- Volume 141, Issue 1, 1995

Expression of the Aspergillus nidulans brlA gene plays a fundamental role in the switch from vegetative growth to asexual reproduction. Using a media-shifting protocol to induce submerged sporulation and brlA-lacZ as an expression marker, it was shown that carbon and nitrogen starvation stress induced brlA transcription to different degrees. Glucose starvation induced brlA rapidly to high levels and resulted in spore formation on reduced conidiophores, whereas nitrogen starvation induced brlA gradually to lower levels and sporulation occurred to a lesser extent but from more complex conidiophores. β-Galactosidase activity paralleled brlAα and brlAβ mRNA. No clear qualitative differences between the two brlA transcripts were found in these starvation conditions, suggesting that the different patterns of sporulation could be explained by quantitative expression differences. Since brlA mRNA did not accumulate in the presence of a high glucose concentration, we investigated the role of other carbon sources on brlA expression. Non-repressing carbon sources such as glycerol, acetate and arabinose were as effective as glucose in preventing brlA mRNA accumulation, suggesting that the glucose effects on brlA expression could be explained as a response to nutrient starvation, rather than by carbon catabolite repression. Despite similar low levels of brlA transcripts being detected during growth in glucose or non-repressing carbon sources, conidiophores were formed only in medium containing glycerol, acetate or arabinose. When mycelia were not shifted to starvation conditions, sporulation was not observed in standard minimal medium even after glucose was exhausted, unless the medium was buffered. This and other results suggest that strong deviation from external neutral pH partially prevented full induction and/or function of brlA.

SUMMARY: The morphology and ultrastructure of two strains of Borrelia anserina were investigated by electron microscopy of negatively stained and ultrathin sectioned cells. One was a cultivable strain originally isolated in the USA and the other was originally isolated in Nigeria and maintained in chickens. The cells were regularly helical, 9-21 μm long and 0·22-0·26 μm wide with a helix wavelength of about 1·7 μm. The cells were surrounded by a surface layer and appeared to divide by binary fission. The structure of the cells from each of the two strains was identical except that those of the USA strain possessed seven flagella inserted at each end and those from the Nigerian strain had eight.

The capacity of Campylobacter jejuni to generate genetic diversity was determined for its flagellar region. Recombination within a genome, as well as recombination after the uptake of exogenous DNA, could be demonstrated. The subunit of the flagellar filament of C. jejuni is encoded by two tandem genes, flaA and flaB, which are highly similar and therefore subject to recombination. A spontaneous recombination within this locus was demonstrated in a bacterial clone containing an antibiotic-resistance gene inserted in flaA. A recombinant was isolated in which the antibiotic-resistance gene had been repositioned into flaB, indicating that genetic information can be exchanged between the two flagellin genes of C. jejuni. The occurrence of recombinational events after the uptake of exogenous DNA by naturally competent bacteria was demonstrated with two mutants containing different antibiotic-resistance markers in their flagellin genes. Double-resistant transformants were formed when purified chromosomal donor DNA was added to a recipient strain, when the two bacterial cultures were mixed under conditions that induce natural competence, or when the two strains were co-cultured. Both mechanisms of recombination may be used by the pathogenic organism to escape the immunological responses of the host or otherwise adapt to the environment.

To identify host genes that might influence nod (nodulation) gene expression in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carrying nodD) was introduced into a chemically mutagenized population of a R. leguminosarum strain lacking a symbiotic plasmid. The transconjugants were screened for expression of alkaline phosphatase (PhoA) on plates containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified. When the nodC-phoA plasmid was cured from the mutant and the symbiotic plasmid pRL1jl introduced, the mutant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal. When the nodC-phoA allele was introduced on pRL1jl a low level of nod gene induction was found. The reduced nodC expression appeared to be caused by a decrease in expression of the regulatory gene nodD, since expression of a nodD-lacZ fusion was also lower in the mutant than in the control. These mutant phenotypes and the low nitrogen fixation were complemented with a plasmid (plJ1848) from a R. leguminosarum cosmid library. DNA hybridization confirmed that plJ1848 was not from the symbiotic plasmid and showed that a DNA insertion was present in the mutant. The complementing region of plJ1848 was defined by transposon mutagenesis; DNA sequencing revealed that it carried the dicarboxylic acid transport (dct) genes. However, the mutant grew well with succinate as sole C-source. Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mutation caused the reduction in nod gene expression. The effect was allele-specific since other mutations in dctB did not influence nod gene expression. Surprisingly, the mutant had a constitutive high level of succinate transport, indicating that the mutation caused unregulated expression of dctA the structural gene for dicarboxylic acid transport. This in some way appears to have lowered the expression of nodD, indicating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB.

