Showing papers in "Microbiology in 1986"

The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth.

Abstract: Summary: Nongrowing bacteria evade the bactericidal activity of βbT-lactam antibiotics. We sought to determine if slow growth rate also alters bactericidal activity. The bactericidal activity of two βbT-lactams on Escherichia coli grown in glucose limited chemostats was compared for generation times ranging from 0·7 to 12 h. The degree of killing varied with drug structure and with E. coli strain. However, all killing rates were a constant function of the bacterial generation time: slowly growing bacteria became progressively more phenotypically tolerant to βbT-lactam antibiotics as the generation time was extended.

Phospholipid Ester-linked Fatty Acid Biomarkers of Acetate-oxidizing Sulphate-reducers and Other Sulphide-forming Bacteria

Abstract: Summary: The phospholipid ester-linked fatty acids were examined in four Desulfobacter strains (2ac9, AcBa, 3ac10 and 4ac11), a Desulfobacter-like ‘fat vibrio’ (AcKo) and Desulfotomaculum acetoxidans (5575), which are all sulphate-reducing bacteria that oxidize acetate. A thermophilic sulphate reducer, Desulfovibrio thermophilus, and two sulphur-reducing bacteria, Desulfuromonas acetoxidans (11070) and a Campylobacter-like spirillum (5175), were also studied. The Desulfobacter spp. were characterized by significant quantities of 10-methylhexadecanoic acid. Other 10-methyl fatty acids were also detected in Desulfobacter spp. No 10-methyl fatty acids were detected in the other organisms examined, supporting the use of 10-methylhexadecanoic acid as a biomarker for Desulfobacter. High levels of cyclopropyl fatty acids, including two isomers of both methylenehexadecanoic (cy17:0) and methyleneheptadecanoic (cy18:0) acids, were also characteristic of Desulfobacter spp. The influence of the volatile fatty acids (VFA) propionate, isobutyrate, isovalerate and 2-methylbutyrate on the lipid fatty acid distribution was studied with two Desulfobacter strains (2ac9, AcBa) and Desulfotomaculum acetoxidans. Although these sulphate reducers cannot oxidize the VFA, their presence in the acetate growth medium caused a shift in the fatty acid distribution in favour of odd-numbered and branched chains by apparent direct incorporation into the fatty acids as chain initiators. The Desulfobacter strains were distinguished from other sulphide-forming bacteria by the percentage of unsaturated and the percentage of branched fatty acids.

Protein degradation by human intestinal bacteria.

Abstract: Summary: Analysis of human gut contents showed that substantial quantities of soluble protein, ammonia and branched chain volatile fatty acids occurred throughout the large intestine [0·1-24·4 g (kg contents)−1, 7·7-66·0 mmol (kg contents)−1 and 1·5-11·1 mmol (kg contents)−1 respectively]. The presence of these metabolites suggested that substantial proteolysis was occurring. In vitro studies showed that casein and bovine serum albumin were partly degraded in slurries of human faeces over a 96 h incubation period, to produce TCA-soluble peptides, ammonia and volatile fatty acids. Proteolytic activity detected in the stools of five individuals ranged from 3·5 to 19·8 mg azocasein hydrolysed h−1 (g faecal material)−1. Washed cell and washed particulate faecal fractions accounted for 24-67% of total activity. The predominant proteolytic bacteria in the faecal samples examined were identified as Bacteroides spp. [1·0 × 1011-1·3 × 1012 (g dry wt faeces)−1] and Propionibacterium spp. [1·2 × 108-1·0 × 1010 (g dry wt faeces)−1]. Other proteolytic bacteria which occurred in lesser numbers were identified as belonging to the genera Streptococcus, Clostridium, Bacillus and Staphylococcus. These results demonstrate that the gut microflora could potentially play a major role in proteolysis in the human colon.

