- Volume 132, Issue 11, 1986

SUMMARY: A non-inducing medium (NID) was defined for studying the induction of methanethiol production by Brevibacterium linens CNRZ 918. The lowest l-methionine concentration capable of inducing maximal methanethiol production by the cells was 7 mM. The peptides l-Ala-l-Met and l-Met-l-Ala induced greater methanethiol production than free l-methionine. D-Methionine, l-cysteine, S-methyl-l-cysteine and l-ethionine were poor inducers. Culture temperature affected the duration of induction. An Na+ concentration of 1 M in the culture medium led to maximal methanethiol production capacity of both cells and cell extracts. l-Methionine and l-ethionine were the best substrates for the crude soluble cells extract (with release of methanethiol and ethanethiol respectively). Neither the derivatives of l-methionine that acted as inducers, nor D-methionine, were substrates for demethiolation. Demethiolation activity of the crude extract was thermolabile, not stimulated by Na+ and strongly inhibited by Zn2+, Mn2+ and Cu2+. The shortest generation time obtained for B. linens CNRZ 918 in NID medium + l-methionine was 4 h at 26 °C. Only coccoid forms were present when the culture temperature was 30 °C. The presence of l-methionine in the medium favoured their appearance. The strain grew best in the presence of 1% NaCl but tolerated concentrations up to 15%. The induction of methanethiol production was due to the induction of l-methionine-γ-demethiolase. The level of induction was probably related to the intracellular concentration of l-methionine. The transport system of l-methionine by B. linens CNRZ 918 was constitutive and Na+ dependent.

SUMMARY: The lipopolysaccharides (LPSs) from four isolates of Erwinia amylovora were examined. One isolate was virulent and capsulate, and it produced extracellular polysaccharide (EPS). Three were avirulent, of which one was capsulate and produced EPS; the remainder were non-capsulate and did not produce EPS. LPS was recovered from the cells and from the culture medium. Material extracted from the cells using phenol-water was indistinguishable from LPS extracted with phenol-chloroform-light petroleum. Acid hydrolysis released lipid A which contained in all cases glucosamine, phosphate and the fatty acids 12:0, 14:0 and 3-OH 14:0. The carbohydrate released by mild acid hydrolysis was resolved by gel permeation chromatography into three components: I, a partially included species identified as core substituted with a short sidechain of fucose, galactose and glucose; II, an unsubstituted core oligosaccharide containing heptose, glucose, uronic acid, amino compounds and 3-deoxy-2-octulosonic acid (KDO); III; a totally-included low M r fraction containing KDO, phosphate and amino compounds. The sidechain component was missing from LPS of one of the non-capsulate strains that did not produce EPS. This strain is believed to lack UDP-glucose-4-epimerase, a key enzyme in the biosynthesis of galactose. Galactose (with glucose and uronic acid) is known also to be a component of EPS. Defects in galactose synthesis may therefore affect the assembly of LPS as well as EPS.

SUMMARY: Enzymes were assayed in extracts of Bacillus sphaericus harvested late in the exponential phase from batch cultures in a minimal (acetate plus salts) medium. Aspartokinase was repressed and inhibited by threonine; lysine alone had no effect, though it increased the inhibition (but not the repression) by threonine. Aspartic β-semialdehyde dehydrogenase was slightly repressed by lysine. Dihydrodipicolinate synthase was inhibited non-competitively by lysine, and dihydro-dipicolinate reductase was partly repressed by lysine. Diaminopimelate dehydrogenase (with tetrahydrodipicolinate as substrate) was inhibited by meso-diaminopimelate. Lysine did not repress diaminopimelate decarboxylase, and only slightly inhibited this enzyme. An auxotrophic mutant that required threonine and methionine excreted lysine after growth had stopped with a limited concentration of threonine.

SUMMARY: A spoIIA:: lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. β-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known spoO loci but on none of the “later” loci tested. The β-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage II mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spoOJ, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.

SUMMARY: A spoV AA:: lacZ gene fusion has been used to study expression of the spoV A operon during sporulation in Bacillus subtilis. β-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoV A mutations are manifested. spoV A expression is dependent on all of the known spoO and spoII loci and on some of the “early” spoIII loci, but not on “later” loci. Several lines of evidence suggest that spoV A expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.

SUMMARY: The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the β-galactosidase gene, lacZ, of Escherichia coli so that the expression of β-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of β-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID:: lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spoO lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make β-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA [spoIIG, spoIIE] [spoIID, spoIIB, spoIIF].

