- Volume 146, Issue 4, 2000

Sulphate permeases in the plasma membrane are responsible for uptake of environmental sulphate used in the sulphate assimilation pathway in bacteria and plants. Here it is reported that the ORF designated cysP, located on the Bacillus subtilis chromosome between cysH and five putative genes involved in sulphate assimilation, encodes a sulphate permease. cysP is able to complement Escherichia coli cysteine auxotrophs with mutations affecting either the membrane or periplasmic components of the sulphate-thiosulphate permease. Transport studies with cell suspensions of a cysA97 E. coli strain transformed with a plasmid expressing the B. subtilis cysP gene indicated that CysP catalyses sulphate uptake. Analysis of the primary sequence showed that CysP (354 amino acids, estimated molecular mass 24 kDa) is a highly hydrophobic protein which has 11 putative transmembrane helices. Sequence comparisons revealed that CysP, together with the phosphate permease of Neurospora crassa, Pho-4, and E. coli PitA, belongs to the family of related transporters, the inorganic phosphate transporter (Pit) family. Among the putative phosphate permeases, CysP shows a similar size and the same domain organization as the archaeal transporters. This is the first report of a sulphate permease in a Gram-positive organism.

A mutation was isolated in the Rhizobium leguminosarum gene fhuA, which appears to specify the outer-membrane receptor for the siderophore vicibactin. The mutant was defective in iron uptake and accumulated the siderophore vicibactin in the extracellular medium. Expression of fhuA was regulated by Fe3+, transcription being higher in iron-depleted cells. Transcription of fhuA was independent of a functional copy of rpoI, a neighbouring gene that specifies a putative ECF σ factor of RNA polymerase and which is involved in siderophore production in Rhizobium. Mutations in fhuA did not detectably affect symbiotic N2 fixation on peas. An fhuA::gus fusion was expressed by bacteria in the meristematic zone of pea nodules but not in mature bacteroids. Some other strains of R. leguminosarum also contain a pseudogene version of fhuA. The sequences of some of these and the ‘real’ fhuA genes were determined.

In rhizobia, besides the constitutive acyl carrier protein (AcpP) involved in the biosynthesis and transfer of common fatty acids, there are at least three specialized acyl carrier proteins (ACPs): (1) the flavonoid-inducible nodulation protein NodF; (2) the RkpF protein, which is required for the biosynthesis of rhizobial capsular polysaccharides; and (3) AcpXL, which transfers 27-hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis. Whereas the nucleotide sequences encoding the three specialized ACPs are known, only the amino acid sequence of the AcpP of Sinorhizobium meliloti was available. In this study, using reverse genetics, the genes for the constitutive AcpPs of S. meliloti and of Rhizobium leguminosarum were cloned and sequenced. Previously, it had been shown that NodF and RkpF can be overproduced in Escherichia coli using the T7 polymerase expression system. Using the same system, the constitutive AcpPs of S. meliloti and of R. leguminosarum, together with the specialized ACP AcpXL, were overproduced and purified. All the known ACPs of rhizobia can be labelled in vivo during expression in E. coli with radioactive β-alanine added to the growth medium due to their modification with a 4’-phosphopantetheine prosthetic group. The availability of all functionally different ACPs should help to unravel how different fatty acids are targeted towards different biosynthetic pathways in one organism.

Two types of Tn1732-induced mutants defective in extracellular amylase activity were isolated from the moderate halophile Halomonas meridiana DSM 5425. Type I mutants displayed amylase activity in the periplasm, and were unable to use any of the carbon sources tested, including starch and its hydrolysis product maltose. The type II mutant was affected in the gene responsible for the synthesis of the extracellular α-amylase. This gene (amyH) was isolated by functional complementation of mutant II and sequenced. The deduced protein (AmyH) showed a high degree of homology to a proposed family of α-amylases consisting of enzymes from Alteromonas (Pseudoalteromonas) haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals. AmyH contained the four highly conserved regions in amylases, as well as a high content of acidic amino acids. The amyH gene was functional in the moderate halophile Halomonas elongata and, when cloned in a multicopy vector, in Escherichia coli. AmyH is believed to be the first extracellular-amylase-encoding gene isolated from a moderate halophile, a group of extremophiles of great biotechnological potential. In addition, H. meridiana and H. elongata were able to secrete the thermostable α-amylase from Bacillus licheniformis, indicating that members of the genus Halomonas are good candidates for use as cell factories to produce heterologous extracellular enzymes.

