Nutrient Transport by Ruminal Bacteria: A Review · cies can ferment the same substrates (Hungate, 1966). Furthermore, because polymer degradation is often slow, soluble nutrient

Nutrient Transport by Ruminal Bacteria: A Review’

Scott A. Martin

Departments of Animal and Dairy Science and Microbiology, The University of Georgia, Athens 30602-2771

ABSTRACT: Fermentation pathways have been elucidated for predominant ruminal bacteria, but information is limited concerning the specific transport mechanisms used by these microorganisms for C, energy, and N sources. In addition, it is possible that changes in ruminal environmental conditions could affect transport activity. Five carrier-mediated soluble nutrient transport mechanisms have been identified in bacteria: 1 ) facilitated diffusion, 2 ) shock sensitive systems, 3 ) proton symport, 4 ) Na+ symport, and the 5 ) phosphoenolpyruvate phosphotransferase system (PEP-PTS). Several regulatory mechanisms are also involved at the cell membrane to coordinate utilization of different sugars. Recent research has shown that predominant ruminal bacteria are capable of trans- porting soluble nutrients by several of the mechanisms outlined above. Megasphaera elsdenii, Selenomo-

nus ruminantium, and Streptococcus bovis transport glucose by the PEP-PTS, and S. ruminantium and S. bouis also possess PEP-PTS activity for disaccharides. Glucose PTS activity in S. bouis was highest a t a growth pH of 5.0, low glucose concentrations, and a dilution rate of .10 h-l. The cellulolytic ruminal bacterium Fibrobacter succinogenes uses a Na+ symport mechanism for glucose transport that is sensitive to low extracellular pH and ionophores. Sodium also stimulated cellobiose transport by F. succinogenes, and there is evidence for a proton symport in the transport of both arabinose and xylose by S. ruminantium. A chemical gradient of Na+ seems to play an important role in A4 transport in several ruminal bacteria. Studying nutrient transport mechanisms in ruminal bacteria will lead to a better understanding of the ruminal fermentation.

Key Words: Rumen, Bacteria, Nutrient Transport

Introduction

The rumen is a diverse and unique microbial ecosystem composed of three types of microorganisms, the bacteria, protozoa, and fungi. Approximately 200 species of bacteria have been isolated and more than 20 types inhabit the rumen in the range of lo7 to 1O1O cells/mL of ruminal fluid. Values greater than lo7 cells/mL have been used to classify ruminal bacterial species as being predominant (Bryant, 1959). More than 20 species of protozoa have been identified and numbers in the rumen are approximately lo6 cells/mL (Williams, 1986). Even though the number of protozoa is less than the number of bacteria, the protozoa are larger and under certain dietary conditions can account for approximately half of the ruminal biomass (Williams, 1986). Compared with the bacteria, relatively little is known about the metabolism and physiology of the protozoa and anaerobic fungi.

lPresented at a symposium titled “Current Aspects of Microbiol- ogy of the Digestive Tract” a t the MAS 85th Annu. Mtg., Spokane, WA.

Received November 5, 1993. Accepted March 9, 1994.

J. h i m . Sci. 1994. 72:3019-3031

Because fungi have been found closely associated with the more slowly digested fractions of plants, they may act as initial colonizers of lignocellulose and increase the rate of bacterial digestion by disrupting lignified plant cell walls (Bauchop, 1979).

Much interest has developed in recent years regarding the use of genetic engineering of ruminal bacteria to manipulate the ruminal fermentation (Forsberg et al., 1986; Russell and Wilson, 1988). However, a better understanding of basic physiological functions, such as nutrient transport mechanisms, as well as factors that affect these functions may be required before these microorganisms can be altered to improve the efficiency of feedstuff utilization. Therefore, the objective of this paper is to review recent progress that has been made in understanding nutrient transport mechanisms as well as factors that affect transport in predominant ruminal bacteria.

Why Study Nutrient Transport?

Ruminal bacterial growth is dependent on the availability of a suitable C and energy source, and the success of most ruminal bacteria relates to their

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Published December 11, 2014

3020 MARTIN

ability to degrade and ferment feedstuffs. Bacteria possess transport systems capable of taking up low molecular weight soluble compounds (i.e., sugars, peptides, AA) across their cell membranes, but feedstuffs are primarily composed of large, relatively insoluble, and sometimes complex polymers (i.e., starch, protein, cellulose). However, these polymers must be degraded by extracellular enzymes to low molecular weight substances before they can be metabolized by the bacteria. Even though fermentation pathways have been identified for the predominant ruminal bacteria, little is known concerning the mechanisms and regulation of nutrient transport in these microorganisms.

Distinct differences are observed between the energy yields of aerobic respiration and anaerobic fermentation. During respiration, glucose is oxidized to carbon dioxide and water, with oxygen serving as the terminal electron acceptor. For each molecule of glucose oxidized there is a maximum total yield of 28 to 38 ATP (Gottschalk, 1986). This compares with a yield of only 2 to 5 ATP per molecule of glucose metabolized by an anaerobic fermentation, because oxygen cannot serve as an electron acceptor and consequently oxidation is less complete. Because ATP is limiting, energetically efficient substrate transport mechanisms would be especially beneficial in anaerobic bacteria. Study of nutrient transport systems in ruminal bacteria is of interest for several reasons. Before bacterial growth can occur, soluble substrates must be transported across the membrane barrier that separates the extracellular medium from the cell interior. In addition, the rumen is a competitive environment that is inhabited by many different microorganisms as described above, and several species can ferment the same substrates (Hungate, 1966). Furthermore, because polymer degradation is often slow, soluble nutrient concentrations are usually low in the rumen (Van Soest, 1982; Czerkawski, 19861, so only bacteria with efficient and high-affinity transport systems will be able to compete successfully. Another consideration is that active transport processes require energy, and this expenditure is especially important in anaerobic microorganisms such as ruminal bacteria because energy is often limiting.

