- Volume 140, Issue 11, 1994

A number of bacterial strains capable of degrading the bacterial exopolysaccharide gellan® have been isolated by standard enrichment procedures. They include several pink-pigmented Gram-negative rod-shaped bacteria. A red-pigmented Gram-positive bacillus earlier found to degrade the exopolysaccharide xanthan fromXanthomonas campestrisalso showed slight gellanase activity. All the Gram-negative bacteria are non-fermentative, motile and amylase-producing. The gellan degradation in each case is due to eliminase-type enzymes(lyases) which appear to be extracellular enzymes cleaving the sequence … β-D-glucosyl-(1→4)-β-D-glucuronosyl… in the tetrasaccharide repeat unit of the substrate polysaccharides. Although in some isolates these enzymes appear to be exo-acting, it appears from the loss in viscosity of the alternative substrate deacetylated rhamsan that they are predominantly endoenzymes. The enzyme activity is inducible: it is almost absent from glucose-grown cells. Associated with the ‘gellanase’ activity, all the Gram-negative bacterial isolates possess intracellular α-L-rhamnosidase and β-D-glucosidase activities apparently located in the periplasm. The enzymes are highly specific and fail to cause significant degradation of most of the other bacterial exopolysaccharides which have been shown to be structurally related to gellan. As well as acting on gellan, they exert similar degradative activity against the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan. The enzymes only have relatively slight activity against the natural, acylated gellan-like polysaccharides from the bacteria now designated as strains of Sphingomonas paucimobilis.

Ca2+transport has been measured across plasma membrane vesicles isolated from cells of Ustilago maydis. This transport was found to be ATP- (or to a lesser extent GTP) and Mg2+-dependent. Inconsistent release of Ca2+from intact vesicles was obtained using the calcium ionophore A23187. However, Ca2+was released by Triton X-100 in a concentration-dependent manner. Transport was inhibited by vanadate (>50%) and erythrosin B (about 50%), I 50being about 10 μM for both inhibitors. In the presence of the protonophores CCCP or gramicidin, partial inhibition of Ca2+transport (about 20%) was observed, but the Ca2+-channel blockers, nifedipine, diltiazem and verapamil had no effect, although the latter inhibited proton transport. The results indicate that Ca2+transport in U. maydisis regulated by a P-type ATPase with similar properties to that found in higher plants.

Three actinomycetes (Streptomycessp. EC22, Streptomyces viridosporusT7A and Thermomonospora fuscaBD25) were assessed for their ability to degrade ball-milled wheat straw. All gave maximum levels of solubilized lignocellulose products (APPL) at the beginning of the stationary phase of growth (72-96 h). Low-molecular-mass aromatic compounds extracted from the APPL were analysed by reverse-phase and gas chromatography. Although the number of chromatographic peaks detected made identification of the products difficult, p-coumaric acid (4-hydroxycinnamic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid), gallic acid methyl ester (methyl-3,4,5-trihydroxybenzoate) and 4-methoxyphenol were recognized. The infrared spectra of the three strains were similar to the spectra of humic acids, with all APPL extracts showing carbonyl, amino, carboxyl, aliphatic and aromatic group vibrations. Also detected were peptide linkages of proteins. The results suggest a role for actinomycetes in the formation of humic substances in soils and composts.

The insect pathogenic fungus Metarhizium anisopliaeproduces several extracellular cuticle-degrading proteases and evidence is consistent with one of these, PR1, which is a chymoelastase, being a determinant of pathogenicity. We have shown previously that PR1 production is regulated by both carbon catabolite and nitrogen metabolite repression and also by specific induction under derepressed conditions by insect cuticle. In the present work we have established that an enzymically released proteinaceous component(s) of insect cuticle is capable of inducing PR1 (based on appearance of extracellular activity). Cuticle of the desert locust Schistocerca gregariatreated with KOH to remove protein failed to induce PR1 production, whereas cuticle treated with either chloroform or ether to remove lipids still induced PR1. Cuticle digested with either PR1 or the trypsin-like PR2 of M. anisopliaereleased peptides mainly in the range 150-2000 Da; addition of these peptides generated by PR1 or PR2 at 3 μg alanine equivalents ml-1induced PR1 production to a level similar (75%) to that obtained with untreated insect cuticle. Several amino acids and peptides which are abundant in insect cuticular protein (Ala, Gly, Ala-Ala, Ala-Ala-Ala, Ala-Pro and Pro-Ala) were tested at a range of concentrations and in restricted cultures for their ability to induce PR1. None induced the protease to the levels seen with cuticle or peptides enzymically released from cuticle, although some dimers and notably the monomers Ala and Gly gave 2-2-7-fold enhanced PR1 activity above derepressed basal levels (up to 48-57% of that achieved with induced synthesis on cuticle). There was evidence for more efficient uptake and/or catabolism by M. anisopliaeof alanine di-and tripeptides than of monomer amino acids.