Cuticle-degrading Enzymes of Entomopathogenic Fungi: Regulation of Production of Chitinolytic Enzymes

Abstract: Summary: Synthesis of chitinase and chitosanase by the entomopathogenic fungus Metarhizium anisopliae is regulated by products of chitin and chitosan degradation through an inducer-repressor mechanism. Slow-feeding with sugars or alanine (about 20 μg ml−1 h−1) in a carbon deficient medium to prevent catabolite repression (restricted cultures) demonstrated that the most effective inducers of chitinase and chitosanase were the principal monomeric constituents of chitin (N-acetylglucosamine) and chitosan (glucosamine) respectively. Increasing the rate of release of N-acetylglucosamine decreased chitinase synthesis by about 87% while causing a sevenfold increase in growth. In batch cultures high chitinase activities were present only in chitin-containing medium. There was a negative correlation between accessibility and amount of chitin substrates, levels of free N-acetylglucosamine in culture fluids and chitinase production. Addition of carbohydrates, lipid or proteins to chitin-grown cultures repressed chitinase production. Basal levels of chitinase were produced in non-inducing media. Production of chitobiase (N-acetylglucosaminidase) was enhanced from high basal levels by amino sugars, but was less inducible and less susceptible to catabolite repression than chitinase.

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae.

Abstract: Summary: The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since (i) inactivation did not occur during carbon starvation, (ii) when a fermentable sugar was added to starved cells, inactivation began, (iii) when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, (iv) when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.

Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers.

Abstract: SUMMARY: Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.

Effect of Growth Conditions and Surface Characteristics of Aquatic Bacteria on Their Attachment to Solid Surfaces

Abstract: Summary: The physico-chemical basis for the effects of nutrient conditions on the attachment of four freshwater isolates, Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp. and Flexibacter sp., to hydrophobic (PD) and relatively hydrophilic (TCD) polystyrene surfaces was investigated. Different nutrient conditions and growth rates resulted in changes in the physico-chemistry of the bacterial surfaces, measured either by liquid contact angles on lawns of cells or by hydrophobic and electrostatic interaction chromatography of cells, and in different levels of attachment to the substrata. The phenotypic changes in cell surfaces and the levels of attachment were different for each species. Levels of bacterial adhesion differed for the two substrata, indicating different adhesion interactions with PD and TCD surfaces. Treatment of attached cells with chloramphenicol did not cause detachment of any of the bacteria from PD or TCD, whereas periodate and protease treatment removed some attached cells, the degree of detachment depending on the species. The presence of complex organic molecules, in the liquid phase and conditioning the solid surface, influenced the extent of bacterial attachment, the effect depending on the substratum, organic concentration and bacterial species. The results suggest that changes in nutrient conditions in natural aquatic habitats will affect the attachment of individual bacterial species differently, thus influencing the population structure of developing biofilms.

Ethanol dissipates the proton-motive force across the plasma membrane of Saccharomyces cerevisiae

Abstract: Summary: Populations of Saccharomyces cerevisiae NCYC 431, harvested after 16 h incubation from self-induced anaerobic cultures, were more tolerant to the inhibitory effect of ethanol on fermentation rate and viability than organisms harvested from 8 h cultures. Ethanol increased the rate of passive influx of protons into de-energized organisms at a rate which was greater with organisms from 8 h compared with 16 h cultures. Rates of passive influx of protons into spheroplasts were significantly greater than into intact organisms, although culture age did not affect rates of ethanol-induced influx of protons into spheroplasts. Ethanol retarded both the initial net rate of proton efflux and the final extent of acidification produced by suspensions of energized organisms, both effects being more pronounced with organisms from 8 h as compared with 16 h cultures. The magnitude of the proton-motive force (Δp) was decreased by ethanol in both energized and de-energized organisms. Although culture age did not affect the extent of ethanol-induced decrease in Δp in de-energized organisms, in energized organisms harvested from 8 h cultures ethanol produced a significantly greater decrease in Δp as compared with organisms from 16 h cultures. If the ability of ethanol to decrease the Δp value is important in its inhibitory effect on growth, it is suggested that some phenomenon other than proton uncoupling is involved.

Transformation of the methylotrophic yeast Hansenula polymorpha

Abstract: Summary: A mutant of Hansenula polymorpha defective in β-isopropylmalate dehydrogenase was isolated. It was transformed with YEp13, a shuttle vector containing the Saccharomyces cerevisiae LEU2 gene, either using the protoplasting method or by treatment with LiCl. Leu+ transformants were recovered at a frequency of 30-60 μg DNA)-1. The transformants were shown to contain an autonomously replicating plasmid by stability tests, hybridization experiments and by the recovery of YEp13 from transformed cells via transformation of Escherichia coli. YEp13 linearized by BamHI digestion was also effective in transformation; the linearized plasmid was shown to recircularize and replicate autonomously in the transformants.

