- Volume 148, Issue 6, 2002

Of 830 Neisseria meningitidis isolates obtained from healthy carriers in Bavaria, Germany, 136 (16·4%) lacked the operons necessary for the synthesis, lipid modification, and transport of capsular polysaccharide. These operons were replaced by a non-coding intergenic region either 113 or 114 bp in length, termed here the capsule null locus (cnl). Comparisons of the nucleotide sequence of this region in the meningococcus and its acapsulate relatives, Neisseria gonorrhoeae and Neisseria lactamica, revealed six distinct sequence variants (cnl-1 to cnl-6), with a total of 10 nucleotide substitutions and three indels. With the exception of one 4 bp insertion, which was unique to a gonococcal isolate, all of the individual sequence changes were present in the N. lactamica isolates examined. The meningococcal isolates with a cnl belonged to one of four otherwise genetically diverse genetic groupings: the ST-53 and ST-1117 complexes (75 isolates); the ST-845 complex (12 isolates); the ST-198 and 1136 complexes (46 isolates), and the ST-44 complex (one isolate). These data demonstrated that a substantial proportion of carried meningococci were incapable of capsule production, that the cnl circulated within Neisseria populations by horizontal genetic exchange, and that the expression of a polysaccharide capsule was not a requirement for person-to-person transmission of certain meningococcal lineages.

The Escherichia coli RecA protein is one of the best-studied enzymes, but less is understood about how RecA homologues of other species are similar to or different from the E. coli RecA. In the Gram-negative pathogen Neisseria gonorrhoeae (the gonococcus; Gc), the causative agent of gonorrhoea, RecA is involved in DNA transformation, pilin antigenic variation, and DNA repair. By expressing the recA genes from Gc and E. coli under control of lac regulatory sequences in E. coli, the authors have shown that the Gc RecA fully complements an E. coli recA mutant for homologous recombination, but only partially complements for survival to DNA damage. By expressing similar constructs in Gc, it was shown that the E. coli RecA complements for pilin antigenic variation, partially complements for DNA transformation, but does not complement for survival to DNA damage, suggesting that species-specific interactions are important for DNA repair, but not for homologous recombination. Co-expression of the E. coli recA and recX genes in Gc suggests that in this heterologous system RecX modulates RecA-mediated processes.

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Nine lgt genes at three chromosomal loci (lgt-1, 2, 3) encoding the glycosyltransferases responsible for the biosynthesis of LOS oligosaccharide chains were examined in 26 Neisseria meningitidis, 51 Neisseria gonorrhoeae and 18 commensal Neisseria strains. DNA hybridization, PCR and nucleotide sequence data were compared to previously reported lgt genes. Analysis of the genetic organization of the lgt loci revealed that in N. meningitidis, the lgt-1 and lgt-3 loci were hypervariable genomic regions, whereas the lgt-2 locus was conserved. In N. gonorrhoeae, no variability in the composition or organization of the three lgt loci was observed. lgt genes were detected only in some commensal Neisseria species. The genetic organization of the lgt-1 locus was classified into eight types and the lgt-3 locus was classified into four types. Two types of arrangement at lgt-1 (II and IV) and one type of arrangement at lgt-3 (IV) were novel genetic organizations reported in this study. Based on the three lgt loci, 10 LOS genotypes of N. meningitidis were distinguished. Phylogenetic analysis revealed a gene cluster, lgtH, which separated from the homologous genes lgtB and lgtE. The lgtH and lgtE genes were mutually exclusive and were located at the same position in lgt-1. The data demonstrated that pathogenic and commensal Neisseria share a common lgt gene pool and horizontal gene transfer appears to contribute to the genetic diversity of the lgt loci in Neisseria.

A search for genes encoding enzymes involved in cobalamin (vitamin B12) production in the commercially important organism Propionibacterium freudenreichii (P. shermanii) has resulted in the isolation of an additional 14 genes encoding enzymes responsible for 17 steps of the anaerobic B12 pathway in this organism. All of the genes believed to be necessary for the biosynthesis of adenosylcobinamide from uroporphyrinogen III have now been isolated except two (cbiA and an as yet unidentified gene encoding cobalt reductase). Most of the genes are contained in two divergent operons, one of which, in turn, is closely linked to the operon encoding the B12-dependent enzyme methylmalonyl-CoA mutase. The close linkage of the three genes encoding the subunits of transcarboxylase to the hemYHBXRL gene cluster is reported. The functions of the P. freudenreichii B12 pathway genes are discussed, and a mechanism for the regulation of cobalamin and propionic acid production by oxygen in this organism is proposed.