We isolated four azide-resistant secA mutants of Bacillus subtilis and found that all of them were the result of a single amino acid replacement of threonine 128 of SecA by alanine or isoleucine. In the presence of 1·5 mM sodium azide, cell growth and protein translocation of the wild-type strain were completely inhibited, but those of the azide-resistant mutant strains were not. Wild-type and two mutant SecA proteins were purified. Both the basal level and the elevated ATPase activity of the mutant SecA proteins were threefold higher than those of the wild-type SecA. The elevated ATPase activity of the SecA mutants was reduced upon the addition of 1·5 mM sodium azide by only 5-10% as compared with 40% for that of the wild-type. These results indicate that the elevated ATPase activity of the SecA mutants is resistant to sodium azide and that it is also required for the protein translocation process of B. subtilis.

A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55·5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods and immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.

SUMMARY: An Escherichia coli K12 mutant defective in both serine biosynthesis (serA) and glycine transport (cycA) was found to exhibit a glycine cleavage negative (GCV-) phenotype, i.e. was unable to use glycine as a serine source. While [2-14C]glycine uptake and induction of a λgcvT::lacZ fusion were greatly reduced in a cycA mutant compared to the wild-type, both strains exhibited parallel increases in uptake and induction with increasing exogenous glycine concentrations. A plasmid carrying the wild-type cyc region complemented the GCV-phenotype and restored both glycine uptake and glycine-inducible gcvT::lacZ expression. Wild-type and cycA strains grown in the presence of either a glycine-containing tripeptide or threonine, which can be degraded internally into glycine, exhibited similar induction of the gcvT::lacZ fusion. However, when a gcv mutation, which causes glycine to accumulate within the cell, was introduced into the cycA strain, there was increased induction of the gcvT::lacZ fusion, but induction was less than that observed in a gcv cycA+ strain. It is proposed that cyc serves primarily in the regulation of gcv by transporting glycine into the cell, which endogenously induces gcv expression. However, the possibility of some form of exogenous regulation of gcv, mediated by the cyc-encoded glycine transport system, exists.

A gene (ansP), which encodes an L-asparagine permease, has been isolated from a cosmid library of Salmonella enterica during screening for recombinant clones which encode L-asparaginase. Nucleotide sequence analysis reveals that the gene product is a polypeptide of 497 amino acid residues, containing 12 putative transmembrane segments. The calculated molecular mass is 54 kDa, although maxicell analysis by SDS-PAGE gave an apparent molecular mass of 37 kDa. Comparison of the deduced amino acid sequence with sequence databases showed significant homology with a family of basic and aromatic amino acid permeases. Strains containing the cloned ansP gene demonstrated a many-fold increase in L-asparagine uptake in comparison with control strains.

The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca 2+ , but not by fluoride or tartrate, and is enhanced by Mg 2+ , Co 2+ and Zn 2+ . At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

SUMMARY: Using a DNA probe from the gene encoding squalene-hopene cyclase (SHC, EC 5.4.99.−) from the Gram-positive bacterium Alicyclobacillus acidocaldarius, we have cloned a 4·3 kb Hindll fragment of chromosomal DNA from Zymomonas mobilis. An open reading frame of 1977 bp was detected that could encode a protein of 658 amino acids with a calculated molecular mass of 74077 Da. Under the control of lac or tac promoters, this gene, shc, was expressed in Escherichia coli K12 strains and its product had squalene-hopene cyclase activity. Sequence alignments with the A. acidocaldarius SHC, the lanosterol cyclase of the yeast Candida albicans, and the cycloartenol synthase of the plant Arabidopsis thaliana revealed six highly conserved regions (mainly in the C-terminal part) of the proteins. These regions contained the core motif Gln-X-X-X-Gly-X-Trp.

