- Volume 144, Issue 1, 1998
Volume 144, Issue 1, 1998
- Review Article
-
- Sgm Special Lecture
-
- Microbiology Comment
-
- Biochemistry
-
-
-
Isoschizomers of the restriction endonuclease Taql (T/CGA) requiring different metal ion concentrations and having a range of thermal stabilities from Thermus species from different continents
More LessSummary: One-hundred-and-fifty-two isolates of the genus Thermus, collected from hot springs on four continents, were screened for evidence of the presence of the thermophilic Type II restriction endonuclease Taql (T/CGA). The presence of isoschizomers of Taql in 27 of the isolates, originating from hot springs in New Zealand, Iceland, USA, Japan, mainland Portugal and the island of Sao Miguel in the Azores, is reported. Six of the Taql-containing isolates from diverse geographical locations, identified by means of DNA/DNA homology and 16S rRNA sequence alignment as belonging to the Thermus species T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus and T. brockianus, were selected for comparative studies. The Taql isoschizomers from each of the six isolates were partially purified. They differed in their magnesium ion requirements, isoelectric points, subunit molecular masses and thermal stability.
-
-
-
-
An RNA polymerase preparation from Methylobacterium extorquens AM1 capable of transcribing from a methylotrophic promoter
More LessSummary: RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The β, β', α and ω subunits were approximately the same size as those of Escherichia coli, and the identity of the ω subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be σ factors, a 40 kDa polypeptide that shared identity with σ32 homologues, and a 97 kDa polypeptide that shared identity with σ70 homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli σ70. The same complement of putative σ factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible σ factor is present in this strain. Run-off assays showed that the purified RNAP was capable of initiating transcription specifically at the transcriptional start site of a methylotrophic gene, mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.
-
- Development And Structure
-
-
-
A mutation in the Rho1-GAP-encoding gene BEM2 of Saccharomyces cerevisiae affects morphogenesis and cell wall functionality
More LessSummary: Saccharomyces cerevisiae strain V918 was previously isolated in a search for thermosensitive autolytic mutants and found to bear a recessive mutation that caused the development of multinucleate swollen cells undergoing cell lysis. The BEM2 gene has been isolated by complementation of the phenotype of a V918 segregant. BEM2 encodes a Rho-GTPase-activating protein (GAP) which is thought to act as a modulator of the Rho1 small GTPase. It is shown that the mutation causing the morphogenetic and autolytic phenotype in strain V918 and its segregants lies in the BEM2 gene, defining a new mutant allele, bem2-21. Mutants in the BEM2 gene have been reported to display loss of cell polarity and depolarization of the actin cytoskeleton, causing a bud-emergence defect. Low resistance to sonication and to hydrolytic enzymes proved that the cell wall is less protective in bem2-21 mutants than in wild-type strains. Moreover, bem2-21 mutants are more sensitive than the wild-type to several antifungal drugs. Transmission electron microscopy revealed the development of abnormally thick and wide septa and the existence of thin areas in the cell wall which probably account for cell lysis. The depolarization of actin in bem2-21 mutants did not preclude morphogenetic events such as cell elongation in homozygous diploid strains during nitrogen starvation in solid media, hyperpolarization of growth in a background bearing a mutated septin, or sporulation. Multinucleate cells from bem2-21 homozygous diploids underwent sporulation giving rise to multispored asci (‘polyads’), containing up to 36 spores. This phenomenon occurred only under osmotically stabilized conditions, suggesting that the integrity of the ascus wall is impaired in cells expressing the bem2-21 mutation. It is concluded that the function of the BEM2 gene product is essential for the maintenance of a functional cell wall.
