- Volume 151, Issue 3, 2005

During Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the asymmetric prespore septum. Here, the author studied the localization of other PBPs, fused to green fluorescent protein (GFP), during spore formation. Fusions to PBPs 4, 2c, 2d, 2a, 3, H, 4b, 5, 4a, 4* and X were expressed during vegetative growth, and their localization was monitored during sporulation. Of these PBPs, 2c, 2d, 4b and 4* have been implicated as having a function in sporulation. It was found that PBP2c, 2d and X changed their localization, while the other PBPs tested were not affected. The putative endopeptidase PbpX appears to spiral out in a pattern that resembles FtsZ redistribution during sporulation, but a pbpX knockout strain had no distinguishable phenotype. PBP2c and 2d localize to the prespore septum and follow the membrane during engulfment, and so are redistributed to the prespore membrane. A similar pattern was observed when GFP–PBP2c was expressed in the mother cell from a sporulation-specific promoter. This work shows that various PBPs known to function during sporulation are redistributed from the cytoplasmic membrane to the prespore.

Gram-negative bacteria use a wide variety of complex mechanisms to secrete proteins across their membranes or to assemble secreted proteins into surface structures. As most archaea only possess a cytoplasmic membrane surrounded by a membrane-anchored S-layer, the organization of such complexes might be significantly different from that in Gram-negative bacteria. Five proteins of Sulfolobus solfataricus, SSO0120, SSO0572, SSO2316, SSO2387 and SSO2680, which are homologous to secretion ATPases of bacterial type II, type IV secretion systems and the type IV pili assembly machinery, were identified. The operon structures of these putative secretion systems encoding gene clusters and the expression patterns of the ATPases under different growth conditions were determined, and it was established that all five putative ATPases do show a divalent cation-dependent ATPase activity at high temperature. These results show that the archaeal secretion systems are related to the bacterial secretion systems and might be powered in a similar way.

Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.

The heat-shock response is conserved amongst practically all organisms. Almost invariably, the massive heat-shock protein (Hsp) synthesis that it induces is subsequently down-regulated, making this a transient, not a sustained, stress response. This study investigated whether the heat-shock response displays any unusual features in the methylotrophic yeast Hansenula polymorpha, since this organism exhibits the highest growth temperature (49–50 °C) identified to date for any yeast and grows at 47 °C without either thermal death or detriment to final biomass yield. Maximal levels of Hsp induction were observed with a temperature upshift of H. polymorpha from 30 °C to 47–49 °C. This heat shock induces a prolonged growth arrest, heat-shock protein synthesis being down-regulated long before growth resumes at such high temperatures. A 30 °C to 49 °C heat shock also induced thermotolerance, although H. polymorpha cells in balanced growth at 49 °C were intrinsically thermotolerant. Unexpectedly, the normal transience of the H. polymorpha heat-shock response was suppressed completely by imposing the additional stress of hypoxia at the time of the 30 °C to 49 °C temperature upshift. Hypoxia abolishing the transience of the heat-shock response appears to operate at the level of Hsp gene transcription, since the heat-induced Hsp70 mRNA was transiently induced in a heat-shocked normoxic culture but displayed sustained induction in a culture deprived of oxygen at the time of temperature upshift.

LasX regulates the transcription of the divergent operons lasXY and lasA–W, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasA–lasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA–lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of P lasA–W , and a third imperfect copy of this motif, overlapping the −10 element of P lasA–W , were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA–lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

Porphyromonas gingivalis, an obligately anaerobic bacterium, is implicated as a major pathogen in the development and progression of chronic periodontitis. Although expression of several virulence factors of the bacterium has been found to be affected by environmental stress such as entrance into the stationary growth phase and heat, there is relatively little information on the mechanisms that may operate in the bacterium in response to environmental stress. In this study, a novel protein (UstA) was investigated that was initially identified following two-dimensional gel analysis. Expression of UstA was upregulated in stationary phase or by exposure to atmospheric oxygen. N-terminal sequencing and database analysis with the P. gingivalis genome sequence revealed that the UstA-encoding gene (ustA) was located upstream of a homologue of the usp gene encoding the universal stress protein on the chromosome. The ustA gene appeared to be transcribed in a monocistronic fashion, as revealed by primer extension and Northern blot analysis. To elucidate the role of UstA in the bacterium, chromosomal mutants carrying a disruption of the ustA gene were constructed. The ustA mutant grew slower than the wild-type parent strain in rich medium, resulting in a lower yield in stationary phase. Furthermore, in this mutant, expression levels of the P. gingivalis homologues of superoxide dismutase, thiol peroxidase and thioredoxin were markedly higher than those in the wild-type, especially in stationary phase. The ustA mutant was more resistant to diamide, a thiol-specific oxidant, than the wild-type. In addition, the ustA mutation suppressed hypersensitivities of the oxyR mutant to diamide, metronidazole and mitomycin C. These results suggest that UstA may play a significant role in oxidative stress responses in the bacterium.

