Alan J. McCarthy

The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences

Partial sequences of the 16S ribosomal RNA genes of eleven autotrophic ammonia-oxidizing bacteria were determined by PCR amplification from small amounts of heat-lysed biomass followed by direct sequencing of PCR products. The sequences were aligned with those of representative Proteobacteria and phylogenetic trees inferred using both parsimony and distance matrix methods. This confirmed that the autotrophic ammonia-oxidizers comprise two major lines of descent within the Proteobacteria. Nitrosomonas spp., Nitrosococcus mobilis, and strains of Nitrosovibrio, Nitrosospira and Nitrosolobus were located in the beta-subdivision. The recovery of Nitrosococcus oceanus strains as a deep branch in the gamma-subdivision supported the RNA catalogue data which had indicated that the genus Nitrosococcus is polyphyletic. The autotrophic ammonia-oxidizing bacteria of the beta-Proteobacteria formed a coherent group which is interpreted as representing a single family. Within this clade, the genera Nitrosovibrio, Nitrosospira and Nitrosolobus exhibited very high levels of homology in their 16S ribosomal RNA gene sequences and can be accommodated within a single genus. Separation of these genera is currently based entirely on gross morphological differences and these can now be considered more appropriate for the identification of species within this group. It is therefore proposed that Nitrosolobus, Nitrosovibrio and Nitrosospira strains be reclassified in a single genus for which the name Nitrosospira has priority.

Actinomycetes as agents of biodegradation in the environment — a review ☆

The diversity of form in the Actinomycetales is well-recognised, due to the sustained generation of environmental isolates for pharmaceutical screening. Actinomycetes isolated from soil and related substrates show primary biodegradative activity, secreting a range of extracellular enzymes and exhibiting the capacity to metabolise recalcitrant molecules. Composting is one process which relies heavily on such prolific actinomycete activity. Amongst actinomycetes in soil, there are examples of different strategies, from cycles of rapid proliferation and sporulation to the maintenance of populations by prolonged slow growth and scavenging, and the evidence for this is examined. The mechanisms of lignocellulose degradation by actinomycetes are discussed in relation to functional conservation within the group, and correlations with those described in other bacteria and fungi.

Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment

Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes. The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples. Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe. Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers. Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe. Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes. This demonstrates that Nitrosospira spp. are widespread in the environment. The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed.

Molecular Biological Detection and Characterization of Clostridium Populations in Municipal Landfill Sites

ABSTRACT Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

ABSTRACT A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number ofShigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

The Fibrobacteres : an Important Phylum of Cellulose-Degrading Bacteria

The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a “third” way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.

Towards elucidation of the lignin degradation pathway in actinomycetes.

Six biodegradative actinomycete strains were grown on a dimeric model lignin compound of the β-aryl ether type. Although only two strains, Thermomonospora mesophila and Streptomyces badius, utilized the compound as a carbon and energy source and produced substantial amounts of monomeric products, all of the strains could demethylate the substrate and oxidize Cα on the phenylpropane side-chain. Streptomyces sp. EC1 produced small amounts of aromatic acids and unidentified lignin-derived products when grown on straw. This organism also produced cell-bound demethylase requiring H2O2 and Mn2+, protocatechuate 3,4-dioxygenase and β-carboxymuconate decarboxylase activity in response to growth on low-molecular-mass aromatic compounds but not lignocellulose or its polysaccharide components. Extracellular peroxidase and catalase activity were detected in all of the strains. These data are used to propose a scheme by which actinomycete attack of the lignin component of plant biomass can be envisaged.

Screening for lignin-degrading actinomycetes and characterization of their activity against [14C]lignin-labelled wheat lignocellulose

SUMMARY: A sedimentation chamber and Andersen sampler were used to isolate a range of actinomycetes on selective and non-selective media. The occurrence of different actinomycete groups in natural substrates was compared and strains were screened for the ability to degrade ball-milled straw or to grow on lignin-related phenolic compounds. Evidence for ligninolytic activity in representatives of several genera was obtained by assaying 14CO2 evolution from [14C]lignin-labelled wheat lignocellulose. Most of the straw-degrading isolates were assigned to the genera Thermomonospora and Micromonospora, but only representatives of the latter were found to be active against [14C]lignin. Of the non-straw-degrading strains also examined, some which could utilize phenolic substrates produced 14CO2 from the [lignin-14C]lignocellulose, and two of these were selected for further study. These strains, Thermomonospora mesophila and a Streptomyces sp., attacked [14C]lignin yielding 14CO2 and water-soluble 14C-labelled compounds during primary growth. This activity was not accounted for by the utilization of phenolic acids linked to the carbohydrate fraction of wheat lignocellulose and was unaffected by cultural parameters known to influence lignin degradation by white-rot fungi.

Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens.

ABSTRACT The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx1 or stx2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage λ, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

Analysis of ammonia-oxidizing bacteria populations in acid forest soil during conditions of moisture limitation

R.C. HASTINGS, C. BUTLER, I. SINGLETON, J.R. SAUNDERS and A.J. McCARTHY.2000.Ammonia‐oxidizer numbers decreased under conditions of moisture limitation in litter, fermentation and humus layers of forest soil in the field, but the extent of regrowth after rehydration varied between layers. Nitrosospira 16S rRNA genes were amplified from all layers, regardless of moisture content or soil pH which varied between 4·1 and 5·2. Nitrosomonas spp. were detected less often, but appeared to exhibit more rapid recovery than the Nitrosospira spp. when drought conditions were relieved by rainfall.