-
-
- Environmental Microbiology
-
-
-
In situ identification of nocardioform actinomycetes in activated sludge using fluorescent rRNA-targeted oligonucleotide probes
More LessSummary: Hitherto, few environmental samples have been investigated by a ‘full cycle rRNA analysis’. Here the results of in situ hybridization experiments with specific rRNA-targeted oligonucleotide probes developed on the basis of new sequences derived from a previously described comparative 16S rRNA analysis of nocardioform actinomycetes in activated sludge are reported. Application of the specific probes enabled identification and discrimination of the distinct populations of nocardioform actinomycetes in activated sludge. One of the specific probes (DLP) detected rod-shaped bacteria which were found in 13 of the 16 investigated sludge samples from various wastewater treatment plants, suggesting their importance in the wastewater treatment process. Another probe (GLP2) hybridized with typically branched filaments of nocardioforms mainly found in samples from enhanced biological phosphorus removal plants, suggesting that these bacteria are involved in sludge foaming. The combination of in situ hybridization with fluorescently labelled rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy improved the detection of nocardioform actinomycetes, which often showed only weak signals inside the activated-sludge flocs.
-
-
- Genetics And Molecular Biology
-
-
-
Genetic characterization of a phospholipase C gene from Candida albicans: presence of homologous sequences in Candida species other than Candida albicans
More LessSummary: Phospholipase C (PLC) enzymes are essential in regulating several important cellular functions in eukaryotes, including yeasts. In this study, PCR was used to identify a gene encoding PLC activity in Candida albicans, using oligonucleotide primers complementary to sequences encoding highly conserved amino acid regions within the X domains of previously characterized eukaryotic phospholipase C genes. The nucleotide sequence of the C. albicans gene, CAPLC1 (2997 bp), was determined from a recombinant clone containing C. albicans 132 A genomic DNA; it encoded a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC1) exhibited many of the features common to previously characterized PLCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 18-19 amino acid residues within the X domain and an unusually long N-terminus which did not contain a recognizable EF-hand Ca2+-binding domain. An overall amino acid homology of more than 27% with PLCs previously characterized from Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that the CAPLC1 protein is a δ-form of phosphoinositide-specific PLC (PI-PLC). PLC activity was detected in cell-free extracts of both yeast and hyphal forms of C. albicans 132A following 7 h and 24 h growth using the PLC-specific substrate p-nitrophenylphosphorylcholine (p-NPPC). In addition, CAPLC1 mRNA was detected by reverse transcriptase PCR in both yeast and hyphal forms of C. albicans 132A at the same time intervals. Expression of CAPLC1 activity was also detected in extracts of Escherichia coli DH5x harbouring plasmids which contained portions of the CAPLC1 gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses revealed that all C. albicans and Candida dubliniensis isolates examined possessed sequences homologous to CAPLC1. Sequences related to CAPLC1 were detected in some but not all isolates of Candida tropicalis, Candida glabrata and Candida parapsilosis tested, but not in the isolates of Candida krusei, Candida kefyr, Candida guillermondii and Candida lusitaniae examined. This paper reports the first description of the cloning and sequencing of a PLC gene from a pathogenic yeast species.
-
-
-
-
Protein F, a fibronectin-binding protein of Streptococcus pyogenes, also binds human fibrinogen: isolation of the protein and mapping of the binding region
More LessSummary: During screening of a gene library of Streptococcus pyogenes type M15 for fibrinogen-binding material, a protein of approximately 100 kDa, encoded outside the vir region, was found. DNA sequencing revealed this component to be identical to protein F, a fibronectin-binding protein. Isolation of the recombinant protein, termed F15, was performed by the use of fibrinogen affinity chromatography. The affinity constant (K a) of protein F15 for fibrinogen, 1.25 x 107 mol−1, was lower than that for fibronectin, 1.8 x 108 mol−1. The fibrinogen-binding domain was located in the N-terminal part of the molecule, while the fibronectin-binding domains, as previously determined, were in the C-terminal portion of protein F. To examine the amino acid sequence heterogeneity of protein F, the 5′ part of the prtF gene, corresponding to the N-terminal variable region of the protein, was amplified by PCR from 12 strains of S. pyogenes belonging to six different M-types. Alignment of these nucleotide sequences indicated that the 5′ portion of the prtF gene had probably undergone a number of intragenic recombination and horizontal gene transfer events, allowing a pattern of structural diversity of protein F observed earlier for some other streptococcal virulence factors. There was no strict correlation between M-type and nucleotide sequence of the variable region of the prtF gene and, compared to streptococcal M protein, the overall variation observed for protein F appeared more limited.
