- Volume 142, Issue 8, 1996
Volume 142, Issue 8, 1996
- Review Article
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- Antigens And Immunity
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Evidence for different mannosylation processes involved in the association of β-1,2-linked oligomannosidic epitopes in Candida albicans mannan and phospholipomannan
More LessA monoclonal antibody specific for β-1,2-linked oligomannosides was used to study the association of these residues with Candida albicans mannan and phospholipomannan (PLM) in relation to growth conditions and in mannan mutant strains. Double immunofluorescence assays performed on cells grown under standard conditions indicated a highly heterogeneous cell surface expression of these epitopes in comparison with the homogeneous expression of α-linked oligomannosidic epitopes. Growth in the presence of tunicamycin, which inhibits mannan N-glycosylation, resulted in an absence of β-1,2-oligomannosidic epitopes on the cell surface, although PLM synthesis still occurred as shown by autoradiography. Similarly, growth in acidic conditions, which inhibits the incorporation of β-1,2-oligomannosides in mannan, resulted in an absence of β-1,2-oligomannosidic epitopes at the cell surface, although they still associated with PLM as shown by Western blotting. Western blots of C. albicans mutant strains with reduced amounts or an absence of phosphorus and acid-labile β-1,2-oligomannosides in their mannan confirmed that the association of β-1,2-linked oligomannosides with mannan and with PLM involves different mannosylation processes.
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- Biochemistry
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Evidence for a novel class of microbial 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa
More LessThe tryptophan-sensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit M r of 54000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa , their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.
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The occurrence of carboxymycobactin, the siderophore of pathogenic mycobacteria, as a second extracellular siderophore in Mycobacterium smegmatis
More LessCarboxymycobactin, in which the usual intracellular mycobactin siderophore is modified by possession of a carboxylic acid group, has been isolated as a second extracellular siderophore from culture filtrates of Mycobacterium smegmatis grown under iron-deficient conditions. (The primary siderophore is an exochelin which is a trihydroxamate, pentapeptide derivative.) There may be up to 12 similar molecules produced with differing chain lengths that can be recognized by HPLC or HPTLC. The amount of carboxymycobactin is about 20 times higher when cultures are grown with glycerol instead of glucose. Formation is maximal with an initial pH of the medium of about 8·4. The proportion of carboxymycobactin to the total siderophores produced - mainly exochelins - is maximally 10% (usually 10–25 μg ml−1). Formation of both extracellular siderophores (exochelin and carboxymycobactin) and of the intracellular mycobactin is maximal at the same initial concentration of iron added to the medium, 0·05-0·1 μg Fe ml−1, though exochelin is synthesized 24 h in advance of both carboxymycobactin and mycobactin.
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Oxygen-dependent low-temperature Δ12 (n6)-desaturase induction and alteration of fatty acid composition in Acanthamoeba castellanii
More LessThe influence of dissolved oxygen on the synthesis and activity of Δ12-desaturase in Acanthamoeba castellanii was investigated. A decline in oxygen concentration during batch growth at 30° was correlated with a decline in the degree of cellular fatty acid unsaturation. Chilling of early-stationary-phase cultures to 15° led to increased dissolved oxygen levels (from < 1 μM to 305 μM) and increased fatty acid unsaturation, which has been shown previously [Avery, S. V., Harwood, J. L. & Lloyd, D. (1994) Microbiology 140, 2423–2431] to be due mainly to δ12-desaturase induction. In contrast, chilling of mid-exponential-phase cultures, where the dissolved oxygen concentration prior to chilling was high (> 160 μM), gave no change in cellular fatty acid unsaturation. Measurement of [1-14C]acetate incorporation by oxygen-limited A. castellanii revealed that labelling of the Δ12-desaturase product, linoleate (18:2), increased with oxygen concentration. Microsomal levels of the Δ12-desaturase enzyme were found to increase by up to 10-fold during aeration of A. castellanii cultures; a transient elevation in oxygen was sufficient to induce Δ12-desaturase synthesis that was still fully detectable 1 h later. In addition, the activity of pre-existing Δ12-desaturase, measured in isolated microsomal membranes, increased by up to fivefold with increases in the oxygen concentration of assay mixtures. These results demonstrate for the first time that (i) oxygen availability alone can regulate de novo Δ12-desaturase synthesis in A. castellanii, and that (ii) oxygen can limit the activity of pre-existing Δ12-desaturase. These responses can occur independently of temperature changes.
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- Bioenergetics And Transport
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The modE gene product mediates molybdenum-dependent expression of genes for the high-affinity molybdate transporter and modG in Azotobacter vinelandii
More LessThe Azotobacter vinelandii mod locus, which is involved in high-affinity molybdate transport and the early events in Mo metabolism, consists of two divergently transcribed operons, modG and modEABC. modA, modB and modC encode the components of the high-affinity molybdate transporter, and modG encodes a Mo-binding protein. High concentrations of Mo repressed transcription of both operons. The modEABC operon was also repressed by tungstate and to a lesser extent by vanadate. modE, the first gene in the modEABC operon, controlled the Mo-dependent transcription of both operons. It was not involved in the metal regulation of alternative nitrogenase gene expression. Although a modE mutant constitutively expressed genes encoding the molybdate transporter, it had a reduced rate of Mo accumulation.
