- Volume 143, Issue 5, 1997
Volume 143, Issue 5, 1997
- Sgm Special Lecture
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- Microbiology Comment
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- Biochemistry
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Increased pyruvate orthophosphate dikinase activity results in an alternative gluconeogenic pathway in Rhizobium (Sinorhizobium) meliloti
More LessThe formation of phosphoenolpyruvate (PEP) is a major step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars. In Rhizobium (now Sinorhizobium) meliloti this step is catalysed by the enzyme PEP carboxykinase (PCK) which converts oxaloacetate to PEP. R. meliloti Pck-mutants grow very poorly with TCA cycle intermediates as the sole source of carbon. Here, the isolation and mapping of suppressor mutations which allow Pck-mutants to grow on succinate and other TCA cycle intermediates is reported. Tn5 insertions which abolished the suppressor phenotype and mapped to the suppressor locus were located within the pod gene encoding pyruvate orthophosphate dikinase (PPDK). Strains carrying suppressor mutations had increased PPDK activity compared to the wild-type. The suppressor phenotype was dependent on the combined activities of malic enzyme and PPDK, which thus represent an alternative route for the formation of PEP in R. meliloti. PPDK activity was not required for symbiotic N2 fixation.
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Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase
More LessThis work reports the role of both superoxide dismutases - CuZnSOD (encoded by SOD1) and MnSOD (encoded by SOD2) - in the build-up of tolerance to ethanol during growth of Saccharomyces cerevisiae from exponential to post-diauxic phase. Both enzyme activities increase from the exponential phase to the diauxic shift and from the diauxic shift to the post-diauxic phase. The levels of mRNA-SOD1 and mRNA-SOD2 increase from the exponential phase to the diauxic shift; however, during the post-diauxic phase mRNA-SOD1 levels decrease while mRNA-SOD2 levels remain unchanged. These data indicate the existence of two regulatory mechanisms involved in the induction of SOD activity during growth: synthesis de novo of the proteins (until the diauxic shift), and post-transcriptional or post-translational regulation (during the post-diauxic phase). Ethanol does not alter the activities of either enzyme in cells from the diauxic shift or post-diauxic phases, although the respective mRNA levels decrease in post-diauxic-phase cells treated with ethanol (14% or 20%). Results of experiments with sod1 and sod2 mutants show that MnSOD, but not CuZnSOD, is essential for ethanol tolerance of diauxic-shift and post-diauxic-phase cells. Evidence that ethanol toxicity is correlated with the production of reactive oxygen species in the mitochondria is obtained from results with respiration-deficient mutants. In these cells, the induction of superoxide dismutase activity by ethanol is low; also, the respiratory deficiency restores the capacity of sod2 cells to acquire ethanol tolerance.
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- Bioenergetics And Transport
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Brucella abortus strain 2308 putative glucose and galactose transporter gene: cloning and characterization
More LessThe gene for the putative transporter for glucose and galactose from Brucella abortus strain 2308 was isolated by functional complementation of Escherichia coli strains lacking either glucose or galactose transport systems. The same two plasmid clones were isolated from each screen. These clones restored glucose and galactose transport to the respective E. coli strains. The sequence of the 1806 bp overlap between these two plasmids was determined. A 1242 bp ORF whose disruption eliminated complementation of both E. coli strains showed 36% identity with the E. coli fucP gene encoding a fucose transporter. These two transporters are members of the major facilitator superfamily, in which they represent a previously undescribed family. In addition, an incomplete gene similar to E. coli hisG was found. One of the plasmids complemented E. coli hisG mutants.
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Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen
More LessEscherichia coli flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of O2 reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0⋅ 5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(III)-hydroxamate K from Rhizobium leguminosarum bv. viciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH-cytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of O2 at the haem. The V max of cytochrome c reduction fell from 7.1 s-1in the presence of 0⋅5 mM CO to 5⋅0 s-1in the presence of 100 μM O2with no significant change in Km for cytochrome c (6⋅8 to 7⋅3 μM, respectively). O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 μM O2 Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globin-like haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of O2 since, in the absence of O2 electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in vivo is not known and alternative models for O2 sensing are also considered.