Differences in actin localization during bud and hypha formation in the yeast Candida albicans.

Abstract: Summary: Stationary phase cells of Candida albicans can form either a bud or a hypha, depending upon the pH of the medium into which they are released. At low pH, cells form an ellipsoidal bud and at high pH, cells form an elongated hypha. By staining cells with rhodamine-conjugated phalloidin, we have compared the dynamics of actin localization during the formation of buds and hyphae. Before evagination, actin granules were distributed throughout the cytoplasmic cortex in both budding and hypha-forming cells. Just before evagination, actin granules clustered at the site of evagination, then filled the early evagination in both budding and hypha-forming cells. With continued bud growth, the actin granules then redistributed throughout the cytoplasmic cortex. In marked contrast, with continued hyphal growth, the majority of actin granules clustered at the hyphal apex. This distinct difference in actin granule localization may be related to the distinct differences in the expansion zones of the cell wall recently demonstrated between growing buds and hyphae. The spatial and temporal dynamics of the large neck actin granules and of actin fibres are also described.

The effect of protein II and pili on the interaction of Neisseria gonorrhoeae with human polymorphonuclear leucocytes.

Abstract: Summary: Colonial variants of Neisseria gonorrhoeae strain P9 expressing different pili and/or outer membrane protein II (P.II) were investigated with respect to their interaction with human polymorphonuclear leucocytes (PMN). Two assay systems were used. A phagocytic killing assay measured the intracellular survival of gonococci, and PMN chemiluminescence (CL) was used to determine the initial surface interactions. All variants expressing P.II were killed effectively by PMN and also greatly stimulated PMN CL. The P.II− variants, on the other hand, were resistant to phagocytic killing and stimulated a much lower CL response. The presence of different P.II species was associated with different CL profiles and therefore different modes of interaction with the PMN membrane. A P.II-specific monoclonal IgG was opsonic and greatly increased PMN CL in contrast to F(ab')2 prepared from the same antibody, which inhibited it, thus confirming the role of P.II in the PMN interaction. Phagocytic killing assays revealed that with the loss of P.II, gonococcal variants acquired resistance to killing. Comparison of piliated and non-piliated pairs of variants with the same P.II profile showed that PMN–gonococcal interactions are dominated by the nature of the P.II species present whereas pili have little effect.

Extracellular Vesicle Formation and Biosurfactant Production by Serratia marcescens

Abstract: Summary: Pigmented and non-pigmented strains of Serratia marcescens produced extracellular vesicles and had wetting activity when grown at 30°C but not at 37°C. Light microscopy showed that the red pigment was present in vesicles and intracellular granules. Electron microscopy revealed the presence of vesicles surrounded by the bacterial membrane. Three lipids having the wetting activity, W1, W2 and W3, were isolated by thin-layer chromatography of lipids from different strains of S. marcescens. Dispersions of the isolated wetting agents had small contact angles on a polystyrene surface and the ability to lower surface tension. Wetting agent W1 was the aminolipid serratamolide. Wetting agents W2 and W3 were also aminolipids but were shown to be different from serratamolide by chemical analyses. Wetting agent and prodigiosin (in a pigmented strain) were the main lipids of isolated vesicles.

Orthanilic Acid and Analogues as Carbon Sources for Bacteria: Growth Physiology and Enzymic Desulphonation

Abstract: Summary: Carbon-limited aerobic batch enrichment cultures were grown and 17 bacteria able to degrade orthanilic acid (2-aminobenzenesulphonic acid), sulphanilic acid, sulphonamide, 4-sulphobenzoic acid, and benzene-, toluene- and phenolsulphonic acids were isolated. The organisms could each use one to three of the substances. Strain O-1, a Pseudomonas sp., which utilized three of these compounds, was studied in detail. A complete mass balance was obtained for the growth of the organism in medium containing, for example, orthanilic acid, and a specific growth rate of 0·1 h-1 was observed. Cell extracts desulphonated six aromatic sulphonates. The enzyme(s) was soluble and was not synthesized in succinate-grown cells. Enzyme activity [about 40 μkat (kg protein)-1] was dependent on the presence of catalytic amounts of NAD(P)H.

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis.

Abstract: SUMMARY: A spoIIA:: lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. β-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known spoO loci but on none of the “later” loci tested. The β-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage II mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spoOJ, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.