-
-
-
Altered adherence properties of a Streptococcus gordonii hppA (oligopeptide permease) mutant result from transcriptional effects on cshA adhesin gene expression
More LessSummary: Cell-surface polypeptide CshA (259 kDa) mediates multiple adherence interactions of Streptococcus gordonii. By generating a chromosomal cshA promoter (p-cshA)-cat gene fusion and measuring both CAT enzyme activity and cat mRNA levels, it was shown that cshA is expressed maximally in cells in the late exponential phase of growth in batch culture. The expression of CAT enzyme activity from the p-cshA-cat promoter fusion was 28% decreased in early stationary phase cell extracts of mutant strain OB528 in which the hppA (oligopeptide-binding lipoprotein) gene was insertionally inactivated. This effect was correlated with proportionally reduced cell-surface expression of CshA protein and with impaired adherence of hppA mutant cells to cells of an oral Actinomyces naeslundii strain. cshA promoter activity was enhanced in streptococcal cells that were incubated in conditioned culture medium as opposed to fresh medium, but this did not occur in an hppA genetic background. It is suggested that HppA is necessary for the response of cells to an extracellular factor that modulates cshA transcription, and hence affects cell-surface CshA expression and streptococcal cell adherence properties.
-
-
-
The phase-variable pilus-associated protein PilC is commonly expressed in clinical isolates of Neisseria gonorrhoeae, and shows sequence variability among strains
More LessSummary: PilC is a phase-variable protein associated with pilus-mediated adherence of pathogenic Neisseria to target cells. In this study, 24 strains of Neisseria gonorrhoeae with known epidemiological data were examined for expression of PilC. All strains produced PilC independently of serovar and site of isolation. To investigate whether the PilC protein is conserved or variable among gonococcal strains, the complete nucleotide sequence of pilC in four strains, isolated from either rectum, throat or blood, was determined. The deduced amino acid sequence in these strains differed from each other and from the two PilC proteins of N. gonorrhoeae MS11. These data demonstrate that PilC is commonly expressed, but the PilC sequence may vary among gonococcal strains.
-
-
-
Recombinational reassortment among opa genes from ET-37 complex Neisseria meningitidis isolates of diverse geographical origins
Summary: Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. ET-37 complex meningococci, defined by multilocus enzyme electrophoresis, have been isolated on different continents. Twenty-six different Opa proteins have been observed within strains of the ET-37 complex isolated between the 1960s and the 1980s, although individual strains have only four opa genes per chromosome. In this work the opa genes of four closely related ET-37 complex N. meningitidis strains recently isolated from Mali, West Africa were characterized and compared with the opa genes of strain FAM18, an ET-37 complex isolate from the USA. DNA sequence analysis and Southern blot experiments indicated that recombinational reassortment, including gene duplication and import by horizontal genetic exchange, has occurred in the opa genes within the ET-37 complex, resulting in two partially different Opa repertoires being present in FAM18 and the Mali isolates. Using synthetic peptides derived from the hypervariable (HV) regions of opa genes, the epitopes for nine mAbs were mapped. These bacteria, isolated on different continents, contain both shared and unique opa HV regions encoding epitopes recognized by mAbs and show evidence of recombinational reassortment of the HV regions.
-
-
-
pqqA is not required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1
More LessSummary: Methylobacterium extorquens AM1 is a facultative methylotroph that oxidizes methanol via the pyrroloquinoline quinone (PQQ)-linked enzyme methanol dehydrogenase. In M. extorquens AM1 and other PQQ-synthesizing bacteria, several genes are involved in the synthesis of PQQ and one of these, pqqA, has been proposed to encode a peptide precursor of PQQ. In other PQQ-synthesizing bacteria, pqqA is required for PQQ production. In this study, it is shown that both deletion and insertion mutants of pqqA in M. extorquens AM1 grow normally on methanol and produce PQQ. The level of PQQ production is reduced in the insertion mutant, but it is sufficient to allow normal growth on methanol. These results suggest either that a different peptide in M. extorquens AM1 can substitute for PqqA in pqqA mutants, or that PqqA-like peptides may not be obligatory precursors of PQQ. In addition, it is shown that the methanol oxidation transcriptional regulator gene, mxbM, is required for normal methanol induction of PQQ synthesis.