Candida albicans, like other pathogens, has to compete with the host for a limited supply of available iron. Consequently, iron acquisition is likely to be an important factor for the growth, survival and virulence of this organism. It was previously demonstrated that C. albicans has a surface-associated ferric reductase similar to that of Saccharomyces cerevisiae. Therefore, functional rescue of a S. cerevisiae fre1 mutant was used to isolate a C. albicans ferric reductase gene (CFL1). This gene has been previously identified. However, the workers had not observed any functional reductase activity associated with the gene. The discrepancy with the findings in this report appears to be due to the clone previously reported carrying a non-contiguous piece of C. albicans DNA. Results shown in this paper demonstrate that CFL1 transcription is regulated in response to levels of iron and copper. This is the first demonstration of a functional ferric reductase gene from C. albicans.

The heat inducibility of the yeast heat-shock response (HSR) pathway has been shown to be critically dependent on the level of unsaturated fatty acids present in the cell. Here the inducibility by heat or salt of the independently regulated general stress response (GSR) pathway is shown to be affected in the same way. An increase in the percentage of unsaturated fatty acids in heat- or salt-acclimated cells correlated with a decrease in the induction of a general stress-response-promoter-element (STRE)-driven reporter gene by either stress. Despite inducing reporter gene expression, sorbic acid treatment did not confer salt cross-tolerance on the cells. This failure correlated with a failure to increase the percentage of unsaturated fatty acids in the cells, suggesting that GSR pathway induction, in the absence of lipid changes, is insufficient for the induction of cross-tolerance. Cells grown with fatty acid supplements under anaerobic conditions provided further evidence for a potential role for lipids in the acquisition of stress resistance. These cells contained different fatty acid profiles depending on the fatty acid supplement supplied, exhibited differential sensitivity to both heat and salt stress, but had not undergone STRE induction. These results suggest that heat- and salt-stress induction of the GSR are sensitive to the level of unsaturated fatty acids present in the cell and that stress cross-tolerance may be a lipid-mediated phenomenon. Given that an increased level of unsaturated fatty acids also down-regulates heat induction of the HSR pathway, these observations lead to the provocative hypothesis that lipid modifications, rather than HSR or GSR pathway induction, are a major contributor to the induced heat and salt tolerance of yeast cells.

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pathovar campestris (Xcc) is subject to co-ordinate regulation by a cluster of genes called rpf (for regulation of pathogenicity factors). These genes are located within a 21·9 kb region of the chromosome isolated as the cosmid clone pIJ3020. The genes in the left-hand section of this region of the chromosome have previously been characterized. This paper reports on the genes in the right-hand section and on the phenotypes of mutants with transposon insertions in these genes. Sequence analysis identified eight genes or ORFs with the gene order rpfD–orf1–orf2–orf3–orf4–recJ–rpfE–greA. RecJ and GreA have established functions in recombination and transcriptional elongation, respectively. rpfD encoded a protein with some amino acid sequence relatedness to a hypothetical protein from Caulobacter crescentus and an autolysin response regulator in Bacillus subtilis. The predicted protein products of orf1, 2 and 3 were related to each other and had substantial amino acid sequence relatedness to hypothetical proteins from C. crescentus. Transposon insertions in orf1, 2 and 3 had no effect on the synthesis of extracellular enzymes or EPS. The predicted proteins RpfE and Orf4 showed the highest amino acid sequence relatedness to hypothetical proteins from Bordetella pertussis and Klebsiella pneumoniae, respectively. Transposon insertions in rpfE led to reduced levels of some extracellular enzymes (endoglucanase and protease) and increased levels of others (polygalacturonate lyase). Transposon insertions in orf4 had no effect on polygalacturonate lyase but led to reduced levels of protease and endoglucanase. Levels of EPS were reduced in both rpfE and orf4 mutants. These alterations in the levels of extracellular enzymes, which were relatively modest (between two- and threefold), did not affect the pathogenicity of Xcc on turnip. It is proposed that the gene designation should be rpfI for orf4.

