- Volume 141, Issue 5, 1995

The products of anaerobic metabolism of glucose and its derivatives sorbitol, gluconate and glucuronate by Bacillus licheniformis have been determined by proton NMR. Glucose was fermented through mixed-acid fermentation pathways to acetate, 2,3-butanediol, ethanol, formate, lactate, succinate and pyruvate. However, the bacterium was incapable of fermenting the three glucose derivatives. When B. licheniformis cells were incubated anaerobically with glucose in the presence of nitrate, the reduced products and formate did not appear and acetate was formed as the major metabolite. Growth and formation of acetate was also observed when B. licheniformis cells were incubated anaerobically with each of the three glucose derivatives, in the presence of nitrate. A formate-nitrate oxido-reductase system was induced under anaerobic conditions, with increased activities when nitrate was added to the anaerobic growth medium. However no activity was detected when cell; were grown in the presence of molecular oxygen. Formate-nitrate oxido-reductase activity was absent in chlorate-resistant mutants isolated spontaneously or following Tn917 insertional mutagenesis. The spontaneous mutants fermented glucose in the presence of nitrate suggesting that they were incapable of nitrate respiration, due to a deficiency in one or more components of the formate-nitrate oxido-reductase system. Two insertional mutants exhibited elevated β-galactosidase activity when grown in the presence of nitrate.

Pulsed-field gel electrophoresis (PFGE) was used to examine chromosomal DNA from various strains of Thiobacillus ferrooxidans: these were the reference strain VKM-458, strains isolated from different environments and pilot plants for processing gold-bearing concentrates, and strains experimentally adapted to high zinc and arsenic concentrations in growth medium. The restriction endonuclease Xbal digested T. ferrooxidans VKM-458 chromosomal DNA into a number of fragments sufficient for identification of their size and calculation of the size of the entire genome (2855/pm44 kb). Restriction fragment length polymorphism of the chromosomal DNA in various strains suggests the usefulness of this approach for analysis of the diversity of T. ferrooxidans strains and for the study of strain stability under conditions of industrial utilization. A comparison of Xbal-restriction patterns in parent strains and in strains with acquired enhanced resistance to zinc or arsenic revealed amplification of certain fragments in the resistant strains, i.e. a 98 kb fragment in strain TFZ and a 28 kb fragment in strain VKM-458As2. We suggest that the enhanced resistance to toxic metals in T. ferrooxidans is gained through increase of the copy number of resistance genes and enhanced synthesis of proteins involved in resistance.

The carbamoyl-phosphate synthase (CPS) enzyme in prokaryotes is a heterodimer, encoded by genes commonly called carA and carB. In most prokaryotes examined, these genes are separated by up to 24 bp and are cotranscribed. In Pseudomonas aeruginosa, carA and carB are also co-transcribed, but are separated by 682 bp. We have determined the complete DNA sequence of the carA and carB genes of Neisseria gonorrhoeae strain CH811. carA (1125 bp) and carB (3237 bp) are similar in size and sequence to other prokaryotic CPS genes, however they are separated by an intervening sequence of 3290 bp which has no similarity to the intervening sequence between other CPS genes; furthermore, putative transcription terminators are found downstream of both carA and carB. Several neisserial repetitive sequences were identified within the 9 kb sequenced, as well as novel 120 and 150 bp repeats (designated RS6 and RS7, respectively) which were found within the intervening sequence between carA and carB. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was not unusual, the sequence between carA and carB was amplified by PCR from 30 isolates of N. gonorrhoeae. The intervening sequence was found to vary in size, from approximately 2·2 to 3·7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS enzyme. A similar large, variably sized intervening sequence was also found between the carA and carB genes of 12 isolates of N. meningitidis and 18 commensal Neisseria isolates comprising nine species. This unexpected organization of the CPS genes in N. gonorrhoeae is therefore widespread throughout the genus Neisseria.

The dnal gene of Bacillus subtilis, previously identified through the isolation of the dnal2 mutant, was found to be the second gene of the dnaB operon. The nucleotide substitution in the dnal2 mutant gene was determined.