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- Development And Structure
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β-Glucosylated proteins in the cell wall of the black yeast Exophiala (Wangiella) dermatitidis
More LessWild-type cells of the pathogenic black yeast Exophiala (Wangiella) dermatitidis grown in a low-pH ascorbate medium became less melanized and less resistant to Zymolyase. This was accompanied by increased staining with fluorescently labelled concanavalin A. The sugar composition of wild-type and mutant cell walls was, except for the presence of galactose, similar to that of Saccharomyces cerevisiae. Digestion of mutant cell walls with laminarinase released galactomannoproteins. In addition, the released cell wall proteins contained glucose and reacted with affinity-purified 1,6-β-glucan antiserum, indicating that they are linked to 1,6-β-glucan. It is proposed that 1,6-β-glucosylated cell wall proteins generally occur among ascomycetes.
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- Environmental Microbiology
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Near-UV-induced absorbance change and photochemical decomposition of ergosterol in the plasma membrane of the yeast Saccharomyces cerevisiae
More LessWhen cells of the yeast Saccharomyces cerevisiae were exposed to near-UV (300-400 nm), their absorption spectra changed slightly within the range 220-300 nm with increasing dosage. Difference spectra, calculated by subtracting the curve recorded in cells exposed to near-UV from the curve of unexposed cells, decreased with increasing dosage over a broad band with peaks at 272, 282 and 295 nm and a shoulder at 265 nm. These peaks were in agreement with the absorption maxima of ergosterol, which is one of the major components of the plasma membrane of yeast. Near-UV radiation induced a simultaneous decrease in absorption spectra and reduction of ergosterol content in the plasma membrane. Photochemical decomposition of ergosterol by near-UV radiation was revealed in vivo, although ergosterol is generally known to be photoconverted to previtamin D2 industrially by UV radiation in vitro. In order to remove photosensitizers, liposomes were prepared from phospholipids and glycolipids, with or without ergosterol from purified yeast plasma membranes. Liposomal ergosterol in the orientated state was photochemically decomposed by near-UV radiation but ergosterol in the disorientated state in a homogeneous solution was not. Near-UV radiation also induced a decrease in activity of membrane-bound ATPase. Dose-response curves for the reduction of ATPase activity were similar to that for decomposition of ergosterol, suggesting that near-UV caused membrane function damage.
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- Genetics And Molecular Biology
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Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn1696 as reporter
More LessA promoter-probe shuttle plasmid (pGL7011) containing the promoterless aminoglycoside-O-acetyltransferase I gene (aacC1) of Tn1696 was used to isolate DNA fragments from Streptomyces ghanaensis phage I19 that possessed promoter activity in Streptomyces lividans TK23. Analysis of gentamicin (Gm) resistance levels in Escherichia coli and in S. lividans TK23, and of aacC1 mRNA levels in S. lividans, identified a fragment (F14) that exhibited a high level of promoter activity in both species. Subsequent analysis revealed that the promoter activity of SF14 (a subcloned fragment of F14) was about twice that of ermEp*, one of the strongest characterized actinomycete promoters. SF14 contained two tandemly arranged promoters, 14-1p and p14-llp, with overlapping and adjacent -10 and -35 regions, respectively. Both promoters appear to be recognized with different efficiencies by the major RNA polymerase holoenzyme (Eshrdb) of Streptomyces coelicolor A3(2).
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Identification by PCR of genes encoding multiple response regulators
More LessEnvironmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component sytems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators. Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria. Sequence analysis revealed that 22 DNA fragments, which clearly originated from response regulator genes, were amplified from Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis and Lactobacillus bulgaricus. In each of these four species the receiver module of putative response regulator genes, which do not seem to be related to any of the already characterized genes, was identified. This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.