Analysis of a region of chromosomal DNA lying between jadR 1 and jadI in the gene cluster for jadomycin biosynthesis in Streptomyces venezuelae ISP5230 detected an ORF encoding 584 amino acids similar in sequence to the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) components of acyl-coenzyme A carboxylases. Multiple sequence alignments of the deduced Jad protein with acyl-coenzyme A carboxylases from various sources located the BC and BCCP components in the N- and C-terminal regions, respectively, of the deduced polypeptides. The organization and amino acid sequence of the deduced polypeptide most closely resembled those in other Gram-positive bacteria broadly classified as actinomycetes. Disrupting the gene, designated jadJ, severely reduced but did not eliminate jadomycin production. The disruption had no effect on growth or morphology of the organism, implying that the product of jadJ is not essential for fatty acid biosynthesis. It is concluded that jadJ supplies malonyl-coenzyme A for biosynthesis of the polyketide intermediate that is eventually processed to form the antibiotic jadomycin B.

The chromosome of Streptomyces lividans shares 15·4 kb homology with one end of the linear plasmid SLP2, consisting of a 10·1 kb terminal sequence followed by the 5·3 kb transposable element Tn4811. The 10·1 kb terminal sequence was determined. The mean G+C content of this sequence is 67·9 mol% with a striking G vs C bias in the last kb. The terminal 232 nt contained 10 palindromic sequences with potential to form complex secondary structures. One typical Streptomyces coding sequence (designated ORF1) of 2643 bp was predicted in the determined sequence. The amino acid sequence of the ORF1 product contained a DEAH helicase motif, and exhibited similarity to type I restriction enzyme HsdR subunits in the database, suggesting a possible role in replication of the telomeres. However, all the ORF1 sequences on the chromosome and SLP2 could be simultaneously knocked out by targeted recombination without affecting the viability of the cells and the linearity of the chromosome and SLP2. This ruled out ORF1 as an essential component in the maintenance of the linear chromosome and plasmids.

High-copy-number amplification of the AUD1 element is frequently associated with the large chromosomal deletions responsible for genetic instability in Streptomyces lividans TK64. Five ORFs were found in a 7 kb region directly adjacent to AUD1. The putative products of ORF1, ORF2 and ORF3 showed similarities to ATP-binding cassette (ABC) sugar transporters, the deduced protein sequence of ORF4 displayed similarities to α-glucosidases whilst no homology to proteins with known functions was found for ORF5. ORF4 (renamed aglA) was expressed in Escherichia coli and the protein purified and characterized. An α-glucosidase activity was detected using the synthetic α-glucoside p-nitrophenyl α-D-glucopyranoside. Of the many oligosaccharides tested, only sucrose was hydrolysed at a measurable rate [specific activity 32·4 units (mg protein)−1] but no growth of S. lividans TK64 on sucrose was observed. A strain in which aglA was disrupted showed the same low α-glucosidase activity as strain TK64 and in both strains no stimulation of activity was seen by sucrose, trehalose or maltose; dextrin increased α-glucosidase activity about 10-fold. This probably resulted from induction of a second α-glucosidase-encoding gene. The AUD1 element contains three 1 kb repeats which encode DNA-binding proteins necessary for high-frequency amplification. In strains with a unique 1 kb repeat, disruption of the repeat led to a significant increase in the α-glucosidase activity. These results strongly suggest that the 1-kb-repeat-encoded proteins of AUD1 have a dual function: they are the repressors of the agl genes and they promote amplification of AUD1.