The surface ultrastructure of three anaerobic Gram-positive cocci frequently encountered in oral infections, Peptostreptococcus micros, P. magnus and P. anaerobius, was studied. The type strains of P. micros (DSM 20468) and P. anaerobius (ATCC 27337), several clinical isolates of both species and the type strain of P. magnus (DSM 20470) were included. Thin-sectioned cells studied by electron microscopy revealed a homogeneous layer outside the peptidoglycan layer in P. anaerobius. In P. micros and P. magnus a more amorphous layer was present. No periodic structures were seen in negatively stained whole cells of these three species. However, in freeze-etched cells of P. anaerobius a crystalline surface protein layer (S-layer) was detected. No periodicity was seen in any of the P. micros strains or the magnus type strain by the methods used, but a periodic pattern was observed in negatively stained specimens of cell wall fragments of sonicated P. anaerobius cells. No capsular material was visible outside the S-layer in P. anaerobius. The cells of the Peptostreptococcus spp. were extracted for 30 min with detergents and urea. One er cent SDS and 6 M urea both extracted a major 78 kDa protein from all strains of P. anaerobius. Extraction of P. micros and P. magnus cells did not reveal any major protein bands comparable to that of P. anaerobius. Surface biotinylation of cells followed by Western blotting and detection by alkaline-phosphatase-conjugated extravidin showed strong staining of the 78 kDa band in P. anaerobius, further indicating that this molecule is located on the surface of the cell and is the S-protein. Another protein, of 127 kDa, was also detected by surface biotinylation in all P. anaerobius strains. No differences were detected in colony morphology or cell surface structures between the four strains. Isoelectric focusing of the proteins of P. anaerobius revealed that the S-proteins of individual strains have different pl values, ranging from 5.3 to 6.9.

The strictly anaerobic Gram-negative beer spoilage bacteria Megasphaera cerevisiae, Pectinatus cerevisiiphilus and P. frisingensis were subjected to cellular fatty acid analysis, employing acid- and base-catalysed cleavage, gas chromatography and mass spectrometry. M. cerevisiae contained 12:0, 16:0, 16:1, 18:1, 17:cyc, 19:cyc, 12:0(3OH), 14:0(3OH) as the main fatty acids, and alk-1-enyl chains instead of acyl chains were detected to a considerable extent (14% of total fatty acids), indicating the presence of plasmalogens. The fatty acid pattern of M. cerevisiae was almost identical to that of M. elsdenii, the only species previously assigned to this genus. P. cerevisiiphilus and P. frisingensis yielded fatty acids that were heavily dominated by odd-numbered chains; 11:0, 15:0, 17:1, 18:cyc and 13:0(3OH) were the main fatty acids detected in both species. Alk-1-enyl chains with similar chain lengths were also found. Both Pectinatus species contained six different 3-hydroxy fatty acids with chain lengths between 11 and 15 carbons, 13:0(3OH) being dominant and the others accounting for generally less than 1% of total fatty acids. Among the minor components, an unsaturated 3-hydroxy fatty acid was detected which was shown to be 13:1(30H). In addition, fatty acid analysis was shown to be applicable to detection of bacterial contamination of beer.

Following the removal of the rodlet layer from aerial or submerged conidia of the entomopathogenic deuteromycetous fungus Beauveria bassiana, SDS-insoluble, formic-acid-extractable proteins were found in the residual cell wall material. Two major proteins (12·8 and 14·0 kDa) were extracted with formic acid from fractured aerial and submerged conidia but not from blastospores. Oxidation of the sample extracted by formic acid resulted in a single protein band (15·4 kDa) as judged by SDS-PAGE. Antibodies against this cell wall protein (cwp1) did not cross-react with cell wall extracts from the entomopathogenic deuteromycetous fungi Verticillium lecanii or Metarhizium anisopliae. Western blot analysis of two-dimensional gels revealed at least three acidic isoforms (pl 4·0-4·8) of cwp1. Immunohistological studies revealed that the cwp1 was primarily localized in the cell wall of aerial and submerged conidia but not in blastospores, Immunolocalization was possible only if the conidia were previously boiled in 5% (v/v) β-mercaptoethanol. The N-terminal sequence of cwp1 showed no similarities with other published sequences. Our results suggest that at least two major species of SDS-insoluble, formic-acid-extractable proteins exist in cell walls of B. bassiana aerial or submerged conidia; one is the hydrophobin which occurs in the outermost rodlet layer and the other, cwp1, occurs primarily next to the rodlet layer.

A second NAD-dependent valine dehydrogenase (VDH) of Streptomyces fradiae was detected and purified to homogeneity by affinity chromatography on Reactive-Blue 2 Sepharose followed by gel filtration and Mono Q fast protein liquid chromatography. The relative molecular masses of the native enzyme and its subunits were determined to be 80000 and 41000, respectively, indicating that the enzyme is a homodimer. The enzyme was the only active VDH in S. fradiae; its activity was significantly induced by L-valine, but was repressed by ammonia. Among branched- and straight-chain amino acids that serve as enzyme substrates, L-2-aminobutyrate and L-valine are preferred. Significant activities were found with deamino-NAD+ and 3-pyridinealdehyde-NAD-. The molecular and catalytic properties of the enzyme distinguish it from the enzyme previously purified, and thus indirectly indicate the existence of two VDHs in S. fradiae.

