- Volume 132, Issue 9, 1986

SUMMARY: The lipid compositions of two azole-sensitive (A and B2630) and two azole-resistant (AD and KB) strains of the opportunistic fungal pathogen Candida albicans were studied by using several lipid extraction procedures: no differences were observed between the lipid content or total phospholipid/neutral lipid ratios of the four strains. All contained phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylserine as major phospholipids, with smaller amounts of phosphatidylglycerol and diphosphatidylglycerol; the relative proportions of these lipids differed between all four strains. The fatty acid composition of each major phospholipid within each strain differed, and there were also interstrain differences. A marked effect of culture growth phase in batch culture on lipid composition was observed. The major neutral lipids in each strain were triacylglycerol, non-esterified sterol and non-esterified fatty acid. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids, and there were also interstrain differences. All strains possessed (lyso)phospholipase activity, which was non-specific. The proportions of triacylglycerol and non-esterified fatty acid did not vary between strains, but the azole-resistant strains AD and KB contained more non-esterified sterol, giving them a phospholipid/sterol ratio approximately half that of azole-sensitive strains. There appeared to be a relationship between the phospholipid/sterol ratio of exponentially growing sensitive strains and their ability to take up azole; this did not extend to the resistant strains, which either did not take up azole (AD and KB) or took it up at a faster rate (Darlington) than sensitive strains.

Summary: The low-M r lipopolysaccharide produced by Nocardia otitidis-caviarum is composed of 6-O-methyl-D-glucose (11 mol), D-glucose (8 mol) and 3-O-methyl-D-glucose (1 mol). Glyceric acid was also found as a constituent. Methylation and periodate oxidation analyses suggested that the backbone was formed by (1.d4)-linked glucose and O-methylglucose residues. Glucose (1 mol) and 6-O-methylglucose (1 mol) served as branching points. Acetic acid was the major acyl substituent esterifying glucose residues. The lipopolysaccharide was isolated from the cytoplasmic fraction. Several other Nocardia strains were examined; all possessed the same lipopolysaccharide in their extracts.

Summary: One strain each of Xanthomonas campestris and “Xanthomonas phaseoli”, three strains of “Xanthomonas oryzae”, and five strains each of Escherichia coli and Klebsiella pneumoniae were found to produce a mixture of (1.2)-β-D-gluco-oligosaccharides and/or low-M r (1.2)-β-D-glucans in their culture media. The saccharides from the strains of Xanthomonas were all composed of unbranched, linear (1.2)-β-D-glucosaccharides with degrees of polymerization (DPs) of 8 to about 20, and a cyclic (1.2)-β-D-glucan (DP 16) containing one (1.6)-linkage and one α-linkage. The saccharides produced by the five strains of E. coli and four strains of K. pneumoniae were glucans with branches at O-6, and DPs of 10 to 15, whereas one strain of K. pneumoniae produced unbranched linear (1.2)-β-D-glucosaccharides with DPs of 6 to about 20.

Summary: Alkaline phosphatase activity in Vibrio cholerae strain 569B grown in low-phosphate medium was stimulated if glucose or glycerol was used as the carbon source. No such stimulation was observed, however, if tricarboxylic acid cycle intermediates like succinate or citrate were used. Experiments using specific enzyme inhibitors strongly indicated that the metabolic reactions of the glycolytic pathway from glyceraldehyde 3-phosphate to 2-phosphoglycerate play a key role in the stimulation process.

Summary: Dihydroxyacetone kinase (ATP: dihydroxyacetone phosphotransferase) from Schizosaccharomyces pombe has been purified and characterized. It has an M r of 160000 and consists of four polypeptides of M r 45000. It shows high substrate specificity, dihydroxyacetone being its only phosphoryl group acceptor and ATP its only phosphoryl group donor; the apparent K m value for dihydroxyacetone was 16 μm and that for ATP 550 μm. It has a pH optimum of 6.5, requires Mg2+ or Ca2+ (the slightly more active ion), and is inhibited by fluoride or ADP but not by dihydroxyacetone phosphate or fructose 1,6-bisphosphate. The isolated enzyme is relatively unstable.

