- Volume 140, Issue 6, 1994

The biosynthetic pathway of glucosylglycerol (GG), the osmoprotective compound of Synechocystis sp. PCC 6803, was found to proceed from ADP-glucose and glycerol 3-phosphate via glucosylglycerol phosphate in a two-step reaction. Using an in vitro assay it was shown that the GG-forming enzyme system required activation, which could be initiated in vivo by hypertonic salt concentrations and osmotic shock, or in vitro by NaCl addition at the stage of enzyme extraction or assay.

The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of γ-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to γ-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, γ-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an Ω-chlorofatty acid. This is degraded by β-oxidation to 4-chlorobutyric acid which is chemically lactonized to γ-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C14:0, C16:0, C16:1 and C18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.

Carnocin UI49, a lantibiotic produced by Carnobacterium piscicola shows bactericidal activity against many lactic acid bacteria. This paper describes the results of a study on the mode of action of carnocin UI49. It has previously been observed that nisin-producing Lactococcus lactis subsp. lactis strains are at least 10-fold more sensitive to carnocin UI49 relative to other lactic acid bacteria. Addition of carnocin UI49 to cells of L. lactis subsp. lactis NZ9700 resulted in a dissipation of the membrane potential and a rapid hydrolysis of internal ATP. These results suggest that carnocin UI49 may act at the cytoplasmic membrane. The correlation between production of and/or immunity to nisin and sensitivity to carnocin of L. lactis subsp. lactis strains was further investigated. Several transformed L. lactis subsp. lactis strains carrying a fragment of the nisin gene cluster were tested for their sensitivity to carnocin UI49 and their immunity to nisin. The results suggest that NisP, which is one of the membrane-associated proteins involved in the production of nisin acts as receptor for carnocin UI49. This may facilitate the binding and/or insertion of carnocin UI49 into the cytoplasmic membrane thus increasing its bactericidal activity. Lantibiotic-producing and non-producing mutants of Staphylococcus epidermidis and Lactobacillus sake did not show a difference in sensitivity to carnocin UI49. The proposed receptor-mediated action of carnocin UI49 at the cytoplasmic membrane therefore seems to be specific for the nisin-producing strains.

Growth of Streptomyces ambofaciens in the presence of valine led to a greater production of spiramycin and excretion of short-chain fatty acids compared with growth on ammonium ions as nitrogen source. We determined the activities of enzymes that lead to the formation of malonyl-CoA, a key intermediate in the biosynthesis of spiramycin. In valine-grown cultures, the activities of acetyl-CoA carboxylase and pyruvate dehydrogenase were increased during the antibiotic production phase. During this idiophase, oxaloacetate dehydrogenase activity was much higher in cultures growing on ammonium compared with valine. Two pathways are proposed concerning the mode of malonyl-CoA formation: a route via oxaloacetate dehydrogenase following growth on ammonium, and a route via acetyl-CoA carboxylase after growth on valine.

Streptomyces thermoviolaceus is a thermophilic actinomycete that was found to produce relatively large amounts of extracellular peroxidase activity when grown on xylan as primary carbon source. The activity was due to multiple isoforms of peroxidase, of which two, designated P-3 and P-5, were predominant. The two proteins were purified to homogeneity by a combination of ultrafiltration, ammonium sulphate precipitation, anion-exchange chromatography, gel filtration and preparative gel electrophoresis. The peroxidases were found to be haemoproteins that catalysed the oxidation of a range of substrates in the presence of hydrogen peroxide. Both are monomeric acidic proteins (P-3: 82 kDa, pl 5·0; P-5: 60 kDa, pl 4·75) but with some differences in substrate specificity, P-3 exhibiting the broader substrate range. Peroxidase activity was optimal at pH values close to neutrality, and both enzymes were robust, exhibiting activity at elevated temperatures in the presence of denaturing agents such as SDS or 8 M urea. Peroxidase P-3 was stable at 50° for more than 24 h and had a half-life of 70 min at 70°. Polyclonal antibodies prepared against each isoform cross-reacted, indicating that the proteins were antigenically related. No cross-reactions were detected against horseradish peroxidase or crude peroxidase preparations from two other thermophilic streptomycetes.