Bacterial Transport Mechanisms

Five carrier-mediated soluble carbohydrate transport mechanisms have been identified in bacteria: 1) facilitated diffusion, 2) shock sensitive systems, 3 ) proton symport, 4 ) Na+ co-transport, and 5) the phosphoenolpyruvate-dependent phosphotransferase system (Figure 1; Dills et al., 1980; Saier, 1985). These transport systems can be classified according to the energy source that is coupled to the transport process. Facilitated diffusion involves a carrier protein and catalyzes the rapid equilibration of a substrate

IN

ADP

S

S

S

S

H+

S Na+

S

OUT

Facilitated Diffusion

Phosphate-bond Driven or Shock Sensitive

Proton Symport

Sodium Symport

PEP-PTS

Figure 1. Transport mechanisms that have been identified in bacteria.

across the cytoplasmic membrane (Dills et al., 1980). Because metabolic energy is not used, the substrate is not concentrated within the cell. For many years glycerol was the only carbohydrate known to be transported by this mechanism in bacteria (Lin, 1976). However, the bacterium Zymomonas mobilis was shown to transport glucose by a stereospecific carrier-mediated facilitated diffusion system (DiMarco and Romano, 1985).

Maltose transport in the Gram-negative bacterium Escherichia coli is an example of a shock-sensitive active transport system (Kellerman and Szmelcman, 1974; Ferenci et al., 1977). In this system, a periplasmic maltose-binding protein interacts with a malto-dextrin porin in the outer membrane as well as two integral cytoplasmic membrane proteins to transport maltose intracellularly (Saier, 1985). Cold osmotic shock of cells abolishes maltose uptake by releasing the periplasmic maltose binding protein. Many shock sensitive transport systems have been described for other carbohydrates (arabinose, ribose, methyl-/3-galactose) as well (Dills et al., 1980). Because chemical energy (ATP, phosphoenolpyruvate [PEP], or acetyl-phosphate) is directly coupled to the uptake process, shock sensitive systems are referred to as primary active transport mechanisms (Ferenci et al., 1977). These systems are inhibited by arsenate, which decreases the intracellular ATP and PEP pools (Klein and Boyer, 1972).

NUTRIENT TRANSPORT BY RUMINAL BACTERIA 3021

A transport process involving the obligatory coupling of two or more ions in parallel is termed symport or co-transport (Nicholls, 1982). The best understood transport system that utilizes a substrate-proton symport is the lactose permease (Hengge and Boos, 1983; Kaback, 1983). The transport stoichiometry of lactose to proton is 1:1 as originally proposed by Mitchell (1963, 1967). Transmembrane translocation of lactose without a proton does not occur under normal conditions (Dills et al., 1980; Saier, 1985). Most bacterial symporters seem to be coupled to protons, but Na+ symport mechanisms have been found in E. coli (Tsuchiya et al., 1977; Chen et al., 19851, marine bacteria (Droniuk et al., 19871, and alkalophilic bacteria (Krulwich, 1986). Another transport system that utilizes sugar-cation symport is the melibiose permease. Although the lactose permease functions by lactose-proton symport, the melibiose permease combines sugar uptake to the co-transport of Na+, proton (H+), or lithium (Li+), depending on the experimental conditions, sugar, and the bacterial strain (Tsuchiya and Wilson, 1978; Tsuchiya et al., 1978).

With all four of the transport systems described up to now, the soluble carbohydrate enters the cell unphosphorylated. To trap the sugar within the cell and initiate glycolysis a phosphorylation step mediated by a kinase is required. The fifth type of carrier- mediated carbohydrate transport mechanism is a group translocation process that phosphorylates the substrate during transport. An example of this process is the PEP-dependent phosphotransferase system (PEP-PTS) that was first described in E. coli by Kundig et al. (1964). This system catalyzes the transport and phosphorylation of a number of carbohy-

OUT IN

drates with PEP serving as the phosphoryl donor in a wide variety of bacterial species.

The general sequence of the PEP-PTS is shown in Figure 2. The first step involves the phosphorylation of a general sugar nonspecific, cytoplasmic protein, enzyme I, via dephosphorylation of PEP. Phos- phorylated enzyme I then transfers the phosphoryl group to a second sugar nonspecific cytoplasmic heat- stable phosphocarrier protein, HPr. In some organ- isms, HPr-P transfers its phosphoryl group to another soluble protein, enzyme 111, which is sugar-specific (Saier, 1985). Enzyme I11 serves as a phosphocarrier to the membrane-bound enzyme 11, but in many cases HPr-P transfers its phosphoryl group directly to enzyme I1 without the involvement of enzyme 111. Enzymes I1 exhibit some substrate specificity, but they can sometimes carry different sugars.

The PTS is widely distributed among facultative and strictly anaerobic bacteria that metabolize sugars by the Embden-Meyerhof-Parnas ( EMP) pathway (Romano et al., 1970; Romano et al., 1979; Dills et al., 19801, and it is advantageous under energy-limited conditions. Hexoses that are metabolized by the EMP pathway result in 2 mol of PEP per hexose. If 1 mol of PEP is utilized by the PTS, the other mole can be used for ATP synthesis via pyruvate kinase. Because ATP is not expended in a glucokinase or hexokinase reaction, the overall ATP yield would be 2 ATP per mole of glucose. Therefore, this mechanism of sugar transport is energetically favorable to anaerobic bacteria that obtain low yields of ATP per mole of hexose fermented because ATP is not expended in a glucokinase or hexokinase reaction.

In some bacteria disaccharides (lactose, maltose, sucrose) are also transported and phosphorylated by

Figure 2. The phosphate transfer chain of the phosphoenolpyruvate phosphotransferase system. PEP, phosphoenolpyruvate; PRY, pyruvate; EI, enzyme I; HPr, heat-stable phosphocarrier protein; EIII, enzyme 111; EII, enzyme 11.

