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The diversity of sulfate-reducing microorganisms was investigated in two contrasting mudflats of the Seine estuary, by PCR amplification, cloning and sequencing of the genes coding for parts of the alpha and beta subunits of dissimilatory sulfite reductase (dsrAB). One site is located in the mixing-zone and shows marine characteristics, with high salinity and sulfate concentration, whereas the other site shows freshwater characteristics, with low salinity and sulfate concentration. Diversity and abundance of dsrAB genes differed between the two sites. In the mixing-zone sediments, most of the dsrAB sequences were affiliated to those of marine Gram-negative bacteria belonging to the order of Desulfobacterales, whereas in the freshwater sediments, a majority of dsrAB sequences was related to those of the Gram-positive bacteria belonging to the genus Desulfotomaculum. It is speculated that this is related to the salinity and the sulfate concentration in the two mudflats.

SUMMARYThe complete nucleotide sequence of beet necrotic yellow vein virus RNA-1 is presented. The RNA molecule is 6746 nucleotides long excluding the poly(A) tail and has one long open reading flame encoding a polypeptide of M r 237 389. The 3' terminal 60 residues of BNYVV RNA-1 display extensive sequence homology with the corresponding portions of BNYVV RNA-2, -3 and -4. Additional 3' terminal homology exists between RNA-1 and -2. The sequence of the M r 237389 RNA-l-encoded polypeptide shares domains of amino acid homology with polypeptides thought to be involved in replication of RNA from tobacco mosaic virus and several other viruses. Amino acid sequence homologies between two open reading frames of BNYVV RNA-2 and two frames of RNA-fl from barley stripe mosaic virus have also been detected.

The presence of Yersinia ruckeri in a French fish farm was investigated. Y. ruckeri was isolated mainly from algae and sediment samples rather than from water. Twenty-two Y. ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification. These strains were able to adhere to solid supports. This characteristic was correlated with flagellum-mediated motility. Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts. Our results demonstrate that surface colonization of fish farm tanks by Y. ruckeri biofilms is a potential source of recurrent infection for extended periods of time.Yersiniosis or enteric redmouth disease (ERM), caused by Yersinia ruckeri, is a serious infectious disease in the rainbow trout farming industry that causes economic problems in many countries (3). ERM is characterized by the presence of congestive or hemorrhagic zones in various tissues and organs, particularly around the mouth and in the intestines. Its mode of transmission has been related to wild or farmed carrier fish and other putative vectors, such as aquatic invertebrates and birds (27). The pathogen has been isolated from the feces of carrier fish 2 months after an ERM outbreak (18). Therefore, the ability of Y. ruckeri to survive and remain infective in the aquatic environment must be considered a major determinant in the spread of the disease (19). It is now accepted that bacterial biofilms are prevalent on most wet surfaces in nature (7). Very few (if any) studies on the ability of Y. ruckeri to form biofilms have been performed, however.In this study, we investigated the presence of Y. ruckeri in a rearing tank on a French fish farm during routine monthly sampling for 1 year. Y. ruckeri was recovered from water, algae, and sediment samples. The genetic diversity among Y. ruckeri environmental isolates was studied by using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The ability of the majority strain to adhere to wood supports was then investigated. The motility of environmental strains was compared to that of a reference strain. The resistance of fixed cells to oxolinic acid (an antibiotic used routinely to treat yersiniosis) was also compared to that of their free-floating counterparts. MATERIALS AND METHODSSampling. Water, sediment, and alga samples were collected monthly from the outdoor rearing tank of a French fish farm from October 1999 to September 2000. This farm is located at the source of the Cailly River, an oligotrophic river that flows into the Seine River in Rouen, France. Water samples were collected aseptically in sterile glass bottles at a depth of 10 cm. Temperature and pH were determined with hand-held probes. During the investigation period, the pH and temperature of the water were uniform (pH 7.5 and 11°C, respectively). Surficial sediments in the tank were collected with a dredge. Algae located on tank walls were removed with a scraper. Three rainbow t...

Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies.

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In this study, we describe a competitive polymerase chain reaction (PCR) for the quantification of the sequences of dsrAB in sulfatereducing microorganisms. We used the dsr1F/dsr4R set of primers, previously designed by Wagner et al. (1998), and a competitor sequence was constructed from the dsrAB genes of Desulfovibrio vulgaris. The detection limit of competitive PCR corresponded to 45 copies of the dsrAB genes per ng of extracted DNA, and most of the dsrAB sequences amplified and cloned from DNA extracted from Seine estuary sediments were amplified with a similar efficiency. Competitive PCR was then used to assess the abundance of dsrAB genes in the total DNA extracted from the sediment of the Seine estuary mudflats. We observed that the abundance of dsrAB coincided with the variation in the sulfate reduction rate with the depth of the sample, confirming the importance of 'dsrAB' sulfate-reducing microorganisms in sulfidogenesis in anoxic environments. We obtained values ranging from 0.045U10 3 to 6.63U10 3 copies of dsrAB per ng of extracted DNA, and values of the sulfate reduction rate ranging from 35 to 158 nmol cm 33 day 31 . These results are similar to those obtained in other studies using molecular biology techniques.