. 10, 1990
2998
RUSSELL ET AL.
penneable to cell membranes. Recent work (45) has indicated that marine bacteria use active transport mechanisms for ammonium ion, but this accumulation is opposed by alkaline intracellular pH If pH is greater inside than outside, the dissociation of ammonium ion favors ammonia efflux. When ammonium is taken up by active transport, as many as six ions may cycle through the cell membrane before a single molecule is assimilated (45). Satter and Slyter (83) indicated that approximately 3 mM ammonia was required for optimal microbial protein production, and Mehrez et al. (59) argued that more than 14 rnM was needed for optimal DM disappearance in some cases. In contrast, Schaefer et al. (84) reported that pure cultures of mminal bacteria had affinity constants ranging from 6 to 50 J.IM. The disparity between mixed culture observations and the pure culture affinity constants may be related to bacterial selection or induction. Schaefer et al. (84) grew their bacteria-in' ammonia-limited continuous cultures with an excess of glucose. Because poor quality diets usually limit energy as well as nitrogen, the selection pressure on ruminal bacteria in vivo is not so constant. When mixed ruminal bacteria were provided with decreasing ammonia and fed growth ratelimiting amounts of mixed carbohydrates on an hourly basis: 1) more than 3 rnM ammonia was required for maximal protein synthesis, 2) intracellular ammonia increased as a linear function of extracellular ammonia, 3) there was only a small concentration gradient of ammonia across cell membranes, 4) the bacteria left more than .7 rnM of ammonia even though carbohydrates still were available, and 5) it appeared that ammonia was assimilated by passive diffusion rather than active transport (78). When a substrate is taken up by passive diffusion, the first enzyme in the pathway of substrate utilization determines affinity. Glutamate dehydrogenase is involved in ammonia assimilation, and the affinity constant for ammonia is relatively high [approximately 5 rnM, (32)]. Tempest et al. (93) revealed a new enzyme, glutamate synthase, which has a higher affinity for ammonia (.2 rnM), and this enzyme was able to work in conjunction with glutamine synthetase as a means of ammonia assimilation. The glutamate synthase and glutamine synthetase cycle, however, requires an input of ATP Journal of Dairy Science Vol. 73,
No. 10, 1990
and is energetically less favorable than glutamate dehydrogenase. Glutamate dehydrogenase is common in ruminal bacteria (1), but there is much less information on glutamate synthase and glutamine Succinovibrio dextrinosolvens, synthetase. Selenomonas ruminantium, and Bacteroides (Ruminobacter) amylophilus had glutamine synthetase activities which were induced by ammonia deprivation (32). The latter two organisms had glutamate synthase activities, but induction by ammonia limitation could only be demonstrated in Bacteroides (Ruminobacter) amylophilus. Because ruminal ammonia concentrations in vivo are rarely if ever less than .7 rnM (62), it is unlikely that the glutamate synthase and glutamine synthetase cycle is an important pathway of ammonia assimilation. VolaUle Fatty Acids
Many of the ruminal bacteria require acetate even though they may produce it (35). Because acetate in its undissociated form can enter the cell by passive diffusion, an alkaline intracellular pH would favor its uptake. Acetate concentrations in the rumen usually are greater than 60 roM, and it is unlikely that acetate ever is limiting. Many of the mminal bacteria [most notably the cellulolytics, (8)] also require branched-chain VFA (isobutyrate, isovalerate, and 2-methylbutyrate), and these acids are found at much lower concentrations in the rumen (2). Even though the permeability coefficient of short-chain fatty acids increases as the aliphatic chain lengthens (89), the availability of branched-chain VFA may at times limit microbial protein synthesis and cellulose digestion (25, 77). FACILITATED DIFFUSION
Many solutes are too lipophobic or large to pass freely across cell membranes, but they may be taken up by diffusion mechanisms involving specific carrier proteins. Facilitated diffusion, like passive diffusion, only allows net flux if the solute passes down a concentration gradient. However, because a carrier protein is involved, facilitated diffusion can exhibit saturation kinetics and directionally asymmetric velocities (affinities). Nonenergized cells of Streptococcus bovis were able to produce ammonia from glutamine, a highly hydrophilic
2999
SYMPOSIDM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
...... 100 c::
°e..... c::
-
secondary Active Transport
40
80
0i; 0
L-
1
V 20
60
a.
0-
E
.....
0
40
0
(;
E c:: ........ >
20
2
ViS x 10 3
...
0 0
4
...
4 2 6 6 10 VIS (nmol/mg protein/m;n/uM)
Figure 2. An Eadie-Hofstee plot of glutamine transport by glucose-energized cells of S. bovis (12). Ammonia production by I10Ilenetgized cells is shown in the iDset. [Reprinted from 1. Bacterial. 171:298 with permission of the publisher.]
solute (12), and the rate of ammonia production was proportional to the extracellular glutamine concentration (Figure 2). Facilitated diffusion of glutamine and glutamate into a recently isolated amino acid fennenting ruminal bacterium also was noted (Russell and Chen, unpublished results)" but it should be realized that amino acid concentrations in ruminal fluid are usually low (100). Streptococcus bovis has a phosphotransferase system (PfS) for sugars (see below), but the glucose PTS could not account for the glucose consumption of exponentially growing cells (57). Recent work has indicated that S. bovis also has a facilitated diffusion mechanism for glucose intake (Russell, unpublished results).