3022 MARTIN

the PTS (McKay et al., 1969; St. Martin and Wittenberger, 1979; Keevil et al., 1984) by the following mechanism:

disaccharide + PEP PTs > disaccharide-phosphate + pyruvate [ l l

hydrolase disaccharide-phosphate > hexose + glucose 6-phosphate [21

glucokinase

glucose 6-phosphate [31 hexose + ATP >

The product of the PEP-dependent phosphorylation reaction is a disaccharide-phosphate with the phosphate group generally located on the glucose moiety (reaction 1) . After it is inside the cell the disaccharide-phosphate is cleaved by a hydrolase to one phosphorylated sugar and one free sugar (reaction 2). Because one of the sugars is phosphorylated during transport, only half as much ATP is needed in a kinase reaction (reaction 3 1.

Regulation of Bacterial Transport

Because transport is the first event in the metabolic process and because bacteria are generally capable of using more than one soluble sugar, one would expect the transport step to be controlled in order to coordinate the utilization of different sugars. If E. coli is exposed to glucose and a second sugar simultane- ously, glucose is used preferentially and as a result growth is biphasic (Gottschalk, 1986). Monod (1947) observed that glucose was the preferred sugar in enteric bacteria and referred to this exclusion phenomenon as the “glucose effect.” Later, the general term “catabolite repression’’ was used because the same growth behavior was measured for a variety of substrate combinations (Magasanik, 196 1).

Catabolite repression occurs a t the level of tran- scription and is caused by the binding of a cyclic adenosine monophosphate ( AMP) receptor protein complex to DNA (Gottschalk, 1986). Early research showed that substrate preferences could also be mediated at the level of transport (McGinnis and Paigen, 1967, 1973). Based on years of research with Gram-negative and Gram-positive bacteria, several regulatory mechanisms associated with soluble sugar transport in bacteria have been identified (Dills et al., 1980; Saier, 1985; Reizer and Peterkofsky, 1987).

R e p la tory Mechanisms

Seven regulatory mechanisms are known to control soluble carbohydrate uptake a t the cell membrane in

bacteria (Dills et al., 1980; Saier, 1985; Reizer and Peterkofsky, 1987). These include the following:

1.

2.

3.

4.

Inhibition due to competition between two sugars for the binding site of a permease protein (i.e., enzyme 11). Some enzymes I1 of the PTS have a broad substrate specificity, and these can bind various sugars with different affinities. If two sugar substrates of the same enzyme I1 are both present in the growth medium, they will compete (Postma and Lengeler, 1985). One sugar will eventually exclude the other, depending on the sugar concentration and relative affinity constants of the enzyme I1 for each sugar. Inhibition of carbohydrate uptake via intracellular carbohydrate phosphates. The intracellular accumulation of sugar phosphates has been associated with decreased activity in a wide variety of carbohydrate permeases (Dills et al., 1980; Postma and Lengeler, 1985). However, the mechanism for this inhibition has not been elucidated. I t has been proposed that the intracellular carbohydrate phosphates could interact directly with an allosteric regulatory site on the cytoplasmic sur- face of the cell membrane, inhibiting permease activity (Dills et al., 1980). One PTS sugar inhibits the uptake of another PTS sugar due to competition by the corresponding enzymes I1 for the common phosphoryl donor, phospho-HPr. Because all enzymes I1 or enzyme II/enzyme I11 complexes use enzyme I and HPr during PTS-mediated transport, they must compete for the general PTS proteins. Consequently, the uptake of one PTS sugar can inhibit the uptake of another (Saier, 1985). If one enzyme I1 or enzyme Wenzyme I11 complex has a higher affinity for phospho-HPr than a competing enzyme I1 or enzyme II/enzyme I11 complex, the second enzyme(s) will have lower activity (Dills et al., 1980; Postma and Lengeler, 1985). Chemiosmotic control of transport activity due to the sensitivity of a permease system to the membrane potential and(or) the intracellular pH. Enzymes I1 of the PTS are inhibited by chemiosmotic energy or the imposition of a transmembrane proton gradient (Saier, 1985; Reizer and Peterkofsky, 1987). One possible explanation for this observation is that the permease proteins interact with the membrane potential or transmembrane proton gradient in a way that in- fluences the conformation of the protein (Saier, 1985). Uptake of a-methylglucoside, a non- metabolizable substrate of the PTS, was inhibited in intact cells of E. coli by the imposition of a proton gradient across the cell membrane (Reider et al., 1979). When the proton gradient was disrupted by the addition of electron transport inhibitors and proton-conducting uncouplers, the uptake of a-methylglucoside was stimulated.


5 . PTS-mediated regulation involving the inhibition of non-PTS permease activity by a protein of the PTS (i.e., enzyme 111). When sugars are transported by the PTS, the majority of enzyme I11 proteins are largely dephosphorylated and in this configuration enzyme 111 can bind directly to the sugar permease and inhibit active transport (Reizer and Peterkofsky, 1987). If PTS sugars are not available, phosphorylation of enzyme 111 occurs and it dissociates from the allosteric regulatory site of the permease. The PTS also regulates the activity of adenylate cyclase, the cyclic AMP biosynthetic enzyme, by an apparently coordinate mechanism (Saier and Feucht, 1975).

6. Regulation of PTS activity by an ATP-dependent HPr kinase. This type of regulation seems to be unique to Gram-positive bacteria and involves modification of HPr by an ATP-dependent membrane-associated protein kinase (Reizer and Peterkofsky, 1987). When PEP is the phosphoryl donor for the PTS, HPr is phosphorylated via enzyme I on a histidine residue (Reizer and Peterkofsky, 1987). However, the HPr kinase uses ATP rather than PEP to phosphorylate a serine residue instead of histidine, and the phosphorylated serine-HPr cannot replace phosphorylated histidine-HPr as a phosphoryl donor to PTS sugars (Reizer and Peterkofsky, 1987). This modification of HPr on a serine residue seems to control the rate of sugar transport and determines which PTS sugar will be transported over another.

