- Volume 133, Issue 9, 1987

SUMMARY: The cloned Bacteroides fragilis glutamine synthetase (GS) subunit produced in Escherichia coli had the same apparent M r of approximately 75000 as the GS subunit from B. fragilis cells. The B. fragilis GS enzyme had an apparent M r of approximately 490000 and it is concluded that the GS is a hexamer. The cloned GS did not appear to be regulated by adenylylation and deadenylylation and the cloned enzyme was inactivated by snake venom phosphodiesterase. The pH profiles of the cloned GS, assayed by the γ-glutamyl transferase (GGT) assay were similar for NH4 +-shocked and unshocked cell extracts and an isoactivity point was not obtained from these curves. The cloned GS was subject to feedback inhibition by amino acids but not by AMP. The GGT activity of the cloned GS in NH4 +-shocked and unshocked cell-free extracts was inhibited by Mg2+. Mn2+ stimulated the cloned GS GGT activity of NH4 +-shocked cell-free extracts. Western blotting indicated that GS production was regulated by nitrogen in B. fragilis cells but cell extracts showed no GGT activity. Cloned B. fragilis GS produced in E. coli was specifically and irreversibly inactivated by B. fragilis cell extracts.

SUMMARY: NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8·5. The K m values for isocitrate and NADP were 74 and 53 μm, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70°C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.

SUMMARY: Cells from oxygen-limited cultures of Acetobacter pasteurianus NCIB 6428 contained cytochrome d, a 596 nm absorbance (in reduced minus oxidized difference spectra at room temperature) similar to cytochrome a 1, and b-type cytochromes, including a cytochrome o-like pigment. Oxygen-sufficient cells lacked the d and a 1-like components. Pyridine haemochrome spectra suggested the presence of haem a. Photolysis with white light at −126°C of an anoxic suspension of reduced oxygen-limited cells, into which CO had been bubbled, elicited a photodissociation spectrum that revealed the cytochrome o-like species, but not cytochromes d or a 1. When a similar suspension, but to which O2 had been added at −23°C, was photolysed at −126 to −132°C, using white light or irradiation with a He-Ne laser, an additional, intense absorbance at 648 nm (relative to the CO-liganded, reduced form) was observed, attributable to an oxygenated (Fe2+O2 or Fe3+O2 -) complex of cytochrome oxidase d. Photolysis at progressively warmer temperatures, or successive scans of a sample photolysed at −91°C, revealed (i) increasing depth of a trough (relative to the CO-liganded, reduced form) at 598 nm attributed to the a 1-like haemoprotein and (ii) cytochrome b oxidation. Laser photolysis of the cytochrome d-CO complex at −81°C in the presence of oxygen suggested that oxidation of cytochrome a 1 preceded that of cytochrome(s) b. It is proposed that, in this strain of Acetobacter, a cytochrome a 1,-like pigment mediates electron transfer from cytochrome(s) b to cytochrome oxidase d, but that cytochrome a 1 is not itself an oxidase.

SUMMARY: When Micrococcus strain 12B grown on o-phthalate was incubated with 3-methylphthalate, three compounds accumulated. These were shown to be 2-pyrone-3-methyl-4,6-dicarboxylic acid, 3,4-dihydroxy-6-methylphthalic acid, and 5-hydroxy-3-methylphthalic acid, all previously undescribed. A pathway for the formation of these compounds is proposed.

SUMMARY: Two acetamidoaldehyde dehydrogenases were identified in Candida boidinii grown on putrescine as sole nitrogen source with glucose as carbon source. One of them, enzyme A, although present when cells were grown on ammonium or l-lysine, increased in activity when cells were grown on putrescine or spermidine. The other, enzyme B, was absent when the putrescine was replaced by l-lysine or ammonium, but was present if the nitrogen source was spermidine or acetylputrescine. Both dehydrogenases were active with NAD+ or NADP+ as electron acceptor. Apparent K m values for 3-acetamidopropionaldehyde and 4-acetamidobutyr-aldehyde were respectively 0·83 mM and 0·041 mM for enzyme A and 0·077 mM and 0·015 mM for enzyme B. Enzyme A was competitively inhibited by chloral hydrate with a K i of 0·6 mM, whiile enzyme B was unaffected. Both enzymes were slightly (20%) stimulated by 50 mM-KCl. Although both enzymes catalysed the oxidation of a range of aldehyde substrates, and are thus both general aldehyde dehydrogenases, it is suggested that acetamidoaldehyde dehydrogenase B is more probably specifically involved in putrescine degradation. Subcellular fractionation of spheroplast lysates showed that enzyme B was cytosolic, remaining unsedimented at 100000 g, while enzyme A co-sedimented with mitochondrial marker enzymes in a sucrose density gradient. It was also shown that acetamidoalkanoate deacetylase and acetylputrescine oxidase activities, two other key enzymes in the breakdown of putrescine and spermidine, were respectively cytosolic and peroxisomal in their location in the cell.

SUMMARY: An antibiotic complex active against multiply resistant strains of staphylococci and other Gram-positive bacteria was isolated from cultures of Streptomyces albus G. Silica gel and Sephadex LH-20 column chromatography gave two congeners with M r values of 786 and 772, which differed by one -CH2-group. The two homologues contained an isothiocyanate group, and proved to be identical with paulomycins A and B produced by Streptomyces paulus; the FAB mass spectra, in addition, proved the same two congeners to be present in proceomycin obtained from Streptomyces alboniger.

SUMMARY: The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined. Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts. The hexokinase PII inactivation induced by d-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D. 308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid. This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting. We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.

SUMMARY: A fragment of Bacillus subtilis DNA 3490 bp long, capable of complementing spoIIIE mutations, was sequenced. The region of the fragment that encodes functions required for sporulation was delimited using integrational plasmids. Sequencing showed that this region contained an operon with two open reading frames together with associated ribosome-binding sites. The deduced translation products would be polypeptides of 518 and 252 amino acid residues. Several sequences resembling promoters recognized by RNA polymerase containing σ29 occur in the region preceding the larger open reading frame. Although no transcription-termination signal was identified downstream of the smaller coding region, analysis with integrational plasmids and determination of the size of spoIIIE messenger RNA suggest that the locus does not contain a third gene.

SUMMARY: A mutation, spo-87, in the spo0J locus of Bacillus subtilis allows appreciable transcription of spoIIA, spoIID and spoIIG and later operons, even though most of the cells are morphologically blocked at stage 0 and the incidence of heat-resistant spores is about 1 per 104 cells. This mutation therefore appears to disengage the genetic control of sporulation from the morphological changes to which it should be connected. spoIIA and spoIIG are transcribed independently of one another. However, the products of both operons are needed for the activation of spoIID which occurs later. This indicates a convergence of parallel pathways of operon expression. We have also shown that nonsense mutations in spoIIAC (which codes for a sigma factor of RNA polymerase) prevent transcription of spoIID; by contrast, missense mutations in the same gene allow transcription of spoIID.

SUMMARY: The predicted polypeptide products of two genes, spoIID and gerE, which appear to be concerned in the regulation of spore formation in Bacillus subtilis have been compared by modelling methods with known DNA-binding proteins. The results indicate that both polypeptides may have DNA-binding properties and the conclusion is drawn that this may account for their regulatory action.

SUMMARY: Mating with Escherichia coli strain SM10 carrying the Tn5 vector pSUP2011 was used to mutagenize Pseudomonas syringae pv. pisi strain 299A. The resulting transconjugants were each tested by stem-inoculation into several pea (Pisum sativum) cultivars. Three classes of mutant, which probably resulted from insertion of part or all of RP4-2-Tc:: Mu into the genome of strain 299A, showed reduced virulence towards one or more pea cultivars. The single class I mutant was avirulent on all pea cultivars tested and had lost the ability to induce a hypersensitive response in tobacco (Nicotiana tabacum) cv. White Burley; the single class II mutant induced a hypersensitive response on all pea cultivars and tobacco; class III mutants showed reduced virulence towards pea cv. Early Onward, while remaining fully virulent towards other normally susceptible pea cultivars, and inducing a hypersensitive response in tobacco.

SUMMARY: In contrast to the established systems of plasmid-coded homologous ribosomal DNA (rDNA) cistrons in Escherichia coli little is known about the fate of heterologous rRNA. In order to study expression of foreign rDNA, rRNA cistrons from Proteus vulgaris were cloned in phage vector Charon 35, subcloned in pBR322 and transformed in E. coli. The inserts of two clones (pPM2 and pPM14) were characterized by restriction analysis and Southern hybridization. Each of them harboured a complete rrn cistron. The location of rRNA genes of clone pPM2 was also verified by R-loop analysis. The 5’ flanking region of the 16S rRNA of pPM2 was sequenced and compared to the E. coli counterparts. High-level homologies exist in the functional parts of this region, e.g. promoters, box A and RNAase III recognition site. The copy number of pPM2 and pPM14 was estimated to be 8 and 10, respectively. Clones showed a markedly reduced growth rate (generation time about 57 to 70 min) as compared to the non-transformed cells (generation time 40 min). rDNA cistrons of P. vulgaris were properly expressed and the transcripts are processed as demonstrated by the presence of 16S rRNA from P. vulgaris in both ribosomes and 30S ribosomal subunits isolated from the transformed E. coli cells. The fraction of heterologous rRNA in ribosomes was about 25%.

SUMMARY: Site-directed mutagenesis was used to replace the codon for the N-terminal cysteine residue of pColE2-P9-encoded mature lysis protein (CelB) by an arginine codon. In contrast to the wild-type CelB protein, the product of the mutated gene, which has an altered signal peptidase cleavage site, was neither processed nor acylated. However, the mutant protein retained sufficient residual activity to cause partial, Mg2+-suppressible lysis and could activate envelope phospholipase Al-A2 and promote colicin release, albeit with reduced efficiency compared to the wild-type protein. We propose that the uncleaved signal peptide of the mutant protein acts as the functional equivalent of the fatty acyl groups normally linked to the N-terminal cysteine residue of the wild-type protein, thereby anchoring the protein in the cell envelope where it exerts its various effects.

SUMMARY: Random insertions of mini-MudII1734 lac gene fusion bacteriophage were constructed on plasmid pLW06, which carries the fdhF gene coding for benzyl-viologen-linked formate dehydrogenase. They allowed us to limit the size of the gene to a 3 kb fragment and to define its direction of transcription. The identification of the fdhF product as a 85 kDa protein was achieved after expression of derivative plasmids in a maxicell system.

SUMMARY: Conjugational recombination in Escherichia coli was investigated by comparing the effects of recN, recO, ruv and lexA mutations on the formation of recombinants in crosses with strains lacking RecBCD enzyme. The results presented reveal that recN and ruv mutations do not abolish residual recombination in a recB mutant, and have only a rather modest effect on recombination in recBC sbcA strains; in these respects they are quite different from recF, recJ and recO mutations. The differences between these two groups of genes are discussed in relation to the molecular exchanges needed to produce viable recombinants.

SUMMARY: Sexual development (crossing) in Physarum polycephalum occurs when two haploid amoebae fuse to form a diploid plasmodium. The imz locus influences the maximum pH at which crossing can occur. A new allele of imz has been identified, bringing the total number of alleles to three. Contrary to earlier findings, it has been shown that all homoallelic combinations of imz alleles display a similar pH limit for crossing, which is lower than that for imz-heteroallelic combinations. It is concluded that imz is a mating compatibility locus; thus the mating-type system of P. polycephalum comprises three multiallelic loci: matA, matB and imz. It is proposed that imz be renamed matC.

SUMMARY: The six plasmid species of Zymomonas mobilis ATCC 10988 were isolated, separated and purified. Evidence is presented that the 1·6 kb (pZMO1) and 1·9 kb (pZMO2) species can co-migrate on agarose gels, contain single sites for BglII and HindIII, or EcoRI and EcoRV respectively, and are related at the nucleotide level. The 16·7 kb species (pZMO5) appears to be a tetramer and is partially homologous with pZMO1 and pZMO2. The 2·7 kb species (pZMO3) has a single SphI site and does not show sequence homology with pZMO1 or pZMO2. Structural features of the 7·3 kb (pZMO4) and 31·3 kb (pZMO6) molecules are discussed.