History and Theory. The study of bacterial transport was hastened by the identification of the E. coli galactoside pennease in 1956 and the use of nonmetabolizable sugars (13). The Monod school proposed that one ATP was needed to transport one molecule of solute, but the exact mechanism of energy coupling was unclear. Horecker et at. (34) showed that dinitrophenol could inhibit galactOSide accumulation, but the participation of proton gradients in bacterial transport was not fully appreciated. The Mitchell hypothesis of energy transduction (60) and membrane vesicle studies by Kaback (40) provided a workable model and experimental proof for proton-solute symport. According to the Mitchell hypothesis, most bacteria use membrane-bound ATPases or electron transport chains to expel protons. When protons are translocated across cell membranes, a chemical gradient of protons is established (ApH). The movement of this positive ion also leaves the cell interior more negative than the exterior, and this difference in charge creates an electrical gradient (A¥). The driving force of chemical and electrical gradients can be predicted from the Nemst equation: -2.3 (RT/F) x log[in]/[out] Because -2.3 (RT/F) is equal to 60 mV at 25"C (62 mV at 39"C), the A¥ has a force of:
Because pH is already a log scale, the chemical gradient of protons (ZApH) becomes:
ACTIVE TRANSPORT
Active transport can accumulate solute against extremely high gradients (107) and this work may be driven by ion gradients or the hydrolysis of chemical bonds (e.g., ATP). PrimaI)' active transport is driven directly by the chemical bond energy; systems coupled to ion gradients are known as secondary active transport.
lReference updated in proof: Ou:n, G., and 1. B. Russell. 1990. Transport and deaminAtion of amino acids by a gram-positive, moneosin-scnsitive ruminal bectaium. Appl. Environ. Miaobiol. 56:2186.
~
mV x ApH
where UpH is by convention a negative value. The total driving force across the membrane (ApI the protonmotive force) is the sum of electrical and chemical gradients: Ap
= A¥ -
ZApH
Positively charged solutes can be drawn in by the A¥, but neutral or negatively charged species can only be transported in symport with cations that respond to A¥. If transport involves proton symport, ZApH can also contrib1ouma1 of Dairy ScieDce Vol. 73,
No. 10, 1990
3000
RUSSElL ET AL. DistrIbution Equfttion
Cone. Gre;dient
C
.10
"c:
.OB
'E
5
60 log [51; / [51 0 = 0
100
60 log [5-];/ [5-]0= 60
10 1
~
L
a.
.06
60 log [5'1; / [5'1 = 120 0
10 2
60 log [51; / [51 0 = 120 , 60
10 3
5
z 60 log (5- I; / [5-]0 = 120 '120
10
4
10
6
ute to the driving force. Accumulation ratios of Ap driven transport can be as great as 1(ji (Figure 3), but the relationship between driving force and the rate of transport is not necessarily linear. When Lactobacillus casei cells were energized with an artificial potassium diffusion potential (Figure 4), little if any uptake was observed until the potential was greater than 80 mV (91). Similar results were noted in Streptococcus cremoris, but the threshold was not as great (14). Enzyme kinetics often have been used to describe the relationship between pH and transport activity. Active sites often contain amino acids with ionizable groups whose dissociation is dependent on pH. Such models may, however, be too simplistic to describe the effect of pH on carriers exposed to two environments (extracellular versus intracellular space). Recent work by Driessen et al. (20) suggested that the dissociation of protons from the leucine carrier at the internal membrane surface was the ratelimiting step in transport. Cosubstrates. Boyer (6) proposed that cations interact with the active site of carrier proteins by forming coordination complexes. It had generally been assumed that proton symporters interacted with H+, but it is possible that the transported species is actually H30+. No. 10, 1990
100
50
mY
•
•
.02
::::J
Figure 3. Transport of solute (S) by various mechanisms where [S); and [S]o are the concentration of solute inside and outside the bacterial cell, respectively. The final equilibrium distribution is calculated from the Nemst equation (Z log [S]/[S]o = m (A¥ + ZApH)}, where Z at 2S'C is 60 mV, A\! is 120 mY, ZApH is 60 mV, and m is the net number of translocated cations. The concentration (cone.) gradient is calculated from the antilog of log [S]i/[S]o'
Journal of Dairy Science Vol. 73,
a
U
UJ ...J
60 log [51; / [51 0 = 2( 120) , 2 (60)
-7
.04
-5 UJ
5
•
-6
CT.
E "'0 E
IZJ
-4 - -5
.....0 Q)
5
-3
0
0
50
100
150
ARTIFICIAL POTENTIAL (mV)
Figure 4. Relationship between membrane potential (A¥) and leucine transport rate in Lactobacillus casei (91). An artificial A\! was imposed by loading eelb; with potassium and diluting into buffer containing 0 to 20 roM potassium chloride. The logarithmic relation between velocity and A\! is shown in the inset. [Reprinted from J. Bacteriol. 171:280 with permission of the publisher.]