7. Regulation of sugar accumulation by expulsion of intracellular sugars. This mechanism involves the intracellular dephosphorylation of sugar-phosphate followed by expulsion of the intracellular free sugar out of the cell in Gram-positive bacteria (Reizer and Peterkofsky, 1987). Expulsion of the nonpreferred substrate, such as a nonmetaboliza- ble sugar analog, may provide a protective mechanism that allows bacteria to survive with an overload of sugar-phosphates. Research with Streptococcus pyogenes showed that expulsion of unphosphorylated sugar analogs can occur fairly quickly ( 10 s 1 but will vary depending on the type and concentration of sugar used (Reizer and Peterkofsky , 198 7).

Sugar Transport in Ruminal Bacteria

Russell and Baldwin (1978) reported that several predominant ruminal bacteria ( PrevotelZa [Bac- teroides] ruminicola, Butyrivibrio fibrisolvens, Megasphaera elsdenii, Selenomonas ruminantium, Streptococcus bouis) exhibited substrate preferences or catabolite regulatory mechanisms when each organism was grown in batch culture. However, the biochemical mechanisms involved were not evaluated

to determine whether regulation was occurring at the level of transport. When affinities for C and energy sources were compared, the ruminal bacteria had much higher affinities for some sugars than for others, and the afinity for the same substrate differed among species (Russell and Baldwin, 1979). Based on these results, research was initiated to characterize the mechanism(s) of glucose transport in P. ruminicola B14, B. jibrisolvens 49, Fibrobacter (Bacteroides) succinogenes S85, M. elsdenii B159, S. ruminantiurn HD4,and S. bovis JB1 (Martin and Russell, 1986). Additional research has been conducted over the past 8 yr, and Table 1 summarizes soluble carbohydrate transport mechanisms that have been identified in ruminal bacteria.

Cellobiose, glucose, maltose, and sucrose are transported by the PEP-PTS in S. bovis JB1 (Table 1). The glucose system is constitutive, whereas the disaccharide systems are inducible and it seems that separate enzymes I1 exist for glucose, maltose, and sucrose (Martin and Russell, 1987). Glucose PEP-PTS activity was also detected in S. bovis strains 581AXY2, G1, and 45S1 (Moore and Martin, 1991). Glucose is also transported by facilitated diffksion in strain J B 1 (Russell, 1990), and maltose can also be catabolized by an inducible maltase and a maltose phosphorylase (Martin and Russell, 1987). The metabolic rationale for this redundancy of transport and phosphorylation systems for glucose and maltose in this bacterium is not clear, but it may be due to the sensitivity of the PEP-PTS to changing environmental conditions within the rumen (see below).

S. ruminantium HD4 possesses constitutive and inducible PEP-PTS activity for glucose and sucrose, respectively (Table 1 ) . Maltose is hydrolyzed by an inducible extracellular/membrane-associated maltase and the resulting glucose monomers are then available for transport by the constitutive glucose PTS (Martin and Russell, 1988). Russell and Baldwin (1978) demonstrated that glucose and sucrose are preferentially used before maltose in strain HD4. Because the maltase is competitively inhibited by glucose and sucrose (Martin and Russell, 19881, this regulation seems to occur at the cell membrane. S. ruminantium HD4 seems to have an inducible active transport system for xylose (Martin and Russell, 1988; Williams and Martin, 19901, and this is consistent with the absence of pentose PEP-PTS in other bacteria (Dills et al., 1980; Lam et al., 1980). In addition, strain HD4 has a glucokinase that is capable of utilizing ATP to phosphorylate the glucose analog 2-deoxyglucose, and this is somewhat unusual in bacteria (Martin and Russell, 1986).

Recently, Strobe1 ( 1993a) reported that SeZenomo- nas ruminantium D has a glucose PEP-PTS and proton symport systems for arabinose and xylose and similar to strain HD4 xylose transport was inducible (Table 1). Furthermore, glucose was preferentially

3024 MARTIN

Table 1. Soluble carbohydrate transport mechanisms in ruminal bacteriaa

Soluble Transport Constitutive/ Bacterium Strain carbohydrate mechanismb inducible Reference

Streptococcus bouis JB1

Streptococcus bovis 58lAXY2 Streptococcus bouis G1 Streptococcus bovis 45S1 Selenomonas ruminantium HD4

Selenomonas ruminantium D

Selenomonas ruminantium GA192 Selenomonas H18 Megasphaera elsdenii ?

Megasphaera elsdenii B159 Prevotella ruminicola B 14

Fibrobacter succinogenes S85

Ruminococcus albus B199 Ruminococcus flavefaciens FD-1

Glucose Cellobiose Maltose

Sucrose Glucose Glucose Glucose Glucose Glucose Sucrose Maltose Xylose Glucose

Arabinose Xylose Glucose Glucose Fructose Glucose Glucose Glucose

Arabinose, Xylose Glucose

Cellobiose Glucose

Cellobiose

PTS PTS PTS

Maltase Maltose phosphorylase

PTS Facilitated diffusion

PTS PTS PTS PTS PTS

Maltase Active transport

PTS Proton symport Proton symport

PTS PTS ETS FTS PTS

Active transport Proton symport

Sodium symport Sodium symport Active transport

Phosphate-bond driven Active transport

Constitutive Inducible Inducible Inducible Inducible Inducible

-

- - -

Constitutive Inducible Inducible Inducible

Constitutive Inducible

-

- -

Inducible Inducible

Constitutive -

-

Constitutive Constitutive Constitutive Constitutive

-

7

12 11

8

8, 17 14

10

2

6 9

13

15 1, 3 5

16 4

aReferences: ( 1) Chow and Russell, 1992; ( 2 Dills e t al., 1981; (3 Franklund and Glass, 1987; ( 4 ) Helaszek and White, 1991; (5) Maas and Glass, 1991; (6 ) Martin and Russell, 1986; ( 7 ) Martin and Russell, 1987; (8) Martin and Russell, 1988; ( 9 ) Martin, 1992a; ( 1 0 ) Martin, 1993; ( 11) Moore and Martin, 1991; ( 1 2 ) Russell, 1990; ( 13) Russell, 1993; ( 1 4 ) Strobel, 1993a; ( 1 5 ) Strobel, 1993b; ( 1 6 ) Thurston et al., 1993; ( 1 7 ) Williams and Martin, 1990.

bPTS, phosphotransferase system.

utilized over pentose sugars, apparently due to catabolite inhibition by the glucose PEP-PTS (Strobel, 1993a). Glucose PTS activity has also been detected in Selenomonas ruminantium GA192 and the recent ruminal selenomonad isolate strain H18 (Martin, 1993).