-
-
-
A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens
More LessSummary: Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carl double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL-independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.
-
-
-
Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum
More LessSummary: The protomer forming the S-layer of Clostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall. Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SIpA. The amino acid composition of SIpA matches the composition of a previously described exocellular glycoprotein. SIpA shared extensive similarity with the S-layer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brevis. In addition, the amino-terminal region of SIpA contained a segment presenting similarities with segments termed SLH (S-layer homologous), which are found in several bacterial exoproteins. A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C. thermocellum cells.
-
-
-
Rhodobacter capsulatus genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbLS) and neighbouring genes were acquired by a horizontal gene transfer
More LessSummary: Analysis of the nucleotide sequence of the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes (cbbL and cbbS) of the non-sulfur purple bacterium Rhodobacter capsulatus indicated that the deduced amino acid sequence of the large subunit was not closely homologous to the large subunit from related organisms. Indeed, phylogenetic analysis suggested that the large subunit protein (CbbL) more closely resembled the enzyme from αlβlγ purple bacteria and cyanobacteria and is within a ‘green-like’ radiation of the RubisCO phylogenetic tree, well separated from CbbL of the related organism Rhodobacter sphaeroides. A cbbQ gene was discovered downstream of cbbS in Rh. capsulatus, a gene arrangement which also appears to be limited to certain organisms containing a ‘green-like’ RubisCO. Upstream, and divergently transcribed from cbbLSQ, is a gene (cbbR I) that encodes a LysR-type transcriptional activator. Phylogenetic analysis of the deduced amino acid sequence of CbbRI also suggests that this protein is quite distinct from the Rh. sphaeroides CbbR protein, and is even distinct from the previously described CbbRII protein, the gene of which is upstream and divergently transcribed from the cbb II operon of Rh. capsulatus. Interestingly, Rh. capsulatus CbbRI is more closely related to CbbR from bacteria whose RubisCO falls within the ‘greenlike’ radiation of the CbbL tree. These studies suggest that the cbbR I-cbbL-cbbS-cbbQ genes were acquired by Rh. capsulatus via horizontal gene transfer from a bacterial species containing a ‘green-like’ RubisCO.
-
- Pathogenicity And Medical Microbiology
-
-
-
Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli
Summary: Osmoregulatory transporters ProP and ProU mediate the use of betaines as osmoprotectants by Escherichia coli. Glycine betaine and proline betaine are present in mammalian urines. Betaine uptake may therefore facilitate the growth of E. coli in the urinary tract, an environment of fluctuating osmolality. ProP transporter activity was approximately threefold higher in a pyelonephritis isolate, E. coli HU734, than in E. coli K-12. The growth rate of E. coli HU734 in aerated minimal salts medium was reduced twofold by 0.2 M NaCl in the absence and by 0.55 M NaCI in the presence of glycine betaine. Maximal growth rate stimulation was achieved when glycine betaine was added at a concentration as low as 25 μM. Deletion of the proP locus impaired the growth rate of E. coli HU734 in human urine but not in minimal medium supplemented with NaCI (0.4 M), with or without glycine betaine (0.1 mM). The expression of pyelonephritis-associated (P) pili was reduced when E. coli HU734 was cultured in a rich culture medium (LB) of elevated salinity. The proP lesion had no influence on P pilus expression in vitro or on the recovery of bacteria from the kidneys of inoculated mice. However, it did reduce their recovery from the bladders of inoculated mice 100-fold. These data provide the first direct evidence that osmoprotective betaine accumulation and transporter ProP are pertinent to both growth in human urine and colonization of the murine urinary tract by uropathogenic E. coli.