Identification, mapping, cloning and characterization of a gene (sbmA) required for microcin B17 action on Escherichia coli K12.

Abstract: Summary: We have identified mutations in three different chromosomal genes of Escherichia coli K12 which reduce sensitivity to microcin B17 Mutations in ompF and ompR genes affected production of an outer membrane porin protein, OmpF, and resulted in reduced sensitivity to a number of other agents (colicins, bacteriophages) besides microcin B17 The third class of mutants were specifically and highly resistant to microcin B17 The mutations in these strains were mapped to a gene (sbmA), located at 8·7 min on the E coli K12 chromosome, which is closely linked to phoA The wild-type sbmA allele was cloned into multiple copy number plasmids, and its location within the cloned DNA fragment was further defined by mutagenesis with MiniMudII1681 These insertion mutations resulted in in-frame fusions between the sbmA and lacZ genes, thereby allowing us to determine the direction of sbmA gene transcription Plasmids carrying these gene fusions produced low levels of β-galactosidase, indicating that the sbmA gene is poorly expressed We have been unable to identify the sbmA gene product, but indirect evidence indicates that it might be an envelope protein involved in microcin uptake

Structure, Distribution and Function of Wax Esters in Acinetobacter calcoaceticus

Abstract: SUMMARY: The wax esters of Acinetobacter calcoaceticus strains NCIB 8250 and NCIB 10487 harvested at stationary phase from N-limited batch cultures were extracted and shown to consist of C14 to C18 saturated and mono-unsaturated alkan-l-ols randomly esterified with C14 to C18 saturated and mono-unsaturated fatty acids. The mono-unsaturated components contained a cis Δ9 double bond. Wax ester content of strain NCIB 8250 increased under conditions of low growth rate in N-limited continuous culture with carbon and energy source in excess. The high content of wax ester in N-limited cultures of strain NCIB 8250 was lowered by incubation in the absence of a carbon and energy source and the wax ester was converted to water-soluble materials and CO2. It is proposed that in A. calcoaceticus NCIB 8250 the endogenous wax ester present in N-limited cells can serve as an energy reserve. All 19 strains of A. calcoaceticus tested contained some wax ester and as 16 of these strains had increased wax ester contents when harvested from stationary phase N-limited batch cultures, it appears that wax esters are widespread, but not universal, energy storage components in the genus Acinetobacter.

Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae

Abstract: Summary: Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osm there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osm cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.

Transcriptional regulation of the mercury-resistance genes of transposon Tn501.

Abstract: Expression of the mercury-resistance (mer) genes of the transposon Tn501 is positively and negatively controlled by the product of the merR gene. DNA sequence analysis has identified three open reading frames as potential candidates for this gene, one of which is oriented divergently with respect to the mercury-resistance genes. We have demonstrated that although RNA polymerase will bind to fragments containing the potential control regions for all three reading frames, only the control region for this divergent reading frame shows detectable promoter activity in vivo. Transcription of this reading frame is required for repression and induction of mer transcription. We have also shown that the Tn501 merR gene product negatively regulates its own synthesis, and have identified the start point of the transcript for this reading frame and for the mercury-inducible transcript of the mercury-resistance genes.

Migration of the Rhizosphere Bacteria Azospirillum brusilense and Pseudomonas fluorescens Towards Wheat Roots in the Soil

Abstract: Migration of the rhizosphere bacteria Azospirillum brasilense and Pseudomonas Puorescens towards wheat seedlings grown in soil was studied under various environmental conditions. The main factor affecting motility was soil moisture; of secondary importance were the soil type and the duration of plant growth prior to bacterial application. Migration was initiated following a lag of 24 h and was characterized by a bacterial band migrating through the soil towards the plant roots. Migration was significantly stimulated by various wheat genotypes and by synthetic attractants, though they did not differ in the intensity of their effect. It is proposed that these two rhizosphere bacteria can be stimulated to migrate non-specifically towards growing wheat plants.

An Accurate Method for Estimating Sizes of Small and Large Plasmids and DNA Fragments by Gel Electrophoresis

Abstract: SUMMARY: Several regression methods were tested for estimating the sizes of a wide range of plasmids (1·37–312 MDa) and restriction fragments (2·2–14·2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates (≤ 3·0 ± 1·5%). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.