Limited research with M. elsdenii has shown that this bacterium possesses PEP-PTS activity for glucose and fructose (Table 1) . Dills et al. (1981) demonstrated that this activity was inducible for both sugars in an unidentified strain of M. elsdenii, but it is not known whether glucose activity in strain B159 is inducible or constitutive (Martin and Russell, 1986). No significant PEP-PTS activity could be detected in P. ruminicola B14 for glucose (Martin and Russell, 1986), but glucose uptake is constitutive and sensitive to metabolic inhibitors, suggesting that active transport, probably a proton symport system, may be involved (Martin, 1992a; Russell, 1993). Further- more, based on work with a glucose-resistant mutant, Russell (1992) recently proposed that P. ruminicola B14 is unable to regulate glucose transport and

utilization when growth is limited by ammonia. Arabinose and xylose transport by P. ruminicola B14 is sensitive to oxygen and dependent on a chemical gradient of Na+ (Strobel, 199313).

Similar to P. ruminicola B14, both of the fiber- degrading bacteria B. fibrisolvens 49 and F. succinogenes ,985 had only low glucose PEP-PTS activity, high glucokinase or hexokinase activities, and seemed to use active transport rather than the PEP-PTS (Martin and Russell, 1986). More recently it has been shown that glucose uptake by F. succinogenes S85 is stimulated by Na+ (Franklund and Glass, 1987) and seems to involve a Na+ symport (Chow and Russell, 1992). An active transport mechanism also seems to play a role in cellobiose transport by F. succinogenes S85 (Maas and Glass, 1991). Cellobiose transport by another cellulolytic ruminal bacterium, Ruminococcus flavefaciens FD-1, seems to involve active transport and the intracellular cellobiose is then hydrolyzed by a cellobiose phosphorylase (Helaszek and White, 1991). Neither cations or the PTS are involved in glucose transport by Ruminococcus albus B 199; transport


Table 2 . Amino acid transport mechanisms in ruminal bacteriaa

Bacterium Amino Transport Kin,

Strain acid mechanism P M Vmaxb Reference

Streptococcus boois

Streptococcus bovis

Selenomonas

Peptos treptococcus

Strain F

Strain SR

JBl Alanine Serine

Threonine J B 1 Glutamine

H18 Aspartate Glutamine

Lysine Phenylalanine

Serine Valine

C Leucine Isoleucine

Valine Glutamate

Glutamine

Histidine Serine

Arginine

Lysine

Sodium symport Sodium symport Sodium symport

Phosphate-bond driven+ Facilitated diffusion

Sodium symport Sodium symport Sodium symport sodium symport Sodium symport Sodium symport Sodium symport Sodium symport Sodium symport

Sodium symport + Facilitated diffusion Sodium symport +

Facilitated diffusion Sodium symport Sodium symport

Sodium symport + Facilitated diffusion

Facilitated diffusion + Sodium symport

8.8 8.6 6.3 -

- - - - - -

77 33 55 4

11

- -

< 1.0

-

3 4 7 10

1 -

5 - - - - - -

328 2 194 243 82 3

50

- -

16 6

aReferences: ( 1 Chen and Russell, 1989a; (2) Chen and Russell, 1989b; ( 3 ) Chen and Russell, 1990; ( 4 ) Russell et al., 1988; ( 5 ) Strobel

bNanomoles/milligram of protein per minute. and Russell, 1991b; ( 6 ) Van Kessel and Russell. 1992.

(high affinity) is apparently driven by ATP hydrolysis (Thurston et al., 1993). Furthermore, R. albus B199 preferentially utilizes cellobiose over glucose, and this preference seems to be related to repression of glucose uptake in cellobiose-grown cells (Thurston et al., 1993).

Amino Acid Transport in Ruminal Bacteria

Active transport can mediate the transport of AA as well as sugars, and this type of transport may be driven by ion gradients L e . , H+, Na+) or the hydrolysis of chemical bonds (i.e., ATP). Even though it is well-known that most of the predominant ruminal bacteria require a N source for growth, information is limited regarding the mechanisms used by these bacteria to accumulate AA, ammonia, and(or) peptides intracellularly. Based on research with mixed ruminal bacteria, it has been suggested that ammonia is assimilated by passive diffusion rather than by active transport (Russell and Strobel, 1987). Passive diffusion is similar to facilitated diffusion except that a carrier protein is not involved (Russell et al., 1990). There is some evidence suggesting that ruminal bacteria prefer peptides to free AA (Chen et al., 19871, but recent research has shown that several ruminal bacteria readily transport AA (Table 2). In S. bouis JB1, alanine, serine, and threonine are transported by Na+-dependent mechanisms (Russell et al.,

1988). Sodium symport also is involved in the transport of aspartate, glutamine, lysine, phenylalanine, serine, and valine by the ruminal selenomonad strain H18 (Strobel and Russell, 1991b). Transport of glutamine by S. bouis JB1 seems to be driven by phosphate-bond energy (i.e., ATP) rather than ions, and facilitated diffusion also seems to be involved (Chen and Russell, 1989a). Once transported into the cell, glutamine is converted by JB1 to ammonia and pyroglutamate (Chen and Russell, 1989a). Additional research with mixed ruminal bacteria suggests that pyro-glutamate may be a significant end product of glutamine degradation when ruminants are treated with monensin (Russell and Chen, 1989).