The response of derepressed cells of Schizosaccharomyces pombe to the addition of glucose included a marked and reversible activation of neutral trehalase that was not produced in repressed cells. The protein synthesis inhibitor cycloheximide, the protonophore 2,4-dinitrophenol or the uncoupler sodium azide also enhanced trehalase activity in derepressed cells provided glucose was present in the incubation assays. However, only 2,4-dinitrophenol or cycloheximide was able to induce trehalase activation in repressed cells. Stimulation of trehalase by these compounds was preceded in all cases by a rapid increase in adenosine 3′-5′-cyclic monophosphate (cAMP) content. Since exogenous cAMP can activate trehalase both in repressed and derepressed growing cells, the results provide evidence for the existence of an induced cAMP signalling pathway in the fission yeast with several entries for trehalase activation. The correlation between cAMP increase and trehalase activation was not maintained when the enzyme was heat-shock-activated, supporting the concept that trehalase activity can be also enhanced in cells by another mechanism in which cAMP does not act as second messenger.

Analysis of data from multilocus enzyme electrophoresis has revealed that Neisseria gonorrhoeae populations are non-clonal. Fifteen percent of 227 isolates of N. gonorrhoeae had an identical multilocus genotype (ET1) and were recovered world-wide over a 26 year period. The recovery of isolates of identical multilocus genotype from geographically and temporally unassociated hosts is a common criterion of a clonal population structure. However, in a recombining (non-clonal) population, isolates with the same multilocus genotype can arise by the random association of the most common alleles in the population. Analysis of the variation in two further enzymes, in the restriction patterns obtained from the glutamine synthetase gene, and in the DNA fragments obtained using an arbitrarily primed polymerase chain reaction, was used to show that members of ET1 were almost as variable as randomly selected N. gonorrhoeae isolates. Unlike the situation in a strongly clonal species, the 26 ET1 isolates examined were increasingly sub-divided to give 19 distinguishable groups as variation at further loci was examined, and 24 distinguishable groups when auxotypes were also considered. We conclude that, as expected of a non-clonal population, the most commonly encountered N. gonorrhoeae multilocus genotype does not define a clone.

DNA sequence analysis and Southern hybridizations, together with complementation experiments, were used to study relationships between lactococcal plasmid replicons. pWVO2, pWVO4 and pWVO5, which co-exist in Lactococcus lactis subsp. cremoris Wg2, and pIL7 (isolated from another strain) all contained a functional replication region which appeared to be very similar to that of some known lactococcal plasmids. They contain a gene encoding a highly conserved RepB protein (60–80% amino acid identity between pWVO2, pWVO4 and pWVO5), which is essential for replication. When supplied in trans, repB of pWVO2 complemented a repB deficiency of pWVO5. Upstream of the repB gene, all these plasmids contain a strongly conserved region including a 22 bp sequence tandemly repeated three-and-a-half times, and an A/T-rich region. The similarity with pWVO2, which is known to replicate via a theta mechanism, suggests that all plasmids of this family are capable of theta replication. Southern hybridizations revealed that many lactococcal strains contain plasmids of this family.

The conversion of pyruvate to lactate is a key feature of lactococcal strains. The enzyme which facilitates this conversion, L-lactate dehydrogenase (LDH), and the gene which encodes it (ldh), are therefore of great significance. This paper presents the cloning and DNA sequence analysis of three further lactococcal genes which are of key importance in the genetic manipulation of commercial starter strains. The ldh gene from Lactococcus lactis subsp. lactis biovar diacetylactis BU2-60 has been isolated from a lambda library and sequenced. The ldh gene from L. lactis subsp. cremoris NCDO 762 and that from L. lactis subsp. lactis IL1403 have been amplified by the polymerase chain reaction (PCR) and sequenced. These DNA sequences and deduced amino acid sequences have been compared with those from L. lactis subsp. lactis MG1363. The LDHs from L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris NCDO 762 are 99·4% homologous. The LDHs from L. lactis subsp. lactis MG1363 and L. lactis subsp. lactis IL1403 are 96·4% homologous. The LDHs from L. lactis subsp. lactis IL1403 and L. lactis subsp. lactis biovar diacetylactis BU2-60 are 99,9% homologous. Our results provide further evidence that L. lactis subsp. lactis MG1363 and other L. lactis subsp. lactis NCDO 712 derived strains should be reclassified as Lactococcus lactis subsp. cremoris

