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Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.

Background

Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed.

Results

C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms.

Conclusions

Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.

A monensin-sensitive ruminal peptostreptococcus was able to grow rapidly (growth rate of 0.5/h) on an enzymatic hydrolysate of casein, but less than 23% of the amino acid nitrogen was ever utilized. When an acid hydrolysate was substituted for the enzymatic digest, more than 31% of the nitrogen was converted to ammonia and cell protein. Coculture experiments and synergisms with peptide-degrading strains of Bacteroides ruminicola and Streptococcus bovis indicated that the peptostreptococcus was unable to transport certain peptides or hydrolyze them extracellularly. Leucine, serine, phenylalanine, threonine, and glutamine were deaminated at rates of 349, 258, 102, 95, and 91 nmol/mg of protein per min, respectively. Deamination rates for some other amino acids were increased when the amino acids were provided as pairs of oxidized and reduced amino acids (Stickland reactions), but these rates were still less than 80 nmol/mg of protein per min. In continuous culture (dilution rate of 0.1/h), bacterial dry matter and ammonia production decreased dramatically at a pH of less than 6.0. When dilution rates were increased from 0.08 to 0.32/h (pH 7.0), ammonia production increased while production of bacterial dry matter and protein decreased. These rather peculiar kinetics resulted in a slightly negative estimate of maintenance energy and could not be explained by a change in fermentation products. Approximately 80% of the cell dry matter was protein. When corrections were made for cell composition, the yield of ATP was higher than the theoretical maximum value. It is possible that mechanisms other than substrate-level phosphorylation contributed to the energetics of growth.

Day, Lawrence E., (Michigan State University, East Lansing) and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. II. Maturation of forespores. J. Bacteriol. 88:695–701. 1964.—Clostridium botulinum, strain 62-A, did not sporulate endotrophically, but forespores matured to refractile, heat-resistant spores when replaced in solutions containing l-alanine and l-proline, l-isoleucine and l-proline, or l-alanine and l-arginine. Solutions of l-arginine or l-citrulline would not support the maturation process. Acetate, CO2, and δ-amino valeric acid were produced during sporulation in a replacement solution of l-alanine and l-proline, indicating the operation of the Stickland reaction. There was no large uptake of either exogenous l-alanine or acetate during this process. Similarly, there was no apparent protein or nucleic acid synthesis, since high levels of chloramphenicol, 8-azaguanine, or mitomycin C failed to inhibit, and no significant amount of P32 was incorporated into the spore nucleic acids. Dipicolinic acid (DPA) was synthesized during forespore maturation. It is believed that these final steps in sporulation of C. botulinum require only an exogenous source of energy which can be obtained via the Stickland reaction system, and that the synthesis of DPA and other unknown materials relies primarily on endogenous substrates.

Dental plaque samples from monkeys (Macaca fascicularis) were shown to contain proline reduction activity in coupled Stickland reactions with other amino acids and also with certain end products of bacterial glucose metabolism. The unusually high concentration of bound and free proline in the oral environment may be of importance in both the production of base and in the removal of acid from the tooth surface after dietary carbohydrate ingestion.

Growing and nongrowing cells of Clostridium sporogenes fermented betaine with l-alanine, l-valine, l-leucine, and l-isoleucine as electron donors in a coupled oxidation-reduction reaction (Stickland reaction). For the substrate combinations betaine and l-alanine and betaine and l-valine balance studies were performed; the results were in agreement with the following fermentation equation: 1 R- CH(NH2)-COOH + 2 betaine + 2 H2O → 1 R-COOH + 1 CO2 + 1 NH3 + 2 trimethylamine + 2 acetate. Growth and production of trimethylamine were strictly dependent on the presence of selenite in the medium. With cell suspensions it was shown that C. sporogenes was unable to catabolize betaine as a single substrate. Betaine, however, was reduced to trimethylamine and acetate under an atmosphere of molecular hydrogen. For the reduction of betaine by cell extracts of C. sporogenes, dimercaptans such as 1,4-dithiothreitol could serve as electron donors. No betaine reductase activity was detected in cells grown in a complex medium without betaine. The pH optimum of betaine reductase was at pH 7.3. When C. sporogenes was cocultured with Methanosarcina barkeri strain Fusaro on betaine together with l-alanine, an almost complete conversion of the two substrates to CH4, NH3, and presumably CO2 was observed.