The H30+, Na+, or Li+ may form stable complexes with appropriately positioned oxygen and nitrogen atoms in a manner similar to cation binding by cyclic polyethers (ionophores). Kaback (42) has presented a different model in which the lactose carrier operates as a charge relay chain rather than a coordination complex. Most bacterial symporters appear to be coupled to protons, but sodium symport mechanisms have been found in E. coli (10, 94), marine bacteria (22), and alkalophilic bacteria (48, 92). In S. hovis, serine, threonine, and alanine were taken up by sodium-dependent mechanisms (80). Transport was driven by either A¥ or a chemical gradient of sodium, and the affinity for sodium was very low (Figure 5a). Because the transport protein had more than one sodium-binding site (Figure 5b), it appeared that there was a low affinity allosteric site as well as a catalytic site. Recently isolated amino acid-fermenting ruminal bacteria, which were sensitive to monensin, also used sodium gradients to drive amino acid transport (11). Some stimulation of glucose transport by Bacteroides (Fibrobacter) succinogenes was noted when sodium or lithium was added (24), but it is uncertain whether either of these ions was used as a cosubstrate. Sodium and lithium have very similar atomic radii, and in E. coli, sodium symporters
SYMPOSIUM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
3001
(Fo). The FIFo ATPase is blocked by dicyclohexylcarbodiimide (DeCO) and is insensitive 'E • to vanadate, an inorganic phosphate analog. --. 2.0 c:; The Na/K ATPases common to animals show reversed sensitivities and are not directly in"1.5 "'volved in proton pumping. • --.E"" According to the chemiosmotic theory of '0 1.0 Mitchell (60), certain components of electron E c:; '-" transport chains can pump protons across bio..... :z .5 logical membranes in response to decreased a: ..... electron potentials. Electrons remain associated with membrane components and are ultimately 20 40 60 80 100 120 involved in the consumption of oxygen and formation of water at the internal membrane Neel (mM) surface. Because oxygen is not available as a terminal electron acceptor in anaerobic environ1.0 b ments like the rumen, and since anaerobic mi...... croorganisms produce products other than car.5 > I X bon dioxide and water, it was generally CD n 8pP = 2 assumed that they lacked electron transport sys0 E > tems. More recent work, however, showed that ...... > -.5 -..... certain anaerobes, including ruminal bacteria, C) had cytochromes and electron transport compo0 ...J -1.0 nents (54, 98). • Konings proposed that lactate efflux from S. - 1.5 +--..--,--....--r---r--,--....--r-..........---l cremoris, an organism used in cheese making, o .5 1.0 1.5 2.0 2.5 could generate a protonmotive force so long as the proton-lactate stoichiometry was greater Log Ne than 1 (47). Because lactate formation generFigure 5. The effect of sodium concentration on (a) the ates one proton intracellularly, the creation of a rate of serine transport by S. bovis, and (b) a Hill plot of the proton motive force could only occur if the pH data, which indicated that the carrier had more than one binding site for sodium (80). [Reprinted from J. Bacteriol. was significantly greater than 6.0 and extracel170:3535 with permission from the publisher.] lular lactate was less than 20 mM. Streptococcus bovis and S. ruminantium also produce lactate, but ruminal conditions would rarely if also can use lithium. A recently isolated rumi- ever favor electrogenic proton pumping via lacnal peptostreptococcus had a branched-chain tate efflux. Rhodopsin, a light harvesting proamino acid carrier, which used lithium as well tein (64), is a primary proton pump in photoas sodium (11), but the neutral amino acid trophs, but one should note that the rumen is carrier of S. bovis (80) and the glutamate and normally very dark. Sodium Pumps. Mitchell predicted that soglutamine carriers of another ruminal amino dium-proton antiporters would be needed to acid fermenting bacterium were only active if prevent cation accumulation at the negatively sodium was present (Chen and Russell, unpubcharged interior of cens (61), and this activity lished results).l Some marine bacteria also are was subsequently demonstrated in E. coli (97). able to discriminate between sodium and Sodium-proton antiporters have been shown in lithium (55). a variety of organisms, including Streptococcus Proton Pumps. The FIFo ATPase was origi- faecalis, Bacillus alkalophilus, halobacteria, nally isolated from mitrochondria, but it is and marine vibrios (87). Under natural condiubiquitous in bacteria. It is composed of a tions, sodium-proton antiporters catalyze the globular head piece (FI), which protrudes from influx of protons and efflux of sodium, and if the cell membrane, and a component that acts the exchange is electroneutral (one for one) as a proton channel through the cell membrane UpH is the driving force. In organisms with .-.. c:;
2.5
el
:e 0
f/)
Journal of Dairy Science Vol. 73, No. la, 1990
3002
RUSSELL ET AL.
low or reversed UpH, the exchange is sometimes electrogenic (2H+ per Na+), and A¥ is the primary driving force. In 1980, Dimroth reported that oxaloacetatefennenting bacteria used biotin-linked decarboxylation reactions to create a sodium gradient in the absence of a protonmotive force (18). These biotin-linked decarboxylation reactions: 1) only occurred if sodium was present, 2) created an electrogenic sodium gradient, and 3) allowed growth even though the fermentation contained no site of substrate level phosphorylation. The ruminal bacterium S. ruminantium decarboxylates succinate via a biotin-linked reaction (85), but sodium pumping has not yet been demonstrated. Acidaminococcus fermentans uses a glutamate decarboxylation reaction to establish a sodium gradient (9), and a recently isolated amino acid-fennenting ruminal bacterium had a similar sodium-dependent pathway of glutamate fermentation (Chen and Russell, unpublished results).l Until recently, it was generally accepted that membrane-bound ATPases of bacteria only translocated protons. Heefner and Harold (31) noted that everted vesicles of S. faecalis had a sodium-stimulated ATPase, which established a sodium gradient at alkaline pH in the absence of a protonmotive force. Streptococcus bovis exhibited rapid efflux of sodium when cells were energized with glucose (90). Because glucose-dependent efflux was resistant to protonophores that collapsed both UpH and A¥, there was no evidence for a sodium-proton antiporter. Proof for a sodium ATPase was obtained from experiments that showed sodium-dependent ATP synthesis, which was DeeD-resistant (Figure 6). The S. bovis sodium ATPase was active at acidic pH. Membrane Conductance
Natural Permeability. Aside from the fact that many bacterial habitats, including the rumen, have a high concentration of sodium, the use of sodium as a coupling agent for transport has several advantages. According to the model of Skulachev (87), bacterial membranes have a high capacitance and the efflux of even a few protons (1 nmol/mg protein) can create a very large electrical potential (A¥). Although the potential may be great, subsequent ion flux and the capacity to do work is small. Potassium 10urnal of Dairy Science Vol. 73,
No. 10, 1990
400
~
,\uH ,\uH+OCCD '\uNa ,\uNa+OCCD
300
2a.
