- Volume 146, Issue 2, 2000

The gene for cytochrome cd 1 nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS. This gene is shown to be flanked by genes previously recognized in other organisms to encode proteins involved in the control of its transcription (nirI) and the biosynthesis of the d 1 cofactor (nirE). Northern blot analysis has established under anaerobic conditions that a monocistronic transcript is produced from nirS, in contrast to observations with other denitrifying bacteria in which arrangement of flanking genes is different and the messages produced are polycistronic. The lack of a transcript under aerobic conditions argues against a role for cytochrome cd 1 in the previously proposed aerobic denitrification pathway in Pa. pantotrophus. A putative rho-independent transcription termination sequence immediately following nirS, and preceding nirE, can be identified. The independent transcription of nirS and nirE indicates that it should be possible to produce site-directed mutants of nirS borne on a plasmid in a nirS deletion mutant. The transcript start point for nirS has been determined by two complementary techniques, 5′-RACE (Rapid amplification of cDNA 5′ ends) and primer extension. It is 29 bp upstream of the AUG of nirS. An anaerobox, which presumably binds Nnr, is centred a further 41·5 bp upstream of the transcript start. No standard σ70 DNA sequence motifs can be identified, but a conserved sequence (T-T-G/C-C-G/C-G/C) can be found in approximately the same position (−16) upstream of the transcript starts of nirS and nirI, whose products are both involved in the conversion of nitrite to nitric oxide.

The causative agent of gas gangrene, Clostridium perfringens, is a Gram-positive anaerobe which produces a number of extracellular toxins and enzymes. The production of several of these toxins is regulated by the VirS/VirR two-component signal transduction system. The sensor histidine kinase, VirS, contains motifs that are conserved amongst sensor histidine kinases, although not in the same relative positions. In this study, the conserved histidine residue (H255), the GXGL and DXGXG motifs, and two glutamate residues located in putative transmembrane domains were altered by site-directed mutagenesis to examine their significance for VirS function. Introduction of the mutated virS genes into the virS::Tn916 mutant, JIR4000, showed that the altered virS genes were not able to complement the host mutation. These results demonstrate that the conserved motifs, including the cytoplasmic DXGXG motif which is located between the putative transmembrane domains 4 and 5, are functional. Furthermore, it is concluded that charged residues located within two of these transmembrane domains are also required for the structural or functional integrity of the VirS sensor kinase.

The whiB sporulation gene of Streptomyces coelicolor was shown [Davis, N. K. & Chater, K. F. (1992) R8 . Mol Gen Genet 232, 351–358] to encode a small, cysteine-rich putative transcription factor unlike any that had been described previously. The large database of DNA sequences of mycobacteria (like Streptomyces, members of the Actinomycetales) has revealed a family of genes encoding proteins related to WhiB. Mycobacterium tuberculosis contains at least six such genes (whiB homologues in mycobacteria: whmA–F) and a likely seventh, whmG. Using conserved features of Whm proteins, a PCR-based approach led to the discovery that S. coelicolor A3(2) contains several similar genes. Cloning and sequencing of these whiB-like (wbl) genes revealed likely orthologues of four of the whm genes of M. tuberculosis. In all, S. coelicolor contains at least five wbl genes in addition to whiB itself. All five were shown by RT-PCR to be transcribed. A Southern blotting survey using each wbl gene as a probe showed that nearly all of a series of representatives of ten actinomycete genera (including morphologically simple organisms) contain close homologues of several wbl genes, suggesting that the ancient progenitor of all these organisms already contained a family of such genes, which have not been found in any other organisms.

One of the long-chain alkyl esters of AMP, adenosine 5′-hexadecylphosphate (AMPC16), exhibited a cytotoxic growth inhibitory effect on cells of various yeast strains. The growth inhibitory effect of AMPC16 on Saccharomyces cerevisiae cells was observed only in medium containing Mg2+, which accelerated cellular uptake of the nucleotide analogue. In the presence of Mg2+, AMPC16 completely inhibited glucose-induced extracellular acidification by the intact cells and also interfered with activation of the plasma membrane ATPase, but did not directly inhibit the ATPase activity itself. AMPC16 treatment prevented cells from increasing their intracellular sn-1,2-diacylglycerol (DAG) level in response to glucose, whereas the inhibition of proton extrusion by the cells could be largely reversed by the coaddition of a membrane-permeable DAG analogue. The DAG analogue, a physiological activator of protein kinase C (PKC), was not protective against the inhibition of glucose-induced proton extrusion by staurosporine, which is capable of directly interfering with the action of PKC. These results implied that AMPC16 caused a Mg2+-dependent cytotoxic effect on Sac. cerevisiae cells by interfering with a phosphatidylinositol type of signal that mediates activation of the plasma membrane proton pump.

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

The primitive free-living protozoon Hexamita inflata was found to maintain a cell volume of approximately 260 fl under standard culture conditions. On increasing the extracellular osmolality the volume decreased and the cells remained shrunken for >30 min. By contrast, a decrease in the external osmolality resulted in a transient increase in cell volume which was followed by an efficient ‘regulatory volume decrease’ (RVD). H. inflata contains high concentrations of amino acids, with alanine constituting over 70% of the total amino acid pool. Exposure to hypo-osmotic medium resulted in the loss from the cell of both amino acids and K+, via one or more swelling-activated pathways. The efflux of amino acids and K+, together with a charge-balancing counter-anion, accounted almost fully for the observed RVD. The pharmacological properties of the swelling-activated pathways differ from those of volume-sensitive transporters and channels described previously in other cell types.