-
-
-
-
Haemolysin production by strains of Verocytotoxin-producing Escherichia coli
More LessSummary: Twenty-one strains of Verocytotoxin-producing Escherichia coli (VTEC) that hybridized with DNA probe CVD419 were examined for the ability to produce haemolysin. With solid media, all strains produced most haemolysin when grown in blood agar tubes and least when grown on blood agar plates incubated in air. Haemolysin production was increased considerably by incubating blood agar plates in an atmosphere comprising 8% carbon dioxide, 40% hydrogen and 52% nitrogen at 37 °C for 16 h, followed by 6 h at 21 °C in air. Haemolysin production was also increased when strains were grown on L-agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) prior to subculture on blood agar. Intracellular haemolysin was detected in five out of the 21 strains of E. coli grown on L-agar in the atmosphere described above, but haemolysin was not detected in L-broth culture supernatants. The haemolysins lysed guinea pig, mouse and ferret erythrocytes, but not human, rabbit, rat, turkey or chicken erythrocytes. Also, the addition of calcium ions to culture media was not required for haemolytic activity. It was concluded that haemolysins produced by VTEC appear to be quite distinct from E. coli α-haemolysin and resemble a form of β-haemolysin.
-
-
-
Lectin reactivity and virulence among strains of Listeria monocytogenes determined in vitro using the enterocyte-like cell line Caco-2
More LessSummary: Forty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested for lectin agglutination using a four-lectin set. Lectin reactivity (i.e. agglutination by one or more of the four lectins) was observed in all 12 clinical isolates, 16 of the 23 food isolates and eight of the 11 collection strains. Virulence was evaluated in vitro based on strains' ability to invade the human enterocyte-like cell line Caco-2. In gentamicin survival experiments, recovery of viable intracellular bacteria among lectin-unreactive strains was usually 100-1000-fold lower than among lectin-reactive haemolytic strains, and lower than among nonhaemolytic strains. Considerable cytopathogenic effects were produced by lectin-reactive haemolytic strains in trypan-blue-stained cell monolayers, whereas lectin-unreactive and nonhaemolytic strains produced no detectable cytopathogenic effect. Among lectin-reactive strains, the number of listerial cells associated with Caco-2 monolayers was more than tenfold greater than among lectin-unreactive strains. Cell invasion was inhibited by pretreatment of Caco-2 cells with sugars recognized by the lectins or of listeriae with enzymes which removed the same sugars from the bacterial surface. The results suggest that the study of lectin interactions could be helpful in understanding the pathogenicity potential of isolates of food and environmental origin.
-
-
-
Seroreactive species-specific lipooligosaccharides of Mycobacterium mucogenicum sp. nov. (formerly Mycobacterium chelonae-like organisms): identification and chemical characterization
Summary: Strains of the new species Mycobacterium mucogenicum exhibit physiological and biochemical features very similar to those of the other species of the Mycobacterium fortuitum complex. To define taxonomic criteria for easy identification of M. mucogenicum, the glycolipid patterns of the reference strains and of 32 environmental and clinical isolates were examined by TLC. It was concluded that all M. mucogenicum strains of smooth colony morphology contained species-specific alkali-labile glycoconjugates. Three different patterns were observed among the strains of the smooth colony type. Fractionation followed by conventional chemical analyses of the purified glycolipids showed the specific glycolipids to be lipooligosaccharides (LOS). The three LOS showed a similar fatty acid composition consisting of straight chain (dodeca-, tetradeca-, hexadecanoyl and hexadecenoyl) and methyl-branched (2,4-dimethyleicosanoyl and 2,4-dimethyleicosenoyl) fatty acyl substituents. The most commonly encountered LOS (present in 76% of the smooth strains) contained a tetraacylated pentasaccharide composed of four moles of glucose and one mole of a 2,4-di-O-methylhexose. A LOS composed of arabinose, glucose and mannose was present in 20% of the smooth strains, whereas the newly proposed type strain of M. mucogenicum (ATCC 49650) was the only strain that contained a LOS composed of glucose and galactose. Serological studies clearly differentiated most of the strains of M. mucogenicum from those of the other members of the M. fortuitum complex, and demonstrated the existence of serovars within the former species. Altogether, these data confirm the validity of the new species but show ATCC 49651 to be the most representative strain.