Soil fungi that attacked methionine required a utilizable source of energy such as glucose for growth. This is an example of co-dissimilation. Experiments with one of the fungi, representative of the group, are reported. In the absence of glucose, pregrown mycelium, even when depleted of energy reserves, oxidatively deaminated methionine with accumulation of α-keto-γ-methyl mercapto butyric acid and α-hydroxy-γ-methyl mercapto butyric acid. When glucose was provided, all of the sulfur of methionine was released as methanethiol, part of which was oxidized to dimethyl disulfide. No sulfate, sulfide, or hydrosulfide products were detected. Evidence was obtained that deaminase and demethiolase were constitutive. Deamination preceded demethiolation and α-keto butyric acid accumulated as a product of the two reactions. Other carbon residues were α-hydroxy butyric acid and α-amino butyric acid. Inability of the fungus to metabolize α-keto butyrate was responsible for its inability to utilize methionine as a source of carbon and energy. Several other fungi isolated from soil grew on α-amino butyrate but could not grow on methionine owing to inability to demethiolate it.

The oxidative decarboxylation of amino acids by a system consisting of myeloperoxidase-hydrogen peroxide-chloride has been demonstrated previously by others and the process has been considered to be part of the microbicidal armamentarium of some phagocytic leukocytes. We were able to translate these earlier observations, made on model systems, to intact guinea pig granulocytes. We could demonstrate differences in the cellular handling of peptide-linked amino acids as particles, compared with free amino acids. Specific inhibitors were used to explore two routes of oxidative decarboxylation: (a) the myeloperoxidase-catalyzed direct decarboxylation-deamination reaction, and (b) oxidation of alpha-keto acids after transamination of amino acids. These inhibitors were cyanide, azide, and tapazole for the former pathway, and amino-oxyacetate for the latter. Amino-oxyacetate profoundly inhibited the decarboxylation of free 14C-amino acids (alanine and aspartate) in both resting and stimulated cells, but had only a minimal effect on 14CO2 production from ingested insoluble 14C-protein. On the other hand, the peroxidase inhibitors cyanide, azide, and tapazole dramatically inhibited the production of 14CO2 from ingested particulate 14C-protein, but had only small effects on the decarboxylation of free amino acid. Soluble, uniformly labeled 14C-protein was not significantly converted to 14CO2 even in the presence of phagocytizable polystyrene beads. These observation suggest that the amino acids taken up by phagocytosis (e.g., as denatured protein particles) are oxidatively decarboxylated and deaminated in the phagosomes by the myeloperoxidase-hydrogen peroxide-chloride system; soluble free amino acids that enter the cytoplasm by diffusion or transport are oxidatively decarboxylated after transamination by the normal cellular amino acid oxidative pathway.

Background

The genetic code is brought into action by 20 aminoacyl-tRNA synthetases. These enzymes are evenly divided into two classes (I and II) that recognize tRNAs from the minor and major groove sides of the acceptor stem, respectively. We have reported recently that: (1) ribozymic precursors of the synthetases seem to have used the same two sterically mirror modes of tRNA recognition, (2) having these two modes might have helped in preventing erroneous aminoacylation of ancestral tRNAs with complementary anticodons, yet (3) the risk of confusion for the presumably earliest pairs of complementarily encoded amino acids had little to do with anticodons. Accordingly, in this communication we focus on the acceptor stem.

Results

Our main result is the emergence of a palindrome structure for the acceptor stem's common ancestor, reconstructed from the phylogenetic trees of Bacteria, Archaea and Eukarya. In parallel, for pairs of ancestral tRNAs with complementary anticodons, we present updated evidence of concerted complementarity of the second bases in the acceptor stems. These two results suggest that the first pairs of "complementary" amino acids that were engaged in primordial coding, such as Gly and Ala, could have avoided erroneous aminoacylation if and only if the acceptor stems of their adaptors were recognized from the same, major groove, side. The class II protein synthetases then inherited this "primary preference" from isofunctional ribozymes.

Conclusion

Taken together, our results support the hypothesis that the genetic code per se (the one associated with the anticodons) and the operational code of aminoacylation (associated with the acceptor) diverged from a common ancestor that probably began developing before translation. The primordial advantage of linking some amino acids (most likely glycine and alanine) to the ancestral acceptor stem may have been selective retention in a protocell surrounded by a leaky membrane for use in nucleotide and coenzyme synthesis. Such acceptor stems (as cofactors) thus transferred amino acids as groups for biosynthesis. Later, with the advent of an anticodon loop, some amino acids (such as aspartic acid, histidine, arginine) assumed a catalytic role while bound to such extended adaptors, in line with the original coding coenzyme handle (CCH) hypothesis.

Reviewers

This article was reviewed by Rob Knight, Juergen Brosius and Anthony Poole.

Ruminal amino acid degradation is a nutritionally wasteful process that produces excess ruminal ammonia. Monensin inhibited the growth of monensin-sensitive, obligate amino acid-fermenting bacteria and decreased the ruminal ammonia concentrations of cattle. 16S rRNA probes indicated that monensin inhibited the growth of Peptostreptococcus anaerobius and Clostridium sticklandii in the rumen. Clostridium aminophilum was monensin sensitive in vitro, but C. aminophilum persisted in the rumen after monensin was added to the diet. An in vitro culture system was developed to assess the competition of C. aminophilum, P. anaerobius, and C. sticklandii with predominant ruminal bacteria (PRB). PRB were isolated from a 10(8) dilution of ruminal fluid and maintained as a mixed population with a mixture of carbohydrates. PRB did not hybridize with the probes to C. aminophilum, P. anaerobius, or C. sticklandii. PRB deaminated Trypticase in continuous culture, but the addition of C. aminophilum, P. anaerobius, and C. sticklandii caused a more-than-twofold increase in the steady-state concentration of ammonia. C. aminophilum, P. anaerobius, and C. sticklandii accounted for less than 5% of the total 16S rRNA and microbial protein. Monensin eliminated P. anaerobius and C. sticklandii from continuous cultures, but it could not inhibit C. aminophilum. The monensin resistance of C. aminophilum was a growth rate-dependent, inoculum size-independent phenomenon that could not be maintained in batch culture. On the basis of these results, we concluded that the feed additive monensin cannot entirely counteract the wasteful amino acid deamination of obligate amino acid-fermenting ruminal bacteria.

Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum to obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.

Clostridium difficile is a nosocomial pathogen whose incidence and importance are on the rise. Previous work in our laboratory characterized the central role of selenoenzyme dependent Stickland reactions in C. difficile metabolism. In this work we have identified, using mass spectrometry, a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. X-ray absorption spectroscopy supports the structure that we proposed based on mass spectrometric data. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. This study demonstrates that targeting selenium metabolism provides a new avenue for antimicrobial development against C. difficile and other selenium-dependent pathogens.

The metabolic pathways utilized by an obligately anaerobic marine spirochete (strain MA-2) to ferment branched-chain amino acids were studied. The spirochete catabolized l-leucine to isovaleric acid, l-isoleucine to 2-methylbutyric acid, and l-valine to isobutyric acid, with accompanying CO2 production in each fermentation. Cell extracts of spirochete MA-2 converted l-leucine, l-isoleucine, and l-valine to 2-ketoisocaproic, 2-keto-3-methylvaleric, and 2-ketoisovaleric acids, respectively, through mediation of 2-ketoglutarate-dependent aminotransferase activities. The branched-chain keto acids were decarboxylated and oxidized to form isovaleryl coenzyme A, 2-methylbutyryl coenzyme A, and isobutyryl coenzyme A, respectively, in the presence of sulfhydryl coenzyme A and benzyl viologen. The acyl coenzyme A's were converted to acyl phosphates by phosphate branched-chain acyltransferase enzymatic activities. Branched-chain fatty acid kinase activities catalyzed formation of isovaleric, 2-methylbutyric, and isobutyric acids from isovaleryl phosphate, 2-methylbutyryl phosphate, and isobutyryl phosphate, respectively. Adenosine 5′-triphosphate was formed during conversion of branched-chain acyl phosphates to branched-chain fatty acids. The results indicate that conversion of l-leucine, l-isoleucine, and l-valine to branched-chain fatty acids by spirochete MA-2 results in adenosine 5′-triphosphate generation. The metabolic pathways utilized for this conversion involve amino acid amino-transferase, 2-keto acid oxidoreductase, phosphate acyltransferase, and fatty acid kinase activities.

Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/α-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

Escherichia coli tRNA Arg was treated with sodium bisulfite to convert exposed cytosine residues to uracil. This treatment resulted in the loss of amino acid acceptance of the tRNA Arg with pseudo first-order reaction kinetics. The active and inactive molecules were separated after about 60e active and inactive molecules were separated after about 60 percent inactivation and analyzed for U in various positions by finger-printing of the oligonucleotides produced by nucleases. The results show that C to U base transitions in the dihydrouridine loop and in the CCA terminus have no effect on the aminoacylation of this tRNA. Deamination of a cytosine residue at the second position of the anticodon resulted in the loss of amino acid acceptor activity of arginine transfer RNA.

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When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.

Aminoacyl tRNA synthetases are ancient proteins that interpret the genetic material in all life forms. They are thought to have appeared during the transition from the RNA world to the theatre of proteins. During translation, they establish the rules of the genetic code, whereby each amino acid is attached to a tRNA that is cognate to the amino acid. Mistranslation occurs when an amino acid is attached to the wrong tRNA and subsequently is misplaced in a nascent protein. Mistranslation can be toxic to bacteria and mammalian cells, and can lead to heritable mutations. The great challenge for nature appears to be serine-for-alanine mistranslation, where even small amounts of this mistranslation cause severe neuropathologies in the mouse. To minimize serine-for-alanine mistranslation, powerful selective pressures developed to prevent mistranslation through a special editing activity imbedded within alanyl-tRNA synthetases (AlaRSs). However, serine-for-alanine mistranslation is so challenging that a separate, genome-encoded fragment of the editing domain of AlaRS is distributed throughout the Tree of Life to redundantly prevent serine-to-alanine mistranslation. Detailed X-ray structural and functional analysis shed light on why serine-for-alanine mistranslation is a universal problem, and on the selective pressures that engendered the appearance of AlaXps at the base of the Tree of Life.

Nitrogen-starved yeast derepress a general amino acid permease which transports basic and hydrophobic amino acids. Although both groups of amino acids are metabolized, the derivatives of the basic amino acids are retained by the cells, whereas those of the hydrophobic amino acids are released as acidic and neutral deaminated derivatives. The release of the deaminated derivatives of the hydrophobic amino acids only occurs in the presence of glucose, which presumably produces amino acceptors. The accumulation of intracellular amino acids results in trans-inhibition of the uptake of exogenous amino acids whether the intracellular amino acid is a basic amino acid or the product of intracellular transamination from a hydrophobic amino acid. Variation of permease and transaminase activity was measured during growth under repressed (ammonia-grown) and derepressed (proline-grown) conditions. Maximum levels for both activities occurs at the mid-exponential phase.

Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation. Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction. Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids. Propionibacterium freudenreichii, Lactococcus lactis, and Enterococcus cecorum all shifted their fermentation patterns towards more oxidized products when humic acids were present; P. freudenreichii even oxidized propionate to acetate under these conditions. When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts. These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction. This information needs to be considered in future studies of electron flow in soils and sediments.

The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.

Clostridium sporogenes was found to have an absolute requirement for selenium to utilize glycine but not proline as oxidant in Stickland-type fermentations. No glycine reductase activity was detectable in cells from media without added selenium. The data indicate that this organism could be used for microbiological assays for very low levels of selenium in certain forms.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family Enterobacteriaceae. The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.

Hydrogen gas (H2) produced by bacterial fermentation of biomass can be a sustainable energy source. The ability to produce H2 gas during anaerobic fermentation was previously thought to be restricted to a few species within the genera Clostridium and Enterobacter. This work reports genomic evidence for the presence of novel H2-producing bacteria (HPB) in acidophilic ethanol-H2-coproducing communities that were enriched using molasses wastewater. The majority of the enriched dominant populations in the acidophilic ethanol-H2-coproducing system were affiliated with low-G+C-content gram-positive bacteria, Bacteroidetes, and Actinobacteria, based on the 16S rRNA gene. However, PCR primers designed to specifically target bacterial hydA yielded 17 unique hydA sequences whose amino acid sequences differed from those of known HPB. The putative ethanol-H2-coproducing bacteria comprised 11 novel phylotypes closely related to Ethanoligenens harbinense, Clostridium thermocellum, and Clostridium saccharoperbutylacetonicum. Furthermore, analysis of the alcohol dehydrogenase isoenzyme also pointed to an E. harbinense-like organism, which is known to have a high conversion rate of carbohydrate to H2 and ethanol. We also found six novel HPB that were associated with lactate-, propionate-, and butyrate-oxidizing bacteria in the acidophilic H2-producing sludge. Thus, the microbial ecology of mesophilic and acidophilic H2 fermentation involves many other bacteria in addition to Clostridium and Enterobacter.