Two human faeces carriage isolates of Listeria monocytogenes (H1 and H2) were compared to reference strains (ScottA and LO28) with regard to their lethality in 14-day-old chick embryos, their haemolytic and phospholipase (phosphatidylcholine-phospholipase C and phosphatidylinositol-phospholipase C) activities and their invasiveness towards Caco-2 cells. Experimental infection of chick embryos allowed discrimination of the strains into those exhibiting high virulence (ScottA and H2), those exhibiting slightly attenuated virulence (LO28) and those exhibiting low virulence (H1). A similar percentage mortality and time to death for embryos was observed when they were infected with H2 as was seen with infection by the reference strain ScottA. Therefore, human carriage strain H2 was considered potentially pathogenic. In contrast to H2 and ScottA, H1 exhibited low virulence. Using the tissue-culture cell-line model, it was found that carriage strain H1 was unable to enter Caco-2 cells efficiently, even though it was similar to the virulent strains in terms of the enzymic activities involved in pathogenicity. Detection of the internalins InlA and InlB, involved in the internalization of L. monocytogenes in the host cells, by immunoblot indicated that a truncated form of InlA was produced by H1. Taken together, these data provide a starting point for the study of the behaviour of two types of human faeces carriage strains and their characterization.

An important step in infections associated with biliary drains is adhesion of micro-organisms to the surface. In this study the role of three surface proteins of Enterococcus faecalis (enterococcal surface protein, aggregation substances 1 and 373) in the adhesion to silicone rubber, fluoro-ethylene-propylene and polyethylene was examined. Four isogenic E. faecalis strains with and without aggregation substances and one strain expressing enterococcal surface protein were used. The kinetics of enterococcal adhesion to the materials was measured in situ in a parallel plate flow chamber. Initial deposition rates were similar for all strains, whereas the presence of surface proteins increased the total number of adhering bacteria. Nearest neighbour analysis demonstrated that enterococci expressing the whole sex-pheromone plasmid encoding aggregation substances 1 or 373 adhered in higher numbers through mechanisms of positive cooperativity, which means that adhesion of bacteria enhances the probability of adhesion of other bacteria near these bacteria. Enterococci with the enterococcal surface protein did not adhere through this mechanism. These findings indicate that the surface proteins of E. faecalis play a key role in the adhesion to bile drains and bile drain associated infections.

This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previously shown to be underproduced by a spontaneous adhesion-defective mutant D5 of Ruminococcus albus 20. An antiserum against wild-type strain 20 was adsorbed with the mutant D5 to enrich it in antibodies ‘specific’ to adhesion structures of R. albus 20. The resulting antiserum, called anti-Adh serum, blocked adhesion of R. albus 20 and reacted mainly with GP25 in bacterial and extracellular protein fractions of R. albus 20. The N-terminal sequence of purified GP25 was identical to that of CbpC, a 21 kDa cellulose-binding protein (CBP) of R. albus 8. The nucleotide sequence of the gp25 gene was determined by PCR and genomic walking procedures. The gp25 gene encoded a protein of 165 aa with a calculated molecular mass of 16940 Da that showed 72·9% identity with CbpC and presented homologies with type IV pilins of Gram-negative pathogenic bacteria. Negative-staining electron microscopy revealed fine and flexible pili surrounding R. albus 20 cells while mutant cells were not piliated. In addition, immunoelectron microscopy showed that the anti-Adh serum probing mainly GP25, completely decorated the pili surrounding R. albus 20, thereby showing that GP25 was a major pilus subunit. This study shows for the first time the presence of pili at the surface of R. albus and identifies GP25 as their major protein subunit. Though GP25 was not identified as a CBP, isolated pili were shown to bind cellulose. In conclusion, these pili, which belong to the family of type IV pili, mediate adhesion of R. albus 20 to cellulose.

