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The Dithiol:Disulfide Oxidoreductases DsbA and DsbB of Rhodobacter capsulatus Are Not Directly Involved in Cytochrome c Biogenesis, but Their Inactivation Restores the Cytochrome c Biogenesis Defect of CcdA-Null Mutants. Meenal Deshmukh, 2003.The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of Escherichia coli, are thought to supply the reducing equivalents required for the biogenesis of c-type cytochromes that occurs in the periplasm of gram-negative bacteria . CcdA-null mutants of the facultative phototroph Rhodobacter capsulatus are unable to grow under photosynthetic conditions (Ps-) and do not produce any active cytochrome c oxidase (Nadi-) due to a pleiotropic cytochrome c deficiency . However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps+ Nadi+ colonies that produce c-type cytochromes despite the absence of CcdA . Complementation of a CcdA-null mutant for the Ps+ growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant . No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered . Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA . DNA sequence data revealed that the dsbA allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA . Indeed, a dsbA ccdA double mutant was shown to be Ps+ Nadi+, establishing that in R . capsulatus the inactivation of dsbA suppresses the c-type cytochrome deficiency due to the absence of ccdA . Next, the ability of the wild-type dsbA allele to suppress the Ps+ growth phenotype of the dsbA ccdA double mutant was exploited to isolate dsbA-independent ccdA revertants . Sequence analysis revealed that these revertants carried mutations in dsbB and that their Ps+ phenotypes could be suppressed by the wild-type allele of dsbB . As with dsbA, a dsbB ccdA double mutant was also Ps+ Nadi+ and produced c-type cytochromes . Therefore, the absence of either DsbA or DsbB restores c-type cytochrome biogenesis in the absence of CcdA . Finally, it was also found that the DsbA-null and DsbB-null single mutants of R . capsulatus are Ps+ and produce c-type cytochromes, unlike their E . coli counterparts, but are impaired for growth under respiratory conditions . This finding demonstrates that in R . capsulatus the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome c biogenesis even though they are important for respiration under certain conditions . Characterization of Staphylococcus aureus SarA Binding Sites. Kristen M. Sterba, 2003.The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes . Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified . In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna . The results suggest the existence of multiple high-affinity binding sites within the cna promoter region . Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments . These fragments were subsequently cloned and sequenced . Randomly chosen clones were also examined by EMSA . These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA . The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome . Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones . By using the same criteria, consensus binding sites were also identified upstream of the S . aureus genes spa, fnbA, sspA, agr, hla, and cna . With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene . Molecular Characterization of Legionella Populations Present within Slow Sand Filters Used for Fungal Plant Pathogen Suppression in Horticultural Crops. Leo A. Calvo-Bado, 2003.The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products . One dominant band had sequence homology to Legionella species, indicating that these bacteria were a large component of the SSF bacterial community . Populations within experimental and commercial SSF units were studied by using Legionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses . In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a single Legionella strain was predominant in each sample . Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample . Sequence analysis of these PCR products showed that several Legionella species were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples . Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number of Legionella cells decreased, resulting in a lower number of cells in the outflow water . In the commercial SSF, legionellae were also detected in the sand column samples . Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth of Legionella and should be avoided . Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation .
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