A ruminal peptostreptococcus transports the branched-chain AA leucine, isoleucine, and valine by a common carrier protein that is dependent on Na+ (Table 2). Two recent ruminal isolates capable of fermenting AA rapidly, strain F and strain SR, have also been shown to transport A4 via a Na+ symport. Strain F has separate Na+ symport transport systems for glutamate, glutamine, histidine, and serine, and this bacterium also seems to transport glutamate and glutamine by facilitated diffusion (Chen and Russell, 1990). Arginine transport by strain SR can be driven by a chemical gradient of Na+ or facilitated diffusion, and facilitated diffusion is the dominant mechanism for lysine transport (Van Kessel and Russell, 1992). It should be noted that all three of these new isolates are sensitive to monensin (Chen and Russell, 198913; Chen

3026 MARTIN

E 18 r

I C 16

8 14 P a 12

.-

.c : 1 0 - - E 8 -

E 6 - d 4 -

3 2 -

= o

Y

a

and Russell, 1990; Van Kessel and Russell, 1992). Before the isolation of these three AA fermenting bacteria (peptostreptococcus, strain F, strain SR), it was assumed that all the predominant AA-utilizing bacteria had been isolated from the rumen. However, these predominant bacteria could not account for in vivo AA degradation rates or the "protein-sparing" effect attributed to monensin treatment (Chen and Russell, 1989b). Due to the ability of the peptostreptococcus, strain F, and strain SR to grow rapidly on AA or peptides and their sensitivity to monensin, it has been suggested that these bacteria may play an important role in ruminal AA degradation (Chen and Russell, 1989b; Chen and Russell, 1990; Van Kessel and Russell, 1992). More research is needed to determine whether these bacteria are found in significant numbers in ruminants throughout the United States and the world. Based on phenotypic criteria and 16s rRNA sequences, these three high ammonia- producing bacteria have been identified as Peptostrep- tococcus anaerobius (peptostreptococcus), Clostridium aminophilum (strain F), and Clostridium sticklandii (strain SR) (Paster et al., 1993).

- - - -

n n rT, r-l

Organic Acid Transport in Ruminal Bacteria

In addition to soluble carbohydrates and AA, some ruminal bacteria are capable of using organic acids as C and energy sources. S. ruminantium HD4 is unique in that when grown in batch culture with glucose, a homolactic fermentation occurs; however, after the glucose is depleted from the medium, S. ruminantium HD4 then utilizes the lactate as a C and energy source (Scheifinger et al., 1975). Because fewer ruminal microorganisms are able to utilize lactate than are able to utilize soluble carbohydrates, this ability to ferment lactate apparently provides S. ruminantium HD4 with some type of survival advantage within the rumen.

Linehan et al. (1978) demonstrated that S. ruminantium HD4 requires L-aspartate, COz, p- aminobenzoic acid, and biotin for growth in a lactate- salts medium. The requirement for aspartate could be

0 25 50 100

NaCI, rnM

Figure 3. Effect of different concentrations of NaCl on L-lactate uptake by whole cells of Selenomonas ruminantium HD4 in the absence (0) and presence 1.1 of 10 mM L-malate (Nisbet and Martin, 1994).

replaced by L-malate or fumarate. No growth on L- aspartate, fumarate, or L-malate occurred in the absence of lactate in the medium. L-Lactate uptake by S. ruminantium HD4 was stimulated by 10 mM L- aspartate (fourfold), fumarate (fourfold), L-malate (10-fold), and a combination of all three compounds increased uptake 10-fold (Nisbet and Martin, 1990). Different concentrations ( .03 to 10 mM) of L-malate stimulated L-lactate uptake in a dose-response fashion with 10 mM L-malate giving the greatest response (Nisbet and Martin, 1991). Recent research showed that L-lactate uptake in the absence of L-malate was low regardless of the Na+ concentration, but Na+ concentrations between 25 and 100 mM stimulated L- lactate uptake in the presence of 10 mM L-malate (Figure 3). The stimulation of L-lactate uptake by L- malate in the absence of added Na+ is most likely due to the use of the disodium salt of L-malate (Nisbet and Martin, 1994). Similar concentrations of K+ had little effect on L-lactate uptake (Nisbet and Martin, 1994). These results suggest that both L-malate and Na+ play a role in stimulating L-lactate utilization by

Table 3. Organic acid transport mechanisms in ruminal bacteriaa

Organic Transport Constitutive/ Bacterium Strain acid mechanism inducible Reference

Selenomonas ruminantium HD4 Lactate Active transport Inducible 2 Selenomonas H18 Fumarate Proton symport -

Proton symport - Succinate Proton symport -

Megasphaera elsdenii B159 Lactate Active transport - Selenomonas ruminantium HD4 Succinate Sodium symport -

Selenomonas ruminantium HD4 Succinate Proton symport -

3 3 4 5 1 4

Malate

aReferences: ( 1 1 Michel and Macy, 1990; ( 2 j Nisbet and Martin, 1994; ( 3 j H. J. Strobel, personal communication; (4) Strobel and Russell, 1991a; ( 5 ) Waldrip and Martin, 1993.


S. ruminantium HD4. Because L-malate had little effect on L-lactate uptake for cells grown on soluble carbohydrates compared with lactate-grown cells, it seems that the stimulation due to malate is inducible (Table 3). D-Lactate uptake by S. ruminantium HD4 was also stimulated by 10 mM L-malate (Nisbet and Martin, 1993). Due to sensitivity to several metabolic inhibitors, it has been suggested that proton gradients may be involved in L-lactate uptake in strain HD4, but more research is needed to characterize the bioenergetics involved in the transport of L-lactate (Nisbet and Martin, 1994).