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- Biotechnology
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Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals: factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration
More LessThe effect of process variables on alkalization and removal of typical contaminating toxic metals from a simulated acid leachate by continuous mixed cultures of sulphate-reducing bacteria was studied. It was shown that the amount of metal removed and rise in pH both varied with the amount of sulphate reduction occurring, the residual sulphate concentration being the main determinant of final pH. Factorial experiments showed that sulphate reduction was enhanced by increasing the substrate concentration and inhibited by the initial sulphate concentration. The dilution rate did not exert a primary effect, but the existence of a significant interactive effect between the substrate and sulphate concentrations and the flow rate was indicative of a quantitative modification of the effect of the former two variables by the latter. The biomass concentration in the cultures was only affected by the substrate concentration indicating that the other variables acted by selection for or against sulphate-reducing components of the mixed culture. A response-surface analysis of the yield of sulphate reduction and alkalization against substrate concentration and dilution rate indicated that sulphate reduction (and alkalization) was sensitive to both of these variables where the substrate: sulphate stoichiometry was in the range 1: 1–3: 1. At lower sulphate concentrations complete reduction occurred at all levels while at higher sulphate concentrations washout occurred in all runs, which indicated that the key variable was the substrate:sulphate stoichiometry and its interaction with the dilution rate. Attention is drawn to the efficiency of the experimental designs employed for elucidating these factors.
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- Development And Structure
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SP75 is encoded by the DP87 gene and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins
More LessHighly purified spore coats of Dictyostelium discoideum each contained about 5 × 106 protein molecules as determined by amino acid composition analysis. By two-dimensional gel electrophoresis the coats were found to contain nine major-abundance and numerous minor protein species, most of which were highly enriched relative to the adjacent interspore matrix. Protein was nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that it was not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free thiol groups have been prealkylated, it was concluded that the D. discoideum spore coat proteins are disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this finding was supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned were associated with the outer layer of the coat. In coats missing one or more of four of these proteins as a result of gene disruption, there were physical changes but, with one exception, the other major coat proteins appeared to be incorporated normally. Sequence analysis showed that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH2-terminal subdomain of epidermal growth factor modules and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation.
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Cytoplasmic localization of the white phase-specific WH11 gene product of Candida albicans
More LessCells of Candida albicans WO-1 switch frequently, spontaneously and reversibly between a white and opaque phase. The white-opaque transition involves the regulation of phase-specific genes. In the white budding phase, cells express the white phase-specific gene WH11, which encodes a protein with homology to the heat shock protein Hsp 12 of Saccharomyces cerevisiae . A recombinant Wh11 protein has been synthesized, purified to apparent homogeneity and used to generate a rabbit polyclonal antiserum. The antiserum was used to localize the Wh11 protein in white phase cells. Wh11 is distributed throughout the cytoplasm but appears to be excluded from vesicles, plasma membrane and nucleus. An analysis by Western blotting of Wh11 expression in a number of C. albicans strains and related species suggests a correlation between round budding cell shape and expression.
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A comparative study of the incorporation of a 1,6-β-glucan and an O-glycosylated protein epitope into the cell wall of Candida albicans
More LessThe topological distribution of two epitopes in the cell wall of Candida albicans, the kinetics of their incorporation into the regenerating protoplast wall, and the effect of different antibiotics upon their incorporation and localization have been studied. To do so, two monoclonal antibodies that react against an O-glycosylated mannoprotein (1B12) and against a 1,6-β-glucan epitope (JRR1) were used. The results show that the JRR1 epitope is localized in an internal layer of the cell wall, in contrast to the 1B12 epitope, which is superficial, and that the incorporation of the JRR1 epitope into walls of regenerating protoplasts precedes that of the 1B12 epitope. The JRR1 epitope is normally found in the culture medium of control cells, but not in that of papulacandin-B-treated cells, and tunicamycin interferes with the incorporation of the 1B12 epitope into the cell walls. Finally, the results support the hypothesis that mannoproteins are not 1,6-β-glycosylated before their secretion.
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- Environmental Microbiology
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Rapid invasion by colicinogenic Escherichia coli with novel immunity functions
More LessBacteriocins have been suggested to play an important role in the invasion dynamics of bacteria. Recently, the ‘diversifying selection’ hypothesis has been proposed, which addresses the origin and diversification of one group of bacteriocins, the colicins of Escherichia coli . According to this hypothesis, novel colicin gene clusters arise from mutations generating expanded immunity functions. Positive selection, favouring these novel immunities, then rapidly drives strains carrying the evolved colicin gene clusters to fixation in the local population. To test this fixation step driven by selection, invasion experiments were carried out by introducing novel colicinogenic strains into established colicinogenic populations. In all cases, invasion by strains expressing novel immunity functions occurred rapidly, even when initial frequencies of the invader were quite low. These invasions were attributed primarily to colicin killing effect. Other factors, such as growth rate, level of colicin production and stationary-phase survival rate, were shown to play very minor roles in the invasion process. These results provide direct evidence for the hypothesis of diversifying selection acting on colicin gene clusters and shed light on the ecological role of colicins.
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Attachment of Vibrio alginolyticus to chitin mediated by chitin-binding proteins
More LessVibrio alginolyticus is the only culturable vibrio associated with the chitinaceous carapace of the copepod Tigriopus fulvus (Fisher 1860) living in Ligurian coastal rock pools (Tyrrhenian Sea). The characteristics of the interaction between chitin particles and V. alginolyticus were studied by analysing strains isolated both from the copepod surface and from rock-pool water. The highest degree of attachment to chitin was observed at 20°, in the presence of 3% NaCI. Bacterial treatment with N-acetylglucosamine and pronase E caused a reduction in attachment of 52–62% and 77–94%, respectively. Chitin pretreatment with either wheat germ agglutinin or membrane proteins (MPs) from V. alginolyticus caused a reduction in attachment, of 50–57% and 53–70%, respectively. No inhibition was observed when bacteria were pretreated with d-glucose, d-fucose or d-fructose, or when chitin was pretreated with concanavalin A and Escherichia coli DH5α MPs. V. alginolyticus MPs able to bind chitin were isolated and analysed by SDS-PAGE. Four chitin-binding proteins were visualized in all tested strains (53, 35, 20 and 14 kDa); in vivo these peptides may efficiently mediate V. alginolyticus attachment to chitin-containing substrates.