To develop a rapid and accurate method of typing large numbers of clinical isolates of Staphylococcus aureus, the spacer region C of the rRNA operon [1391-507 (16S-23S)] was enzymically amplified from 322 strains. When the products were separated by denaturing PAGE, 15 variable-length rrn alleles were demonstrated, ranging in size from 906 to 1223 bp. The variable-length HpaII-digested region C [(region E; 1446-196 (16S-23S)] amplification products were cloned into M13mp18RF to sequence separate variable-length alleles. A total of 17 region E inserts were sequenced, aligned and divided into nine alleles by length (938-1174) and sequence properties. The 16S-23S spacer rDNA varied in length (303-551 bp) and in properties; three alleles contained a tRNAlle gene alone, two alleles contained a tRNAlle and a tRNAAla gene, and four alleles lacked tRNA genes. The sequences of two alleles showed less than 1% variation when isolated from two or three S. aureus strains. The 48 penicillin-and methicillin-sensitive strains were divided into 26 ribotypes; in contrast, the 274 methicillin-resistant S. aureus (MRSA) strains were divided into nine ribotypes (A-I) with 97% typing as either ribotype A or B (rrnL was missing in B). The sequence conservation of the rrn operons argues for the use of the 16S-23S spacer region as a stable and direct indicator of the evolutionary divergence of S. aureus strains.

Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.

Although glycerol is not taken up via the phosphotransferase system (PTS) in Bacillus subtilis, some mutations that affect the general components of the PTS impair the ability of cells to grow on glycerol. Five revertants of a pts deletion mutant that grow on glycerol were analysed. They were shown to carry mutations in the glycerol kinase gene. These are missense mutations located in parts of the glpK gene that could encode regions important for the activity of glycerol kinase. The results strongly suggest that the main effect of the PTS on glycerol utilization in B. subtilis is mediated via glycerol kinase.

Plasmids belonging to Escherichia coli incompatibility group P are of particular interest because they can transfer between, and be stably maintained in, almost all Gram-negative bacterial species. The segment of the IncPα plasmid genome between the key regulatory gene korA and the vegetative replication origin, oriV, encodes a series of operons co-regulated with replication and transfer functions by the KorA protein. To determine which of these genes are likely to have an important role in IncP plasmid survival the equivalent region of the distantly related IncPβ plasmid R751 was sequenced. Sequence comparisons show that the kla operon (formerly the kilA locus, which is also responsible for a cryptic tellurite-resistance determinant) is completely absent from R751. Similarly in the kle region, which encodes genes associated with the KilE+ phenotype of unknown function, kleC and kleD, which we proposed arose by a duplication of kleA and kleB, are also completely absent. The genes that are conserved are klcA (formerly kilC, responsible for the KilC+, and recently proposed to be involved in overcoming restriction barriers during transfer), klcB (an ORF interrupted by Tn 1 insertion in RK2), korC (a transcriptional repressor which controls the kleK and kle operons), and kleA, kleB, kleE and kleF. A striking feature of the organization in R751 is the lack of the strong transcriptional termination signals which are present in IncPα plasmids. The degree of divergence between the plasmids facilitates the identification of motifs of probable functional importance in the primary protein sequences.

A Candida albicans peptide transport gene, CaPTR2, was cloned from a C. albicans genomic library by functional complementation of a peptide transport deficient mutant (strain ptr2-2) of Saccharomyces cerevisiae. CaPTR2 restored peptide transport to transformants as determined by uptake of radiolabelled dileucine, growth on dipeptides as sources of required amino acids, and restoration of growth inhibition by toxic peptides. Plasmid curing experiments demonstrated that the peptide transport phenotype was plasmid borne. CaPTR2 was localized to chromosome R of C. albicans by contour-clamped homologous electric field gel chromosome blots. Deletion subclones and frameshift mutagenesis were used to narrow the peptide transport complementing region to a 5:1 kb DNA fragment. DNA sequencing of the complementing region identified an ORF of 1869 bp containing an 84 nucleotide intron. The deduced amino acid sequence predicts a protein of 70 kDa consisting of 623 amino acids with 12 hydrophobic segments. A high level of identity was found between the predicted protein and peptide transport proteins of S. cerevisiae and Arabidopsis thaliana. This study represents the first steps in the genetic characterization of peptide transport in C. albicans and initiates a molecular approach for the study of drug delivery against this pathogen.

