- Volume 146, Issue 11, 2000

Pathogenic yeasts of the genus Candida secrete aspartic proteinases (Sap) which are synthesized as preproenzymes. Expression of the C. albicans SAP1 gene lacking the propeptide-coding region in the methylotrophic yeast Pichia pastoris does not lead to the secretion of the enzyme into the culture supernatant, but results in an accumulation of recombinant protein in the cell. Co-expression in this system of the unattached propeptide from Sap1p, as well as from other Saps, restored Sap1p secretion. A deletion analysis revealed that only a 12 aa sequence in the propeptide, corresponding to a highly conserved region in all Sap propeptides, was necessary and sufficient to produce a large amount of Sap1p in culture supernatant. No Sap1p was secreted when Sap1p was produced with a propeptide carrying an F to D mutation in the identified 12 aa sequence. However, the simultaneous production of equivalent amounts of Sap1p and His-tagged Sap1p (H6-Sap1p) with a mutated and a non-mutated propeptide, respectively, led to the secretion of both proteins in a ratio of approximately 1:2. The restoration of Sap1p secretion occurred at the expense of secretion of H6-Sap1p since the total activity was comparable to that of strains producing only H6-Sap1p with a non-mutated propeptide. In contrast, the proteolytic activity of strains secreting Sap1p and H6-Sap1p both with a functional propeptide was twice that of strains producing either Sap1p or H6-Sap1p alone, and the two enzymes were found in an equivalent amount in the culture supernatant. Altogether, these results show that the propeptide can only function once and that the maturation of recombinant C. albicans secreted aspartic proteinase Sap1p is directed through a combination of intra- and inter-molecular pathways.

Integron-encoded integrases recognize two distinct types of recombination site: attI sites, found in integrons, and members of the 59-base element (59-be) family, found in the integron-associated gene cassettes. The class 1 integron integrase, IntI1, catalyses recombination between attI1 and a 59-be, two 59-be, or two attI1 sites, but events involving two attI1 sites are less efficient than the reactions in which a 59-be participates. The full attI1 site is required for high-efficiency recombination with a 59-be site. It is 65 bp in length and includes a simple site, consisting of a pair of inversely oriented IntI1-binding domains, together with two further directly oriented IntI1-binding sites designated strong and weak. However, a smaller region that contains only the simple site is sufficient to support a lower level of recombination with a complete attI1 partner and the features that determine the orientation of attI1 reside within this region. An unusual reaction between the attI1 site and a 59-be appears to be responsible for the loss of the central region of a 59-be to create a potential fusion of two adjacent gene cassettes.

Some cyanobacteria have been shown to exchange genetic information under laboratory conditions, but it has not been clear whether such genetic exchange occurs in the natural environment. To address this, a population genetic study was carried out on the filamentous diazotrophic cyanobacterium Nodularia in the Baltic Sea. Nodularia filaments were collected from 20 widely distributed sampling stations in the Baltic Sea during June and July 1998. Allele-specific PCR (AS-PCR) was used to characterize over 2000 filaments at three loci: a non-coding spacer between adjacent copies of the main structural gas vesicle gene gvpA (gvpA-IGS), the phycocyanin intergenic spacer (PC-IGS) and the rDNA internal transcribed spacer (rDNA-ITS). The three loci were all found to be polymorphic in the 1998 population: two alternative alleles were distinguished at the gvpA-IGS and PC-IGS loci, and three at the rDNA-ITS locus. All 12 possible combinations of alleles were found in the filaments studied, but some were much more common than others. The index of association (I A) for all possible pairwise combinations of isolates was found to differ significantly from zero, which implies that there is some linkage disequilibrium between loci. The I A values for 16 out of 20 individual sampling stations also differed significantly from zero: this shows that the observed linkage disequilibrium is not due to pooling data from genetically distinct subpopulations. Monte-Carlo simulations with random subsets of the data confirmed that some combinations showed significantly more linkage disequilibrium than expected by chance alone. It is concluded that genetic exchange occurs in the natural Nodularia population, but the frequency is not high enough for the loci to be in linkage equilibrium. The distribution of the 12 genotypes across the Baltic Sea was found to be non-random, but did not correlate with temperature, salinity or major nutrient concentrations. A significant relationship was found between the gene diversity among filaments at each station and the distance of the station from the centre of the sampling area: possible reasons for this trend are discussed.

