- Volume 137, Issue 3, 1991

The region upstream of the coding sequence of the spoVA operon was studied by several techniques to identify the promoter and to determine the start point for transcription of spoVA. The results of plasmid integration analysis in Bacillus subtilis showed that no more than 119 bp upstream of the coding sequence is needed for expression. A comparison of the sequence of this upstream region with the corresponding sequence from Bacillus licheniformis showed four stretches that were perfectly conserved, interspersed with poorly conserved stretches; the second and third of these conserved stretches appeared to represent the ‘–35’ and ‘–10’ regions of a promoter recognized by RNA polymerase containing σG. Primer extension analysis in B. subtilis revealed a spoVA transcript which had apparently initiated 6 bp downstream of the putative ‘–10’ heptanucleotide CATACTA, that is, 27 bp upstream of the coding sequence. This transcript was observed 4 h and 5 h after the initiation of sporulation, but not at earlier times.

A novel assay method for cis-aconitate decarboxylase (EC 4.1.1.6) has been developed. Differential centrifugation of cell-free extracts of Aspergillus terreus prepared by Novozyme treatment/nitrogen cavitation of itaconate-producing mycelia has shown that cis-aconitate decarboxylase, a key enzyme of itaconate biosynthesis, is localized exclusively in the cytosol. The intracellular localization and maximal activities of nine other enzymes putatively involved in itaconate biosynthesis were examined under both producing and non-producing growth conditions. Two striking differences between growth and production mycelia were observed: (a) cis-aconitate decarboxylase was absent from growing mycelia, and (b) aconitase activity was increased at least two-fold in production mycelia. No significant variations in the maximal activities or intracellular localization were observed for the other enzymes investigated. Mitochondrial and cytosolic isoenzymes of NADP:isocitrate dehydrogenase and malate dehydrogenase were detected by cellulose acetate strip electrophoretic analysis, whereas this analysis detected only single isoenzymes of aconitase and citrate synthase. We therefore propose that in itaconate accumulation cis-aconitate is synthesized in the mitochondrion and then transported to the cytosol where it is converted to itaconate by cis-aconitate decarboxylase.

Synthetic heterodisaccharides composed of glucose and xylose were tested as inducers of cellulose- and xylan-degrading enzymes in Aspergillus terreus, and the inducing abilities were compared with those of sophorose and xylobiose or their positional isomers. Measurement of secreted and cell-associated enzyme activities revealed that the heterodisaccharides induced the synthesis of the cellulolytic and xylanolytic enzymes, 2-O-?d-glucopyranosyl d-xylose (Glc?-2Xyl) being the most powerful inducer. Sophorose and 2-O-?d-xylopyranosyl d-xylose (Xy1/?-2Xy1), or their positional isomers, selectively induced the synthesis of cellulases and ?xylanases, respectively. An analysis of the extracellular enzymes (which were separated by isoelectric focusing followed by detection using chromogenic and fluorogenic substrates) showed that Glc?-2Xyl initiated the synthesis of specific endo-1,4-?glucanases and specific endo-1,4-?xylanases identical to those produced separately in response to sophorose or Xy1?-2Xy1. Glc?-2Xy1 also induced specific endo-1,4-?glucanases that hydrolysed 4-methylumbelliferyl ?lactoside at the agluconic bond. The results strengthen the concept of separate regulatory control of the synthesis of cellulases and ?xylanases. The results also suggest that mixed disaccharides, composed of glucose and xylose moieties, which may occur in nature, could play an important role in regulating the synthesis of wood-degrading enzymes.

Membranes of the acidophilic methylotroph Acetobacter methanolicus contained only b- and c-type cytochrome and a CO-binding b-type cytochrome. An azide-sensitive oxidase that oxidizes cytochrome c and ascorbate/TMPD was solubilized from the membrane with a mixture of CHAPS and Zwittergent3-12 (1.7 fold increase in specific activity with 32% yield). The solubilized oxidase is unusually stable with respect to high ionic strength (200 mM-NaCl) and stable between pH 4.0 and 6.8. Of the two soluble c-type cytochromes from A. methanolicus only the typical class I cytochrome (cytochrome c H) was a good substrate, as was equine cytochrome c. The oxidase was partially purified by anion-exchange chromatography but further purification proved impossible. The yield with respect to equine cytochrome c oxidation was 18%, with a 22-fold purification, but during purification most of the activity with respect to cytochrome c H and TMPD was lost. Neutral phospholipids had little effect on activity of the oxidase but the charged phospholipids phosphatidylglycerol and phosphatidylserine stimulated activity up to about fourfold. It proved impossible to incorporate the oxidase into active lipoprotein vesicles. During the purification process the pH optimum for oxidation of cytochrome c H was unchanged (pH 5.6) whereas that for oxidation of equine cytochrome changed from pH 9.5 to 7.5 and the sensitivity of the oxidase to azide changed from non-competitive to competitive during the purification process. The partially-purified oxidase contained only b-type cytochrome, some of which was CO-reactive. It is proposed that the oxidase is a cytochrome co type of oxidase that loses its cytochrome c component during the purification process and is only able to oxidize c-type cytochromes if these can be formed into a ‘reconstituted’ cytochrome co with the partially-purified oxidase.

