- Volume 143, Issue 9, 1997

Summary: Rhodococcus sp. strain IGTS8 (ATCC 53968) is able to utilize dibenzothiophene (DBT) as a sole source of sulphur. The carbon skeleton of DBT is not metabolized and is conserved as 2-hydroxybiphenyl (HBP), which accumulates in the medium. This phenotype is due to the expression of the plasmid-encoded DBT-desulphurization (dsz) operon, which encodes three proteins, DszA, B and C. In this paper it is shown, using [35S]DBT radiolabelling studies, that sulphur is released in the form of inorganic sulphite. The pathway of DBT desulphurization is described in detail. In summary, DszC catalyses the stepwise S-oxidation of DBT, first to dibenzothiophene 5-oxide (DBTO) and then to dibenzothiophene 5,5-dioxide (DBTO2); DszA catalyses the conversion of DBTO2 to 2-(2′-hydroxyphenyl)benzene sulphinate (HBPSi−) and DszB catalyses the desulphination of HBPSi− to give HBP and sulphite. Studies with cell-free extracts show that DszA and DszC, but not DszB, require NADH for activity. 18O2-labelling studies show that each incorporated oxygen atom is derived directly from molecular oxygen. These results are consistent with the role of DszC as a mono-oxygenase, of DszA as an apparently unique enzyme which catalyses the reductive hydroxylation of DBTO2 leading to cleavage of the thiophene ring, and of DszB as an aromatic sulphinic acid hydrolase.

Summary: The in vitro production of different hydrolytic enzyme activities by Verticillium fungicola, a mycoparasite of Agaricus bisporus fruit bodies, was examined in cultures grown with various carbon sources (glucose, fructose, sucrose and A. bisporus cell walls). Several of the identified enzymes were influenced by the carbon source. The results of A. bisporus cell wall digestion by these enzymes in vitro have been compared with V. fungicola infection of A. bisporus fruit bodies in vivo. Evidence supporting mycoparasite enzymic action on the host cell walls is presented.

Summary: A 16S rRNA-based molecular ecological study was performed to search for dominant bacterial sequences in Drentse A grassland soils (The Netherlands). In the first step, a library of 165 clones was generated from PCR-amplified 16S rDNA. By sequence comparison, clone DA079 and two other identical clones could be affiliated to a group of recently described uncultured Actinobacteria. This group contained 16S rDNA clone sequences obtained from different environments across the world. To determine whether such uncultured organisms were part of the physiologically active population in the soil, ribosomes were isolated from the environment and 16S rRNA was partially amplified via RT-PCR using conserved primers for members of the domain Bacteria. Subsequent sequence-specific separation by temperature-gradient gel electrophoresis (TGGE) generated fingerprints of the amplicons. Such community fingerprints were compared with the TGGE pattern of PCR-amplified rDNA of clone DA079 which was generated with the same set of primers. One of the dominant fingerprint bands matched with the band obtained from the actinobacterial clone. Southern blot hybridization with a probe made from clone DA079 confirmed sequence identity of clone and fingerprint band. This is the first report that a member of the novel actinobacterial group may play a physiologically active role in a native microbial community.

Summary: A new Staphylococcus aureus gene termed scdA was found upstream of the autolysis regulatory genes, lytS and lytR, and was shown to potentially encode a hydrophilic 25 kDa protein. Analysis of scdA transcription revealed that it is transcribed as a monocistronic message and is lytSR-independent. A role in cell wall metabolism was indicated by examination of the scdA mutant S. aureus KB323, which had a grossly aberrant cellular morphology and formed large cell clusters when grown in liquid culture medium. Furthermore, KB323 exhibited a reduced rate of autolysis and had increased peptidoglycan cross-linking compared to the parental strain, NCTC 8325-4. These data suggest that scdA plays an important role in staphylococcal cell division.

Summary: The gene homologous to glutathione synthetase of Escherichia coli was inactivated in the cyanobacterium Synechococcus sp. PCC 7942. The region of genomic DNA including the mutation site was isolated from the mutant by plasmid rescue and the native gene of the wild-type was cloned from a genomic DNA library of the wild-type using the flanking DNA as a probe. The wild-type gene, designated gshB, encodes a polypeptide of 323 amino acids with a molecular mass of 35 kDa. The deduced amino acid sequence resembles glutathione synthetases of bacteria, but not those of higher organisms. When gshB was overexpressed in E. coli, glutathione synthetase activity was increased markedly in the E. coli extract. In addition, the Synechococcus sp. PCC 7942 gshB mutants had lost their ability to synthesize glutathione. These findings demonstrate that the gshB gene of Synechococcus sp. PCC 7942 is a structural gene for glutathione synthetase and is involved in the biosynthesis of glutathione.

Summary: The genes for adenosine-5′-phosphosulfate (APS) reductase, aprBA, and sirohaem sulfite reductase, dsrAB, from the sulfur-oxidizing phototrophic bacterium Chromatium vinosum strain D (DSMZ 180T) were cloned and sequenced. Statistically significant sequence similarities and similar physicochemical properties suggest that the aprBA and dsrAB gene products from Chr. vinosum are true homologues of their counterparts from the sulfate-reducing chemotrophic archaeon Archaeoglobus fulgidus and the sulfate-reducing chemotrophic bacterium Desulfovibrio vulgaris. Evidence for the proposed duplication of a common ancestor of the dsrAB genes is provided. Phylogenetic analyses revealed a greater evolutionary distance between the enzymes from Chr. vinosum and D. vulgaris than between those from A. fulgidus and D. vulgaris. The data reported in this study are most consistent with the concept of common ancestral protogenotic genes both for dissimilatory sirohaem sulfite reductases and for APS reductases. The aprA gene was demonstrated to be a suitable DNA probe for the identification of apr genes from organisms of different phylogenetic positions. PCR primers and conditions for the amplification of apr homologous regions are described.