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Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor
More LessAn autotrophic, synthetic medium for the enrichment of anaerobic ammonium-oxidizing (Anammox) micro-organisms was developed. This medium contained ammonium and nitrite, as the only electron donor and electron acceptor, respectively, while carbonate was the only carbon source provided. Preliminary studies showed that the presence of nitrite and the absence of organic electron donors were essential for Anammox activity. The conversion rate of the enrichment culture in a fluidized bed reactor was 3 kg NH4 + m−3 d−1 when fed with 30 mM NH4 +. This is equivalent to a specific anaerobic ammonium oxidation rate of 1000–1100 nmol NH4 +h−1 (mg volatile solids)−1. The maximum specific oxidation rate obtained was 1500 nmol NH4 +h−1 (mg volatile solids)−1. Per mol NH4 + oxidized, 0.041mol CO2 were incorporated, resulting in a estimated growth rate of 0.001 h−1. The main product of the Anammox reaction is N2, but about 10% of the N-feed is converted to NO3 −. The overall nitrogen balance gave a ratio of NH4 −-conversion to NO2 −-conversion and NO3 −-production of 1:1·31±0·06:2·02±0·02. During the conversion of NH4 + with NO2 −, no other intermediates or end-products such as hydroxylamine, NO and N2O could be detected. Acetylene, phosphate and oxygen were shown to be strong inhibitors of the Anammox activity. The dominant type of micro-organism in the enrichment culture was an irregularly shaped cell with an unusual morphology. During the enrichment for Anammox micro-organisms on synthetic medium, an increase in ether lipids was observed. The colour of the biomass changed from brownish to red, which was accompanied by an increase in the cytochrome content. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.
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- Genetics And Molecular Biology
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Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes
More LessA cluster of anthracycline biosynthetic genes isolated from Streptomyces nogalater was expressed in Streptomyces lividans and in Streptomyces galilaeus. A 12 kb DNA fragment cloned from this cluster in pIJ486 caused the production of a novel compound when introduced into S. lividans. The compound is derived from nogalonic acid methyl ester, an early intermediate in nogalamycin biosynthesis. Complementation with the cloned 12 kb fragment of S. galilaeus mutants blocked in aclacinomycin biosynthesis caused the production of hybrid anthracyclines. Cloning of the nogalamycin gene cluster should make possible a detailed study of the biosynthesis of this interesting antibiotic, as well as the production of novel anthracyclines of potential value as cytostatic drugs.
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Bacillus subtilis mutS mutL operon: identification, nucleotide sequence and mutagenesis
More LessThe Bacillus subtilis mutS and mutL genes, involved in the DNA mismatch repair system, have been cloned and characterized. From sequence analysis the two genes appear to be organized in a single operon, located immediately downstream of the cotE gene (approximately 150° on the genetic map). The deduced MutS protein is 49% identical to HexA and MutL is 46% identical to HexB of Streptococcus pneumoniae. Deletion of both mutS and mutL resulted in an increase in the frequency of spontaneous mutations and abolished the marker effect observed in transformation. The expression of the mut operon was studied with the use of a mutSL-IacZ transcriptional fusion. An increase in expression was observed during late exponential growth.
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The phage-like element PBSX and part of the skin element, which are resident at different locations on the Bacillus subtilis chromosome, are highly homologous
More LessPBSX and skin are two unusual genetic elements resident on the Bacillus subtilis chromosome. PBSX is a phage-like element located at approximately 100° which is induced by the SOS response and results in cell lysis with the release of phage-like particles. The phage particles contain bacterial chromosomal DNA and kill sensitive bacteria without injecting DNA. The skin element is located at approximately 230° on the chromosome and is positioned within the sigK open reading frame (ORF). It is excised at a particular stage of sporulation, leading to reconstitution of the complete sigK gene. In this paper, we show that there are phage-like operons present in the skin element which are highly homologous to the region of PBSX comprising part of the control region and the late operon. These operons are similar in terms of their gene organization, the percentage identity of the products of homologous ORFs and the positioning and strengths of ribosome-binding sites for each ORF. Although this high degree of conservation suggests that the phage-like operons in skin can be expressed, expression of the late operon was not detected during exponential growth, during sporulation or after induction of the SOS response. However two non-phage-like operons in the skin element are expressed and have distinct expression profiles that are dependent on the growth and developmental status of the cell.
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A Bacillus subtilis secreted phosphodiesterase/alkaline phosphatase is the product of a Pho regulon gene, phoD
More LessA secreted phosphodiesterase/alkaline phosphatase, APaseD, was purified from a culture of Bacillus subtilis JH646MS. Its phosphodiesterase activity was reminiscent of an APase isolated and characterized previously. Immunoassay and N-terminal sequencing showed the two proteins to be identical. Using the first 20 amino acids of the mature protein, a BLAST search of GenBank was used to find an homologous sequence. An exact match was found but in a putative non-coding region. It was hypothesized that there was a base pair deletion in the phoD gene. A DNA fragment internal to the coding region was generated by PCR using template DNA from a strain which produced APaseD. The PCR fragment was cloned and used to interrupt the gene. Western blot analysis of the parent and the mutated strains showed that APaseD was missing in the mutant. Resequencing of the gene revealed a larger ORF encoding a protein similar in size to the 49 kDa APaseD estimated by SDS-PAGE. The promoter was then cloned, sequenced and used in phoD-IacZ promoter fusions which showed that the gene was phosphate-starvation-induced and dependent on PhoP and PhoR for expression.
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Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis
More LessThe Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used IacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo− genetic backgrounds (spoOA, sigE and sigK) cryIIIA was activated from the end of vegetative growth to stage II of sporulation ( t 3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigE mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the σE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.