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FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites
More LessThe ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at −50.5 and −94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR*(FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR*repressed ndh gene expression both aerobically and anaerobically in vivo. Gel retardation analysis and DNase I footprinting with purified FNR*protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR-sites are needed for efficient repression in vivo. The α-subunit of RNAP interacted with two regions (centred at −105 and −46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at −50.5 prevents the α-subunit of RNAP from docking with the DNA just upstream of the −35 element. Binding of a second FNR*molecule at the −105 site likewise prevents binding of the α-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the α-subunit of RNAP.
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The outer membrane of lipid A-deficient Escherichia coli mutant LH530 has reduced levels of OmpF and leaks periplasmic enzymes
More LessWe have previously described a new Escherichia coli K-12 mutant, LH530, which has a defective outer membrane. LH530 is very sensitive to hydrophobic antibiotics, does not grow at 42 ° and synthesizes reduced amounts of lipid A. Phenotypically LH530 is very similar to the known lipid A biosynthesis mutants of E. coli and Salmonella typhimurium. Its genetic defect is not known, but the defect is suppressed by multiple copies of ORF195. Here we show that at 37 ° LH530 contains a reduced amount of the OmpF porin and that it leaks periplasmic °-lactamase at 37 °. and 42 °. We further show that ORF195, when present at low copy number, restores the antibiotic resistance and lipid A biosynthesis of LH530 at 28 °, but not at higher temperatures. In contrast, OmpF expression is restored at 37 °.
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The flagellin N-methylase gene fliB and an adjacent serovar-specific IS200 element in Salmonella typhimurium
More LessThe cloning and molecular genetic analysis of a locus mapping within the flagellar gene (fli) complex of Salmonella typhimurium is reported. A copy of the insertion element IS200 was located in a noncoding stretch of DNA upstream of the fliA gene. Comparative nucleotide sequence analysis showed that this copy of IS200 was 711 bp long and that its flanking regions contained no features common to other characterized insertion sites of this element. The element was located 37 bp downstream of an ORF whose product was shown by interspecific transfer and amino acid analysis to carry out N-methylation of selected lysine residues in Salmonella flagellin. The sequence and phenotype of this ORF identified it as fliB, encoding the only prokaryotic N-methylase acting on amino groups to have been characterized to date. It was found to be conserved among all clinically significant serovars of Salmonella. The IS200 insertion site is of particular interest since it was conserved in all but two rare evolutionary lines of S. typhimurium, and was absent from 85 Salmonella strains belonging to 37 other serovars. It is thus a phylogenetically significant marker at the serovar level.
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Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae
More LessA protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest. Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein. Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase. The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively. One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases. PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products.
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The flgK motility operon of Borrelia burgdorferi is initiated by a s70-like promoter
More LessA cluster of flagellar genes of Borrelia burgdorferi was identified and sequenced. This cluster comprises an operon, designated the flgK operon, which is initiated by a s70-like promoter. The flgK operon consists of flbF (function unknown), flgK (encoding HAP1), flgL (encoding HAP3) and orfX (function unknown), and maps at 185 kb on the chromosome. In other bacteria, the hook-associated proteins HAP1 and HAP3 connect the flagellar filament to the hook and are required for the last stage of flagellar assembly. Reverse transcriptase-PCR analysis indicated that flbF through to orfX are transcribed as a single mRNA, and primer extension analysis revealed that transcription of the flgK operon is initiated by a s70-like promoter upstream of flbF. Subcloning the flgK promoter element into a promoter probe cat vector revealed that the flgK promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. In addition, when this construct was transformed into a fliA mutant of S. typhimurium which lacked a functional flagellar-specific s28factor, the flgK promoter was still functional. Based on these results, the promoter element of the flagellin gene (fla, hereafter referred to as flaB) was re-examined. flaB encodes the flagellar filament protein, and a sgp33-34-like promoter has been reported to be involved in the transcription of this gene. A transcriptional start point was found 1 bp downstream of the reported start site. The sequence around -10 and -35 are consistent with the presence of a s70-like promoter in addition to the putative sgp33-34-like promoter for flaB. In contrast to the flgK promoter element, no activity was detected after subcloning a flaB promoter element into the promoter probe cat vector. Because a s70-like promoter rather than a unique flagellar sigma factor is involved in the later stage of flagellar assembly, the regulation of B. burgdorferi flagellar genes is evidently different from that of other bacteria.