Ioe(
200
100 oP:=:q=e:;~~$=;~::::::;;a-....-.,........j 12 4 10 o 2 6 8 TIME (min)
Figure 6. 1be use of an artificial protonmolive force (6uH) or an artificial electrochemical gradient of sodium (6uNa) to drive AlP synthesis in whole cells of S. bovis. The FlFo AlPase inhibitor dicyclohexylcarbodiimide (DCCD) was added at .25 JUllol/mg protein (90). [Reprinted from Appl. Environ. Microbiol. 55:2664 with pcnnission from the publisher.]
influx discharges the 11¥, relieves the back pressure on the proton ATPase, and allows an increase in UpH. The UpH has a greater capacity to do work, but bacterial membranes are inherently leaky to protons. Sodium-proton antiporters can convert UpH into a chemical gradient of sodium, anG sodium is much less penneable than protons. Sodium ATPases and decarboxylases have the same end result even though proton currents are not directly involved. Many bacteria maintain a near neutral intracellular pH even if extracellular pH decreases (43). 'The maintenance of a constant intracellular pH without a compensatory decrease in 11¥ would create a very high total Ap and increase proton conductance. Bacteria such as E. coli have circumvented this problem by taking up potassium, decreasing 11¥, and maintaining a high ApH. Bacteroides succinogenes, one of the predominant ruminal cellulolytic bacteria, also interconverts A¥ and UpH, but it is unable to grow at pH below 5.9 (73). The ruminal bacteria S. bovis and Bacteroides ruminicola have a different strategy of intracellular pH regulation, which allows internal pH to decrease as a function of extracellular pH, and these bacteria are among the most acid-resistant species in the rumen (75).
3003
SYMPOSIUM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
OUT
IN
(high Ne+. low K+)
4.0
(high K+. low Ne +)
C
€
---
Ithaca, NY 14853 HERBERT J. STROBEL
Department of Animal Science Comell University Ithaca, NY 14853 SCOTT A. MARTIN Departments of Animal and Dairy Science and Miaobiology University of Georgia Athens 30602 ABSTRACT
The survival of bacteria in natural environments like the romen depends on the ability of the bacteria to scavenge nutrients. It is now evident that mminal bacteria use a variety of transport mechanisms. Hydrophobic substances, such as ammonia and acetate, are permeable to the lipid bilayers of cell membranes and can be taken up by passive diffusion. Hydrophilic compounds (e.g., sugars, amino acids, peptides) do not easily pass through lipid bilayers and must be transported across cell membranes on carrier proteins. Facilitated diffusion can display saturable kinetics but does not result in accumulation of solute. Active transport can establish extremely high concentration gradients, and this work may be driven by the hydrolysis of chemical bonds (e.g., ATP) or ion gradients, which are coupled to solute symport. Many solute symports involve protons, but sodium systems also are common in mminal bacteria. The phosphotransferase system chemically modifies sugars as they pass across the cell membrane, and several ruminal bacteria have this method of group translocation. Many feed additives have either a direct or indirect effect on rumen bacterial transport. For instance,
Received September 15, 1989. Accepted January 19, 1990. 1990 J Dairy Sci 73:2996-3012
ionophores can inhibit transport by destroying (sometimes even reversing) ion gradients, lowering intracellular pH, or causing excessive ATP hydrolysis. (Key words: mminal bacteria, transport, ionophores) INTRODUCTION
It is thought that life began more than 4 billion yr ago by chemical reactions that gave rise to a variety of organic compounds. Although the exact mechanisms of biochemical evolution are still controversial (96, 99), it generally is accepted that the emergence of cells provided order and the possibility of even more complex processes. As cells proliferated, many of the substrates necessary for growth were depleted from the environment, and there was a need to scavenge nutrients. Many present-day bacteria are able to grow when energy sources and other nutrients are available at micromolar or even submicromolar concentrations. The rumen usually contains an abundance of feed, but this material is primarily composed of large, relatively insoluble, and sometimes complex polymers. These polymers must be degraded to low molecular weight substances (sugars, oligosaccharides, amino acids, peptides, etc.) by extracellular enzymes before they can be utilized by the bacteria. Because cell densities in the rumen are very high, and polymer degradation often is slow, the success of individual species and the composition of fermentation end-products is at least partially related to the affinity, specificity, and regulation of bacterial transport systems. 2996
SYMPOSIUM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
lipopolY'8ccheride
Outer
membrane
Protein
[ .-u-"'-Llt"Y"~
Porin
Periplumic :spl!lce
[
Cv to plesmic
• .....fi-I1iJ)...._i!lIII
Bindinq Protein li Poprotein PeptidoqJ'jc.n
[
membnne
Figure 1. Schematic representation of a gram-negative bacterial cell envelope.
