Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized. Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups.

The expression of many virulence determinants in Staphylococcus aureus including ␣-hemolysin-, protein A-, and fibronectin-binding proteins is controlled by global regulatory loci such as sar and agr. In addition to controlling target gene expression via agr (e.g. ␣-hemolysin), the sar locus can also regulate target gene transcription via agr-independent mechanisms. In particular, we have found that SarA, the major regulatory protein encoded within sar, binds to a conserved sequence, homologous to the SarA-binding site on the agr promoter, upstream of the ؊35 promoter boxes of several target genes including hla (␣-hemolysin gene), spa (protein A gene), fnb (fibronectin-binding protein genes), and sec (enterotoxin C gene). Deletion of the SarA recognition motif in the promoter regions of agr and hla in shuttle plasmids rendered the transcription of these genes undetectable in agr and hla mutants, respectively. Likewise, the transcription activity of spa (a gene normally repressed by sar), as measured by a XylE reporter fusion assay, became derepressed in a wild type strain containing a shuttle plasmid in which the SarA recognition site had been deleted from the spa promoter region. However, DNase I footprinting assays demonstrated that the SarA-binding region on the spa and hla promoter is more extensive than the predicted consensus sequence, thus raising the possibility that the consensus sequence is an activation site within a larger binding region. Because the sar and agr regulate an assortment of virulence factors in S. aureus, we propose, based on our data, a unifying hypothesis for virulence gene activation in S. aureus whereby SarA is a regulatory protein that binds to its consensus SarA recognition motif to activate (e.g. hla) or repress (e.g. spa) the transcription of sar target genes, thus accounting for both agr-dependent and agr-independent mode of regulation.

The expression of many virulence determinants in Staphylococcus aureus is controlled by regulatory loci such as agr and sar. We have previously shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus. To define the specific molecular interaction, we overexpressed SarA as a glutathione S-transferase (GST) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector. The purified GST-SarA as well as cleaved SarA were able to bind specifically to the P2, P3, and the combined P2-P3 promoter fragments of agr in gel shift assays. Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16 -124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA 16 -124 ) could activate agr transcription to a level approaching that of the full-length counterpart (SarA 1-124 ). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription.

Fibronectin‐binding proteins (FnBPs) are thought to be important for the attachment of Staphylococcus aureus during infection. The regulation of the genes fnbA and fnbB by the global regulatory loci sar and agr was examined using site‐specific regulatory mutants of S. aureus strain Newman. The results from binding assays using both aqueous and solid‐phase fibronectin as well as ligand blotting with biotinylated fibronectin showed that the expression of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar–agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA‐specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB promoter activity in promoter fusion assays was not affected by either sar or agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post‐transcriptional level. Gel shift assays demonstrated that SarA binds to the fnbA promoter fragments, probably as a dimer. DNase I footprinting assays with SarA revealed a protected area of 102 bp upstream of fnbA.

Determination of the nucleotide sequence of the nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing the 3 end of nifK2 and 1,390 bp of the nifE2-homologous genes. Open reading frame nifK2 is 1,371 bp long including the stop codon TAA and encodes a polypeptide of 456 amino acids. Phylogenetic analysis of the deduced amino acid sequences of the nifK2 and nifE2 gene products from M. barkeri showed that both genes cluster most closely with the corresponding nif-1 gene products from Clostridium pasteurianum, consistent with our previous analyses of nifH2 and nifD2. The nifE gene product is known to be homologous to that of nifD, and our analysis shows that the branching pattern for the nifE proteins resembles that for the nifD product (with the exception of vnfE from Azotobacter vinelandii), suggesting that a gene duplication occurred before the divergence of nitrogenases. Primer extension showed that nifH2 had a single transcription start site located 34 nucleotides upstream of the ATG translation start site for nifH2, and a sequence resembling the archaeal consensus Nitrogen fixation is known to be widespread in both eubacteria and methanogenic archaea. All known nitrogenases consist of two components: component 1, dinitrogenase, or the MoFe protein (except in alternative nitrogenases lacking Mo), an ␣ 2 ␤ 2 tetramer encoded by nifD and nifK, and component 2, dinitrogenase reductase, or the Fe protein, a homodimer encoded by nifH. Related to the nifDK genes are the nifEN genes, which encode a protein resembling component 1 which is believed to serve as a ''scaffold'' for synthesis of the FeMo cofactor, which is then inserted into component 1 (5).Phylogenetic analysis by ourselves (4) and others (11,17,18,30) has indicated that functional nitrogenase genes form three families, cluster I, consisting of most conventional eubacterial MoFe nitrogenases; cluster II, containing eubacterial alternative nitrogenases lacking Mo and several methanogen nitrogenases; and cluster III, containing the Mo nitrogenase (nif-1) genes from the gram-positive eubacterium Clostridium pasteurianum and the nif-2 genes from the archaeon Methanosarcina barkeri. Another recently recognized member of this cluster is nifH from Desulfovibrio gigas (29a). A fourth cluster contains genes from methanogens which are likely to serve in functions other than nitrogen fixation (4). Also related to nifH are bclX, bclL, and chlL genes, which encode Fe proteins involved in bacteriochlorophyll or chlorophyll ring reduction (3). These groupings raise interesting questions about the roles of horizontal gene transfer and ancient gene duplications in nitrogenase evolution.Sibold et al. (25) described two sets of nifH genes in M. barkeri: nifH1, which is a member of cluster II, and nifH2, which was in cluster III. In our previous study (4), we demonstrated that the N-terminal sequence of the purified nitrogenase component 2 from M. barkeri cells grown diazo...