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Analysis of a new dimeric extradiol dioxygenase from a naphthalenesulfonate-degrading sphingomonad
More LessA new extradiol dioxygenase was cloned by screening a gene bank from the naphthalenesulfonate-degrading bacterial strain BN6 for colonies with 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity. A 16 kb DNA fragment was sequenced and an ORF of 954 bp identified. Comparison of the deduced amino acid sequence of DHBPDO II from strain BN6 with previously published sequences showed the closest relationship to a metapyrocatechase (Mpcll) from Alcaligenes eutrophus JMP 222. Thus, the enzyme was only distantly related to the main groups of catechol 2,3-dioxygenases or DHBPDOs. The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-methylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene. Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocatechol with previous reports suggested that this substrate is oxidized by different extradiol dioxygenases either by proximal or distal ring cleavage. The enzyme required Fe2+for maximal activity. In contrast to most other extradiol dioxygenases, the enzyme consisted of only two identical subunits.
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Highly thermostable endo-1,3-β -glucanase (laminarinase) Lam A from Thermotoga neapolitana: nucleotide sequence of the gene and characterization of the recombinant gene product
More LessThe nucleotide sequence of clone pTT26 (3786 bp), containing the gene for 1,3-β -glucanase lamA (laminarinase) from Thermotoga neapolitana, was determined. It contains an ORF encoding a protein of 646 aa (73 328 Da). The central part of the protein is homologous to the complete catalytic domain of bacterial and some eukaryotic endo-1,3-β -D-glucanases and belongs to family 16 of glycosyl hydrolases. This domain is flanked on both sides by one copy on each side of a substrate binding domain homologue (family II). The recombinant laminarinase protein was purified from Escherichia coli host cells in two forms, a 73 kDa and a processed 52 kDa protein, both having high specific activity towards laminarin (3100 and 2600 U mg-1, respectively) and K m values of 2.8 and 2.2 mg ml-1, respectively. Limited activity on 1,3-1,4-β -glucan (lichenan) was detected (90 U mg-1). Laminarin was degraded in an endoglucanase modus, yielding glucose, laminaribiose and -triose as end products. Thus lamA classifies as an endo-1,3(4)-β -glucanase (EC 3.2.1.6). The optimum temperature of the enzymes was 95° (73 kDa) and 85° (52 kDa) at an optimum pH of 6.2. The superior thermostability of the 73 kDa enzyme is demonstrated by incubation without substrate at 100°, where 57% of the initial activity remained after 30 min (82% at 95°). Thus, lamA is the most thermostable 1,3-β -glucanase described to date.
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Molecular and immunological characterization of OprL, the 18 kDa outer-membrane peptidoglycan-associated lipoprotein (PAL) of Pseudomonas aeruginosa
Immunological screening of a Pseudomonas aeruginosa cosmid library led to the identification of clones producing an 18 kDa outer-membrane protein. This protein reacted in Western blots with a polyclonal antiserum against outer-membrane proteins of P. aeruginosa and with a monoclonal antibody (MA1-6) specific for OprL, the peptidoglycan-associated outer-membrane lipoprotein (PAL). Sequencing of pOML7, a subclone expressing oprL, revealed an ORF of 504 bp encoding a polypeptide with a typical lipoprotein signal recognition sequence. Another ORF was found upstream of oprL, with homology to the ToIB protein of Escherichia coli and Haemophilus influenzae. Downstream of oprL, a second ORF, of 321 bp, was found (orf2), encoding a protein with a signal peptide and with no homology with proteins of known biological function. After the stop codon of orf2, a rho-independent terminator sequence was detected which is part of the P. aeruginosa PA01 insertion element IS222. OprL showed homologies with all known PALs from Gram-negative bacteria, especially in the C-terminal part. mAb MA1-6 reacted with P. aeruginosa cells in immunofluorescence, and with E. coli cells expressing oprL, which had an abnormal, elongated morphology, an indication that production of the protein perturbed the division process.