-
- Physiology And Growth
-
-
-
Differential expression of glucose-regulated (grp78) and heat-shock-inducible (hsp70) genes during asexual development of Neurospora crassa
More LessSummary: The expression of a glucose-regulated gene (grp78) changes significantly during the vegetative life cycle of Neurospora crassa: the amounts of grp78 mRNA are low in dormant conidia, increase during germination and exponential growth, decline in young aerial hyphae and reach a maximum in late (15-18 h) aerial hyphae. Heat shock (30 min at 45°C) elevated the mRNA level of this gene especially in early aerial hyphae, whereas no increase above the high constitutive amount was found after heat treatment of late aerial hyphae. The expression of the inducible hsp70 gene after heat shock also varied with the state of development and showed the highest inducibility in late aerial hyphae. Surface mycelium, from which aerial hyphae emerge, showed a similar increase in the amounts of both mRNA species. A developmental mutant (acon-2), which is defective in minor constriction budding of aerial hyphae, showed lower levels of con-2 mRNA as well as of grp78 and hsp70 mRNA (after heat shock) in late aerial hyphae. The acon-2 mutant did not form conidia at this stage. It is concluded that the high constitutive and inducible expression of stress genes in late aerial hyphae is due to a developmental activation of their transcription or, alternatively, to a lower degradation rate of their mRNA during this stage.
-
-
-
-
Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans
More LessSummary: The role of actin in apical growth and enzyme secretion in the filamentous fungus Aspergillus nidulans was studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and at the sites of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence at the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again, which led to a beaded apperance. We also studied the effect of CA on the secretion of α- and β-galactosidase. α-Galactosidase is secreted into the culture medium, whereas β-galactosidase remains in the mycelium, with part of its activity bound to the cell wall. When A. nidulans mycelium was incubated in the presence of CA, a reduction in the secretion of α-galactosidase into the culture medium and a decrease in the α- and β-galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion in A. nidulans. Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.
-
-
-
The role of autolysins during vegetative growth of Bacillus subtilis 168
More LessSummary: A set of isogenic mutants of Bacillus subtilis 168, insertionally inactivated in the genes encoding a number of lytic enzymes and a sigma factor (σD, which controls the expression of a number of autolysins) was constructed. Phenotypic analysis of the mutants determined the individual and combined roles of the autolysins in vegetative growth. The major vegetative autolysins of B. subtilis, LytC (50 kDa amidase) and LytD (90 kDa glucosaminidase), were shown to have roles in cell separation, cell wall turnover, antibiotic-induced lysis and motility. LytC was also shown to have a role in general cell lysis induced by sodium azide. Renaturing SDS-PAGE of cell-wall-binding protein extracts of the mutant strains revealed the presence of a novel autolysin that was previously masked by LytC. This 49 kDa enzyme was shown to be σD-controlled and was identified as a candidate cell separation and cell wall turnover enzyme. A multiple mutant strain, lacking LytC, LytD and the 49 kDa enzyme, retained at least ten bands of autolytic activity. These may correspond to individual or proteolytically processed novel autolysins, the functions of which are unknown. The multiple mutant strains facilitate the study of these, and other lytic enzymes, to determine their cellular functions.
-
-
-
Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and γ-butyrobetaine via the ABC transport system OpuC
More LessSummary: It was found that low concentrations of the naturally occurring and structurally related betaines L-carnitine, crotonobetaine and γ-butyrobetaine conferred a high degree of osmotic tolerance to Bacillus subtilis. Kinetic analysis of L-[N-methyl −14C]carnitine uptake in cells grown in minimal medium revealed the presence of a high-affinity transport system with a K m value of 5 μM and a maximum rate of transport (V max) of 41 nmol min−1 (mg protein)−1. A rise in medium osmolarity moderately increased the maximum velocity [V max 71 nmol min−1 (mg protein)−1] of this transport system, but had little effect on its affinity. Growth and transport studies with a set of strains that carried defined mutations in the previously identified glycine betaine transport systems OpuA, OpuC and OpuD allowed the identification of the ATP-binding cassette (ABC) transport system OpuC as the only uptake route for L-carnitine in B. subtilis. Competition experiments with crotonobetaine and γ-butyrobetaine revealed that the OpuC system also exhibited a high affinity for these trimethylammonium compounds with K i values of 6.4 μM. Tracer experiments with radiolabelled L-carnitine and 13C-NMR tracings of cell extracts demonstrated that these betaines are accumulated by B. subtilis in an unmodified form. In contrast, the β-substituted acylcarnitine esters acetylcarnitine and octanoylcarnitine both functioned as osmoprotectants for B. subtilis but were found to be accumulated as carnitine by the cells. None of these trimethylammonium compounds were used as sole carbon or nitrogen sources. The results thus characterize L-carnitine, crotonobetaine and γ-butyrobetaine as effective compatible solutes for B. subtilis and establish a crucial role of the ABC transport system OpuC for the supply of B. subtilis with a variety of osmoprotectants.