2997
lipid membranes is low, other structures are needed to maintain cellular integrity. Most bacteria have a rigid layer of N-substituted glucosamine and muramic acid linked together by ~ 1,4 bonds. These polysaccharide chains are joined by short peptides, which can include diaminopimelic acid. The peptidoglycan of gram-positive bacteria is approximately 30 om thick, and this rigid matrix can account for 40 to 90% of the cell wall (88). In gram-negative species, the peptidoglycan accounts for only 5 to 10% of the wall. Outer Membrane
BACTERIAL CELL ENVELOPES
Cell Membranes
Cells are defined by a bilayer of phospholipids that contains varying amounts of protein (Figure 1). The cell membranes of all living organisms are morphologically similar, but the observation that fatty acid differences can be used as a tool in bacterial classification illustrates the potential variation (63). Environmental conditions can also influence lipid composition. According to the model of Singer and Nicholson (86), membrane lipids must be maintained in a fluid mosaic. H membranes are to be fluid over a range of temperatures, membrane composition must change. In Escherichia coli, fatty acid synthesis is regulated by a special dehydratase that replaces saturated fatty acids with unsaturated ones as temperature decreases (26). Since unsaturated fatty acids are biohydrogenated by ruminal bacteria, membrane fluidity is controlled by the proportion of branched-ehain fatty acids (44). Membrane proteins may constitute as much as 75% of cell membrane weight (69). Peripheral proteins can be removed easily from the cell membrane by chelating agents, but some membrane proteins are embedded in the lipid bilayer via hydrophobic domains. Proteins that traverse the cell membrane (e.g., transport proteins) often have several hydrophobic regions. Because the membrane is fluid, integral pr0teins can theoretically move in a lateral fashion (7). Whether membrane proteins are spatially organized is still a matter of speculation. Peptidoglycan
Because intracellular solute concentrations are usually very high and the tensile strength of
The outer membrane of gram-negative bacteria serves as a protective barrier that excludes high molecular weight substances and is chemically different from the cell membrane (37). The inner surface consists of phospholipids, but the outer surface is coated with irregular lipopolysaccharide chains, which are stabilized by divalent cations. The outer membrane is joined to the underlying peptidoglycan by lipoproteins. Porins traverse the outer membrane and act as channels for low molecular weight compounds. In E. coli, porins are peptide trimers with a molecular weight cut off of approximately 600 daltons (29). The outer membrane also contains transport proteins for specific high molecular weight substances (maltodextrins, cobalamin, etc.). The region between the cell membrane and the outer membrane is known as the periplasm. In E. coli, the periplasm contains more than 50 proteins at a density which resembles a gel rather than an aqueous solution (7). Periplasmic binding proteins have a high affinity for substrates and donate specific solutes to transport proteins in the cell membrane (3). PASSIVE DIFFUSION
Ammonia
Ammonia has been recognized as a substrate for rumen microbial growth since the 1950s (5), but there was considerable disagreement concerning the optimal concentration. Ammonia can pass across cell membranes by passive diffusion, but at neutral or acidic pH, most of the ruminal ammonia would be present as ammonium ion, a polar species that should not be Journal of Dairy Science Vol. 73, No. 10, 1990
2998
RUSSELL ET AL.
penneable to cell membranes. Recent work (45) has indicated that marine bacteria use active transport mechanisms for ammonium ion, but this accumulation is opposed by alkaline intracellular pH If pH is greater inside than outside, the dissociation of ammonium ion favors ammonia efflux. When ammonium is taken up by active transport, as many as six ions may cycle through the cell membrane before a single molecule is assimilated (45). Satter and Slyter (83) indicated that approximately 3 mM ammonia was required for optimal microbial protein production, and Mehrez et al. (59) argued that more than 14 rnM was needed for optimal DM disappearance in some cases. In contrast, Schaefer et al. (84) reported that pure cultures of mminal bacteria had affinity constants ranging from 6 to 50 J.IM. The disparity between mixed culture observations and the pure culture affinity constants may be related to bacterial selection or induction. Schaefer et al. (84) grew their bacteria-in' ammonia-limited continuous cultures with an excess of glucose. Because poor quality diets usually limit energy as well as nitrogen, the selection pressure on ruminal bacteria in vivo is not so constant. When mixed ruminal bacteria were provided with decreasing ammonia and fed growth ratelimiting amounts of mixed carbohydrates on an hourly basis: 1) more than 3 rnM ammonia was required for maximal protein synthesis, 2) intracellular ammonia increased as a linear function of extracellular ammonia, 3) there was only a small concentration gradient of ammonia across cell membranes, 4) the bacteria left more than .7 rnM of ammonia even though carbohydrates still were available, and 5) it appeared that ammonia was assimilated by passive diffusion rather than active transport (78). When a substrate is taken up by passive diffusion, the first enzyme in the pathway of substrate utilization determines affinity. Glutamate dehydrogenase is involved in ammonia assimilation, and the affinity constant for ammonia is relatively high [approximately 5 rnM, (32)]. Tempest et al. (93) revealed a new enzyme, glutamate synthase, which has a higher affinity for ammonia (.2 rnM), and this enzyme was able to work in conjunction with glutamine synthetase as a means of ammonia assimilation. The glutamate synthase and glutamine synthetase cycle, however, requires an input of ATP Journal of Dairy Science Vol. 73,
