No abstract

Aims: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. Methods and Results: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4 ¢ ,6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were £2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were £2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. Conclusions: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, microorganisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. Significance and Impact of the Study: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.

No abstract

To analyse nod gene expression in Rhizobium leguminosarum, a broad host‐range lacZ protein fusion vector was constructed. Two protein fusions, nodC‐lacZ and nodD‐lacZ, were used to measure the regulation of expression of the promoters of the nodA,B,C and the nodD transcripts by measuring the induced levels of β‐galactosidase activity in R. leguminosarum. In the absence of plant root exudate the nodD‐lacZ hybrid was expressed but the nodC‐lacZ hybrid was not. The expression of the nodD‐lacZ hybrid was repressed in R. leguminosarum strains containing an intact cloned nodD gene indicating that the nodD gene is autoregulatory. The induction of the nodC‐lacZ hybrid required both the nodD gene and a component present in plant root exudate. Therefore the nodD gene acts both as a repressor and as an activator of gene expression. The nodD gene is adjacent to nodA and transcribed divergently from nodA,B,C with only ∼300 nucleotides between the coding regions of nodA and nodD. Within this intergenic region is a unique BclI site and, using nodC‐lacZ or nodD‐lacZ translational fusions with this BclI site as an end point, no induction of nodC‐lacZ or nodD‐lacZ was observed. Therefore the promoters of nodD and nodA,B,C overlap at least at this region, and the regulation of these overlapping promoters appears to be controlled by the nodD protein which becomes an activator only in the presence of a component from plant exudate.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin 0 (My) and 235 rRNA were sequenced for a range of Listeris monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multiloeus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.