Variation in the Expression of Pili and Outer Membrane Protein by Neisseria meningitidis during the Course of Meningococcal Infection

Abstract: 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.

The lipid composition of azole-sensitive and azole-resistant strains of Candida albicans.

Abstract: 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.

Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation.

Abstract: Streptomyces sp. MA406-A-1 produced formycin (a nucleoside antibiotic) in parallel with cell growth in a synthetic medium. When the synthetic medium was supplemented with 1% (w/v) Casamino acids, however, formycin was produced only after the end of exponential growth. The intracellular ppGpp pool increased gradually towards the end of exponential growth and was maximal at the beginning of formycin production. After shift down from Casamino acids medium to synthetic medium, the ppGpp pool increased immediately, while the GTP pool decreased; under such conditions, the ability to produce formycin increased eightfold. Relaxed (rel) mutants, the first isolated for a Streptomyces species, were found at high incidence (10%) among spontaneous thiopeptin-resistant isolates and had severely reduced abilities to accumulate ppGpp. These rel mutants also failed to produce formycin under the usual culture conditions and exhibited numerous pleiotropic effects such as an inability to produce melanin and an extended delay of aerial mycelium formation. Thus Streptomyces sp. exhibited a typical stringent response, and the response initiated (or was needed for) the induction of secondary metabolism. The response may have also participated in the initiation of aerial mycelium formation by decreasing the intracellular GTP pool.

Sucrose-dependent cell adherence and cariogenicity of serotype c Streptococcus mutans

Abstract: 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.

Effect of Growth Conditions on the Production, Composition and Viscosity of Xanthomonas campestris Exopolysaccharide

Abstract: Summary: A streptomycin-resistant variant of Xanthomonas campestris was grown in defined nutrient-deficient media in both batch and continuous culture. The production, composition and viscosity of the extracellular polysaccharide (xanthan) synthesized by this strain were influenced by the fermentation time and nutrient exhaustion in batch culture and by the dilution rate in continuous culture. The specific rate of exopolysaccharide synthesis was maximal during exponential growth in all of the nutrient-deficient media studied although some xanthan was formed during stationary phase. The concentration of exopolysaccharide decreased at later stages of stationary phase in some cultures. Both the extent of acylation and the consistency index of xanthan isolates were low or minimal during exponential growth, maximal in polysaccharide isolated as the growth rate fell and usually lower after this time. Between dilution rates of 0·03 and 0·06 h−1 in chemostat culture, the cell and exopolysaccharide dry weights were independent of dilution rate, the specific rate of xanthan synthesis decreasing at lower growth rates. Although the variation in the acyl content and consistency index was less than that observed in batch culture, exopolysaccharide isolated at higher dilution rates tended to have a higher acetyl content, lower pyruvyl content and lower consistency index.

Ultrastructure and Chemical Composition of Lipopolysaccharide Extracted from Leptospira interrogans serovar copenhageni

Abstract: SUMMARY: Lipopolysaccharide (LPS) from Leptospira interrogans serovar copenhageni was prepared from the aqueous phase of a phenol/water extract. Electron microscopic examination of negatively stained LPS showed a mixture of ribbon-like, round and ring structures. Carbohydrate analysis of the preparations revealed pentoses, hexoses, heptoses, hexosamines, and a 2-keto-3-deoxyonic acid which was chromatographically different from authentic 2-keto-3-deoxyoctonic acid (KDO). The major fatty acids of the LPS were hydroxylauric, palmitic and oleic acids. Although the leptospiral LPS preparations did not contain KDO or hydroxymyristic acid, they were otherwise morphologically and chemically similar to the LPS of other Gram-negative bacteria.

Regulation of Nitrite Reductase in the Cyanobacterium Anacystis nidulans

Abstract: SUMMARY: The effect of the nitrogen source on the activity of ferredoxin-dependent nitrite reductase has been studied in the cyanobacterium Anacystis nidulans. De novo synthesis of nitrite reductase occurred in the absence of an added nitrogen source, although enzyme activity was higher when the medium contained either NO3 − or NO2 −. The positive effect of NO3 − on nitrite reductase was also evident in tungstate-treated A. nidulans, which lacked an active nitrate reductase, indicating that the stimulatory effect was due to NO3 − itself and not to the NO2 − resulting from its intracellular reduction. NH4 + acted as a repressor, overriding any positive effect of NO3 − or NO2 −. Nitrite reductase synthesis was freed from NH4 + repression by L-methionine-DL-sulphoximine, an irreversible inhibitor of glutamine synthetase. NH4 + must therefore be metabolized through glutamine synthetase before repressing nitrite reductase. The kinetics of nitrate reductase and nitrite reductase development were similar in cells transferred from NH4 +- to NO3 −-containing media, suggesting a coordinate regulation of synthesis.