In the complete genome sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 [Kaneko et al. (1996 R19 ). DNA Res 3, 109–136] genes were identified encoding putative group 3 σ-factors SigH (Sll-0856), SigG (Slr-1545) and SigF (Slr-1564) and the regulatory protein RsbU (Slr-2031). Mutations in these genes were generated by interposon mutagenesis to study their importance in stress acclimation. For the genes sigH, sigF and rsbU, the loci segregated completely. However, attempts to mutagenize the sigG locus resulted in merodiploids. Under standard growth conditions only minor differences were detected between the mutants and wild-type. However, cells of the RsbU mutant showed a clear defect in regenerating growth after a nitrogen- and sulphur-starvation-induced stationary phase. After applying salt, heat and high-light shocks, stress protein synthesis was analysed by means of one- and two-dimensional electrophoresis. Cells of the SigF mutant showed a severe defect in the induction of salt stress proteins. Although the acclimation to moderate salt stress up to 684 mM NaCl was not significantly changed in this mutant, its ability to acclimate to higher concentrations of NaCl was reduced. Northern blot experiments showed a constitutive expression of the rsbU and sigF genes. The expression of the sigH gene was found to be stress-stimulated, particularly in heat-shocked cells, whilst that of sigG was transiently decreased under stress conditions. Possible functions of these regulatory proteins in stress acclimation of Synechocystis cells are discussed.

The formation of the Escherichia coli division septum has been well characterized and the majority of the genes involved have been shown to map to the dcw cluster. One exception is ftsK, which lies at 20 minutes, immediately downstream of the global response regulatory gene, lrp. The promoter for ftsK has not yet been assigned. Here, it is reported that ftsK is transcribed from two promoters; the first is located within the lrp reading frame and is dispensable whilst the second is essential and corresponds to dinH, previously characterized as an SOS promoter regulated by LexA. ftsK is the first essential gene to be described that is controlled by an SOS-inducible promoter. A possible mechanism by which dinH may be activated in recA minus strains, or in strains with uncleavable LexA, is discussed.

Expression of the Bacillus subtilis dra–nupC–pdp operon is subject to catabolite repression by glucose. It was shown that a cis-acting catabolite-responsive element (CRE) sequence located 64 bp downstream of the transcription-start site mediated catabolite repression of the dra–nupC–pdp operon as it does for many other B. subtilis genes. Point mutations in the CRE sequence caused the loss of catabolite repression of the operon. Catabolite repression of dra–nupC–pdp expression was relieved in a ccpA mutant and was found to be dependent on both HPr and the HPr-like protein Crh. Furthermore, a transcription-repair coupling factor, Mfd, was also found to be involved in the glucose repression of dra–nupC–pdp expression. By the use of in vitro gel mobility shift analysis, a specific HPr-P dependent binding of CcpA to the dra CRE site was demonstrated.

GcvA binds to three sites in the gcvTHP control region, from base −34 to −69 (site 1), from base −214 to −241 (site 2) and from base −242 to −271 (site 3). Previous results suggested that sites 3 and 2 are required for both GcvA-dependent activation and repression of a gcvT::lacZ fusion. However, the results were less clear as to the role of site 1. To determine the role of site 1 in regulation, single and multiple base changes were made in site 1 and tested for their ability to alter GcvA-mediated activation and GcvA/GcvR-mediated repression. Several of the mutants were also tested for effects on GcvA binding to site 1 and the ability of GcvA to bend DNA at site 1. The results are consistent with site 1 playing primarily a role in negative regulation of the gcvTHP operon.

Expression of the sigF gene encoding a sporulation-specific sigma factor, σF, in Streptomyces aureofaciens is restricted only to sporulation. Gel mobility-shift assays using protein fractions from different developmental stages of S. aureofaciens revealed two different putative proteins specifically bound to the sigF promoter region: a protein (designated RsfA) present in young substrate mycelium, and a protein (designated RsfB) present in the course of sporulation. Based on the characteristic profiles of their appearance during differentiation, RsfA might be a repressor and RsfB an activator of sigF expression. The location of a specific binding site of the repressor-like protein (RsfA) was determined by gel mobility-shift assays of promoter deletion fragments and by DNase I footprinting analysis. The binding site mapped from nucleotides −87 to −25 relative to the transcription start point of the sigF promoter, and overlapped the −35 promoter region. Given the dependence of sigF expression upon whiH, the putative sporulation transcription factor WhiH was overproduced in Escherichia coli and used in the mobility-shift assays with the sigF promoter. However, no specific binding was detected, indicating an indirect dependence of sigF upon whiH.

The Bacillus subtilis 168 csn gene encodes a chitosanase. It was found that transcription of the csn gene was temporally regulated and was not subject to metabolic repression. Chitosanase synthesis was abolished in a csn mutant strain. Csn was overproduced in B. subtilis, partially purified and characterized. The deduced amino acid sequence, K m, and optimal pH and temperature of the B. subtilis enzyme were closer to those of a chitosanase from Streptomyces sp. N174 than to those of chitosanases from other Bacillus strains.