SUMMARY: The sporulation gene spoIIIC from Bacillus subtilis was fused to the lacZ gene from Escherichia coli, so that the transcription of the lacZ gene was under the control of the spoIIIC promoter. Production of β-galactosidase, under conditions of sporulation, was then used as an indicator to study the expression of spoIIIC in relation to other sporulation loci. Expression of spoIIIC, which occurred only in the mother cell compartment, was prevented by mutations in all of the stage 0 and stage II loci, and also by spoIIID, spoIV A and spoIV B mutations. By contrast, the last three operons are not needed for expression of spoV A, which has previously been shown to be spore-specific. In consequence, a branched pathway of gene expression is proposed. One branch leads to expression of spoV A within the spore compartment, the other to expression of spoIIIC in the mother cell.

SUMMARY: The expression of the spoIIA and spoV A sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoV A loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.

SUMMARY: We have determined the nucleotide sequence of a 1496 bp stretch of Bacillus subtilis chromosome that complements the gerE36 mutation. The sequence contains three open reading frames. One of these is part of the sdhC gene, the other two, whose functions are unknown, are capable of encoding proteins with M r values of approximately 17000 and 8500. The use of integrational plasmids to delimit the gerE transcriptional unit shows that the gerE locus consists of one gene, which encodes a polypeptide of 74 amino acid residues and which is switched on after t 3 during sporulation of wild-type B. subtilis 168.

SUMMARY: A segment of DNA from Bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoIID68, in Bacillus subtilis, was cloned into a derivative of the temperate bacteriophage ϕ105. The segment of DNA included an entire structural gene and complemented the mutation spoIID298, in addition to spoIID68, in B. subtilis.

The nucleotide sequence of the gene from B. amyloliquefaciens was determined and compared with that of the B. subtilis gene; 74% homology was found in the coding region. Amino acid primary sequences derived from the nucleotide sequences of the two genes were also compared. The gene from B. amyloliquefaciens coded for a protein of 344 amino acid residues, one more than the protein coded by the corresponding gene from B. subtilis. Comparison of the primary amino acid sequences of the two genes showed that 78% of the residues were completely conserved and 8% were semi-conserved. Variation, however, was not random, i.e. some segments were much more highly conserved than others. Both proteins had a hydrophobic region at the N-terminus.

SUMMARY: The DNA of certain species of halophilic and methanogenic archaebacteria is dam methylated, as shown by restriction endonuclease sensitivities. The Dam+ phenotype appears to be confined to particular taxonomic groupings defined by DNA:rRNA hybridization or 16S RNA oligonucleotide cataloguing.

SUMMARY: Genetic transformation of intact cells of Saccharomyces cerevisiae, achieved by incubating the cells with plasmid DNA in the presence of PEG, could be enhanced, not only by pretreatment of the cells with Li+ and 2-mercaptoethanol, as has been reported previously, but also by pretreatment with proteolytic enzymes. The efficiency of transformation with 2-mercapto-ethanol rose dramatically when the pretreated cells had been handled in osmotically stabilized media. Following all the pretreatments the cells became leaky for nucleic acids as detected by the presence of endogenous RNAs in the medium. The pretreatments evidently facilitated the passage of transforming DNA across the cell wall.

SUMMARY: An Escherichia coli strain unable to use gluconate was isolated by spontaneous curing of λc1857 s7 xis6 b515 b519, λc1857 s7 Δ(A-att) dargI valS lysogens. Two lesions, linked to asd and pyrB markers, respectively, were necessary to produce this phenotype. The asd-linked mutation gnt-17, of regulatory type, seems to affect the expression of the major system of gluconate utilization (min 75) as well as that of 6-phosphogluconate dehydratase (gene edd, min 41), the first enzyme of the Entner-Doudoroff pathway. A closely linked suppressor of gnt-17 causes constitutivity of these activities; this suppressor resembles gntR, which is also in the asd region. Hence, it is possible that gnt-17 is a super-repressing allele of gntR, rather than a positive controlling element. Lesion gnt-17 alone does not prevent the utilization of gluconate; for this, the mutation gnt-18 at 96.9 min is also necessary. This mutation abolishes the thermosensitive gluconokinase activity and thus eliminates the subsidiary ability to catabolize gluconate. Accordingly, gnt-18 seems to be allelic with gntV, the locus postulated as being in the pyrB region specifying the thermosensitive gluconokinase.

SUMMARY: A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24-9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr− mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu− mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of M r 30000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.

SUMMARY: The results of the identification of 933 strains of Gram-negative, aerobic, rod-shaped, fermentative bacteria (Enterobacteriaceae, Pasteurellaceae, Vibrionaceae) by a probabilistic method, in a computer, are given. The identification rate on the matrix was 89.2%. Many of the strains were atypical and had caused difficulty in identification in medical diagnostic laboratories. The results are given for each taxon by genus and species.