The ruminal selenomonad strain H18, a recent isolate, also required dicarboxylic acids to grow on lactate (Strobel and Russell, 1991b). However, strain H18 differed from strain HD4 in that lactate could be used for growth only when Na+ and aspartate were added to the medium. Malate or fumarate could replace aspartate, but Na+ was not required (Strobel and Russell, 1991b). The fact that dicarboxylic acids, especially malate and fumarate, stimulate lactate utilization is consistent with the presence of the randomizing or succinate-propionate pathway that involves a fumarate reductase in this bacterium (Gottschalk, 1986). 5'. ruminantium utilizes the succinate-propionate pathway to make propionate from lactate, and both fumarate and malate are intermediates in this pathway (Gottschalk, 1986). Involvement of a proton symport in the transport of fumarate and malate in strain H18 has been suggested (H. J. Strobel, personal communication).

Unlike S. ruminantium, M. elsdenii converts lactate to propionate and acetate by the acrylate (nonran- domizing) pathway (Gottschalk, 1986). Both Na+ and K+ had little effect on L-lactate uptake by M. elsdenii B159 (Waldrip and Martin, 1993). In addition, L- lactate uptake was inhibited between 34 and 61% by protonophores, compounds capable of disrupting proton gradients across bacterial membranes (Nicholls, 1982). Therefore, protons may be involved in driving L-lactate uptake by an active transport mechanism in M. elsdenii B159 (Table 3 ) .

Even though many ruminal bacteria produce succinate, little succinate ever accumulates in the rumen (Hungate, 1966). Generally, it has been assumed that S. ruminantium is the primary organism responsible for the conversion of succinate to propionate and keeping succinate concentrations low in the rumen. S. ruminantium HD4 can also produce succinate (Mel- ville et al., 1988). Transport must occur in order to decarboxylate succinate and produce propionate and limited information is available detailing succinate transport in S. ruminantium (Table 3 ) . Michel and Macy (1990) reported that S. ruminantium HD4 utilizes a Na+ symport mechanism to transport succinate, but as pointed out by Strobel and Russell (1991a) there was little evidence presented by Michel and Macy (1990) to support involvement of Na+ in

succinate transport. Rather than a Na+ symport, it seems that a proton symport is involved in succinate transport in strain HD4 and strain H18 (Strobel and Russell, 1991a). In addition, succinate transport was strongly inhibited by glucose, indicating that glucose is preferred over succinate (Strobel and Russell, 1991a).

Environmental Factors and Nutrient Transport

The ruminal environment is not static, and conditions vary depending on the type and amount of feed that is fed as well as the frequency of feeding. In general, the dilution rate within the rumen is between .05 and .10 h-l (Hungate, 19661, and the concentrations of soluble sugars in ruminal fluid are usually very low (1.0 g/L or less) except immediately after feeding (Czerkawski, 1986). Previous research with the oral bacterium Streptococcus mutans showed that glucose PTS activity was significant only under conditions of low substrate concentration or low growth rate at or near neutral pH (Hamilton and Ellwood, 1978; Ellwood et al., 1979). Because the ruminal environment is subject to change, it is possible that these changes will have an effect on nutrient transport by ruminal bacteria. Based on growth studies, cellulolytic ruminal bacteria ( B . fibrisolvens, F. succinogenes, R. albus, R. flavefaciens) are more sensitive to acidic pH than noncellulolytic bacteria ( S. bovis, S. ruminantium, M. elsdenii) (Russell et al., 1979; Russell and Dombrowski, 1980). Recent research showed that F. succinogenes S85 is unable to grow a t low extracellular pH (< 6.0) on glucose because the Na+ symport mechanism used for glucose transport is pH-sensitive (Chow and Russell, 1992).

To simulate conditions that may occur within the rumen, experiments were conducted to evaluate the effects of various glucose concentrations ( .5 , 2.0, and 6.0 g/L), dilution rates (.05, .lo, and .20 h-l), and growth pH (6.5, 5.0, and 4.8) on glucose PTS activity in S. bovis JB1 (Moore and Martin, 1991). It has been well documented that S. bovis proliferates and produces lactate under conditions of high sugar concentrations (i.e., high cereal grain diet) and low pH. Glucose PTS activity tended to be highest at a dilution rate of .10 h-l at all three glucose concentrations tested except when the cells were grown with .5 g/L of glucose at pH 5.0 (Table 4). As the glucose concentration was increased from .5 to 6.0 g/L, glucose phosphorylation activity decreased. Furthermore, the culture washed out at a low dilution rate (.05 h-l) with 6.0 g/L of glucose, indicating a greater tolerance of low pH when glucose concentrations were less than 6.0 g/L. Russell and Dombrowski (1980) reported that S bouis JB1 washed out at pH 4.55 with a glucose

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Table 4. Effect of growth conditions on PEP-dependent phosphorylation of glucose by Streptococcus bovis J B l grown in continuous culturea

Glucose, g/L

Dilution rate, h-l

PH .05 .10 .20

.5

2.0

6.5 5.0 4.8 6.5 5.0 4.8

~~ Specific activity, ~

nmol.mg of protein-’.min-l 415.6 506.5 402.7 663.3 665.5 685.4 107.4 399.6 274.9 220.0 235.3 206.6 279.1 281.4 264.4

- b _. 116.3 6.0 6.5 72.3 119.0 86.0

5.0 32.2 37.0 25.1 4.8 - - -

aMoore and Martin (1991). bWashout of culture from chemostat.

concentration of .54 g L and a dilution rate of .158 h-l. Washout refers to the inability of a bacterial culture to grow due to unfavorable environmental conditions within the culture vessel. These results suggest that the S. bouis JB1 glucose PTS has higher activity under conditions of low glucose concentrations and at a dilution rate of .10 h-l. When the cells were incubated with 6.0 g/L of glucose, glucose PTS activity was optimum at a growth pH of 6.5 and dilution rate of .10 h-l.