No. 10, 1990
and is energetically less favorable than glutamate dehydrogenase. Glutamate dehydrogenase is common in ruminal bacteria (1), but there is much less information on glutamate synthase and glutamine Succinovibrio dextrinosolvens, synthetase. Selenomonas ruminantium, and Bacteroides (Ruminobacter) amylophilus had glutamine synthetase activities which were induced by ammonia deprivation (32). The latter two organisms had glutamate synthase activities, but induction by ammonia limitation could only be demonstrated in Bacteroides (Ruminobacter) amylophilus. Because ruminal ammonia concentrations in vivo are rarely if ever less than .7 rnM (62), it is unlikely that the glutamate synthase and glutamine synthetase cycle is an important pathway of ammonia assimilation. VolaUle Fatty Acids
Many of the ruminal bacteria require acetate even though they may produce it (35). Because acetate in its undissociated form can enter the cell by passive diffusion, an alkaline intracellular pH would favor its uptake. Acetate concentrations in the rumen usually are greater than 60 roM, and it is unlikely that acetate ever is limiting. Many of the mminal bacteria [most notably the cellulolytics, (8)] also require branched-chain VFA (isobutyrate, isovalerate, and 2-methylbutyrate), and these acids are found at much lower concentrations in the rumen (2). Even though the permeability coefficient of short-chain fatty acids increases as the aliphatic chain lengthens (89), the availability of branched-chain VFA may at times limit microbial protein synthesis and cellulose digestion (25, 77). FACILITATED DIFFUSION
Many solutes are too lipophobic or large to pass freely across cell membranes, but they may be taken up by diffusion mechanisms involving specific carrier proteins. Facilitated diffusion, like passive diffusion, only allows net flux if the solute passes down a concentration gradient. However, because a carrier protein is involved, facilitated diffusion can exhibit saturation kinetics and directionally asymmetric velocities (affinities). Nonenergized cells of Streptococcus bovis were able to produce ammonia from glutamine, a highly hydrophilic
2999
SYMPOSIDM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
...... 100 c::
°e..... c::
-
secondary Active Transport
40
80
0i; 0
L-
1
V 20
60
a.
0-
E
.....
0
40
0
(;
E c:: ........ >
20
2
ViS x 10 3
...
0 0
4
...
4 2 6 6 10 VIS (nmol/mg protein/m;n/uM)
Figure 2. An Eadie-Hofstee plot of glutamine transport by glucose-energized cells of S. bovis (12). Ammonia production by I10Ilenetgized cells is shown in the iDset. [Reprinted from 1. Bacterial. 171:298 with permission of the publisher.]
solute (12), and the rate of ammonia production was proportional to the extracellular glutamine concentration (Figure 2). Facilitated diffusion of glutamine and glutamate into a recently isolated amino acid fennenting ruminal bacterium also was noted (Russell and Chen, unpublished results)" but it should be realized that amino acid concentrations in ruminal fluid are usually low (100). Streptococcus bovis has a phosphotransferase system (PfS) for sugars (see below), but the glucose PTS could not account for the glucose consumption of exponentially growing cells (57). Recent work has indicated that S. bovis also has a facilitated diffusion mechanism for glucose intake (Russell, unpublished results).
History and Theory. The study of bacterial transport was hastened by the identification of the E. coli galactoside pennease in 1956 and the use of nonmetabolizable sugars (13). The Monod school proposed that one ATP was needed to transport one molecule of solute, but the exact mechanism of energy coupling was unclear. Horecker et at. (34) showed that dinitrophenol could inhibit galactOSide accumulation, but the participation of proton gradients in bacterial transport was not fully appreciated. The Mitchell hypothesis of energy transduction (60) and membrane vesicle studies by Kaback (40) provided a workable model and experimental proof for proton-solute symport. According to the Mitchell hypothesis, most bacteria use membrane-bound ATPases or electron transport chains to expel protons. When protons are translocated across cell membranes, a chemical gradient of protons is established (ApH). The movement of this positive ion also leaves the cell interior more negative than the exterior, and this difference in charge creates an electrical gradient (A¥). The driving force of chemical and electrical gradients can be predicted from the Nemst equation: -2.3 (RT/F) x log[in]/[out] Because -2.3 (RT/F) is equal to 60 mV at 25"C (62 mV at 39"C), the A¥ has a force of:
Because pH is already a log scale, the chemical gradient of protons (ZApH) becomes:
ACTIVE TRANSPORT
Active transport can accumulate solute against extremely high gradients (107) and this work may be driven by ion gradients or the hydrolysis of chemical bonds (e.g., ATP). PrimaI)' active transport is driven directly by the chemical bond energy; systems coupled to ion gradients are known as secondary active transport.
lReference updated in proof: Ou:n, G., and 1. B. Russell. 1990. Transport and deaminAtion of amino acids by a gram-positive, moneosin-scnsitive ruminal bectaium. Appl. Environ. Miaobiol. 56:2186.
~
mV x ApH
where UpH is by convention a negative value. The total driving force across the membrane (ApI the protonmotive force) is the sum of electrical and chemical gradients: Ap
= A¥ -
ZApH
Positively charged solutes can be drawn in by the A¥, but neutral or negatively charged species can only be transported in symport with cations that respond to A¥. If transport involves proton symport, ZApH can also contrib1ouma1 of Dairy ScieDce Vol. 73,
No. 10, 1990
3000
RUSSElL ET AL. DistrIbution Equfttion
Cone. Gre;dient
C
.10
"c:
.OB
'E
5
60 log [51; / [51 0 = 0
100
60 log [5-];/ [5-]0= 60
10 1
~
L
a.