The virulence region of the Salmonella enteritidis 55 kb plasmid pNL2001 was identified by Tn1-insertion mutagenesis, DNA hybridization studies, and Western blot analysis of proteins encoded in the virulence region of the plasmid. DNA hybridization studies showed that the pNL2001 plasmid contained a 6·4 kb SalI-EcoRI fragment homologous to the 6·4 kb SalI-EcoRI Salmonella plasmid virulence (spv) region of the S. choleraesuis 50 kb plasmid (pKDSC50). One of the 247 Tn1-insertion mutants of S. enteritidis, designated strain TA19, showed a reduced mouse lethality, and the Tn1-insertion of strain TA19 was located within this homologous 6·4 kb region, suggesting that the 6·4 kb SalI-EcoRI fragment of pNL2001 contained the spv region. Two contiguous SalI-EcoRI (2·3 kb) and EcoRI-EcoRI (4·1 kb) fragments derived from the 6·4 kb SalI-EcoRI fragment were cloned into the expression vectors. By Western blot analysis using four Spv peptide antisera, each specific for individual proteins encoded in the spvR, spvA, spvB and spvC genes of pKDSC50, four proteins encoded in the 6·4 kb SalI-EcoRI fragment of pNL2001 were identified. Protein SpvR with an apparent molecular mass of 32 kDa was produced from the 2·3 kb SalI-EcoRI fragment, and proteins SpvA, SpvB and SpvC with apparent molecular masses of 32, 70 and 29 kDa, respectively, were produced from the 4·1 kb EcoRI-EcoRI fragment. From the 4·1 kb EcoRI::Tn1 fragment of the TA19 plasmid, proteins SpvA and SpvB were expressed, but not SpvC. It was therefore suggested that the spvC gene may contribute to the expression of virulence of S. enteritidis. Furthermore, the nucleotide sequence of the 6·4 kb SalI-EcoRI fragment encoding these four proteins was determined. Four open reading frames which encoded the four proteins with deduced molecular masses of 33906, 28200, 65349 and 27646 Da were detected. Deduced amino acid sequences of each protein showed a high degree of identity to corresponding sequences in the virulence region of S. choleraesuis, S. dublin and S. typhimurium virulence plasmids. Therefore, we confirmed that the virulence plasmids of Salmonellae including S. enteritidis share the highly conserved region responsible for virulence.

The IncPα plasmid RK2 carries a cryptic tellurite resistance (Ter) determinant. This determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558). The Ter determinant identified as the kilA locus comprises an operon of three genes: kilA, telA and telB [also referred to as klaA, klaB and klaC on RK2(Tes)]. Each of the genes was subcloned into the expression vector pJF118EH behind an inducible tac-promotor using PCR. The PCR primers were used to engineer an efficient ribosome-binding site and adjacent sequence to improve protein expression. Expression plasmids were modified by inclusion of different resistance markers for selection during complementation. The tellurite-resistance phenotype was studied with the overexpressing plasmids. The study provides further evidence that all three genes within the kilAteIAB operon are required for cells to show resistance to potassium tellurite. Additionally, site-directed mutagenesis was carried out on the two cysteine residues in TelB. Changing either Cys125 or Cys132 to a Ser or Ala residue decreased the resistance mediated by the operon. These mutants demonstrate the requirement of cysteine residues within TelB for expression of tellurite resistance.