Summary: Plasma membranes from osmotically lysed protoplasts of Metschnikowia reukaufii were concentrated 21 to 26-fold (using the recovery of [3H]dansyl chloride and ATPase activity at pH 6.5, as criteria). The contamination by mitochondria was approximately 10%. The plasma membrane ATPase was stimulated by K+ by up to 350% at pH 6.5; Na+ had a lesser effect, whereas Li+ had none. The stimulation by K+ was independent of Cl−, NO3 − or CO2 3 − as accompanying anions. The pH optimum of the ATPase was narrowed in the presence of 80 mm-KC1 to a distinct peak at pH 6.5. Under these conditions nucleoside triphosphates other than ATP were hydrolysed at rates less than 5% of that with ATP, and the K m of the ATPase for ATP was lowered from 1.3 to 0.6 mm. Orthovanadate (40 μm) and oligomycin (5 μg ml−1) inhibited the plasma membrane ATPase by 65% and by 20%, respectively. In contrast, the ATPase activity of the mitochondrial fraction had a sharp optimum at pH 8.5 and was not stimulated by added K+.

Summary: We have demonstrated that Rhizobium leguminosarum strain LPR1105 contains a heat stable and a heat labile glutamine synthetase (EC 6.3.1.2) activity similar to those described for other Rhizobiaceae. Most of the activity is heat stable when this strain is grown on glutamine as sole nitrogen source, but most is heat labile when grown on nitrate. Using a gene bank of R. leguminosarum DNA we have isolated two clones, which code for heat stable (p7D9) and heat labile (p4F7) glutamine synthetase activity, by complementing the glutamine auxotrophy of Klebsiella pneumoniae glnA mutants. Cross-hybridization of p7D9 with a fragment of the glnA gene of K. pneumoniae was observed, but no cross-hybridization between p7D9 and p4F7 was found. Since these two regions hybridize to genomic DNA of R. leguminosarum they are probably the structural genes for GSI and GSII, and the availability of these genes will make it possible to test this hypothesis. Clone p4F7 complements an ntrC+ but not an ntrC K. pneumoniae glnA mutant, suggesting that the ntrC gene is required for the complementation of the glutamine auxotrophy by this plasmid.

Summary: cysB, the regulatory gene of the cysteine regulon, is autoregulated. Inhibitors of both gyrase subunits, nalidixic acid and novobiocin, affect the expression of cysB, as monitored by β-galactosidase activity in cysB:: lac fusion strains. In gyrA mutants that are resistant to nalidixic acid, this drug does not affect cysB expression. The amount of mRNA transcribed from the cysB promoter isolated from cultures grown in the presence of gyrase inhibitors was significantly lower than that from the control culture without inhibitors. Urea also decreased cysB expression. These results suggest that DNA topology could play a role in cysB expression.

Summary: An enzyme preparation purified from Micrococcus luteus was shown to be specific for UV-induced pyrimidine dimers and was suitable for the detection of DNA excision repair systems. The wild-type Bacteroides fragilis Bf-2 strain and a mitomycin C-sensitive mutant (MTC25) had constitutive dimer excision systems which functioned efficiently under anaerobic and aerobic conditions. A UV-sensitive mutant (UVS9) had markedly reduced levels of the constitutive dimer excision systems under anaerobic and aerobic conditions. Since liquid holding recovery under aerobic conditions was inhibited by chloramphenicol whereas the final level of excision repair in B. fragilis Bf-2 was not affected, it is concluded that pyrimidine dimer removal is not the process responsible for increased physiological aerobic liquid holding recovery.

Summary: A generalized transducing phage, named CP75, was derived from a lysogenic strain of Pseudomonas cepacia. The frequency of transduction per phage particle ranged from 1.0 x 10−6 to 2.0 x 10−6 for a given marker. About half of the 105 P. cepacia strains tested were sensitive to the phage. The molecular size of the CP75 genome was approximately 52 kb.

Summary: Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.