Starved cultures of the soil amoeba Acanthamoeba castellanii suspended in phosphate-buffered saline exhibit stimulated O2 uptake in response to phagocytosis of either heat-killed yeast or latex beads. This phagocytosis-dependent (cyanide-insensitive) oxidative activity was observed when cells were isolated from either cyanide-stimulated or -inhibited cultures, and was therefore independent of the relative activities of the alternative mitochondrial oxidases known to exist in this organism. The extra O2 consumed during phagocytosis was stoichiometrically converted into O- 2 as detected by the rate of superoxide dismutase-inhibitable cytochrome c reduction. Phagolysosomal membranes isolated after uptake of latex beads were enriched in a b-type cytochrome which was spectrally similar to that present in immune phagocytes. Thus, the biochemical events during phagocytosis by either A. castellanii or immune phagocytes appear similar, suggesting that the ‘respiratory burst’ enzyme(s) responsible for O- 2 generation in these two cell types is structurally related.

The yeast Candida albicans is able to utilize l-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of a-aminoadipate-d-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the e-group of l-lysine was found in this yeast. The enzyme, l-lysine e-dehydrogenase, is strongly induced in cells grown on l-lysine as the sole nitrogen source. The enzyme is specific for both l-lysine and NADP+. The K m values were determined to be 0.87 mM for l-lysine and 0071 mM for NADP+. An apparent M r of 87000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 °C under our assay conditions.

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-l-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent K m for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent K m for S-AdoMet of 8.4 μM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-l-homocysteine and its analogue sinefungin with apparent K i values of 12.9 and 1.6 μm, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.

The relationship between enzyme stability and glycosylation was examined for two different Bacillus (1,3-1,4)-β-glucanases following expression of the corresponding genes in Escherichia coli and in Saccharomyces cerevisiae. Both of the (1,3-1,4)-β-glucanases secreted from yeast cells were glycosylated and a pronounced difference in the type and extent of glycosylation was observed. Thermostability analysis of the glycosylated enzymes and their unglycosylated counterparts synthesized by E. coli disclosed a substantially higher thermotolerance of the glycosylated enzymes. At 70 °C the half-life of the glycosylated form of B. macerans (1,3-1,4)-β-glucanase was 26 min, as compared to 10 min for the unglycosylated form of the enzyme. Using the same conditions, the half-life of the B. amyloliquefaciens-B. macerans hybrid (1,3-1,4)-β-glucanase was 5 min for the unglycosylated enzyme and about 100 min when the enzyme was glycosylated.

Teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teichomyceticus, comprises five main components, denoted T-A2 to T-A2-5, differing in the structure of their acyl side chain, which is linear in T-A2-1 and T-A2-3 and branched in the other components. Production of T-A2-1, characterized by a linear C10:1 acyl moiety, is entirely dependent on the presence of linoleate in the fermentation medium. Addition to the medium of oleic acid esters at 2 g l-1 increases the yields of T-A2-3, characterized by a linear C10:0 acyl chain, about threefold. The antibiotic linear side chains thus appear to originate from C18 unsaturated acid by β-oxidation degradation. The percentage of T-A2-2, T-A2-4 and T-A2-5, bearing the iso-C10:0, anteiso-C11:0 and iso-C11:0 acyl moieties, respectively, is strongly influenced by the presence in the medium of the amino acids known to be precursors of branched-chain fatty acids. Thus, valine increases the production of T-A2-2 whereas isoleucine or leucine increase the relative yields of T-A2-4 or T-A2-5, respectively. Analysis of the total cell lipids upon addition of the same amino acid shows corresponding increases in the proportion of the iso-C16:0, iso-C15:0 or anteiso-C17:0. A mutant A. teichomyceticus strain, which produces a novel teicoplanin with a linear C9:0 chain, differs from the wild strain in the presence of the linear C17:1 acid in its lipids. The relative incorporation of [14C]acetate into teicoplanin acyl moieties is substantially lower when this precursor is added to grown A. teichomyceticus mycelium rather than at the time of inoculation. The results suggest that teicoplanin branched acyl moieties also originate from β-oxidation degradation of cellular long-chain fatty acids.

The cell wall of Lactobacillus helveticus was found to have a three-layered structure when thin sections of whole cells or isolated cell walls were stained by various procedures and examined by electron microscopy. The main feature of the composition of isolated cell walls was a high protein content (about 48% of the cell wall dry weight) with a predominant 52 kDa protein. Peptidoglycan and what was considered as teichoic acids accounted for 36% and 14%, respectively, of the cell wall dry weight. The 52 kDa protein was apparently unglycosylated, located in the outer layer and noncovalently bound to the inner layers. Peptidoglycan was mostly assigned to the innermost layer. The conditions leading to autolysis of whole cells or isolated cell walls were determined, as well as the specificity of the autolysin involved in cell wall degradation.