Linear DNA plasmids were found in the following yeasts: four strains of Kluyveromyces lactis, one of Debaryomyces hansenii, one of Winges robertsiae and four of Pichia etchellsii. In each case, the plasmids were present as a pair of DNA molecules of different sizes. The plasmids of K. lactis strains were associated with a killer activity and their structure was similar to the known killer plasmids pGKL1 and 2. The plasmids from the other three species were different from pGKL plasmids and showed no killer activity against the yeast species tested so far. In all cases, the linear molecules possessed terminal (probably inverted) repeats and their 5′ ends had a protected structure insensitive to λ exonuclease, while the 3′ ends were accessible to exonuclease III. All these strains could be efficiently cured of the plasmids by ultraviolet irradiation. The plasmids from D. hansenii (pDH1 A and B) and from W. robertsiae (pWR1A and B) shared related sequences with some of the K. lactis killer plasmid genes (encoding the supposed DNA polymerases, RNA polymerase and the chitinase), suggesting related genome organization of these plasmids. The pair of plasmids from P. etchellsii (pPE1 A and B) appear to be a distantly related member of the group. This pair showed no sequence homology with other plasmids, except weak homology with the putative RNA polymerase gene of pGKL2. None of the plasmids contained the sequences homologous to ORF3 and ORF4 of pGKL1 encoding the toxin resistance determinant and the toxin γ subunit, respectively.

The activity of BstVI DNA methyltransferase was monitored during the sporulative cycle of Bacillus stearothermophi lus V. Significant methylase activity was found only in bacteria growing vegetatively. This was confirmed by Northern hybridization, which indicated that the bstVIM gene was not transcribed in cells undergoing sporulation. Supporting evidence came from experiments which demonstrated that the RNA polymerase holoenzyme from these cells did not recognize the promoter elements upstream of the bstVIM gene.

Phosphatase activities were investigated in Morganella morganii, which is one of the few enterobacterial species producing high-level phosphateirrepressible acid phosphatase activity (HPAP phenotype), and the gene encoding the major phosphate-irrepressible acid phosphatase was cloned, sequenced, and its product characterized. Using p-nitrophenyl phosphate as substrate, Morganella produced a major phosphate-irrepressible acid phosphatase (named PhoC) which is associated with the HPAP phenotype, a minor phosphate-irrepressible acid phosphatase, and a phosphate-repressible alkaline phosphatase. The presence of the PhoC activity prevented induction of alkaline phosphatase when a PhoC-hydrolysable organic phosphate ester, such as glycerol 2-phosphate, was the sole phosphate source. PhoC is a secreted nonspecific acid phosphatase apparently composed of four 25 kDa polypeptide subunits. The enzyme is resistant to EDTA, Pi, fluoride and tartrate. The M. morganii PhoC showed 84·6% amino acid sequence identity to the PhoN nonspecific acid phosphatase of Providencia stuartii, 45·3 % to the PhoN nonspecific acid phosphatase of Salmonella typhimurium, and 37·8% to the principal acid phosphatase (PhoC) of Zymomonas mobilis. Comparison of sequence data and of regulation of these enzymes suggested a different phylogeny of members of this gene family within the Enterobacteriaceae.

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.

N-terminal sequence analysis of peptides generated by proteolytic treatment of the Pseudomonas fluorescens OE 28.3 major outer-membrane protein OprF, embedded in outer membranes or present in whole cells, indicated a surface-exposed location for the proline-rich region of the protein. This region is absent from the P. aeruginosa and P. syringae OprFs. Evidence was obtained for the presence of additional exposed but less accessible regions in the carboxy half of OprF. Four OprF-specific monoclonal antibodies were all directed to the C-terminal part of the protein but did not recognize a surface-exposed epitope as shown by flow cytometry. Our data support the model previously proposed for P. aeruginosa OprF in which the entire protein is embedded in the outer membrane, unlike the topology proposed for the major outer-membrane protein from Escherichia coli, OmpA, whose carboxy half resides in the periplasmic space. For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes. The proline-rich domain represented the most conserved region of the different P. fluorescens OprFs. Based on the sequence of its oprF gene, it was shown that the mushroom pathogen P. tolaasii is quite closely related to P. fluorescens. Comparative sequence analysis further showed that the carboxy half of OprF contains a sequence motif that is well conserved in the enterobacterial OmpA proteins but is also present in a number of other outer-membrane proteins, including peptidoglycan-associated lipoproteins.