Specific pathogen-free C57BL/6 mice infected with recombinant Mycobacterium smegmatis (rM. smegmatis) bearing BCG genes showed increased splenic survival compared to those receiving the vector control (plasmid DNA only). The mouse-passaged rM. smegmatis (J3R) survived in peritoneal macrophages better than the vector control, regardless of whether the macrophages were infected in vivo or in vitro. When rM. smegmatis J3R was cultured in synthetic Proskauer-Beck-Tween medium, protein bands characteristic of BCG culture filtrates and not present in the vector control preparation were observed. Mice immunized with two doses of heat-killed J3R suspended in Freund's adjuvant were able to limit the growth of virulent Mycobacterium tuberculosis within the lung and spleen compared to that observed in control mice receiving adjuvanted vector control or Freund's adjuvant alone.

Growing bacteria are sensitive to various -lactam derivatives due to their interference with peptidogly can biosynthesis. At low concentrations, penicillin G (benzylpenicillin) blocks cell division without affecting mass growth rate. The MIC for division of Escherichia coli B/r (H266) was found to depend on the growth rate, which was modified by the nutritional conditions. Our hypothesis, that division sensitivity is proportional to the rate of peptidoglycan synthesis for septum formation, as well as to cell circumference, was thus confirmed.

The biosynthesis of carbamoyl phosphate (CP), a metabolic precursor of arginine and the pyrimidines was investigated in the hyperthermophilic archaeon Pyrococcus furiosus. The half-life of CP was found to be less than 2 s in the optimum temperature range of this organism (100-102 °C). The carbamoyl-phosphate synthase (CPSase) of P. furiosus uses ammonia as the nitrogen donor, and not glutamine like all micro-organisms investigated so far. The Mr of the enzyme, which is devoid of regulatory properties, is 70000, at variance with that of known CPSases. The possible significance of these findings with regard to hyperthermophilic nitrogen metabolism is discussed. Competition experiments with P. furiosus crude extracts indicated a marked preference of ornithine carbamoyltransferase (OTCase) for CP synthesized by CPSase rather than for CP added to the reaction mixture. In addition, the bisubstrate analogue -N-phosphonoacetyl-L-ornithine inhibits the formation of citrulline from bicarbonate, ammonia, ATP and ornithine much less than its synthesis from ornithine and CP in the presence of free OTCase. Such results suggest that, in vivo, CPSase and OTCase associate in a complex able to channel CP. Such a channelling may confer protection to CP, thus avoiding the accumulation of toxic amounts of cyanate arising from its decomposition as well as the waste of the two molecules of ATP required for its synthesis.

The nucleotide sequence of a 1:1 kb Xhol-HindIII fragment downstream of the malate dehydrogenase (mdh) gene of Thermus flavus revealed the presence of an ORF and an incomplete ORF lacking its NH2-terminal portion, in the opposite orientation to that of the mdh gene. These two genes overlapped with each other, sharing two base pairs, suggesting that these genes are co-transcribed in a single mRNA. One ORF (termed gpt) encoded a protein of 154 amino acids showing significant amino acid sequence similarity to purine phosphoribosyltransferases, such as xanthine-guanine phosphoribosyltransferase of Escherichia coli and human hypoxanthine phosphoribosyltransferase. Cloning and sequencing of the upstream region of the gpt gene, together with sequence comparison of the gene product encoded by the region upstream of gpt, suggested that the upstream ORF encoded two in-frame overlapping aspartokinase genes, askA, encoding the β-subunit of 405 amino acids, and askB, encoding the β-subunit of 161 amino acids, which was part of the 3′ portion of askA. Consistent with the sequence data, the askAB and the gpt genes conferred the heat-stable enzyme activities of aspartokinase and phosphoribosyltransferase, respectively, on E. coli. Preliminary characterization of these enzymes produced in E. coli is described.

Dimer formation and associated copy number depression is an important cause of multicopy plasmid instability. Natural multicopy plasmids employ site-specific recombination to convert dimers to monomers, thus maximizing the number of independently segregating molecules at cell division. Resolution of dimers of Escherichia coli plasmid ColE1 requires the plasmid cer site and at least four chromosome-encoded proteins: the XerC and XerD recombinases, and accessory factors ArgR and PepA. It has been suggested that ArgR has a role in the initial pairing of recombination sites and we describe here an attempt to detect this process in vivo. Our approach exploits a previous observation that a cer-like site known as the type II hybrid supports inter-molecular recombination and causes extensive multimerization of plasmids. We report that type-II-mediated multimerization can be repressed by a cer site in cis or in trans and propose that this is due to a physical interaction between the sites. If this hypothesis is correct, suppression of multimer formation provides an assay of site pairing. Our results demonstrate that the putative pairing interaction is independent of the topological relationship of the sites and that both PepA and ArgR are involved. Although most recombination-deficient mutant derivatives of ArgR are unable to pair recombination sites, we have found two (ArgR110 and ArgR115*) which retain pairing activity. The validity of the pairing hypothesis is discussed in the light of alternative explanations for our data.