.06
60 log [5'1; / [5'1 = 120 0
10 2
60 log [51; / [51 0 = 120 , 60
10 3
5
z 60 log (5- I; / [5-]0 = 120 '120
10
4
10
6
ute to the driving force. Accumulation ratios of Ap driven transport can be as great as 1(ji (Figure 3), but the relationship between driving force and the rate of transport is not necessarily linear. When Lactobacillus casei cells were energized with an artificial potassium diffusion potential (Figure 4), little if any uptake was observed until the potential was greater than 80 mV (91). Similar results were noted in Streptococcus cremoris, but the threshold was not as great (14). Enzyme kinetics often have been used to describe the relationship between pH and transport activity. Active sites often contain amino acids with ionizable groups whose dissociation is dependent on pH. Such models may, however, be too simplistic to describe the effect of pH on carriers exposed to two environments (extracellular versus intracellular space). Recent work by Driessen et al. (20) suggested that the dissociation of protons from the leucine carrier at the internal membrane surface was the ratelimiting step in transport. Cosubstrates. Boyer (6) proposed that cations interact with the active site of carrier proteins by forming coordination complexes. It had generally been assumed that proton symporters interacted with H+, but it is possible that the transported species is actually H30+. No. 10, 1990
100
50
mY
•
•
.02
::::J
Figure 3. Transport of solute (S) by various mechanisms where [S); and [S]o are the concentration of solute inside and outside the bacterial cell, respectively. The final equilibrium distribution is calculated from the Nemst equation (Z log [S]/[S]o = m (A¥ + ZApH)}, where Z at 2S'C is 60 mV, A\! is 120 mY, ZApH is 60 mV, and m is the net number of translocated cations. The concentration (cone.) gradient is calculated from the antilog of log [S]i/[S]o'
Journal of Dairy Science Vol. 73,
a
U
UJ ...J
60 log [51; / [51 0 = 2( 120) , 2 (60)
-7
.04
-5 UJ
5
•
-6
CT.
E "'0 E
IZJ
-4 - -5
.....0 Q)
5
-3
0
0
50
100
150
ARTIFICIAL POTENTIAL (mV)
Figure 4. Relationship between membrane potential (A¥) and leucine transport rate in Lactobacillus casei (91). An artificial A\! was imposed by loading eelb; with potassium and diluting into buffer containing 0 to 20 roM potassium chloride. The logarithmic relation between velocity and A\! is shown in the inset. [Reprinted from J. Bacteriol. 171:280 with permission of the publisher.]
The H30+, Na+, or Li+ may form stable complexes with appropriately positioned oxygen and nitrogen atoms in a manner similar to cation binding by cyclic polyethers (ionophores). Kaback (42) has presented a different model in which the lactose carrier operates as a charge relay chain rather than a coordination complex. Most bacterial symporters appear to be coupled to protons, but sodium symport mechanisms have been found in E. coli (10, 94), marine bacteria (22), and alkalophilic bacteria (48, 92). In S. hovis, serine, threonine, and alanine were taken up by sodium-dependent mechanisms (80). Transport was driven by either A¥ or a chemical gradient of sodium, and the affinity for sodium was very low (Figure 5a). Because the transport protein had more than one sodium-binding site (Figure 5b), it appeared that there was a low affinity allosteric site as well as a catalytic site. Recently isolated amino acid-fermenting ruminal bacteria, which were sensitive to monensin, also used sodium gradients to drive amino acid transport (11). Some stimulation of glucose transport by Bacteroides (Fibrobacter) succinogenes was noted when sodium or lithium was added (24), but it is uncertain whether either of these ions was used as a cosubstrate. Sodium and lithium have very similar atomic radii, and in E. coli, sodium symporters
SYMPOSIUM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
3001
(Fo). The FIFo ATPase is blocked by dicyclohexylcarbodiimide (DeCO) and is insensitive 'E • to vanadate, an inorganic phosphate analog. --. 2.0 c:; The Na/K ATPases common to animals show reversed sensitivities and are not directly in"1.5 "'volved in proton pumping. • --.E"" According to the chemiosmotic theory of '0 1.0 Mitchell (60), certain components of electron E c:; '-" transport chains can pump protons across bio..... :z .5 logical membranes in response to decreased a: ..... electron potentials. Electrons remain associated with membrane components and are ultimately 20 40 60 80 100 120 involved in the consumption of oxygen and formation of water at the internal membrane Neel (mM) surface. Because oxygen is not available as a terminal electron acceptor in anaerobic environ1.0 b ments like the rumen, and since anaerobic mi...... croorganisms produce products other than car.5 > I X bon dioxide and water, it was generally CD n 8pP = 2 assumed that they lacked electron transport sys0 E > tems. More recent work, however, showed that ...... > -.5 -..... certain anaerobes, including ruminal bacteria, C) had cytochromes and electron transport compo0 ...J -1.0 nents (54, 98). • Konings proposed that lactate efflux from S. - 1.5 +--..--,--....--r---r--,--....--r-..........---l cremoris, an organism used in cheese making, o .5 1.0 1.5 2.0 2.5 could generate a protonmotive force so long as the proton-lactate stoichiometry was greater Log Ne than 1 (47). Because lactate formation generFigure 5. The effect of sodium concentration on (a) the ates one proton intracellularly, the creation of a rate of serine transport by S. bovis, and (b) a Hill plot of the proton motive force could only occur if the pH data, which indicated that the carrier had more than one binding site for sodium (80). [Reprinted from J. Bacteriol. was significantly greater than 6.0 and extracel170:3535 with permission from the publisher.] lular lactate was less than 20 mM. Streptococcus bovis and S. ruminantium also produce lactate, but ruminal conditions would rarely if also can use lithium. A recently isolated rumi- ever favor electrogenic proton pumping via lacnal peptostreptococcus had a branched-chain tate efflux. Rhodopsin, a light harvesting proamino acid carrier, which used lithium as well tein (64), is a primary proton pump in photoas sodium (11), but the neutral amino acid trophs, but one should note that the rumen is carrier of S. bovis (80) and the glutamate and normally very dark. Sodium Pumps. Mitchell predicted that soglutamine carriers of another ruminal amino dium-proton antiporters would be needed to acid fermenting bacterium were only active if prevent cation accumulation at the negatively sodium was present (Chen and Russell, unpubcharged interior of cens (61), and this activity lished results).l Some marine bacteria also are was subsequently demonstrated in E. coli (97). able to discriminate between sodium and Sodium-proton antiporters have been shown in lithium (55). a variety of organisms, including Streptococcus Proton Pumps. The FIFo ATPase was origi- faecalis, Bacillus alkalophilus, halobacteria, nally isolated from mitrochondria, but it is and marine vibrios (87). Under natural condiubiquitous in bacteria. It is composed of a tions, sodium-proton antiporters catalyze the globular head piece (FI), which protrudes from influx of protons and efflux of sodium, and if the cell membrane, and a component that acts the exchange is electroneutral (one for one) as a proton channel through the cell membrane UpH is the driving force. In organisms with .-.. c:;
2.5
el
:e 0
f/)
Journal of Dairy Science Vol. 73, No. la, 1990
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RUSSELL ET AL.