Photoactive retinal pigments in haloalkaliphilic bacteria

Abstract: Light-induced fast transient absorbance changes were detected by time-resolved spectroscopy in 38 of 51 haloalkaliphilic isolates from alkaline salt lakes in Kenya and the Wadi Natrun in Egypt. They indicate the presence of two retinal pigments, Pf and Ps, which undergo cyclic photoreactions with half-times of 2 ms and 500 ms respectively. Pf absorbs maximally near 580 nm and Ps near 500 nm. The pigments differ in their sensitivity to hydroxylamine and detergent bleaching and the photoreactions of Pf are strongly dependent on chloride concentration. Of the 38 pigment-containing strains, 29 possess both Pf and Ps, 9 possess only Ps. Inhibition of retinal synthesis with nicotine blocks pigment formation and addition of retinal restores it. Hydroxylamine-bleached pigments can be reconstituted with retinal or retinal analogues. Their similarity to the retinal pigments of Halobacterium halobium strongly suggests that they are also rhodopsin-like retinyledene proteins. Pf in all properties tested is almost identical to halorhodopsin, the light-driven chloride pump of H. halobium, and may serve the same function in the haloalkaliphiles. Ps has photocycle kinetics similar to sensory rhodopsin and a far-blue-shifted long-lived photocycle intermediate, but its ground state absorption maximum is near 500 nm instead of 587 nm. We have not found a bacteriorhodopsin-like pigment in the haloalkaliphiles.

Outgrowth Patterns of Mycelial Cord-forming Basidiomycetes from and between Woody Resource Units in Soil

Abstract: SUMMARY: Wood blocks colonized by the basidiomycetes Hypholoma fasciculare and Phanerochaete velutina were placed in plastic trays containing moist unsterilized soil. Both fungi grew out radially from the inoculum blocks in the form of networks of mycelial cords. When a second, uncolonized wood block, or set of wood blocks, was provided as a ‘bait’ about 5 cm from the inoculum block, marked changes in the form and growth characteristics of the mycelial network followed contact with the bait. These changes were influenced by the relative size of inoculum and bait and included inhibition of radial extension from the inoculum; stimulation of development of connective mycelium; directed growth responses to the bait; fan-shaped outgrowth with conserved polarity from the bait; eventual regression of non-connective mycelium originating from the inoculum. These effects presumably reflect the capacity of the mycelium to behave as a co-ordinated unit and to economize on biomass when growing between discontinuously supplied resource units.

Transport of Sulphur Dioxide by Saccharomyces cerevisiae

Abstract: SUMMARY: Accumulation of label from a suspension (pH 4·0) of Saccharomyces cerevisiae containing 100 mm-glucose and 1 mm-[35S]sulphite was initially rapid. Net accumulation ceased after 5 min, but at this time [35S]sulphite was still transported by organisms, and could be washed out to an extent that depended on the wash volume. Pre-incubation in the absence of glucose, and omitting glucose from the reaction mixture, had no effect on initial velocity of sulphite accumulation, although it decreased the total amount accumulated. Initial velocity of accumulation was also unchanged when organisms were pre-incubated in the presence of 2-deoxy-d-glucose and this inhibitor was included in the reaction mixture. Initial velocity of sulphite accumulation decreased logarithmically as the pH value of the suspension was increased from 3·0 to 5·0; the decrease closely paralleled the decline in concentration of molecular SO2 over this pH range. Woolf–Hofstee plots for accumulation of SO2, at pH 3·0 or 4·0, gave near-vertical plots. Raising the temperature from 19 to 39°C increased the initial velocity of SO2 accumulation. The initial velocity of transport was not affected by pretreatment of organisms with carbonyl cyanide m-chlorophenylhydrazone, DNP or iodoacetamide but pretreatment with 20 mm-uranyl nitrate increased the initial velocity almost threefold. It is concluded that SO2 is transported into S. cerevisiae by simple diffusion.