The Tra1 region of broad host range IncPα plasmid RK2 encodes proteins essential for its promiscuous conjugative transfer and includes oriT, the site at which nicking occurs to initiate transfer replication. Unregulated expression of the Tra1 region genes would be likely to place a major burden on the host. To investigate the control of these genes the three transcriptional promoters from this region were cloned by PCR and inserted into xylEpromoter probe vectors. The strength of traJpand traKpwas estimated to be six to eightfold less than the strong trfApromoter which is required for expression of genes for vegetative replication of RK2. The traGpromoter was about one-tenth the strength of the other two. These promoters are not repressed by products of the central control operon of RK2. However, traJpand traKp, which are arranged as back to back divergent promoters in the oriTregion, are repressed by TraK which constitutes part of the relaxosome necessary for nicking at oriT.A second relaxosome protein, TraJ, represses traJp. traGpis not repressed by any relaxosome proteins. All three promoters are repressed by TrbA, which is encoded at the start of the trboperon containing the rest of the transfer genes (the Tra2 region). These circuits provide: (i) an autoregulatory way of ensuring production of enough relaxosome proteins without overburdening the host; and (ii) a means of coordinating expression of both blocks of transfer genes.

Many of the changes induced in yeast by sublethal yet stressful amounts of ethanol are the same as those resulting from sublethal heat stress. They include an inhibition of fermentation, increased induction of petites and stimulation of plasma membrane ATPase activity. Ethanol, at concentrations (4-10%, v/v) that affect growth and fermentation rates, is also a potent inducer of heat-shock proteins including those members of the Hsp70 protein family induced by heat shock. This induction occurs above a threshold level of about 4% ethanol, although different heat-shock proteins and heat-shock gene promoters are optimally induced at different higher ethanol levels. In addition ethanol (6-8%) causes the same two major changes to integral plasma-membrane protein composition that result from a sublethal heat stress, reduction in levels of the plasma membrane ATPase protein and acquisition of the plasma membrane heat-shock protein Hsp30.

The temperate bacteriophage AAU2 of ‘Arthrobacter aureus’ C70, isolated from a soil sample, has been purified and characterized. The phage belongs morphologically to the Siphoviridaefamily. Its genome consists of a linear double-stranded DNA of 45.4 kb with cohesive ends. The attPsite and the phage-encoded functions essential for integration were cloned in Escherichia colias a 5.25 kb BglII-EcoRV fragment. The resulting plasmid vector, non-replicating in ‘A. aureus’, is able to integrate at high frequency into the chromosomes of ‘A. aureus’and the industrially used coryneform bacteria Brevibacterium lactofermentumand Corynebacterium glutamicum.

The gerBspore germination operon of Bacillus subtilis168 is a homologue of the gerAspore germination operon. The expression and regulation of the gerBoperon has been examined using a lacZtranscriptional fusion and the transcriptional start defined. The gerBoperon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, σG. This is a further homology to the gerAoperon, which is similarly regulated. It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.

Protoplasts were prepared from two auxotrophic and antibiotic-resistant strains (Met-Nalrand Thr-Strr, respectively) of the facultative alkaliphile Bacillussp. C-125 by treatment with lysozyme. The protoplasts fused effectively in the presence of polyethylene glycol. Fusants obtained between two parental protoplasts were regenerated on solid medium containing the two antibiotics, nalidixic acid (Nal) and streptomycin (Str). Parental protoplasts were regenerated at high frequency (43-97%) on non-selective medium but not on selective medium. The NalrStrrfusants had the form of bacilli. Met and Thr markers segregated among the fusants with a predominantly Met+Thr+phenotype. The exfusants seemed to restore the parental ploidy.