In addition to S bouis, the effects of pH on nutrient transport by several predominant ruminal bacteria have been evaluated. Consistent with the sensitivity of F. succinogenes S85 to acidic pH when grown in continuous culture (Russell and Dombrowski, 19801, glucose uptake by S85 decreased as extracellular pH declined from 7.0 to 5.0 (Chow and Russell, 1992; Martin, 1992b). Similarly, when R. albus B199 was exposed to extracellular pH values less than 7.0, glucose uptake was reduced more than 80% (Thurston et al., 1993). Glucose uptake by P. ruminicola B14 and xylose uptake by S. ruminantium HD4 and S. ruminantium D is fairly resistant to declines in extracellular pH (Williams and Martin, 1990; Martin, 1992a; Strobel, 1993a), whereas arabinose uptake by S. ruminantium D is somewhat sensitive to declines in extracellular pH (Strobel, 1993a). Transport of arabinose and xylose by P. ruminicola B14 is more sensitive to declines in extracellular pH than is glucose transport; uptake of both pentoses was reduced approximately 50% as pH decreased from 7.0 to 6.0 (Strobel, 1993b). Low extracellular pH also decreased the transport of arginine by strain SR (Van Kessel and Russell, 19921, glutamate by strain F (Chen and Russell, 1990), leucine by peptostreptococcus (Chen and Russell, 1989b), and succinate by the ruminal selenomonad strain H18 (Strobel and Rus- sell, 1991a).

Monensin is an ionophore that alters the ruminal fermentation by selectively inhibiting certain microor-

ganisms and is routinely incorporated into the diets of beef cattle (Russell and Strobel, 1989). This com- pound is capable of disrupting Na+ or K+ gradients across bacterial cell membranes (Russell and Strobel, 1989). Sodium is the predominant (90 to 150 mM) extracellular cation within the rumen (Durand and Kawashima, 19801, and seems to play an important role in the transport of soluble nutrients by ruminal bacteria (Tables 1 and 2). Consistent with the involvement of Na+ in the transport of soluble carbohydrates and AA by ruminal bacteria is the observation that several of these transport mechanisms are sensitive to monensin. Amino acid transport by the recent ruminal isolates peptostreptococcus, strain F, and strain SR is inhibited by monensin, and it has been suggested that this helps to explain the decrease in ruminal ammonia in monensin-treated animals (Chen and Russell, 1989b; Chen and Russell, 1990; Van Kessel and Russell, 1992). Monensin also is a strong inhibitor of Natdependent transport of neutral AA by S. bouis JB1 (Russell et al., 1988). In addition to AA, monensin is a strong inhibitor of glucose transport by F. succinogenes S85 (Chow and Russell, 1992) and succinate transport by the ruminal selenomonad strain H18 (Strobel and Russell, 1991a). Collectively, these results suggest that monensin alters the ruminal microbial fermentation by influenc- ing transport of nutrients at the bacterial cell membrane.

Final Thoughts

Over the past 5 to 10 yr there has been significant rhetoric about the “potential” for manipulating the ruminal microflora to enhance feedstuff utilization and improve the efficiency of animal production. However, compared with other microorganisms, we know relatively little about the genetics of ruminal


microorganisms. Genetic transfer mechanisms have not been identified in most of the predominant ruminal bacteria, and information is limited regarding the genes associated with specific physiological functions. In addition, I would argue that even though some progress has been made, we know relatively little about the physiology of ruminal microorganisms. For example, few enzymes have been purified and biochemically characterized. In the case of the ruminal protozoa, information is even more scarce due to our inability to cultivate them for long periods of time in the laboratory.

Even though a ruminal bacterium may be genetically manipulated to have superior cellulose-digesting ability, what effect will the genetic modifications have on other fundamental physiological processes such as nutrient transport? If nutrients are not transported into the cell, then bacterial growth may not be maximal and the modified organism will have diffi- culty surviving in the competitive rumen ecosystem. Because transport is the first step of nutrient metabolism by a bacterium, it is imperative that we fully understand the mechanisms and regulation of these sometimes complex processes if progress is to be made in manipulating the ruminal fermentation.

Research is also needed to elucidate how predominant ruminal bacteria transport molecules larger than AA, monosaccharides, or disaccharides. Several ruminal bacteria can utilize peptides and(or) water- soluble cellodextrins that result from the extracellular hydrolysis of proteins and cellulose, respectively (Russell, 1985; Chen and Russell, 198913; Chen and Russell, 1990). Peptides, cellodextrins, and pentose oligomers are probably more prevalent during feedstuff digestion in the rumen than AA or simple sugars.

As pointed out by Russell et al. (19901, nutri- tionists and microbiologists have to quit treating the rumen as a “black box” and focus on the details of the fermentation. By understanding something as specific as nutrient transport mechanisms in ruminal bacteria, it may be possible to develop new feed additives that are not antibiotics and(or) ionophores. Knowing the details of soluble carbohydrate transport mechanisms in streptococci isolated from the human mouth has led to the use of fluoride and sugar analogues in products ranging from toothpaste to sugar-free gum to prevent tooth decay. Similar products could be developed for use in ruminants. One example of an alternative feed additive might be the use of malate to stimulate lactate utilization by S. ruminantium in cattle fed high-grain diets.

Implications

Progress has been made in identifying how many ruminal bacteria transport nutrients (soluble carbohydrates, AA, organic acids) from the extracellular

medium across the cell membrane and into the cell. Elucidating the biochemistry and genetics associated with nutrient transport mechanisms as well as how environmental factors commonly found within the rumen affect these mechanisms will lead to a better understanding of ruminal fermentation. Over time this type of information could be used to manipulate the fermentation to maximize feedstuff utilization and improve the profitability of ruminant production systems.

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Nutrient Transport by Ruminal Bacteria: A Review · cies can ferment the same substrates (Hungate, 1966). Furthermore, because polymer degradation is often slow, soluble nutrient