Summary: Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy clone of the proY + gene or both a single copy of the proY + gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.

Summary: The omp-31 gene, encoding a major outer-membrane protein in Brucella melitensis, was PCR-amplified from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-31 published sequence. Amplification of omp-31 was achieved from DNA of all Brucella species with the exception of Brucella abortus, the only Brucella species where expression of omp-31 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (4.4-17 kb) containing B. melitensis 16M omp-31 and adjacent DNA of different sizes, with HindIII-digested total DNA showed that a large fragment, comprising the entire omp-31 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specific markers for B. abortus and B. melitensis. At the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-31 revealed specific markers for Brucella ovis, Brucella canis and Brucella suis bv. 2. Using a combination of omp-31 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1, 3, 4 and 5. Results presented in this paper demonstrate the potential of omp-31 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-31 and flanking DNA. In such a large deletion, other genes in addition to omp-31 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Brucella species.

Summary: E colicins are plasmid-coded, protein antibiotics which bind to the BtuB outer membrane receptor of Escherichia coli cells and are then translocated either to the outer surface of the cytoplasmic membrane in the case of the pore-forming colicin E1, or to the cytoplasm in the case of the enzymic colicins E2-E9. Translocation has been proposed to be dependent on a putative TolA box; a pentapeptide (DGSGW) located in the N-terminal 39 residues of several Toldependent colicins. In this study, site-directed mutagenesis was used to change each of the residues of the putative TolA box of colicin E9 to alanines. In the case of the two glycine residues, the resulting mutant proteins were indistinguishable from the native colicin E9 protein in a biological assay; whereas the other three residues when mutated to alanines resulted in a complete loss of biological activity. Mutagenesis of two serine residues flanking the putative TolA box, Ser34 and Ser40, to alanines did not abolish the biological activity of the mutant colicin E9, although the zones of growth inhibition were hazy and slow to form. The size of the zone of inhibition was significantly smaller than the control in the case of the Ser40Ala mutant. The ColE9/lm9 complex was isolated from the three biologically inactive TolA box alanine mutants. In competition assays all three mutant protein complexes were capable of protecting sensitive E. coli cells against killing by the native ColE9/lm9 complex. On removal of the Im9 protein from the three mutant ColE9/lm9 complexes, all three mutant colicins exhibited DNase activity. These results confirm the importance of the putative TolA box in the biological activity of colicin E9, and suggest that the TolA box has a role in the translocation of colicin E9.

Summary: Both alleles of the XOG1 gene of Candida albicans, which encodes a protein with exoglucanase activity, were sequentially disrupted. Enzymic analysis of either cell extracts or culture supernatants of disrupted strains revealed that this gene is responsible for the major exoglucanase activity in C. albicans, although residual exoglucanase activity could still be detected. xog1 null mutants showed similar growth rates in both rich and minimal liquid medium as compared to the wild-type strain, indicating that the enzyme is not essential for C. albicans growth. In addition, no differences were observed between wild-type and xog1 null mutants with respect to their ability to undergo dimorphic transition. However, small but repeatable differences were found between the wild-type and the null mutant with respect to susceptibility to chitin and glucan synthesis inhibitors. Using a murine model of experimental infection, no significant differences in virulence were observed. The xog1 null strain is thus a suitable recipient for studying Candida gene expression using the exoglucanase as a reporter gene.

Summary: The pH-regulated expression of the acid (AXP) and alkaline (AEP) extracellular proteases of the yeast Yarrowia lipolytica 148 was analysed. Expression in batch and continuous cultures was determined at the mRNA level by Northern blotting, and at the enzyme level by enzyme assays and Western blotting. Culture pH regulated AEP and AXP expression predominantly at the level of mRNA content. Highest levels of AEP mRNA were detected at pH 6.5 whereas highest levels of AXP mRNA were detected at pH 5.5. At pH values either side of these maxima AEP and AXP expression were progressively down-regulated. For both enzymes, the variation in mRNA levels with culture pH occurred progressively rather than by discrete steps. AXP expression did not occur above pH 7.0. Some degree of AEP expression occurred at all pH values tested in two unrelated strains of Y. lipolytica.

Summary: The stress-sensing systems leading to the cellular heat shock response (HSR) and the mechanism responsible for the desensitizing of this response in stress-acclimated cells are largely unknown. Here it is demonstrated that there is a close correlation between a 3 ° increase in the temperature required for maximal activation of a heat-shock (HS)-inducible gene in Saccharomyces cerevisiae and an increase in the percentage of cellular unsaturated fatty acids when cells are subjected to extended periods of growth at 37 °. The latter occurs with the same kinetics as HS gene down-regulation during a prolonged HS and is reversed by reacclimation to growth at 25 °. The transient nature of the HS may therefore be due to a lipid-mediated decrease in cellular heat sensitivity. Further evidence that unsaturated fatty acids desensitize cells to heat, with a resultant down-regulation of the HSR, is provided by demonstrating a 9 ° increase in the temperature required for maximal induction of this HS-inducible gene in cells containing high levels of unsaturated fatty acids assimilated during anaerobic growth at 25 °.