Bacterial viruses are important in regulating bacterial population densities in natural aquatic environments, but the dynamics of bacteriophage-bacterial interactions in nature are poorly understood. In this study, the attachment and replication of three Pseudomonas aeruginosa bacteriophages (one temperate and two virulent) were investigated under conditions similar to those found in nature. Attachment and replication of bacteriophages were not impaired at host-cell densities equal to or lower (< 105 c.f.u. ml-1) than those frequently found in aquatic environments when the host cells were physiologically competent to allow phage growth. Attachment (45–93%) to either actively growing or starved cells was not impaired in river water, indicating that attachment is efficient in natural freshwater habitats. However, the replication of bacteriophages was significantly altered in starved cells in river water: the latency period was extended (broth, 70–110 min; river water, 110–240 min), and the burst size was reduced (broth, 27–65; river water 5–7). The findings of this study indicate that phages are likely to affect microbial ecology significantly in freshwater ecosystems.

Chlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1·5 kb labelled DNA probe containing the 3′ region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8·5 kb BglII fragment containing the complete MOMP gene was cloned into λEMBL3. Two hybridizing EcoRI fragments were sub-cloned into the λZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.

The two protein components, II and III, of the 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 were cloned separately into Escherichia coli. Component II was obtained on plasmid pCBSII, containing a 3.0 kbp HindIII fragment, and component III on plasmid pCBSIIIb, containing a 1.3 kbp SalI/PstI fragment. The identities of the two components were confirmed by comparison with the authentic components from Pseudomonas sp. CBS3. Both components were expressed constitutively in E. coli. Neither component alone showed dehalogenating activity. Only in the mixture of crude extracts from both clones was 4-chlorobenzoate dehalogenase detectable. The specific activities in E. coli crude extracts were 2.9 mU (mg protein)−1 for component II and 3.5 mU (mg protein)−1 for component III. Expression analysis by minicell experiments revealed a single polypeptide chain of 29 kDa for component II and of 31 kDa for component III.

Two mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2·6-fold and 4·7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.

Genetic instability in Streptomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.

By screening a cDNA library with a cDNA encoding the Dictyostelium discoideum cAMP-binding protein CABP1, under conditions of reduced stringency, we have isolated clones which code for two closely related molecules. Hybrid selection experiments indicated that these cDNAs encoded polypeptides with molecular masses of 34 (p34) and 31 (p31) kDa, both of which were recognized by anti-CABP1 monoclonal antibodies. Sequence analysis revealed that the clones were identical except for the presence of a 102 nucleotide segment inserted in-frame in the p34 cDNAs, just downstream of the translation initiation codon. DNA blot analysis suggested that p34 and p31 were encoded by the same gene. This hypothesis was strongly supported by the observation that both polypeptides were generated when a single cDNA was expressed under the control of the actin 15 promoter in D. discoideum cells. RNA blot analysis indicated that the cDNAs were complementary to three developmentally regulated transcripts of sizes 1.15 kb, 1.25 kb and 1.4 kb. Comparison of the derived amino acid sequences of p34 and p31 with those of the two subunits of CABP1 indicated that these polypeptides were very closely related, and that the corresponding genes probably arose by duplication followed by sequence divergence. Finally, the carboxy termini of these four polypeptides demonstrated 50% similarity to two polypeptides encoded by a bacterial plasmid which confers resistance to tellurium anions.

The proBA operon, coding for γ-glutamyl kinase (GK) and γ-glutamyl phosphate reductase (GPR), was cloned from the chromosome of the wild-type strain of Serratia marcescens Sr41. From our sequence data the proB (1101 bp) and proA (1472 bp) genes were shown to code for two proteins of M r 39169 and 44640, respectively. Analysis of expression of the lacZ structural gene fused with the proBA promoter showed that the proBA operon is not subject to proline-mediated feedback repression. Amplification of the proBA operon enabled us to determine GK activity, which was inhibited in the presence of a low concentration of l-proline. Comparison of the amino acid sequences of the S. marcescens GK and GPR proteins with those of the GK and GPR proteins from E. coli revealed extensive similarities.

Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using λgt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.

A collection of 72 reference strains of the Salmonella typhimurium complex of clones recovered from a variety of hosts and environmental sources in diverse geographic locations has been established for use in studies of variation in natural populations. Included are strains of the serovars S. typhimurium, S. saintpaul, S. heidelberg, S.paratyphi B (including variety java) and S. muenchen. The strains, which have been characterized by enzyme electrophoresis for allelic variation in 24 chromosomal structural genes and represent 48 distinctive multilocus genotypes (electrophoretic types or ETs), exemplify the full range of genotypic variation in the S. typhimurium complex. Evolutionary genetic relationships among the ETs are indicated in a phylogenetic tree generated by the neighbour-joining method from a matrix of Nei's standard genetic distance.