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Translational control of puf operon expression in Rhodobacter sphaeroides 2.4.1
More LessThe puf operon of Rhodobacter sphaeroides 2.4.1 encodes the β- and α-polypeptides of the B875 complex, the L and M polypeptides of the reaction centre and the pufX gene product. A previous report from the authors′ laboratory indicated the potential existence of a 20-codon open reading frame (orfK, now designated pufK) located immediately upstream of the pufB structural gene. It is now demonstrated that pufK is translated in vivo and that the specific levels or nature of the rare codons within pufK affect the expression of pufK. Using a series of pufK-specific mutations, both in trans as IacZ translational fusions and incorporated into the genome in single copy, evidence has been obtained that translation initiation through pufK may be essential to translation of pufB. Further, the abundance, quality and distribution of rare codons within pufK may serve to ‘gate’ the entry of ribosomes at pufB. The data also suggest that translation of pufB is uncoupled from that of pufA, with the latter capable of being produced in excess of the former. It is also revealed that the secondary structure at the 5′ end of the large and small puf transcripts may play a role in mRNA stability and that stability of the small puf transcript is independent of translation.
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Cloning of the Rhodobacter sphaeroides hisI gene: unifunctionality of the encoded protein and lack of linkage to other his genes
More LessThe Rhodobacter sphaeroides 2.4.1 hisI gene, which encodes a phosphoribosyl AMP-cyclohydrolase that catalyses the third step in the histidine biosynthetic pathway, has been isolated from a genomic library of this phototrophic bacterium by complementation of an Escherichia coli his mutant. Analysis of the nucleotide sequence of the R. sphaeroides hisI gene reveals that it encodes a deduced product of 119 aa with a predicted molecular mass of 13·4 kDa. In contrast to the situation in E. coli, the R. sphaeroides hisI gene encodes a unifunctional protein and it is not linked to the hisE gene. The absence of a single histidine operon like that of E. coli was confirmed by PFGE experiments and complementation analysis of a R. sphaeroides hisI mutant that was constructed by marker exchange. The location of hisI in the R. sphaeroides genome has been determined to be at map co-ordinate 2275±20 of chromosome I.
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Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824
Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.
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Clostridium paradoxum DSM 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences
More LessSequence analysis of the cloned 16S rRNA genes of Clostridium paradoxum DSM 7308T revealed the presence of 15 different sequences in variable region I ( Escherichia coli positions 73–97) of the 16S rDNA. The majority of the cloned genes contained intervening sequences (IVSs), which varied in length from 120–131 nt, and were present in the DNA obtained from single colonies of C. paradoxum. The absence of IVSs in the mature rRNA was demonstrated by Northern hybridization and sequence analysis of the 16S rRNA reverse transcriptase (RT)-PCR product. This finding was supported by the failure of oligonucleotide probes specific for certain IVSs to hybridize to the RT-PCR product obtained from C. paradoxum. Alterations in culture conditions (temperature, pH, salt) or culture age did not lead to expression of RNA containing IVSs, as indicated by the size of RT-PCR products. Hybridization of the restriction-enzyme-digested genomic DNA of C. paradoxum with probes derived from the IVSs demonstrated that the 16S rRNA genes containing different IVSs are located at different sites on the chromosome.
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Acyl carrier protein of Azospirillum brasilense: properties of the purified protein and sequencing of the corresponding gene, acpP
More LessAcyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in Escherichia coli is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene (acpP) from Azospirillum brasilense using conventional methods. Therefore, ACP from A. brasilense was purified to homogeneity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the acpP region generated by two oppositely oriented internal primers designed from the partial acpP gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2·0–3·0 × 104 molecules per cell), (b) compared to E. coli ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.
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16S–23S rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous
More LessThe study of the intra-specific phylogenetic structure of Leuconostoc oenos is essential to understand the participation of several strains in malo-lactic fermentation (MLF). RFLP of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR) was performed in Leuc. oenos and other related species. The RFLP patterns with seven endonucleases were identical for the 37 Leuc. oenos strains, but differed from those obtained for all other species tested. This method could provide an invaluable insight for molecular identification of the wine leuconostocs. The RFLP relationships of members of the genera Leuconostoc and Weissella were highly similar to those previously reported by 16S and 23S rRNA sequencing studies. The 16S–23S rDNA ISR was sequenced in five strains of Leuc. oenos. A single tRNAAla was detected. The ISR sequence seems to be identical in the two rRNA (rrn) operons found in Leuc. oenos and no significant sequence variation was observed between strains that revealed relative differences as previously shown by PFGE. Results from the present study demonstrated that Leuc. oenos is phylogenetically a very homogeneous species (according to DNA-DNA hybridization studies) and sustain that this species is different from the genus Leuconostoc. The extremely conserved ISR of these organisms suggests that Leuc. oenos strains currently isolated and characterized must have spread with the transfer of viticulture rather than coming from indigenous populations.
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In vitro formation of a catabolic plasmid carrying Klebsiella pneumoniae DNA that allows growth of Escherichia coli K-12 on 3-hydroxybenzoate
More LessThe four enzymes needed to convert 3-hydroxybenzoate to pyruvate and fumarate via the gentisate pathway, as well as a putative positive regulator protein, were encoded on an 8 kb SphI fragment of Klebsiella pneumoniae DNA. The five genes were clustered in the order regulator-gentisate dioxygenase-fumarylpyruvate hydrolase-3-hydroxybenzoate monooxygenase-maleylpyruvate isomerase (mhbRDHMI), with the catabolic genes transcribed in the dioxygenase to isomerase direction. 2-Hydroxybenzoate was found to be a non-metabolizable inducer analogue for the mhb genes, supporting the view that gentisate rather than maleylpyruvate was the physiological inducer. The plasmid pNDR20 encoding the full gentisate catabolic pathway endowed Escherichia coli with the ability to grow on 3-hydroxybenzoate but the host cell appeared to be responsible for substrate uptake.
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somA, a novel gene that encodes a major outer-membrane protein of Synechococcus sp. PCC 7942
More LessThe outer membrane of a cyanobacterium (Synechococcus sp. strain PCC 7942) contains only a few major proteins. A gene encoding one of them, somA, was cloned and characterized. Based on the nucleotide sequence, SomA was predicted to comprise 531 amino acids with a calculated molecular mass of 57136 Da. The deduced amino acid sequence of SomA shares similarities with two bacterial cell-surface proteins, the S-layer protein of Thermus thermophilus and the flagellin of Campylobacter coli. The predicted amino acid sequence of SomA revealed also that it contains a signal peptide-like sequence at its N terminus. This signal peptide-like sequence was capable of mediating protein translocation across the cytoplasmic membrane into the outer membrane of Escherichia coli, provided that this sequence was fused to the E. coli outer-membrane protein, OmpF. The signal peptide-like sequence was cleaved upon the translocation of the SomA::OmpF protein. We suggest that SomA is synthesized as a precursor and that its N-terminal 24 amino acid sequence is a cleavable signal peptide involved in protein targeting into the outer membrane. To our knowledge, this is the first example of cleavable signal peptides for proteins transported into the outer membrane of cyanobacteria.
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The non-haem chloroperoxidase from Pseudomonas fluorescens and its relationship to pyrrolnitrin biosynthesis
More LessThe non-haem chloroperoxidase gene (cpoF) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe. Based on the overexpression of cpoF in Escherichia coli and the stabilty of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity. Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs. To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette. Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis.
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Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein
More LessProtein C (OprC) of the outer membrane of Pseudomonas aeruginosa forms small channels, as assayed by the liposome swelling method. We report here that OprC functions as a channel-forming and copper-binding protein. OprC purified to homogeneity formed a channel in planar lipid bilayers with an ion conductance of about 200 pS in 1 M NaCl. Cloning and sequencing of the gene encoding OprC revealed that it specified a polypeptide comprising 723 and 668 amino acid residues for the precursor and mature polypeptides (M r 73372), respectively. The amino acid sequence of OprC showed the highest degree of similarity with that of NosA of Pseudomonas stutzeri (65% sequence identity) which conveys Cu2+ to intracellular acceptor(s). OprC showed high copper-binding activity (K d = 2·6 μM) in aqueous solution containing surfactant. The expression of OprC appeared to be repressed by exogenous Cu2+ and derepressed by anaerobiosis in the presence of nitrate. These results suggest that OprC might be involved in copper utilization.
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Ribosomal protein gene transcription in Saccharomyces cerevisiae shows a biphasic response to nutritional changes
More LessNutrients are major determinants of ribosomal protein (rp-) gene transcription in Saccharomyces cerevisiae . In order to investigate the molecular mechanisms underlying this nutritional control, yeast mutants that display defects in the glucose upshift response of rp-gene transcription were isolated. Interestingly, although growth of these mutants on glucose-containing medium was severely affected an initial increase in rp-gene transcription by nutritional upshift was still observed. However, at later time points, rp-mRNA levels decreased strongly. Various other types of severe growth limitation also did not prevent the initial upshift in transcription. The results suggest that the glucose upshift response of rp-gene transcription comprises two phases: an initial, transient response independent of the actual growth potential, and a sustained response which is dependent on growth and requires both glucose and adequate nitrogen sources. Previously, it was found that protein kinase A (Pka) mediates the initial upshift response, without the need for regulation of Pka activity by cAMP. The present data substantiate that, besides the RAS/adenylate cyclase pathway, an alternative pathway through Pka regulates rp-gene transcription. In addition, evidence is presented that the sustained response does not require Pka activity. Based on these results, taken together, a model is proposed in which rp-gene transcription is dynamically regulated by multiple signal transduction pathways.
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LAG2, a gene that determines yeast longevity
More LessSaccharomyces cerevisiae has a limited life span, measured by the reproductive capacity of the individual cell. Several genes that are differentially expressed during the yeast life span have been isolated. One of these genes, LAG2, has been characterized for its role in longevity. LAG2 is preferentially expressed in young cells. It encodes a predicted 680 amino acid protein with a putative transmembrane helix. The sequence does not show significant similarity to any other DNA or protein sequences in the databases. Deletion of LAG2 in a haploid strain did not affect growth, but it resulted in a 50% decrease in the mean and maximum life span. When LAG2 was overexpressed, the mean and maximum life span of the yeasts was extended by about 36% and 54%, respectively. These results indicate that this is a longevity-assurance gene in yeast.
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- Pathogenicity And Medical Microbiology
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Flagellin gene and protein variation amongst clinical isolates of Pseudomonas aeruginosa
More LessFlagellin gene sequences from 64 clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa were amplified by PCR and subjected to RFLP analysis by using seven restriction enzymes to digest the amplified products. Using this approach the isolates were assigned to one of 13 groups. The method was rapid, reproducible and applicable to all isolates. In contrast, serotyping failed to satisfactorily resolve 49% of the strains tested. The vast majority of clinical isolates generated amplified products of 1·02 kb (type a) or 1·25 kb (type b). Electron microscopical analysis revealed evidence for some flagellar structural variation between P. aeruginosa strains. This study provides further evidence that the flagellin gene is a widely applicable and useful genetic marker for studying genetic variation within populations of closely related bacteria.