The genetic elements specifying six putative two-component regulatory systems (2CSs) were identified on the chromosome of Lactococcus lactis MG1363. These 2CSs appear to represent distinct loci, each containing a histidine kinase and response-regulator-encoding gene pair. Transcriptional analysis of the six 2CSs was performed either by generating transcriptional fusions to a reporter gene or by primer extension. Two of the systems appeared to be expressed constitutively at a high level, whilst the remaining four exhibited growth-phase-dependent expression. Insertional mutagenesis established that the two constitutively expressed 2CSs are necessary for normal cell growth and/or survival. Mutational analysis of the remaining four systems revealed that they are implicated in susceptibility to extreme pH, osmotic or oxidative conditions, or the regulation of phosphatase activity in L. lactis.

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of an O-linked trisaccharide, Gal(β1-4)Gal(α1-3)2,4-diacetimido-2,4,6-trideoxyhexose. In a previous study the authors identified and characterized a gene, pglA, encoding a galactosyltransferase involved in pilin glycosylation. In this study a set of random genomic sequences from N. meningitidis strain MC58 was used to search for further genes involved in pilin glycosylation. Initially, an open reading frame was identified, and designated pglD (pilin glycosylation gene D), which was homologous to genes involved in polysaccharide biosynthesis. The region adjacent to this gene was cloned and nucleotide sequence analysis revealed two further genes, pglB and pglC, which were also homologous with genes involved in polysaccharide biosynthesis. Insertional mutations were constructed in pglB, pglC and pglD in N. meningitidis C311#3, a strain with well-defined LPS and pilin-linked glycan structures, to determine whether these genes had a role in the biosynthesis of either of these molecules. Analysis of these mutants revealed that there was no alteration in the phenotype of LPS in any of the mutant strains as judged by SDS-PAGE gel migration. In contrast, increased gel migration of the pilin subunit molecules of pglB, pglC and pglD mutants by Western analysis was observed. Pilin from each of the pglB, pglC and pglD mutants did not react with a terminal-galactose-specific stain, confirming that the gel migration differences were due to the alteration or absence of the pilin-linked trisaccharide structure in these mutants. In addition, antisera specific for the C311#3 trisaccharide failed to react with pilin from the pglB, pglC, pglD and galE mutants. Analysis of nucleotide sequence homologies has suggested specific roles for pglB, pglC and pglD in the biosynthesis of the 2,4-diacetimido-2,4,6-trideoxyhexose structure.

P27 is an antigenic membrane lipoprotein synthesized by members of the Mycobacterium tuberculosis complex. Northern blotting and RT-PCR experiments indicated that the genes encoding P27 and a putative antibiotic transporter (P55) form an operon. A promoter region was identified and characterized by deletion analysis in Mycobacterium smegmatis. Two transcription initiation points were mapped in Mycobacterium bovis BCG by primer extension analysis to 76 bp and 87 bp upstream of the ATG initiation codon. Putative −10 and −35 promoter consensus sequences associated with these showed 66% similarity to previously identified mycobacterial promoters. These results suggest that the P27/P55 operon is transcribed from two promoters in M. bovis BCG.

Synonymous codon usage in the complete genome of Helicobacter pylori was investigated. The moderate A+T-richness of the genome (G+C=39 mol%) is reflected in the overall synonymous codon usage but the frequencies of some codons cannot be explained by simple mutational biases. A low level of heterogeneity among genes was observed, but this does not appear to be due to varying mutational bias or translational selection. Some of the heterogeneity was due to amino acid composition variation among the encoded proteins, and some may be attributable to recent acquisition of genes from other species. Since Hel. pylori codon usage is not dominated by biased mutation patterns, the absence of evidence for translationally mediated selection among synonymous codons is striking. This has implications with regard to the life history of this species, and in particular suggests that Hel. pylori strains are not subject to periods of competitive exponential growth. Despite the lack of selected codon usage, base composition immediately after the translation initiation site is skewed, consistent with selection against secondary structure formation in this region.