Streptomyces coelicolor A3(2) strain M145 has eight chitinase genes scattered on the chromosome: six genes for family 18 (chiA, B, C, D, E and H) and two for family 19 (chiF and G). In this study, the expression and regulation of these genes were investigated. The transcription of five of the genes (chiA, B, C, D and F) was induced in the presence of colloidal chitin while that of the other three genes (chiE, G and H) was not. The transcripts of the five induced chi genes increased and reached their maximum at 4 h after the addition of colloidal chitin, all showing the same temporal patterns. The induced levels of the transcripts of chiB were significantly lower than those of the other four genes. Dynamic analysis of the transcripts of the chi genes indicated that chiA and chiC were induced more strongly than chiD and chiF. Addition of chitobiose also induced transcription of the chi genes, but significantly earlier than did colloidal chitin. When cells were cultured in the presence of colloidal chitin, an exponential increase of chitobiose concentration in the culture supernatant was observed prior to the induced transcription of the chi genes. This result, together with the immediate effect of chitobiose on the induction, suggests that chitobiose produced from colloidal chitin is involved in the induction of transcription of the chi genes. The transcription of the five chi genes was repressed by glucose. This repression was apparently mediated by the glucose kinase gene glkA.

The napEDABC operon of Paracoccus pantotrophus encodes a periplasmic nitrate reductase (NAP), together with electron-transfer components and proteins required for the synthesis of a fully functional enzyme. Previously, it had been shown that high NAP activity was observed when P. pantotrophus was grown aerobically on highly reduced carbon sources such as butyrate or caproate, but not when cultured on more oxidized substrates such as succinate or malate. The enzyme is not present to any extent when the organism is grown anaerobically under denitrifying conditions, regardless of the carbon source. Transcriptional analyses of the nap operon have now identified two initiation sites which were differentially regulated in response to the carbon source, with expression being maximal when cells were grown aerobically with butyrate. Analysis of a P. pantotrophus mutant (M6) deregulated for NAP activity identified a single C→A transversion in a heptameric inverted-repeat sequence that partially overlapped the proximal promoter. Transcription analysis of this mutant revealed that expression of nap was completely derepressed under all growth conditions examined. Taken together, these findings indicate that nap transcription is negatively regulated during anaerobiosis, such that expression is restricted to aerobic growth, but only when the carbon source is highly reduced.

To determine if cellular functions of the phosphatidylinositol 3-kinase CaVps34p are related to processes governing Candida albicans pathogenicity, both copies of the gene were sequentially disrupted. Homozygous deletion of C. albicans VPS34 resulted in a mutant strain which exhibited defects not only in intracellular vesicle transport processes but also in morphogenesis. The CaVPS34 null mutant was unable to form hyphae on different solid media whilst showing a significantly delayed yeast-to-hyphae transition in liquid media. In addition, the mutant was rendered hypersensitive to temperature and osmotic stresses and had a strongly decreased ability to adhere to mouse fibroblast cells compared to the wild-type strain SC5314. Finally, evidence was obtained that CaVPS34 is essential for pathogenicity of C. albicans as the CaVPS34 null mutant was shown to be avirulent in a mouse model of systemic infection. C. albicans pathogenicity was restored to a near wild-type degree upon reintroduction of CaVPS34 into the chromosome of the null mutant, demonstrating that the observed avirulence corresponded to the loss of CaVPS34. Thus, the results suggest that CaVPS34 may serve as a potential target for antifungal drugs.

Salmonella typhimurium 4/74 is highly virulent for cattle after oral challenge, causing severe diarrhoea, which is sometimes associated with systemic spread of the micro-organism. Although susceptible to oral challenge, groups of cattle were found to be relatively resistant to subcutaneous challenge with this strain. The virulence of S. typhimurium 4/74 harbouring mutations in htrA and purE was also assessed in cattle. Although S. typhimurium 4/74 htrA and purE are attenuated following oral challenge in mice, cattle were highly susceptible to oral challenge with these mutants. As with the parent S. typhimurium 4/74 strain, cattle exhibited greater susceptibility to oral compared to subcutaneous challenge with S. typhimurium htrA and purE mutants. Following subcutaneous challenge with sublethal levels of S. typhimurium 4/74, calves produced significant levels of antibodies to S. typhimurium soluble extract. No correlation was detected between interferon gamma levels in sera and susceptibility to infection by any route. The concentrations of the acute-phase-associated protein haptoglobin were increased in the sera of five of six cattle inoculated subcutaneously, although increases in concentration were smaller in cattle inoculated orally.

To investigate the role of mitogenic factor (MF) in streptococcal pathogenesis, the structural gene (mf) encoding this protein was disrupted in a clinical isolate of Streptococcus pyogenes H293, to yield the isogenic mutant H363. Growth in enriched broth and on blood agar was unaffected by disruption of mf. Cell-free broth supernatants from H293 and H363 demonstrated identical promitogenic activities when co-incubated with human peripheral blood mononuclear cells, even when diluted 100000-fold, showing that MF is not a major streptococcal mitogen compared with other secreted superantigens. Disruption of mf resulted in complete loss of DNase B production and detectable DNase activity in H363 compared with the parent strain, confirming that the single gene mf, which is present in all group A streptococcal M serotypes studied, encodes DNase B. Despite loss of DNase activity, the virulence of S. pyogenes in a mouse model of necrotizing fasciitis and myositis was unaffected.