Journal of general microbiology最新文献

The transport and incorporation of D-glucose into the human pathogen Helicobacter pylori was investigated employing radioactive tracer analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The bacterium was found to utilize D-glucose contrary to the accepted view that it cannot catabolize carbohydrates. Under the experimental conditions employed, the rate of transport of [14C]glucose was 3.24 mmol min-1 (g protein)-1, and the rate of incorporation into the cellular mass was 1.06 mumol h-1 (g protein)-1. The utilization of [13C]glucose showed biphasic characteristics with a slower initial period followed by a phase with a rate of utilization at least an order of magnitude faster. The apparent rates of decline of glucose levels during both phases varied between strains and depended on the growth conditions of the bacteria prior to harvesting. The main product of glucose catabolism was identified as lactate. These findings provide new perspectives into the physiology of H. pylori and have implications for the active search to develop appropriate therapies for the micro-organism.

The spoIIAB gene of Bacillus subtilis encodes an inhibitor of sigma F, a transcription factor that plays a crucial role in the establishment of prespore-specific gene expression during sporulation. The SpoIIAB protein can probably also inhibit a closely related sigma factor sigma G, which determines the later phase of prespore-specific transcription. We have isolated two new missense mutations in the spoIIAB gene. spoIIAB28 behaves like the previously described spoIIAB1 mutation, in that it mainly affects the activity of sigma G. In contrast, the spoIIAB22 mutation seems to be impaired mainly in its ability to inhibit sigma F. All three missense mutations are clustered in the N-terminal coding region of spoIIAB, suggesting that this region of the protein interacts with the sigma factors. The extreme N-terminal part of SpoIIAB may be specifically concerned with the regulation of sigma G activity.

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

Different mechanisms have been proposed previously for the biodegradation of monomethyl sulphate (MMS) in Agrobacterium sp. and Hyphomicrobium sp. Sulphate liberation from MMS in Agrobacterium sp. M3C was previously shown to be O2-dependent, whereas in several Hyphomicrobium spp. the initiating step has been considered hitherto to be hydrolytic and catalysed by methyl sulphatase. In the present study, Agrobacterium and Hyphomicrobium strains were compared for their ability to oxidize MMS and its potential metabolites in the oxygen electrode. MMS-grown Agrobacterium sp. M3C and Hyphomicrobium sp. MS223 oxidized MMS with consumption of 0.5 mol O2 per mol of substrate, but they were unable to oxidize methanol. By repeatedly challenging MMS-grown Hypomicrobium with MMS in the electrode chamber, all the O2 in the electrode became exhausted, at which point SO4(2-) liberation stopped although excess MMS was available. SO4(2-) release resumed immediately when O2 was re-admitted to the electrode chamber. Thus liberation of SO4(2-) from MMS in the oxygen electrode was dependent on the continuing availability of O2. Hyphomicrobium sp. MS223 therefore closely resembled Agrobacterium sp. M3C in its obligatory requirement for O2 in MMS degradation. Unlike Agrobacterium sp. M3C, Hyphomicrobium sp. MS223 was able to grow on methanol and methanol-grown cells oxidized methanol (0.5 mol O2 per mol of substrate) but not MMS. Cyclopropanol, an inhibitor of methanol dehydrogenase, abolished oxidation of methanol by methanol-grown Hyphomicrobium sp. MS223 but did not affect oxidation of MMS by MMS-grown cells. Thus Hyphomicrobium sp. MS223 expresses enzymes for oxidation of methanol when needed for growth on this compound, but not when grown on MMS.(ABSTRACT TRUNCATED AT 250 WORDS)

A 5.4 kb HindIII fragment of Lactococcus lactis subsp. lactis was identified using a homologous dnaK probe generated by PCR and cloned in Escherichia coli. Upstream sequences were generated by inverse PCR. The two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orf1-grpE-dnaK-orf4. orf1 encodes a 39 kDa protein of unknown function which shows considerable sequence homology with the Orf39 and Orfa proteins of Bacillus subtilis and Clostridium acetobutylicum, respectively. The downstream ORFs showed high homology to the grpE and dnaK genes of other prokaryotes. The DnaK protein has a characteristic 24-amino-acid deletion exhibited by all the known DnaK proteins of Gram-positive species. In many bacteria the dnaK and dnaJ genes are found as part of the same operon. The L. lactis dnaK operon is unusual in that the dnaK gene is followed by a putative transcription terminator and a fourth large ORF which shares no homology with the dnaJ genes of other bacteria but has a small degree of homology with various membrane proteins. Vegetative promoter sequences are found upstream of both orf1 and orf4. A 12 bp inverted repeat is found upstream of the putative promoter of orf1 and an 8 bp inverted repeat is found between this promoter and the orf1 initiation codon. These repeats are thought to be involved in regulation of the heat-shock genes. The DnaK homologue is induced approximately 3-fold on heat shock at 42 degrees C.

A temperature-sensitive mutation in a new Escherichia coli gene, located at 62.5 min on the linkage map and designated lytF, resulted in bacteriolysis at the restrictive temperature. Temperature sensitivity and lytF-mediated lysis were simultaneously suppressed by either of two previously described unlinked mutations designated smhA1 and smhB1. The smhA1 and smhB1 alleles were originally isolated as specific extragenic suppressors of temperature-sensitive mutations in three other genes known as murH (99 min), lytD (13 min) and lytE (25 min) which conferred lysis phenotypes indistinguishable from that of the lytF mutation. The murH, lytD and lytE genes have been proposed to be related on the bases of phenotypic similarities and the specificities of their extragenic suppressors. It is now further proposed that lytF belongs to this group. The isolation of new alleles of smhA and smhB as extragenic suppressors of lytF further supports this proposal.

A gene encoding the extracellular alpha-amylase of Aeromonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Escherichia coli JA221 carrying pCA101 revealed that approximately 60% of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an alpha-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete alpha-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several alpha-amylases were also present in A. hydrophila alpha-amylase at the corresponding positions.

A tryptophanase gene from Enterobacter aerogenes SM-18 was cloned and sequenced. The structural gene for tryptophanase, tnaA, consisted of 1389 bp encoding 462 amino acid residues, and its nucleotide sequence and deduced amino acid sequence showed significant homology to those of tnaA from Escherichia coli K12. A short open reading frame consisting of 31 amino acid residues was found upstream of tnaA, and it showed some similarity to the E. coli tnaC gene known to be a cis-acting regulatory element for transcription. A partial open reading frame homologous to the 5' end of E. coli tnaB was observed at the 3'-flanking region of tnaA. These genes may thus constitute an operon as in E. coli.