low or reversed UpH, the exchange is sometimes electrogenic (2H+ per Na+), and A¥ is the primary driving force. In 1980, Dimroth reported that oxaloacetatefennenting bacteria used biotin-linked decarboxylation reactions to create a sodium gradient in the absence of a protonmotive force (18). These biotin-linked decarboxylation reactions: 1) only occurred if sodium was present, 2) created an electrogenic sodium gradient, and 3) allowed growth even though the fermentation contained no site of substrate level phosphorylation. The ruminal bacterium S. ruminantium decarboxylates succinate via a biotin-linked reaction (85), but sodium pumping has not yet been demonstrated. Acidaminococcus fermentans uses a glutamate decarboxylation reaction to establish a sodium gradient (9), and a recently isolated amino acid-fennenting ruminal bacterium had a similar sodium-dependent pathway of glutamate fermentation (Chen and Russell, unpublished results).l Until recently, it was generally accepted that membrane-bound ATPases of bacteria only translocated protons. Heefner and Harold (31) noted that everted vesicles of S. faecalis had a sodium-stimulated ATPase, which established a sodium gradient at alkaline pH in the absence of a protonmotive force. Streptococcus bovis exhibited rapid efflux of sodium when cells were energized with glucose (90). Because glucose-dependent efflux was resistant to protonophores that collapsed both UpH and A¥, there was no evidence for a sodium-proton antiporter. Proof for a sodium ATPase was obtained from experiments that showed sodium-dependent ATP synthesis, which was DeeD-resistant (Figure 6). The S. bovis sodium ATPase was active at acidic pH. Membrane Conductance
Natural Permeability. Aside from the fact that many bacterial habitats, including the rumen, have a high concentration of sodium, the use of sodium as a coupling agent for transport has several advantages. According to the model of Skulachev (87), bacterial membranes have a high capacitance and the efflux of even a few protons (1 nmol/mg protein) can create a very large electrical potential (A¥). Although the potential may be great, subsequent ion flux and the capacity to do work is small. Potassium 10urnal of Dairy Science Vol. 73,
No. 10, 1990
400
~
,\uH ,\uH+OCCD '\uNa ,\uNa+OCCD
300
2a.
Ioe(
200
100 oP:=:q=e:;~~$=;~::::::;;a-....-.,........j 12 4 10 o 2 6 8 TIME (min)
Figure 6. 1be use of an artificial protonmolive force (6uH) or an artificial electrochemical gradient of sodium (6uNa) to drive AlP synthesis in whole cells of S. bovis. The FlFo AlPase inhibitor dicyclohexylcarbodiimide (DCCD) was added at .25 JUllol/mg protein (90). [Reprinted from Appl. Environ. Microbiol. 55:2664 with pcnnission from the publisher.]
influx discharges the 11¥, relieves the back pressure on the proton ATPase, and allows an increase in UpH. The UpH has a greater capacity to do work, but bacterial membranes are inherently leaky to protons. Sodium-proton antiporters can convert UpH into a chemical gradient of sodium, anG sodium is much less penneable than protons. Sodium ATPases and decarboxylases have the same end result even though proton currents are not directly involved. Many bacteria maintain a near neutral intracellular pH even if extracellular pH decreases (43). 'The maintenance of a constant intracellular pH without a compensatory decrease in 11¥ would create a very high total Ap and increase proton conductance. Bacteria such as E. coli have circumvented this problem by taking up potassium, decreasing 11¥, and maintaining a high ApH. Bacteroides succinogenes, one of the predominant ruminal cellulolytic bacteria, also interconverts A¥ and UpH, but it is unable to grow at pH below 5.9 (73). The ruminal bacteria S. bovis and Bacteroides ruminicola have a different strategy of intracellular pH regulation, which allows internal pH to decrease as a function of extracellular pH, and these bacteria are among the most acid-resistant species in the rumen (75).
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SYMPOSIUM: RUMEN MICROBIAL ECOLOGY AND NUTRITION
OUT
IN
(high Ne+. low K+)
4.0
(high K+. low Ne +)
C
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