- Volume 132, Issue 10, 1986

SUMMARY: Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.

SUMMARY: When an Arg-mutant of Streptomyces fradiae was grown on sucrose-nitrate agar supplemented with L-citrulline, it produced only non-sporulating aerial mycelia. Sporulation could be induced by adding 0.1 to 10.0 mM-L-methionine or 0.01 mM-cyanocobalamin; several other compounds including homocysteine, other amino acids, vitamins and precursors of nucleic acids were ineffective. Growth of the mutant did not differ significantly with or without methionine. The mutant was more sensitive to L-norleucine (a methionine analogue) than the parent strain. Spontaneous mutants, resistant to this analogue, were obtained. They all sporulated well without exogenously added methionine. In medium-exchange experiments, methionine was required for sporulation only after 48 h cultivation. The phenomenon reported here is most probably based on partially impaired methionine biosynthesis caused by an as yet uncharacterized non-auxotrophic mutation, since Arg+ revertants retained their methionine requirement for sporulation, and were also sensitive to L-norleucine.

SUMMARY: Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli. This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.

SUMMARY: Antibiotic-resistant and auxotrophic mutants of Bordetella pertussis were isolated. These were used as recipients for the uptake from Escherichia coli of broad-host-range R plasmids R68.45, RP1, and RP1 and RP4 carrying transposons Tn501 and Tn7 respectively. B. pertussis transconjugants from these crosses were used as donors to mobilize StrR, NalR, thr + and gly + chromosomal markers to B. pertussis or to B. parapertussis recipient strains. The frequency of plasmid transfer varied and depended on the donor and recipient strains used. Differences in chromosome mobilization frequencies of individual markers were observed and appeared to depend on the presence or absence of transposons Tn501 and Tn7 on the plasmid. Linkage was detected between the gly + and NalR markers.

SUMMARY: A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B. fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified: it had an apparent subunit M r of approximately 75000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.

SUMMARY: The modification status of DNA throughout the life cycle of Bacillus coagulans strain 26 was analysed by restriction analysis with methylation-sensitive enzymes. A significant fraction of the GATC sequences (dam target) in spore DNA contain N 6-methyladenine, a modification that is lacking during the vegetative phase. From the modulation of the modification pattern of GATC sites, the existence of a de novo methylase may be inferred. Spore DNA was more sensitive than vegetative cell DNA to BamHI, HpaI, SalI and XhoI, indicating that the sites for these enzymes are modified during the vegetative growth phase.

SUMMARY: Phage Fo lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid Fo lac. A host range mutant, phage Fo lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS:: Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage Fo lac h. Phages Fo lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.

SUMMARY: A restriction map for the 87 kbp IncP9 naphthalene catabolic plasmid pWW60-1 is presented. Transposon mutants were obtained using direct Tn5 insertion or using an indirect method involving the intermediate formation of unstable cointegrates between RP4 and pWW60-1. Insertions which affected expression of the early enzymes of the pathway (naphthalene to salicylate) were separated from inserts which affected expression of salicylate hydroxylase (nahG) or catechol 2,3-oxygenase (nahH). nahABC were cloned on a contiguous region of the plasmid on either a 6.9 kbp HindIII fragment (HE) or a 5.7 kbp XhoI fragment (XD). nahGH were cloned on a region situated about 30 kbp from nahABC on an XhoI fragment (XC), but nahH was only expressed on the corresponding but smaller XhoI fragments from two derivative plasmids of pWW60-1 with a deletion in that region. The detailed restriction map of nahH shows no similarities with the restriction maps of the genes for catechol 2,3-oxygenases from TOL plasmids, although the cloned gene did hybridize with genes for catechol 2,3-oxygenase from two different TOL plasmids. The organization of the catabolic genes on pWW60-1 suggests a separation into two operons as also described for the naphthalene catabolic plasmid NAH7 (alternatively called pIG7), but the relative directions of their transcription differs from that in NAH7.

SUMMARY: The biological properties and genome structure of two actinophages, 121 and SE-5, infecting Streptomyces erythraeus were characterized. They had the same host range (limited to S. erythraeus) and similar DNA G + C contents (around 60 mol %). Restriction maps of their genomes also showed many similarities. The close relationship between the two phages was confirmed by DNA hybridization experiments: large parts of their genomes were homologous, except for a segment in the middle of the map, where no hybridization was detected.

SUMMARY: The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (108 bacteria per 105 cells). Washed UEC, retained on 8 μm pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.

SUMMARY: Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S. mutans clinical variant, MT6801R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG14 and NG7183, were induced from strain MT6801R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to saliva-coated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801R and NG14, but not NG7183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de novo glucan synthesis is necessary for the accumulation of serotype c S. mutans cells on the tooth surface and the induction of dental caries.

SUMMARY: Monoclonal antibodies (MAb) raised to intact Streptococcus mutans P-4 cells (serotype e) were used to demonstrate the presence of shared antigenic determinant(s) between S. mutans BHT (serotype b) cell membranes and human heart tissue. MAb binding to both BHT membrane and human heart tissue was demonstrated by ELISA. Common antigens were identified by immunoblot analysis following separation of BHT membrane components and human heart antigens by SDS-PAGE. MAb 22C4 recognized three polypeptides from the BHT membrane preparation, having molecular masses of 42, 56 and 85 kDa. MAb 22C4 also recognized an 85 kDa component and a 200 kDa component from human heart tissue. MAb D159 was specific for a single 82 kDa polypeptide in BHT membrane, and also bound to two high molecular mass components in human heart (165 and 200 kDa). When both MAb D159 and 22C4 were first absorbed with S. mutans P-4 cells, subsequent reactivity to the aforementioned BHT membrane components was inhibited, indicating that these cross-reactive components are found in S. mutans P-4 as well as in S. mutans BHT micro-organisms. Competitive binding analysis showed that both MAb D159 and MAb 22C4 bound to myosin, indicating that S. mutans BHT membrane, human heart tissue and myosin share at least one immunodeterminant. This indicates that myosin could be the cross-reactive tissue component in human heart.

SUMMARY: Intestinal damage to mice caused by an enterotoxin from a coatless spore mutant of Clostridium perfringens type A (8-6) was examined by scanning electron microscopy. Two distinct types of damage were observed, both of which could be correlated with animal age. Damage appeared to occur in a specific sequence similar to that found in previous studies in rabbits. We conclude that the type of ileal tissue damage reflects the mode of toxin incorporation from the gut, which is a function of animal age.

SUMMARY: Rhizobium leguminosarum MNF3201 is a mutant defective in C4 dicarboxylic acid transport (dct). which forms ineffective nodules on pea. The Tn5-induced mutants MNF3041 (ribokinase-negative), MNF3045 (arabinonate dehydratase-negative), MNF3064 (defective in the “Entner-Doudoroff enzymes”) and MNF3070 (pyruvate carboxylase-negative) nodulate peas and fix N2. The carbohydrate-utilization mutations of these strains were each transduced into MNF3201 using phage RL38. The double mutants were defective in the utilization of both C4 dicarboxylic acids and the respective carbohydrates. All the double mutants formed nodules on peas, although these were still ineffective. Thus it appears that R. leguminosarum can use carbon sources other than dicarboxylic acids or common classes of sugars to fuel the nodulation process.