Mycobacterial α-, methoxy- and keto-mycolic acid methyl esters were separated by argentation chromatography into mycolates with no double bond, with one trans double bond or with one cis double bond. Meromycolic acids were prepared from each methyl mycolate fraction by pyrolysis, followed by silver oxide oxidation, and analysed by high-energy collision-induced dissociation/fast atom bombardment MS to reveal the exact locations of the functional groups within the meromycolate chain. The locations of cis and trans double bonds, cis and trans cyclopropane rings, methoxy and keto groups, and methyl branches within the meromycolate chain were determined from their characteristic fragment ion profiles, and the structures of the meromycolic acids, including those with three functional groups extracted from Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG and Mycobacterium microti, were established. Meromycolic acids with one cis double bond were structurally closely related to those with one cis cyclopropane ring, whereas the meromycolic acids with one trans cyclopropane ring were closely related to the corresponding meromycolic acids with one cis cyclopropane ring. A close relationship between methoxy- and keto-meromycolic acids was also implied. The relationship between the meromycolic acids with a trans double bond and the other meromycolic acids was not clearly revealed, and they did not appear to be immediate substrates for trans cyclopropanation.

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) have been shown to be of major importance in the promotion of cardiovascular health, proper human development and the prevention of some cancers. A high proportion of bacterial isolates from low-temperature and high-pressure marine environments produce EPA or DHA. This paper presents the sequence of a 33 kbp locus from the deep-sea bacterium Photobacterium profundum strain SS9 which includes four of the five genes required for EPA biosynthesis. As with other bacterial pfa (polyunsaturated fatty acid) genes, the deduced amino acid sequences encoded by the SS9 genes reveal large multidomain proteins that are likely to catalyse EPA biosynthesis by a novel polyketide synthesis mechanism. RNase protection experiments separated the SS9 pfa genes into two transcriptional units, pfaA–C and pfaD. The pfaA transcriptional start site was identified. Cultivation at elevated hydrostatic pressure or reduced temperature did not increase pfa gene expression despite the resulting increase in percentage composition of EPA under these conditions. However, a regulatory mutant was characterized which showed both increased expression of pfaA–D and elevated EPA percentage composition. This result suggests that a regulatory factor exists which coordinates pfaA–D transcription. Additional consideration regarding the activities required for PUFA synthesis is provided together with comparative analyses of bacterial pfa genes and gene products.

The ability of methylotrophic α-proteobacteria to grow with dichloromethane (DCM) as source of carbon and energy has long been thought to depend solely on a single cytoplasmic enzyme, DCM dehalogenase, which converts DCM to formaldehyde, a central intermediate of methylotrophic growth. The gene dcmA encoding DCM dehalogenase of Methylobacterium dichloromethanicum DM4 was expressed from a plasmid in closely related Methylobacterium strains lacking this enzyme. The ability to grow with DCM could be conferred upon Methylobacterium chloromethanicum CM4, a chloromethane degrader, but not upon Methylobacterium extorquens AM1. In addition, growth of strain AM1 with methanol was impaired in the presence of DCM. The possibility that single-carbon (C1) utilization pathways in dehalogenating Methylobacterium strains differed from those discovered in strain AM1 was addressed. Homologues of tetrahydrofolate-linked and tetrahydromethanopterin-linked C1 utilization genes of strain AM1 were detected in both strain DM4 and strain CM4, and cloning and sequencing of several of these genes from strain DM4 revealed very high sequence identity (96·5–99·7%) to the corresponding genes of strain AM1. The expression of transcriptional xylE fusions of selected genes of the tetrahydrofolate- and tetrahydromethanopterin-linked pathways from strain DM4 was investigated. The data obtained suggest that the expression levels of some C1 utilization genes in M. dichloromethanicum DM4 grown with DCM may differ from those observed during growth with methanol.

Partial sequences of farnesyl diphosphate (FPP) synthase genes derived from the Rhodobacter–Rhodovulum group and from the Rhodopseudomonas palustris–Bradyrhizobium japonicum group of the α-Proteobacteria were subjected to phylogenetic analysis to investigate the relationships of phototrophic and non-phototrophic bacteria in the α-Proteobacteria. The four Rhodovulum species formed a monophyletic group within the Rhodobacter cluster, and Agrobacterium ferrugineum IAM 12616T intermingled with the Rhodobacter species. This topology is in good agreement with the 16S rRNA phylogeny, although the FPP synthase gene was more divergent than the 16S rRNA. On the other hand, strains of the phototrophic Rps. palustris formed a cluster far from that of the non-phototrophic Bradyrhizobium japonicum strains. Moreover, Rps. palustris strains were differentiated from the nodule-forming B. japonicum, Mezorhizobium loti MAFF 303099 and Sinorhizobium sp. NGR 234 in the FPP synthase phylogeny. This relationship does not agree with the 16S rRNA phylogeny, wherein Rps. palustris was more closely related to B. japonicum than to strains of the Rhodobacter–Rhodovulum group. These results suggest that the FPP synthase gene of Rps. palustris diverged from that of B. japonicum.

Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.

Benzaldehyde and its metabolic intermediates were effectively degraded by the brown-rot basidiomycetes Tyromyces palustris and Gloeophyllum trabeum. The pathway of benzaldehyde degradation was elucidated by the identification of fungal metabolites produced upon the addition of benzaldehyde and its metabolic intermediates. The oxidation and reduction occurred simultaneously, forming benzyl alcohol and benzoic acid as major products. Hydroxylation reactions, which seemed to be a key step, occurred on benzaldehyde and benzoic acid, but not on benzyl alcohol, to form corresponding 4-hydroxyl and 3,4-dihydroxyl derivatives. 1-Formyl derivatives were oxidized to 1-carboxyl derivatives at several metabolic stages. All of these reactions resulted in the formation of 3,4-dihydroxybenzoic acid. This was further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which may be susceptible to the ring-fission reaction. Ring-U-14C-labelled benzaldehyde and benzoic acid were effectively mineralized, clearly indicating that the brown-rot basidiomycetes are capable of metabolizing certain aromatic compounds to CO2 and H2O, despite the fact that brown-rot fungi cannot degrade polymeric lignin. Inhibitor experiments, using hydroxyl radical scavengers, catalase and cytochrome P450 inhibitors, strongly suggested that the aromatic hydroxylation reactions found in the brown-rot fungi are catalysed by intracellular enzyme(s), but not by Fenton-reaction-derived hydroxyl radicals.

To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase–oxygenase (RuBisCO; EC 4.1.1.39). To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes. The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced. For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing. Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis. This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp. used in this study. Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp. endosymbiont was closely related to the genus Rhodobacter, a member of the α-Proteobacteria. For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR. A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences. The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria. To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp. trophosome was visualized by in situ hybridization (ISH), using specific probes. Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules. Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no cbbM-specific signals. The RuBisCO form I gene (cbbL) was neither amplified by PCR nor detected by ISH. This is the first demonstration of chemoautotrophic symbiosis in the deep-sea gutless tubeworm, based on sequence data and in situ localization of both the 16S-rRNA- and RuBisCO-encoding genes.

NH3/ was the principal product from soybean bacteroids, prepared by various procedures, when assayed in solution in a flow chamber under N2 fixation conditions. In addition, small quantities of alanine were produced (reaching 20% of NH3/ under some conditions). Some 15N was assimilated by bacteroids purified from soybean root nodules on Percoll density gradients and shaken with 15N2 and 0·008 atm O2. Under these conditions, accounted for 93% of the 15N fixed into the soluble fraction. This fraction contained no measurable [15N]alanine. Neither these bacteroids nor those prepared by the previously used differential centrifugation method, when incubated with exogenous alanine under non-N2-fixing conditions, gave rise to NH3 from alanine. Therefore, contamination of bacteroid preparations with enzymes of plant cytosolic origin and capable of producing NH3 from alanine cannot explain the failure to detect [15N]alanine [as reported elsewhere: Waters, J. K., Hughes, B. L., II, Purcell, L. C., Gerhardt, K. O., Mawhinney, T. P. & Emerich, D. W. (1998). Proc Natl Acad Sci USA 95, 12038–12042]. Cell-free extracts of the bacteroids as used in the 15N experiments contained alanine dehydrogenase and were able to produce alanine from pyruvate and . Other experiments with alanine dehydrogenase in extracts of cultured rhizobia and bacteroids are reported and discussed in relation to the 15N experiments. Possible reasons for the differences between laboratories regarding the role of alanine are discussed. It is concluded that NH3 is the principal soluble product of N2 fixation by suspensions of soybean bacteroids ex planta and that should continue to be considered the principal product of N2 fixation which is assimilated in vivo in soybean nodules.