-
-
-
Metabolism is required for chemotaxis to sugars in Rhodobacter sphaeroides
Summary: Chemotaxis towards carbohydrates is mediated, in enteric bacteria, either by the transport-independent, methylation-dependent chemotaxis pathway or by transport and phosphorylation via the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). This study shows that Rhodobacter sphaeroides is chemotactic to a range of carbohydrates but the response involves neither the classical methyl-accepting chemotaxis protein (MCP) pathway nor the PTS transport pathway. The chemoattractant fructose was transported by a fructose-specific PTS system, but transport through this system did not appear to cause a chemotactic signal. Chemotaxis to sugars was inducible and occurred with the induction of carbohydrate transport systems and with substrate incorporation. A mutation of the glucose-6-phosphate dehydrogenase gene (zwf) inhibited chemotaxis towards substrates metabolized by this pathway although transport was unaffected. Chemotaxis to other, unrelated, chemoattractants (e.g. succinate) was unaffected. These data, in conjunction with the fact that mannitol and fructose (which utilize different transport pathways) compete in chemotaxis assays, suggest that in R. sphaeroides the chemotactic signal is likely to be generated by metabolic intermediates or the activities of the electron-transport chain and not by a cell-surface receptor or the rate or mode of substrate transport.
-
-
-
Carbon and energy flux constraints in continuous cultures of Alcaligenes eutrophus grown on phenol
More LessSummary: The growth behaviour of Alcaligenes eutrophus on phenol was investigated in continuous cultures to identify the phenomena limiting both growth efficiency and substrate degradation rates. It was shown that the fixed stoichiometry of the meta pathway of phenol degradation, leading to equimolar quantities of pyruvate and acetate, and the structure of the central pathways, which do not allow gluconeogenesis of acetate during growth on phenol, provoke the accumulation of polyhydroxybutyrate (PHB) under certain growth conditions. Acetate is predominantly used as an energy source and PHB accumulates when the cells are carbon-limited rather than energy-limited. The maximum rates of phenol degradation can be attributed to the expression of the enzymes of the catabolic pathway. This is particularly true of phenol hydroxylase and 2-hydroxymuconate semialdehyde (2-hms) dehydrogenase, whose substrates accumulated to physiologically significant concentrations at high growth rates. Indeed the concentration of 2-hms that accumulated in the medium indicated that this enzyme was substrate-saturated at maximum growth rates. However, the specific activity profiles of other catabolic enzymes associated with phenol degradation were close to the estimated flux through the pathway. This suggests a complex control structure in which several enzymes contribute to the control of pathway flux, as would be expected in a catabolic pathway.
-
- Genome Analysis
-
-
-
Physical identification of a chromosomal locus encoding biosynthetic genes for the lipopeptide calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2)
Summary: Putative peptide-synthetase-encoding DNA fragments were isolated from the Streptomyces coelicolor A3(2) chromosome using a PCR-based approach and mapped to a single ∼ 35 kb segment. In integrative transformation experiments, DNA fragments from this region disrupted production of the calcium-dependent antibiotic (CDA) and had sequences characteristic of non-ribosomal peptide synthetases, thus proving that the cda locus had been cloned.
-
-
- Corrigendum
-
- Guidelines For Authors
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)