Summary: Δψ-reduced amiA mutants of Streptococcus pneumoniae were shown to be resistant to the positively charged antitumoral drugs 2-N-methylellipticinium (NME) and 2-N-methyl-9-hydroxyellipticinium (NMHE). Conversely, mutants selected for their resistance to NMHE were mapped within the amiA locus and exhibited the pleiotropic AmiA− phenotype. This shows that Δψ is a critical parameter in determining resistance to these drugs in S. pneumoniae and suggests that they are accumulated within this bacterium in response to Δψ. As a consequence NME and NMHE appear to be valuable tools for selecting Δψ-reduced mutants in S. pneumoniae.

Summary: Operon fusions were isolated between Mu dX (lac CmR ApR) and btuB, the gene encoding the multivalent vitamin B12 outer membrane receptor. Using these fusions, vitamin B12-mediated repression of btuB in Escherichia coli was demonstrated. Mutations in metH, metE and OmpR as well as exogenous methionine, membrane pertubants, high osmolar conditions and temperature had no major effect on the expression of the btuB gene.

Summary: The occurrence of antigenic shift during meningococcal infection has been investigated by comparison of paired isolates obtained from the blood, cerebrospinal fluid or nasopharynx of patients. Isolates from any individual produced identical DNA "fingerprints" and showed stability in expression of both class 2 outer membrane protein and an antigen common to pathogenic Neisseria, confirming their origin as a single strain. One of the four strains examined produced variants which differed in the molecular mass of their class 5 outer membrane proteins. Three of the strains produced pili containing the epitope recognized by monoclonal antibody SM1 and two of these gave rise to variants which expressed pili of differing subunit molecular masses. The two variants of the remaining strain produced pilins lacking the common epitope detected by antibody SM1 but radioimmune precipitation with polyclonal anti-pilus antiserum revealed that variation in the molecular mass of the pilin expressed also occurred with this second class of pili. Antigenic variation in expression of both class 5 outer membrane proteins and pili therefore appears to be a common occurrence during meningococcal infection.

Summary: Passaging of the K88-positive Escherichia coli strain CN6913 through synthetic medium containing immune colostrum gave rise to large numbers of K88-negative CN6913 variants. These K88-negative variants had all lost a single large plasmid known to encode the K88 genetic determinant. Four other large plasmids harboured by this strain were unaffected. Viable K88-positive and K88-negative variants of CN6913 accumulated at a similar rate in synthetic medium and in medium containing non-immune colostrum. In the presence of immune colostrum, viable cells of the K88-negative variant accumulated faster and to a greater extent in cultures than the K88-positive variant if incubated at 37 °C, which favours the phenotypic expression of K88. However, when similar cultures were incubated at 18 °C, a temperature known to inhibit phenotypic expression of K88, the accumulation of viable cells of the two variants was strictly comparable in all media and no loss of plasmid or increase in K88-negative variants was observed. Cells containing a pBR322-based K88-encoding recombinant plasmid were also eliminated by immune colostrum whereas cells containing pBR322 were not. Plasmids encoding the K99 antigen were not readily eliminated from strains passaged through medium containing immune colostrum. K99-negative variants that were detected still harboured the K99-encoding plasmid.

Summary: Two O14:H12 strains of Serratia marcescens with different sensitivities to killing by normal pooled human serum were investigated. Complement binding, studied by measuring hydrophobicity and using rocket immunoelectrophoresis with anti-human C3, showed the sensitive cells (S1220) rapidly bound and fixed complement whereas the resistant cells (4444-60) bound less C3b. The strains had identical membrane protein composition. Crossed immunoelectrophoresis suggested that in S1220 cells the polysaccharide material including LPS was less antigenic and present in smaller amounts than in 4444-60 cells. This was confirmed by examining extracted polysaccharide material chemically and by SDS-PAGE. The resistant strain had 33% more phenol-extractable polysaccharide material than the sensitive strain, possibly comprising LPS with longer. O antigen chain lengths, or a microcapsule of O antigen polysaccharide. Extra polysaccharide material on the surface of the resistant strain prevents complement components binding and reaching the hydrophobic membrane where lytic lesions occur.