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A low-fibronectin-binding mutant of Staphylococcus aureus 879R4S has Tn918 inserted into its single fnb gene
More LessA low-fibronectin-binding mutant of Staphylococcus aureus strain 879R4SSp generated by transposon Tn918 mutagenesis is attenuated in a rat endocarditis model (J. M. Kuypers & R. A. Proctor, 1989, Infect Immun 57, 2306–2312). PCR and Southern hybridization analysis with primers and probes, respectively, for the fnbA and fnbB genes of strain 8325–4 showed that strain 879R4SSp possesses a single fnb gene which is homologous to fnbA. This was confirmed by sequencing 41 bp of 5′ non-coding and 237 bp of 5′ coding DNA, which showed 97% base identity to fnbA. Southern hybridization and sequencing showed that Tn918 was inserted 41 bp 5′ to fnbA in the mutant 879R4SSp/1536, between the promoter and initiation codon. Reduced adherence of the mutant to surface-bound fibronectin correlated with lower expression of a 180 kDa wall-associated fibronectin-binding protein.
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Capnocytophaga gingivalis: effects of glucose concentration on growth and hydrolytic enzyme production
More LessIn chemostat culture, the microaerophilic, CO2 requiring, gingival-plaque-associated bacterium Capnocytophaga gingivalis responded to the addition of glucose (1–6 g l−1) by doubling its growth rate and increasing its biomass yield fivefold. The data suggest that the glucose is catabolized by a fully aerobic route. Rather than repressing hydrolytic enzymes which might be associated with pathogenic properties, glucose enhanced the specific activity of aminopeptidase, trypsin-like protease, acid and alkaline phosphatase and α-glucosidase in comparison with a control culture grown in a tryptone/thiamin medium. Thus, the supply of glucose could be of importance in maximizing the pathogenic potential of this organism.
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A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae
More LessA large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites. Adjacent to the atfL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases. The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity). Genomic analysis of numerous V. cholerae strains (01, non-01 and 0139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase. The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V. cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.
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Effect of monoclonal antibodies directed against Candida albicans cell wall antigens on the adhesion of the fungus to polystyrene
The adhesion of Candida albicans to polystyrene and the effect of three monoclonal antibodies (mAbs) reactive with C. albicans cell wall surface antigens on this process was assessed in vitro with several C. albicans strains. In the absence of mAbs, adhesion of C. albicans to polystyrene increased in parallel with germ-tube formation. However, the growth of the strains in the yeast phase at 25 or the use of an agerminative mutant inhibited adhesion to polystyrene. Serotype A and B strains showed similar kinetics of adhesion to polystyrene and no statistically significant differences in germination or adhesion were observed when strains from the two serotypes were compared. The three mAbs had different effects on both germination and adhesion of C. albicans. mAb 3D9 showed no influence on either germination or adhesion to polystyrene in two C. albicans strains. mAb B9E decreased both adhesion (45·6%) and filamentation (52·6%), and mAb 21E6 decreased filamentation (34·0%) but enhanced adhesion by 23·3%. This enhancement was also observed with the agerminative mutant and it was dose-dependent. It was not related to the binding capacity of the MAb to polystyrene nor to an increase in cell surface hydrophobicity of the antibody-treated cells. In conclusion, both growth phases of C. albicans can adhere to polystyrene, although the conditions for this process seem to be different in each phase. The two types of adhesion of C. albicans to polystyrene might have a role in the colonization of medical implants. The disparate effects shown by mAbs directed against cell wall mannoproteins of C. albicans on the adhesion of the fungus to polystyrene should be taken into consideration when designing strategies to block the adhesion of C. albicans to plastic materials with mAbs.
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The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase
More LessGroup A streptococci bind the serine protease plasmin with high affinity. Previously, a 41 kDa protein was identified as a candidate plasmin receptor protein (Plr) from group A streptococcal strain 64/14. The plr gene encoding Plr was cloned and the deduced amino acid sequence of Plr had significant similarity to glyceraldehyde-3-phosphate dehydrogenases (GAPDHs). In this study we have isolated cytoplasmic GAPDH of streptococcal strain 64/14. This enzyme was examined, on both structural and functional levels, for its relatedness to the Plr of strain 64/14 purified from mutanolysin extract and to recombinant Plr. We report here that no differences were detected between streptococcal Plr and cytoplasmic GAPDH on the basis of antibody reactivity, plasmin-binding activity, GAPDH activity, N-terminal amino acid sequence, peptide map analysis by V8 protease digestion and amino acid composition analysis. Furthermore, the plr gene appears to be present as a single copy in group A streptococci.
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- Physiology And Growth
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Modified peptidoglycan chemical composition in shape-altered Escherichia coli
More LessPeptidoglycan synthesis and its fine chemical composition were studied in dividing cocci of Escherichia coli carrying the lov-1 mutation and in which the coccal shape was obtained either by mecillinam treatment or by transferring a pbpA mutation (penicillin-binding protein 2− phenotype), as compared to normal rods and non-dividing cocci. Synchronously dividing cocci showed peptidoglycan synthesis only in the cell cycle phase corresponding to cell septation. During the phase corresponding to lateral wall elongation, peptidoglycan synthesis was strongly reduced. This type of synthesis suggests that the dividing cocci consisted only of the two poles. Analysis of the muropeptide composition revealed a specific fourfold increase in the tetra-tetra-tetra trimer in dividing cocci as compared to non-dividing cocci or parental rods. We postulate that, in E. coli, the chemical composition of septal peptidoglycan differs from that of lateral wall peptidoglycan.
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Growth rate control of protein and nucleic acid content in Streptomyces coelicolor A3(2) and Escherichia coli B/r
More LessEscherichia coli possesses regulatory mechanisms that coordinate cell growth with the synthesis of essential macromolecules (protein, RNA and DNA). While fundamental differences have been identified in the growth habit and chromosome structure of E. coli and Streptomyces, little is known about these regulatory mechanisms in filamentous bacteria. This paper reports on the relationship between the macromolecule content of S. coelicolor A3(2) and its specific growth rate. The protein, RNA and DNA contents (g per 100 g biomass) of S. coelicolor A3(2) grown in steady-state continuous culture over a range of specific growth rates (0·025–0·3 h−1) were 31–45, 10–22 and 3·5–4·5% (w/w), respectively. This composition is qualitatively similar to that of other microorganisms. Changes in the macromolecular content of S. coelicolor A3(2) and E. coli B/r with specific growth rate appear to be essentially similar. However, the data indicate that the RNA content of S. coelicolor A3(2), grown under the conditions used, exceeds that of E. coli grown at the same specific growth rate. The data also suggest that overlapping rounds of replication are not a feature of DNA synthesis in S. coelicolor A3(2). This may be a function of the organism’s low maximum specific growth rate. Alternatively, it may be a consequence of regulatory mechanisms which act to inhibit the initiation of DNA synthesis in a linear chromosome which is already undergoing replication.
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Evidence of a role for NAD+-glycohydrolase and ADP-ribosyltransferase in growth and differentiation of Streptomyces griseus NRRL B-2682: inhibition by m-aminophenylboronic acid
More Lessm-Aminophenylboronic acid (APBA) inhibited the germination, growth and sporulation of Streptomyces griseus NRRL B-2682 in an age- and concentration-dependent manner in submerged and solid cultures. When added to cells or cell extracts it irreversibly inhibited NAD+-glycohydrolase and ADP-ribosyltransferase activity. ADP-ribosyltransferase was more sensitive, but inhibition was not complete, even in the presence of 10 mM APBA. The in vivo effects of the inhibitor correlated with its in vitro effect on ADP-ribosylation and on the profile of ADP-ribosylated endogenous proteins. The physiological importance of ADP-ribosyltransferase was supported by the observation that APBA strongly inhibited the growth of a non-sporulating and NAD+-glycohydrolase-negative mutant of the parental strain. The resistance of S. griseus NRRL B-2682 strains able to grow in the presence of APBA was due to permeability factors. A comparison of the ADP-ribosylated protein profiles of S. griseus NRRL B-2682 grown under various conditions showed similarities, but also specific differences. The results suggest that the ADP-ribosyltransferase of S. griseus NRRL B-2682 is an indispensable enzyme for growth and differentiation of the strain. It may regulate the activity of key enzymes or developmental proteins by responding to intra- and extracellular conditions.
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End-product control of enzymes of branched-chain amino acid biosynthesis in Streptomyces coelicolor
More LessIn streptomycetes, the branched-chain amino acids leucine, isoleucine and valine may serve as precursors for commercially important polyketides, and it is of interest to investigate whether the availability of these amino acids affects the production of the secondary metabolites derived from them. This paper reports studies on end-product control in the model organism Streptomyces coelicolor of the enzymes acetohydroxy acid synthase (AHAS) and isopropylmalate synthase (IPMS), mediating steps in the pathways to isoleucine-valine and leucine respectively. Specific activities of both enzymes were similarly affected when minimal medium was supplemented with the amino acids singly or in combination. Isoleucine alone caused a 2- to 3-fold increase, while all three amino acids caused a 5- to 8-fold decrease. Growth of an ilv auxotroph in media with limiting isoleucine gave enzyme specific activities 4- to 6-fold higher than in unsupplemented minimal medium. Spontaneous mutants were obtained by growing S. coelicolor on minimal medium containing 4-azaleucine. At least four patterns of end-product control were found among the mutants, one of which showed high constitutive levels of both enzymes (7- and 15-fold above unsupplemented minimal medium values for AHAS and IPMS respectively). It is concluded that the variation in specific activities of the two enzymes under different physiological and genetic conditions spans a range of around 50 to 100, and that S. coelicolor has molecular mechanisms capable of producing this response.
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Decision phase regulation of streptomycin production in Streptomyces griseus
More LessThe streptomycin (Sm) producer, Streptomyces griseus N2-3-11, shows medium-independent biphasic kinetics of the vegetative or exponential growth phase (EGP), reflecting an innate clock-like behaviour of growth and differentiation. The S. griseus growth and development cycle has the following characteristics: (1) after the developmental cycle commences, it cannot be influenced by environmental conditions; (2) the first EGP (decision phase) and its duration seem to be genetically determined, and it is also exhibited in pleiotropic mutants deficient in differentiation and antibiotic production; (3) during this early phase of growth, the decision to produce Sm is established and the fixation of later production and differentiation can only be influenced by effector molecules, e.g. A-factor, during this period; (4) after the onset of the second EGP, the commitment to Sm production cannot be reversed by dilution into fresh medium, nor by effector molecules; (5) the length of time of this effector-insensitive growth phase (second EGP or execution phase) can be extended by dilution into fresh medium; (6) the differentiation cycle of S. griseus is completed on entering stationary phase. The cells of S. griseus then return to a decision-making stage and recover sensitivity to effector molecules. Evidence that this type of phasing is valid for the growth and developmental cycles of all streptomycetes is discussed.
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Bidirectional usage of ferulate by the acetogen Peptostreptococcus productus U-1: CO2 and aromatic acrylate groups as competing electron acceptors
More LessThe influence of CO2 on the ability of Peptostreptococcus) productus U-1 (ATCC 35244) to use an aromatic acrylate group as an energy-conserving electron acceptor during O-methyl-dependent growth was examined. Ferulate (a methoxylated phenylacrylate), unlike hydroferulate (a methoxylated phenylpropionate),supported growth under CO2-limited conditions. Two phases occurred during ferulate utilization in CO2-limited cultures. In phase I (maximum growth), O-methyl-derived reductant was coupled mainly to acrylate group reduction, and acetate synthesis (CO2 as reductant sink) was minimal. In phase II, acetate synthesis increased, but cell yields in this phase were much less than in phase I. In CO2-enriched cultures, distinct phases were not observed; reductant was coupled equally to CO2 and acrylate group reduction. Under CO2-enriched conditions, O-methyl and acrylate groups were incompletely metabolized, and molar growth yields were significantly lower compared to CO2-limited conditions. Resting cell studies indicated that O-demethylase and aromatic acrylate oxidoreductase activities were induced by ferulate. These findings demonstrated that P. productus U-1 can use the aromatic acrylate oxidoreductase system as a sole, energy-conserving, electron-accepting process, but is not able to prevent the simultaneous use of the bioenergetically less favourable acetyl-CoA pathway during O-methyl-dependent growth.
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The assimilation of sulfur from multiple sources and its correlation with expression of the sulfate-starvation-induced stimulon in Pseudomonas putida S-313
More LessConditions were optimized for the batch growth of Pseudomonas putida S-313 under sulfur-limited conditions. P. putida grew exponentially with sulfate as the sole source of sulfur, and growth was concomitant with the utilization of sulfate until it was exhausted. A further 20% of protein was synthesized after the apparent disappearance of sulfate. A mass balance for the utilized sulfate in cell material was calculated, given the observed molar growth yield of about 3·6 kg protein (mol S)−1 and a sulfur content of 0·41% S in dry matter. Similar data were obtained for growth with cysteine and thiocyanate. The organism also grew exponentially with 4-toluenesulfonate (TS) as sulfur source, essentially as observed with sulfate, except that negligible protein formation after exhaustion of TS was observed. Similar data were also obtained with 4-nitrocatecholsulfate (NCS) and ethanesulfonate. Any substrate pair selected from sulfate, cysteine and thiocyanate was utilized simultaneously, and although one of the pair of substrates was always preferred, growth continued at the same rate when only one substrate remained. Growth after substrate exhaustion was observed. Any substrate pair selected from TS, NCS and ethanesulfonate gave similar data, but with less growth after exhaustion of the sulfur sources. If a mixed substrate pair was chosen from the two groups, the sulfur source from the first-named group was initially used exclusively, and the second source of sulfur was utilized subsequently, after a lag phase. The data are considered to reflect the control of scavenging for sulfur and of distribution of sulfur in the cell exerted by the sulfate-starvation-induced stimulon [Kertesz, Leisinger & Cook, J Bacteriol (1993) 175, 1187-1189].
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Physiological effects of nitrogen starvation in an anaerobic batch culture of Saccharomyces cerevisiae
More LessThe effects of nitrogen starvation on the anaerobic physiology of Saccharomyces cerevisiae were studied in cells cultivated in a bioreactor. The composition of the mineral medium was designed such that the nitrogen source became depleted while there was still ample glucose left in the medium. The culture was characterized by acoustic gas analysis, flow injection analysis and HPLC analysis of extracellular substrates and metabolites. During the cultivation, the macromolecular composition of the cells was analysed with respect to the cellular content of RNA, protein, trehalose and glycogen. During exponential growth under anaerobic conditions, the maximum specific growth rate (μmax) of S. cerevisiae CBS 8066 (0·46 h-1) was identical to the μmax determined under aerobic conditions. Depletion of ammonium in the medium led to an abrupt decrease in the flux through glycolysis. Subsequently, a continuous decrease in the carbon dioxide evolution rate, caused by catabolite inactivation of the hexose-transport system, was observed. The apparent half-life of the transport system under nitrogen starvation was 13 h. During the exponential growth phase, the cellular content of RNA and protein was 15% (w/w) and 60% (w/w), respectively. At the end of the cultivation where the cells had been starved of nitrogen for 18 h, the cellular content of RNA and protein had decreased to 4% (w/w) and 22% (w/w), respectively. The intracellular carbohydrate content increased dramatically as trehalose and glycogen accumulated to final concentrations of 7% (w/w) and 25% (w/w), respectively. Glycerol formation during nitrogen starvation was higher than that accounted for by the formation of organic acids, suggesting a protein turnover of approximately 6% h−1. The growth energetics of S. cerevisiae CBS 8066 also changed as a result of nitrogen starvation, and Y xATP was observed to increase from 80 mmol g−1 during the exponential growth phase to more than 130 mmol g−1 towards the end of the cultivation. The presented results illustrate the effect of nitrogen starvation on glycerol formation, protein turnover, catabolite inactivation of the sugar-transport system, the cellular composition, the cell cycle and growth energetics.
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- Plant-Microbe Interactions
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Endophytic taxol-producing fungi from bald cypress, Taxodium distichum
More LessPestalotiopsis microspora occurs as a range of strains in bald cypress, Taxodium distichum. The organisms live as endophytes in the bark, phloem and xylem, and isolates show differences in cultural and microscopic characteristics on common laboratory media. Many of these fungi make taxol as determined by the reactivity of partially purified culture extracts with specific monoclonal antibodies against taxol. In the case of one strain of P. microspora (CP-4), taxol was isolated from culture medium and was shown to be identical to authentic taxol by chromatographic and spectroscopic means.
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- Genome Analysis
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Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome
The standard strategies of genome sequencing based on λ-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli . One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104 109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13695 and 20303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned ir YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.
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A Bacillus subtilis gene cluster similar to the Escherichia coli phosphate-specific transport (pst) operon: evidence for a tandemly arranged pstB gene
More LessWe have determined the complete nucleotide sequence of the Bacillus subtilis homologues of the Escherichia coli phosphate-specific transport (pst) genes in the framework of the international B. subtilis genome sequencing project. The pst genes in E. coli form an operon arranged in the order pstS, pstC, pstA, pstB and phoU . In the case of B. subtilis, there are also five ORFs presumably forming an operon. The deduced amino acid sequences of the products of these ORFs show striking similarities to their E. coli counterparts. Comparison of the organization of the pst operon of B. subtilis with that of E. coli revealed that the gene corresponding to phoU is missing, while there are two genes homologous to pstB in B. subtilis. The pst operon is located at 222° on the B. subtilis chromosome
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