Psychrotrophic bacterial flora of raw ewes' milk, with particular reference to gram negative rods

The microbial flora of 141 samples of raw ewes' milk was determined, before and after storage for 72 h at 4 degrees and 7 degrees C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated (ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.     

Fermentative degradation of glyoxylate by a new strictly anaerobic bacterium

A new strictly anaerobic, gram-negative, nonsporeforming bacterium, Strain PerGlx1, was enriched and isolated from marine sediment samples with glyoxylate as sole carbon and energy source. The guanineplus-cytosine content of the DNA was 44.1±0.2 mol %. Glyoxylate was utilized as the only substrate and was stoichiometrically degraded to carbon dioxide, hydrogen, and glycolate. An acetyl-CoA and ADP-dependent glyoxylate converting enzyme activity, malic enzyme, and pyruvate synthase were found at activities sufficient for growth (0.25 U x mg protein^-1). These findings allow to design a new degradation pathway for glyoxylate: glyoxylate is condensed with acetyl-CoA to form malyl-CoA; the free energy of the thioester linkage in malyl-CoA is conserved by substrate level phosphorylation. Part of the electrons released during glyoxylate oxidation to CO₂ reduce a small fraction of glyoxylate to glycolate.     

The lactoferrin receptor complex in gram negative bacteria

Bacteria that inhabit the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment on the mucosal surface where iron is complexed by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic anti-microbial peptide lactoferricin is produced by proteolysis of lactoferrin. Several members of the Neisseriaceae and Moraxellaceae families express surface receptors, capable of specifically binding host lactoferrin and extracting the iron from lactoferrin as a source of iron for growth. The receptor is comprised of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a largely exposed surface lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for mediating growth using lactoferrin as a sole iron source whereas LbpB only plays a facilitating role. LbpB, with the presence of a large tract of negatively charged residues, appears to protect the bacterial cell from the bactericidal effects of the lactoferricin. The lactoferrin receptors in these species appear to be essential for survival and thus may serve as potential vaccine targets.     

Patterns of LPS synthesis in gram negative bacteria

Lipopolysaccharide (LPS), a lipid based carbohydrate polymer, is found in the outer membrane of Gram negative bacteria where it plays a vital role in its structure and function. It is chiefly responsible for the toxic effects of the bacterial diseases caused by these organisms and plays a role in the organisms defense against host immune attack. In recent experiments using high resolution gel techniques Goldman & Leive (1980) have revealed an unexpected heterogeneity in the distribution of polymer lengths found in the membrane with lengths ranging from 0 (lipid-A-core) to nearly 40 sugar units. Monomer units are an essential element of the synthesis process. Working with mutant strains of E. coli and Salmonella typhimurium, they have also shown that these bacteria will continue to synthesize LPS molecules with very long chains even though monomer unit production is severely reduced. The steps involved in the synthesis of LPS are known and in this paper it is shown that the results of Goldman & Leive cannot be obtained assuming the synthesis process is length independent as has been suggested. Moreover, the paradoxical persistence of long chains in spite of monomer suppression has a simple explanation once length dependence is assumed. These conclusions result from the analysis of a Markov chain model of synthesis.     

Production of an extracellular emulsifier by a gram negative bacterium

Summary Gram negative bacterial isolated PCB-106 produced significant amounts of bioemulsifiers in the late log phase of growth during the shake cultivation in minimal medium with ethanol (115 units/ml). About 3 fold higher emulsification activity was achieved by manipulating the medium components. The emulsifier was found to be more effective towards the mixture of aliphatic and aromatic than individual hydrocarbons.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

Clostridium viride sp. nov., a strictly anaerobic bacterium using 5-aminovalerate as growth substrate,previously assigned to Clostridium aminovalericum

Strain T2–7, a 5-aminovalerate-fermenting bacterium previously classified as Clostridium aminovalericum, was further characterized, both physiologically and phylogenetically. Comparative sequencing analysis of the almost complete 16S rDNA revealed that strain T2–7 forms a distinct lineage within a phylogenetically coherent cluster of gram-positive bacteria currently assigned to the genus Clostridium. Strain T2–7 grew with 5-aminovalerate, 5-hydroxyvalerate, 4-hydroxybutyrate, vinylacetate, and crotonate, and required yeast extract and l-cysteine for growth. Other substrates were not utilized. The fermentation products, depending on the growth substrate, were ammonia, acetate, propionate, butyrate, and valerate. Sulphur was reduced by a mechanism not linked to energy conservation. Other acceptors were not utilized. Cells were gram-positive pointed-ended ovals, motile by means of two subpolar flagella, and possessed a gram-positive cell wall structure with an S-layer of hexagonally arranged subunits of 18.5 nm diameter. The DNA mol% G+C was 41.5. Strain T2–7 (DSM 6836) is proposed as the type strain of a new species, Clostridium viride sp. nov. Dedicated to H. A. Barker on the occasion of his 87th birthday     

A study of the inhibition of an amidase with a wide substrate spectrum and its consequences for the bioconversion of nitriles

Summary TheBrevibacterium sp. R 312 strain possesses a nitrile-hydratase and an amidase, both with a wide substrate spectrum. These two enzymes can be used for the bioconversion of nitriles into the corresponding organic acids: the actions of three types of compounds (nitriles, amides and acids) on the activity of the amidase are reported in the present work.