The dnaX gene of Escherichia coli encodes τ and γ clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in the dnaA gene that partly suppressed the dnaX2016 temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and τ or γ is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.

Candida albicans strain SC5314 contains two ALS3 alleles, which differ in sequence with respect to the number of copies of the 108 bp tandem repeat sequence within the central domain of the coding region. One allele (ALS3(12)) has 12 tandem repeat copies while the other (ALS3(9)) has 9 copies. Wild-type C. albicans (ALS3(12)/ALS3(9)) and those containing various ALS3 alleles (ALS3(12)/als3Δ(9), als3Δ(12)/ALS3(9) and als3Δ(12)/als3Δ(9)) were assayed for adhesion to monolayers of cultured vascular endothelial and pharyngeal epithelial cells. These assays showed obvious adhesive function for the larger Als3p protein, compared to a minor contribution to adhesion from the smaller protein. These functional differences in strain SC5314 prompted examination of ALS3 allelic diversity across the five major genetic clades of C. albicans. This analysis focused on the number of copies of the tandem repeat sequence within the central domain of the coding region and showed a range of alleles encoding from 6 to 19 tandem repeat copies. Clades differed with respect to prevalent ALS3 alleles and allele distribution, but were similar for the mean number of tandem repeat copies per ALS3 allele. Analysis of allelic pairing showed clade differences and the tendency for C. albicans strains to encode one longer and one shorter ALS3 allele. The allelic variability observed for ALS3 and its functional consequences observed in strain SC5314 highlight the importance of understanding ALS allelic diversity in order to draw accurate conclusions about Als protein function.

Nematode-trapping fungi enter the parasitic stage by developing specific morphological structures called traps. The global patterns of gene expression in traps and mycelium of the fungus Monacrosporium haptotylum were compared. The trap of this fungus is a unicellular spherical structure called the knob, which develops on the apex of a hyphal branch. RNA was isolated from knobs and mycelium and hybridized to a cDNA array containing probes of 2822 EST clones of M. haptotylum. Despite the fact that the knobs and mycelium were grown in the same medium, there were substantial differences in the patterns of genes expressed in the two cell types. In total, 23·3 % (657 of 2822) of the putative genes were differentially expressed in knobs versus mycelium. Several of these genes displayed sequence similarities to genes known to be involved in regulating morphogenesis and cell polarity in fungi. Among them were several putative homologues for small GTPases, such as rho1, rac1 and ras1, and a rho GDP dissociation inhibitor (rdi1). Several homologues to genes involved in stress response, protein synthesis and protein degradation, transcription, and carbon metabolism were also differentially expressed. In the last category, a glycogen phosphorylase (gph1) gene homologue, one of the most upregulated genes in the knobs as compared to mycelium, was characterized. A number of the genes that were differentially expressed in trap cells are also known to be regulated during the development of infection structures in plant-pathogenic fungi. Among them, a gas1 (mas3) gene homologue (designated gks1), which is specifically expressed in appressoria of the rice blast fungus, was characterized.

A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by blast searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified solute-binding protein (designated ArtJ) was demonstrated to bind l-arginine with high affinity (K d=0·39±0·06 μM). Competition experiments revealed only partial inhibition by excess l-lysine (38 %) and l-ornithine (46 %), while no inhibition was observed with l-histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus lipids, displayed ATPase activity in the presence of ArtJ only. Addition of l-arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 °C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0·71 μmol Pi min−1 (mg protein)−1 and a K m (ATP) of 1·59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.