Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four 'Pfiesteria-like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the alpha-3 or SAR83 cluster.

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl ؊ . Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl ؉ strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose. Xylose metabolism has been described for a wide array of microorganisms. Extensively characterized xylose-metabolizing (Xyl ϩ ) bacteria include Escherichia coli (24, 46), Lactobacillus spp. (3,4,(26)(27)(28), Bacillus spp. (16,33,38), and Staphylococcus xylosus (44). Free xylose can be transported via low-affinity symporters (XylE or XylT) or high-affinity binding-protein dependent systems (XylFGH) (1, 6, 9, 41). Xylose isomerase (xylA gene) then converts the aldose sugar to xylulose, which is phosphorylated by xylulokinase (xylB gene). Further metabolism of xylulose-5-phosphate occurs via the pentose-phosphate or phosphoketolase pathways. Xylose metabolism is induced by xylose, mediated via XylR. In Salmonella and E. coli strains, XylR is an activator when xylose is present (42,46). In grampositive organisms, XylR is a repressor which is inactivated when xylose binds (13,26,27,37,44,45). The catabolite repression of xylose metabolism by glucose is mediated primarily through the CcpA protein (40).Lactococcus lactis subsp. cremoris and almost all L. lactis subsp. lactis strains cannot metabolize xylose. These organisms have undergone intense selection for use in dairy fermentations, but plants are thought to be their original ecological niche because L. lactis subsp. lactis isolates have been recovered from many different plants (22,34,35). Also, the plant isolate Lactobacillus xylosus has been reclassified as L. lactis subsp. lactis (39). However, L. lactis subsp. cremoris strains are almost exclusively dairy associated (22).We discovered xylose metabolic genes from both plant (Xyl ϩ ) and dairy (Xyl Ϫ ) isolates of L. lactis. The sequencing of xylRAB from L. lactis strains IO-1, 210, a...

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A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.