The murF gene encodes UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloyl-D-Ala-D-Ala synthetase (EC 6.3.2.15), which catalyses the final step in the synthesis of UDP-N-acetylmuramoyl-pentapeptide, the precursor of murein. An open reading frame identified as the murF gene was found in the genome of the cyanobacterium Synechocystis sp. PCC 6803. The murF gene encodes a polypeptide of 454 amino acid residues with a predicted molecular mass of 48 kDa. The murF gene is present as a single-copy gene in the genome of Synechocystis sp. PCC 6803. The amino acid sequence deduced from the murF gene is 39% identical to that of the product of the murF gene of Escherichia coli. The cyanobacterial murF gene complemented a temperature-sensitive mutation in a murF-deficient strain of E. coli, restoring the mutant's ability to synthesize a cell wall and to survive at high sublethal temperatures.

The hydrogenase enzyme of Clostridium acetobutylicum plays a pivotal role in controlling electron flow, and hence carbon flow, during the complex biphasic fermentation of carbohydrates to the neutral solvents acetone and butanol. We report here the cloning and molecular characterization of the hydrogenase-encoding gene (hydA) from C. acetobutylicum P262. This gene was isolated by colony hybridization, using the Clostridium pasteurianum hydrogenase-1 gene as a probe. The DNA sequence encoding the hydA gene from C. acetobutylicum was determined, and revealed an ORF (1722 bp) encoding a 574 amino-acid protein. This C. acetobutylicum hydrogenase protein product has 82% similarity and 67% identity with the C. pasteurianum hydrogenase-1 protein. Northern blot analysis of RNA isolated from C. acetobutylicum indicates that the C. acetobutylicum hydrogenase protein product is translated from a monocistronic operon. RNA was isolated from the different morphological and physiological stages of a batch C. acetobutylicum fermentation, and further Northern blot analyses revealed no differences in the expression of the gene during acidogenesis as opposed to solventogenesis. Primer extension experiments confirmed these results and identified the 5' start of the mRNA transcript. These results correlated well with the physiological need for this organism to dispose of excess reducing equivalents.

SUMMARY: An efficient electrotransformation system for Streptococcus suis type 2 is described. It is demonstrated that vectors based on the broad-host-range plasmid pWVO1 replicate in S. suis type 2. Transformation efficiencies of about 107 transformants per μg of plasmid DNA could be obtained. Derivatives of plasmid pBR322 containing S. suis chromosomal DNA did not replicate but integrated into the chromosome. Southern hybridization analysis revealed that double as well as single cross-over integration events had occurred. Double cross-over events occurred at a frequency of about 15%. With these transformation and integration systems, recombinant DNA technology can now be applied to this important pathogenic species.

SUMMARY: Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity. Approximately 3 mg purified enzyme (specific activity 3300 U mg-1) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pl 4·0-4·4) was observed to exist as a homodimer (Mr 57000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH2-terminus were determined by step-wise Edman degradation. This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR. The probe was used to isolate a molecular clone of tpi from a λGEM11 library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit Mr of 26802 after removal of the NH2-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15-fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene. Northern analysis of L. lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi. The 5′ end of the transcript was determined by primer extension analysis to be a G located 64 bp upstream from the tpi start codon. These transcript analyses indicated that in L. lactis, tpi is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme. The location of tpi on the L. lactis DL11 chromosome map was determined to be between map coordinates 1·818 and 1·978.

Staphylococci grow and cause infection under the iron-restricted conditions found in vivo. They therefore must possess mechanisms to obtain iron for metabolism from this environment. To determine if staphylococci can extract iron bound to human transferrin, we labelled transferrin with 55 Fe and performed uptake assays on cells grown in iron-restricted and iron-plentiful conditions. Growing cultures of Staphylococcus aureus NCTC 8532 could take up radioactive iron during mid- to late-exponential phase of growth. This process was iron-regulated and did not require direct contact between the cell and the labelled transferrin. Siderophore production was detected during this phase, but reductase or protease activity was not. S. epidermidis ATCC 14990 could not access55 Fe bound to transferrin, nor did this isolate produce siderophore, reductase or protease. This difference in the ability to acquire iron bound to transferrin may contribute to the increased virulence of S. aureus when compared to S. epidermidis.