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Fluorescent oligonucleotide rDNA probes that specifically bind to a common nanoflagellate, Paraphysomonas vestita
Nanoflagellates are ecologically important, but morphological identification requires techniques which are not practicable for use in quantitative studies of populations; alternative methods of accurate recognition of nanoflagellate species in mixed populations are therefore desirable. Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as ‘phylogenetic stains’ in the identification of bacteria. In this paper we describe the preparation and application of probes which specifically hybridize with a common nanoflagellate species, Paraphysomonas vestita. The sequence of nucleotides in the SSU rRNA gene of this flagellate was determined and compared with those of related species to select unique P. vestita sequences 18-21 nucleotides in length. Five sequences in different parts of the SSU rRNA gene were used to design 5 -fluorescently labelled oligonucleotide probes. Published sequences were used to make probes that hybridized with all eukaryotes (EUK) or any cellular organism (UNI), and probes were designed not to hybridize with rRNA (CON). Optimum conditions for hybridization were determined. In all cases, UNI probes hybridized with the cells, but CON probes were only bound to a limited extent. All five probes targeted to P. vestita proved to be species-specific; they hybridized well with this species, but not with three other species of the same genus, nor with three more distantly related flagellate species, nor with a ciliate, nor with bacteria. These probes provide a means of quantitatively measuring the proportion of P. vestita cells in samples of mixed protists.
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Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1
More LessA region of 14-2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of genes involved in one-carbon (C1) metabolism, including serine cycle genes, pqq genes, regulatory methanol oxidation genes and the gene for N5,N10-methylene tetrahydrofolate dehydrogenase (mtdA). Fifteen new ORFs have been identified within the new region, and their sequences suggest that they encode the following polypeptides: the C-terminal part of phosphoenolpyruvate carboxylase, malyl-CoA lyase, polypeptides of 9.4 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of mxaF and mxaJ from M. extorquens AM1 and other methylotrophs, a cytochrome c, three enzymes of folate metabolism, and polypeptides of 13 and 20.5 kDa with no homologues in the protein database. Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C1 metabolism. A mutation in mcIA. encoding malyl-CoA lyase, resulted in a C1-minus phenotype, while mutations in the other genes all showed a C1-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, foIC, implying the necessity of these folate synthesis genes for metabolism of C1 and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to mxaG and mxaJ, and other unidentified ORFs produced double-crossover recombinants with a C1-positive phenotype. Promoter regions have been investigated upstream of orf3 and orf4 using the promoter probe vector pHX200. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory mox mutants.
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Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1
More LessFive genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains. These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood. In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xyIE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF. The nucleotide sequence suggests that mxbD encodes a histidiine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator. A xyIE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate. Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression. The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes. xyIE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation. These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation.
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Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA
More LessFive independent clones of the Propionibacterium acnes P-37 lipase gene (gehA) were obtained in Escherichia coli, and the gene was localized to a 2.75 kb Xhol fragment by subcloning. The five clones were shown to contain the same gene by Southern blotting with a DIG-labelled probe to gehA. The nucleotide sequence of gehA was determined, and shown to contain a single ORF of 1017 kb, encoding a protein of 339 amino acids. The predicted molecular mass was 36 kDa. A 33 kDa (PAGE) radiolabeled polypeptide was detected from E. coli minicell preparations harbouring gehA, which could correspond to GehA after cleavage of the putative 26 amino acid residue signal peptide. gehA was overexpressed in E. coli under the control of the bacteriophage T7 promoter, and the corresponding polypeptide was found to be present in insoluble aggregates. Active lipase was produced when the overexpressing strain was incubated at a reduced temperature in the presence of sucrose. Purification of lipase from P. acnes culture supernatant fluids confirmed the production of a 33 kDa (PAGE) lipase.
