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

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 (stx(1) and/or stx(2)) 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 stx(1) or stx(2) 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 lambda, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill.

Inferred amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea, generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously.

Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes.

The protection of mannosylglycerate, at 0.5 M concentration, against heat inactivation of the model enzyme lactate dehydrogenase (LDH) was compared to that exerted by other compatible solutes, namely, trehalose, ectoine, hydroxyectoine, di- myo-inositol phosphate, diglycerol phosphate, and mannosylglyceramide. Mannosylglycerate and hydroxyectoine were the best stabilizers of the enzyme and showed comparable protective effects. Diglycerol phosphate, trehalose, and mannosylglyceramide protected the enzyme to a lower extent. Ectoine conferred no protection, and di- myo-inositol phosphate had a strong destabilizing effect. The superior ability of mannosylglycerate to prevent LDH inactivation was accompanied by a higher efficiency in preventing LDH aggregation induced by heat stress. Moreover, mannosylglycerate induced an increase of 4.5 degrees C in the melting temperature of LDH, whereas the same molar concentration of trehalose caused an increase of only 2.2 degrees C. The effectiveness of mannosylglycerate in protecting LDH was also compared to that of other chemically related compounds: mannose, methyl-mannoside, potassium glycerate, glucosylglycerol, glycerol, and glucose. Mannosylglycerate conferred the highest protection, but glucosylglycerol and potassium glycerate were very efficient; glucose exerted a low degree of protection, glycerol and methyl-mannoside had no significant effect, and mannose caused destabilization. Mannosylglycerate was also a good thermoprotectant of glucose oxidase from Aspergillus niger, an enzyme with a net charge opposite to that of LDH under the working conditions. Given the superior performance of mannosylglycerate as a thermoprotectant of enzyme activity in vitro, it is conceivable that it also fulfills a protein thermoprotective function in vivo.

Development of oligonucleotide probes and PCR primers for detecting phylogenetic subgroups of sulfate-reducing bacteria.

PCR primer sets for the 16S rRNA gene of six phylogenetic groups of sulfate-reducing bacteria (SRB) were designed. Their application in conjunction with group-specific internal oligonucleotide probes was used to detect SRB DNA in samples of landfill leachate. Six generic/suprageneric groups could be differentiated: DESULFOTOMACULUM:; DESULFOBULBUS:; DESULFOBACTERIUM:; DESULFOBACTER:; DESULFOCOCCUS:-DESULFONEMA:-DESULFOSARCINA:; DESULFOVIBRIO:-DESULFOMICROBIUM: The predicted specificities of the PCR primer and oligonucleotide probe combinations were confirmed with DNA from reference strains. In all cases, the PCR primers and probes were specific, the only exception being that the Desulfococcus-Desulfonema-Desulfosarcina (group 5) PCR primers were able to amplify DNA from DESULFOBACTERIUM: (group 3) reference strains but these groups could nevertheless be differentiated with the internal oligonucleotide probes. The proliferation of SRB in landfill sites interferes with methanogenesis and waste stabilization, but relatively little is known about the composition of SRB populations in this environment. DNA was extracted from samples of landfill leachate from several municipal waste landfill sites and used as template in PCR reactions with SRB group-specific primer sets. Group-specific oligonucleotide probes were then used to confirm that the PCR products obtained contained the target SRB 16S rDNA. Both 'direct' and 'nested' PCR protocols were used to amplify SRB 16S rDNA from landfill leachates. Three of the six SRB groups could be detected using the 'direct' PCR approach (DESULFOTOMACULUM:, DESULFOBACTER: and Desulfococcus-Desulfonema-Desulfosarcina). When 'nested' PCR was applied, an additional two groups could be detected (DESULFOBULBUS: and DESULFOVIBRIO:-DESULFOMICROBIUM:). Only DESULFOBACTERIUM: could not be detected in any leachate samples using either direct or nested PCR. The SRB-specific 16S rDNA primers and probes described here can be applied to investigations of SRB molecular ecology in general, and can be further developed for examining SRB population composition in relation to landfill site performance.