The plasmid pTnPF1 containing the transposon Tn917PF1was introduced into the protoplasts of two Bacillus licheniformisstrains in the presence of polyethylene glycol. Transpositions were produced at high temperature which inhibited plasmid replication and kanamycin was used for selection. Transposon Tn917PF1was inserted randomly into the bacterial chromosome, producing different auxotrophic, prophage BLF and bacitracin-non-producing mutants. The auxotrophic mutant phenotypes were characterized by the Holliday-test and some mutations by hybridization with a transposon DNA probe. Insertions for the entire chromosome or for the prophage genophore were found at random, but preferred target sites were detected within limited regions, like the bacitracin synthetase or sulphate reductase genes. The partial physical map of the chromosomal region of bacitracin synthetase was constructed based on the hybridization patterns of insertion mutants.

Malate synthase is one of the key enzymes of the glyoxylate cycle and is essential for growth on acetate as sole carbon source. The aceBgene from Corynebacterium glutamicum, encoding malate synthase, was isolated, subcloned and expressed in Escherichia coliand C. glutamicum. Sequencing of a 3024 bp DNA fragment containing the aceBgene revealed that it is located close to the isocitrate lyase gene aceA. The two genes are separated by 597 bp and are transcribed in divergent directions. The predicted aceBgene product consists of 739 amino acids with an M rof 82362. Interestingly, this polypeptide shows only weak identity with malate synthase polypeptides from other organisms and possesses an extra N-terminal sequence of about 170 amino acid residues. Inactivation of the chromosomal aceBgene led to the absence of malate synthase activity and to the inability to grow on acetate, suggesting that only one malate synthase is present in C. glutamicum. The malate synthase was purified from an aceB-overexpressing C. glutamicumstrain and biochemically characterized. The native enzyme was shown to be a monomer migrating at an M rof about 80000. By sequencing the N-terminus of malate synthase the predicted translational start site of the enzyme was confirmed. The enzyme displayed K mvalues of 30 μM and 12 μM for the substrates glyoxylate and acetyl CoA, respectively. Oxalate, glycolate and ATP were found to be inhibitors of malate synthase activity. The present study provides evidence that the malate synthase from C. glutamicumis functionally similar to other malate synthase enzymes but is different both in size and primary structure.

The gene (nspC) encoding carboxynorspermidine decarboxylase (CANS DC), the last enzyme in norspermidine biosynthesis, in Vibrio alginolyticuswas isolated by immuno-screening and its complete nucleotide sequence was determined. Sequence analysis of the subcloned fragment (2.0 kb) revealed an ORF of 1131 bp encoding a protein of 377 amino acids with a calculated molecular mass of 42008 Da. The sequence of 20 N-terminal amino acids of purified CANS DC was found to be identical to that predicted from the nspCgene. A putative ribosome binding sequence was observed 8 bp upstream from the translation start site (ATG), and promoter- and terminator-like sequences were detected upstream and downstream of the ORF, respectively. Database searches identified no similar proteins, but the deduced amino acid sequence contained a putative pyridoxal 5′-phosphate binding region similar to those of the bacterial meso-2,6-diaminopimelate decarboxylases and eukaryotic ornithine decarboxylases. Another full ORF was found on the opposite strand downstream from the nspCgene. It encoded a protein of 69 amino acids with a calculated molecular mass of 7441 Da, which exhibited some weak similarity to ScrR,a repressor protein of V. alginolyticus, in the helix-turn-helix DNA binding domain, but did not appear to be expressed in the host cells.

This paper reports the first attempt to characterize the penicillin-binding proteins (PBPs) of Shigella dysenteriae,an important human pathogen. The PBP pattern of the membranes of S. dysenteriaeclosely resembles that of Escherichia colimembranes. A 38 kDa PBP which is an important target for the penem SCH34343, the cephamycin cefoxitin and the oxacephem moxalactam, has been purified. This PBP is immunologically related to a PBP of similar molecular mass in E. coliand is present at high levels in stationary-phase cells of S. dysenteriae.