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Control of Neisseria gonorrhoeae pilin gene expression by environmental factors: involvement of the pilA/pilB regulatory genes
More LessThe control of the expression of the pilin gene (pilE) in Neisseria gonorrhoeae under a wide variety of growth conditions has been studied. The expression of pilE was measured using transcriptional fusions between pilE and the gene encoding chloramphenicol acetyltransferase (CAT), and the level of pilin production was measured by Western blot analysis. Many of the conditions tested affected both growth rate and pilin gene expression (e.g. isoleucine, high osmolarity, high temperature, anaerobic growth, pH 6, urea and iron depletion). Changes in the level of many other proteins were also observed, depending on the conditions, indicating that gonococci undergo an adaptive response to environmental variations. Moreover, environment-induced changes in the level of many proteins, including pilin, seem to involve the pilA/pilB regulatory system, which has been previously proposed to modulate the expression of the gonococcal pilin gene.
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Variation in assimilating functions occurs in spontaneous Candida albicans mutants having chromosomal alterations
More LessIn this study, four clinical isolates and over 100 colony morphology mutants, previously derived spontaneously from strain 3153A during growth on glucose medium, were examined for their utilization of 21 carbon and 3 nitrogen sources at various growth temperatures. The results demonstrated extensive variability in the pattern of assimilation among the mutants and strains, including both the gain and loss of assimilating functions. The persistent alterations in assimilation patterns observed in sequentially produced subclones illustrated an extensive ability of C. albicans populations to constantly produce new combinations of assimilating functions. The variability among spontaneous mutants derived from a single strain explains the well documented variability among natural isolates. From these results we established a relationship between the previously documented broad spectrum of spontaneous chromosomal aberrations in these mutants to the expression of genes controlling the utilization of alternative carbon and nitrogen sources. The existence of cryptic genes, responsible for growth on alternative substrates, was previously deduced from the analysis of other mutants obtained as a response to the restrictive condition on media containing non-assimilating carbon sources. Thus, mutants with altered assimilation functions can arise either on glucose medium or by selection on restricted media. Extensive differences between the patterns of chromosomal aberrations and the distribution of correlated phenotypes in the two groups of mutants indicated that the same phenotypes may be produced by two different mechanisms involving the same or different genes.
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- Physiology And Growth
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Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties
More LessThe evolution of Escherichia coli MG1655 mutants was followed over 126 d in a glycerol-limited chemostat at a dilution rate of 0.05 h-1. This corresponds to a total of 217 generations at a doubling time of 13.9 h. After this time, nearly 90% of the chemostat population consisted of evolved mutant strains as determined by their altered colony morphologies on plates. Two mutants were isolated that exhibited generally improved growth phenotypes in batch cultivations on glycerol, glucose or the gluconeogenic substrate acetate. Higher specific growth rates and increased biomass yields were found for both mutants. For one mutant, this behaviour was combined with significantly reduced secretion of overflow metabolites when either glycerol or glucose was the carbon source. Additionally, during all growth phases of a batch cultivation, this mutant exhibited increased resistance to a variety of adverse conditions including heat shock, osmotic stress and nutrient deprivation. It also displayed significantly shorter lag phases.
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Resuscitation of ‘non-culturable’ cells from aged cultures of Campylobacter jejuni
More LessWhen stationary phase batch cultures of Campylobacter jejuni were stored in sealed flasks under static conditions, viable numbers declined from 2 x 109c.f.u. ml-1to around 103-106c.f.u. ml-1within 4-6 weeks. When the aged cultures were sparged with a microaerobic gas mixture, there was a rapid increase in viable numbers accompanied by a change from predominantly coccoid to vibrioid morphology. The most probable number (MPN) technique was used to distinguish resuscitation of injured or dormant cells from multiplication of residual viable cells. MPN estimates using fresh Brucella broth containing 0.2% mucin revealed that plate counts underestimated the true viable count by up to 23-fold. The experiments clearly demonstrated that a proportion of surviving cells in aged cultures were in an injured or latent state that prevented growth on agar plates. It is possible that the size of this fraction is greater than was demonstrated and that much higher recoveries would be obtained under other recovery conditions. Nevertheless, from presently available evidence, it must be concluded that the size of the latent fraction is quite small and that most of the increase in count that occurs on regassing a spent culture comes from multiplication of residual viable cells.
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Paracoccidioides brasiliensis by ajoene is associated with blockade of phosphatidylcholine biosynthesis
In Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans, no significant differences were observed in the phospholipid species of both morphological phases. The species observed were phosphatidylcholine (PC, 30–40%), phosphatidylethanolamine (PE, 27-28%), phosphatidylserine (16–19%), phosphatidylinositol (13–17%) and sphingomyelin (3–5–0025;). The main fatty acids found in the yeast (Y) phase were palmitate (56%), linoleate (18%) and oleate (15%), while linoleate predominated (61 %) in the mycelial (M) phase, followed by palmitate (27%) and oleate (7%). In the Y phase the main free sterol was ergosta-5,22-dien-3β-ol (82%) plus some lanosterol (12%) and ergosterol (6%), while in the M phase, the latter predominated (88%), followed by low levels of ergosta-5,22-dien-3β-ol (12%). Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a platelet aggregation inhibitor derived from garlic, induced alterations in phospholipid and fatty acid proportions such that PC was reduced to about 18% in both phases and PE increased to 38% (Y phase) or 44% (M phase), suggesting inhibition of PC synthesis. Ajoene also reduced saturated fatty acids (16:0 and 18:0) from 67 to 35% in the Y phase, with a corresponding increase in the unsaturated components. This effect was not seen in the M phase.
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A 16 kDa protein family overexpressed by Streptococcus thermophilus PB18 in acid environments
More LessThe one- and two-dimensional protein patterns of Streptococcus thermophilus PB18 in the exponential and stationary phases of growth were analysed. Onedimensional SDS-PAGE showed that a 16 kDa protein was overexpressed in stationary phase as well as 2 h after an acid shock, and that it was not expressed when the bacteria reached the stationary phase in medium with limiting lactose concentrations (5 or 10 g l-1), in which the pH (5∙5) was not as acid as in control cultures (pH 4∙7, lactose 20 g l-1). The results support the idea that this protein is expressed in response to the acidic environment and not in response to the growth phase. Two-dimensional PAGE showed that nine proteins were expressed only during the exponential phase and ten others only during the stationary phase. The 16 kDa band seen in one-dimensional SDS-PAGE corresponded to a 16 kDa protein family observed on two-dimensional SDS-PAGE/IEF gels, whose expression was increased 8∙5-fold when the extracellular pH reached a critical value below 5∙0. The N-terminal sequences of proteins from two spots on the two-dimensional gels (members of the 16 kDa family) were determined and found to be identical. The physiological role of this protein family has not yet been elucidated.
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Catabolism of D-glucose by Pseudomonas putida U occurs via extracellular transformation into D-gluconic acid and induction of a specific gluconate transport system
More LessPseudomonas putida U does not degrade D-glucose through the glycolytic pathway but requires (i) its oxidation to D-gluconic acid by a peripherally located constitutive glucose dehydrogenase (insensitive to osmotic shock), (ii) accumulation of D-gluconic acid in the extracellular medium, and (iii) the induction of a specific energy-dependent transport system responsible for the uptake of D-gluconic acid. This uptake system showed maximal rates of transport at 30 ° in 50 mM potassium phosphate buffer, pH 7.0. Under these conditions the Km calculated for D-gluconic acid was 6.7 μM. Furthermore, a different transport system, specific for the uptake of glucose, was also identified. It is active and shows maximal uptake rates at 35 ° in 50 mM potassium phosphate buffer, pH 6.0, with a K m value of 8.3 μM.
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Respiration of Pseudomonas fluorescens as a function of intracellular substrate concentration
More LessA kinetic method to measure the intracellular concentration of respiratory substrates in short-term starvation-enrichment experiments is proposed. Samples of bacterial suspension from steady-state chemostat cultures were subjected to 25 min starvation, followed by pulse addition of [14C]glucose. Residual substrate utilization rates and respiration rates (uptake of dissolved O2) before and after amendment were recorded. Increases in pool sizes (δL) during transients were calculated on the basis of C balance. The dependence of respiration rate qresp on δL was found to obey modified Michaelis-Menten kinetics: q resp = