Showing papers in "Fems Microbiology Reviews in 1992"

Methanogenesis from acetate: a comparison of the acetate metabolism in Methanothrix soehngenii and Methanosarcina spp.

Abstract: Acetate is the precursor of approximately two-thirds of the methane produced in anaerobic bioreactors. Only two genera of methanogenic archae are known to use acetate as sole energy source: Methanosarcina and Methanothrix. Methanosarcina appears to be a generalist with a high growth rate, but low affinity for acetate. Methanothrix is a specialist having a high affinity for acetate, but low growth rate. Methanothrix shows a much lower minimum threshold for acetate utilization (7–70 μM) than Methanosarcina (0.2–1.2 mM). This is consistent with the evidence that Methanothrix is found in environments with low acetate concentrations. The acetate degradation by acetotrophic methanogens starts with an activation of acetate to acetyl-coenzyme A. In Methanosarcina spp. this activation is catalysed by an acetate kinase/phosphotransacetylase system at the expense of one ATP. Acetyl-coenzyme A synthetase activates acetate in Methanothrix, with concomitant hydrolysis of one ATP to AMP and PPi. Both enzyme systems have been purified and comparison of the kinetic properties confirmed the hypothesis that low acetate concentrations favour Methanothrix. The gene encoding for acetyl-CoA synthetase of Methanothrix was isolated from a genomic library and actively expressed in Escherichia coli. The deduced amino acid sequence showed homology to proteins with similar function and contained two putative ATP binding sites. The most characteristic and complex enzyme involved in the acetate degradation by acetotrophic methanogens is carbon monoxide dehydrogenase. The enzyme has been purified from both Methanothrix and Methanosarcina, and represents 5–10% of the soluble protein of these microorganisms. CO dehydrogenase is proposed to catalyse both the cleavage of acetyl-CoA in a methyl-, carbonyl- and CoA-moiety, and the oxidation of the carbonyl group to CO2. This multifunctional redox enzyme contains several iron, acid-labile sulfur and nickel atoms. These atoms are arranged into several paramagnetic complexes, which have been studied by EPR spectroscopy. The low spin recovery of the different paramagnetic centers makes statements about structure and functions difficult. There are good spectroscopic and genetic indications that the CO dehydrogenase of Methanothrix contains at least one ferrodoxin-like [4Fe-4S] cluster, which could play a role in the electron transfer of the CO oxidation. Further, in EPR spectra of concentrated samples of CO dehydrogenase from Methanothrix a very unusual signal was observed, which showed great similarity to putative [6Fe-6S] prismane clusters. The final step in the methanogenesis from acetate, the reduction of methyl-coenzyme M, is catalysed by methyl-coenzyme M methylreductase. The enzyme purified from Methanothrix and Methanosarcina showed great homology with the methyl-CoM methylreductase of other methanogenic archae, although the specific activity was rather low (60–125 nmol min−1 mg−1). The reduction of the heterodisulfide between coenzyme M and component B is proposed to be the common site for energy conservation in all methanogens. Acetoclastic methanogens, however, need additional sites of energy conservation to compensate for their high energy input in acetate activation. The oxidation of CO to CO2 could form one possible site. The partially membrane-associated pyrophosphatase of Methanothrix could form another site of energy conservation.

Microbial breakdown of halogenated aromatic pesticides and related compounds

Abstract: Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.

The Entner-Doudoroff pathway: history, physiology and molecular biology

Abstract: The Entner-Doudoroff pathway is now known to be very widely distributed in nature. Biochemical and physiological studies show that the Entner-Doudoroff pathway can operate in a linear and catabolic mode, in a ‘cyclic’ mode, in a modified mode involving non-phosphorylated intermediates, or in alternative modes involving C1 metabolism and anabolism. Molecular and genetic analyses of the Entner-Doudoroff pathway in Zymomonas mobilis, Escherichia coli and Pseudomonas aeruginosa have led to an improved understanding of some fundamental aspects of metabolic controls. It can be argued that the Entner-Doudoroff pathway is more primitive than Embden-Meyerhof-Parnas glycolysis.

Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria

Abstract: The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus, purple non-sulfur bacteria, such as Rhodospirillum rubrum, purple sulfur bacteria, such as Chromatium vinosum, pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens, and for the Gram-positive bacterium Rhodococcus ruber. Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.

Structural and functional relationships among the RTX toxin determinants of gram-negative bacteria.

Abstract: The RTX (repeats in toxin) cytolytic toxins r represent a family of important virulence factors that have disseminated widely among Gram-negative bacteria. They are characterised by a series of glycine-rich repeat units at the C-terminal end of each protein. They also have other features in common. Secretion from the cell occurs without a periplasmic intermediate by a novel mechanism which involves recognition of a signal sequence at the C-terminus of the toxin by membrane-associated proteins that export the toxin directly to the outside of the cell. The structural gene for each protein encodes an inactive toxin which is modified post-translationally to an active cytotoxic form by another gene product before secretion. The genes for toxin synthesis, activation and secretion are for the most part grouped together on the chromosome and form an operon. The toxins all create pores in the cell membrane of target cells leading to eventual cell lysis and they appear to require Ca2+ for cytotoxic activity. Although the toxins have a similar mode of action, they vary in target cell specificity. Some are cytotoxic for a wide variety of eukaryotic cell types while others exhibit precise target cell specificity and are only active against leukocytes from certain host species. The characteristic glycine-rich repeat units have been identified in other exoproteins besides those with cytotoxic activity and it is likely that the novel secretory mechanism has been harnessed by a variety of pathogens to release important virulence-associated factors from the cell or to locate them on the cell surface.

Gene expression in Lactococcus lactis

Abstract: Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis. A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis. This feature allowed the expression of a number of L. lactis-derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.

Biodegradation of polyhydroxyalkanoates

Abstract: Degradation of poly(3-hydroxybutyrate) and copolymers with 3-hydroxyvaleric acid was investigated in natural environments, and the microorganisms involved were isolated and identified. The influence of abiotic and biotic factors on the degradation is discussed.

Protein secretion in Pseudomonas aeruginosa

Abstract: The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to habe disseminate widely among Gram-negative bacteria.

On the relative importance of specific and non-specific approaches to oral microbial adhesion

Abstract: In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physico-chemical forces are responsible for so-called ‘non-specific’ and ‘specific’ binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereo-chemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucanbinding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods. during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.

Intestinal, segmented, filamentous bacteria.

Abstract: Segmented, filamentous bacteria (SFBs) are autochthonous, apathogenic bacteria, occuring in the ileum of mice and rats. Although the application of formal taxonomic criteria is imposible due to the lack of an in vitro technique to culture SFBs, microbes with a similar morphology, found in the intestine of a wide range of vertebrate and invertebrate host species, are considered to be related. SFBs are firmly attached to the epithelial cells of the distal ileal mucosa, their preferential ecological niche being the epithelium covering the Peyer's patches. Electron microscopic studies have demonstrated a considerable morphological diversity of SFBs, which may relate to different stages of a life cycle. Determinants of SFB colonization in vivo are host species, genotypical and phenotypical characteristics of the host, diet composition, environmental stress and antimicrobial drugs. SFBs can survive in vitro incubation, but do not multiply. On the basis of their apathogenic character and intimate relationship with the host, it is suggested that SFBs contribute to development and/or maintenance of host resistance to enteropathogens.

Polyhydroxyalkanoate production in recombinant Escherichia coli.

Abstract: The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-beta-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-3HV).

Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi

Abstract: The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-β-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-β-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes). All fungal strains tested were able to grow on all three media. Growth, activity and clarity of PHA-degradation, however, differed depending on the medium used. For screening of BIOPOL degradation the mineral medium with 0.1% BIOPAL, 0.01% yeast extract and 0.01% peptone proved most satisfactory and showed about 55% of the fungal strains to be active, regardless of systematic and ecological groups.

Biological complexes of poly-β-hydroxybutyrate

Abstract: Short-chain complexed poly-β-hydroxybutyrate, 130–170 monomer units, is a ubiquitous constituent of cells, wherein it is usually associated with other macromolecules by multiple coordinate bonds, or by hydrogen bonding and hydrophobic interactions. This conserved PHB has been isolated from the plasma membranes of bacteria, from a variety of plant tissues, and from the plasma membranes, mitochondria, and microsomes of animal cells. In bacterial membranes, PHB has been found complexed to the calcium salts of inorganic polyphosphates, and to single-stranded DNAs. The ability of PHB to solvate salts, consisting of cations having high solvation energies and large delocalized anions, is in accordance with its molecular characteristics, that of a flexible linear molecule possessing a large number of electron-donating ester oxygens suitably spaced to replace the hydration shell of cations. In turn, PHB may be rendered soluble in aqueous media by complexation to water-soluble proteins, such as serum lipoproteins and albumin. Such solvates are highly resistant to hydrolytic enzymes. These findings suggest that the physiological roles of this unique biopolymer may include the solvation of salts of polymeric anions to facilitate their movement through hydrophobic barriers, and the protection of cellular polymers from enzymatic degradation.

Microbial degradation of natural and of new synthetic polymers

Abstract: In landfills, deposited waste material is usually faced with strictly anoxic conditions. This means that the design of new biodegradable polymers must take into consideration that degradation should be possible especially in the absence of molecular oxygen. Poly-β-hydroxybutyrate is depolymerized by the anaerobic fermenting bacterium Ilyobacter delafieldii through an extracellular hydrolase. Monomers are degraded inside the cells through classical β-oxidation. Polyalkanoates containing odd-numbered or branched-chain acid monomers should he degraded in an analogous manner; in most cases the final mineralization of these residues requires special pathways. A comparison of the chemistry of natural polymer biodegration leads to the conclusion that synthetic biodegradable polymers should be designed in the future to contain linkages which can be cleaved by extracellular hydrolytic enzymes. Recent findings on aerobic and anaerobic bacterial degradation of synthetic polyethers suggest that natural evolution of new depolymerizing enzymes, perhaps from existing hydrolases, could be possible in a reasonable amount of time, provided that the monomers are likely energy sources for a broad variety of microbes.

Biodegradability of polymers in the environment: complexities and significance of definitions and measurements

Abstract: There is a world-wide research effort to develop biodegradable polymers as a waste-management option for polymers in the environment. This effort may prove to be fruitless unless we can agree on a definition and test protocols—what do we expect biodegradable polymers to do in the environment and how do we demonstrate that they do what we expect? Establishing a definition and test protocols is not trivial; the task is made complex by the wide range of disciplines involved directly in or interested in the subject, including polymer scientist, biochemists, environmentalists, legislators, and laypeople, all with their own perspectives and expectations. In this paper, I present arguments in favor of biodegradable polymers and indicate what remains to be done to satisfy detractors that they represent a viable option for polymer waste-management.

Can the excretion of metabolites by bacteria be manipulated

Abstract: Bacteria can release metabolites into the environment by various mechanisms. Excretion may occur by passive diffusion or by the reversal of the uptake process when the internal concentration of the metabolite exceeds the thermodynamic equilibrium level. In other cases, solutes are excreted against the concentration gradient by special extrusion systems. Their mode of energy coupling is different to that of the well-studied group of uptake systems. A thorough understanding of the transport processes will help to improve the excretion of metabolites of commercial interest, allow a more efficient production of metabolites in bulk quantities, and permit their exploitation to establish new markets.

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Abstract: Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.

Poly-β-hydroxybutyrate in staphylococci

Abstract: Staphylococci—chemoorganotrophic bacteria whose main habitats are human and animal organisms—can accumulate poly-β-hydroxybutyrate (PHB) in their cells. The polymer is metabolized in endogenous turnovers preceding degradation of aminoacids, proteins and RNA. PHB depolymerase was not found in staphylococci but β-hydroxybutyrate dehydrogenase was estimated, purified and characterized.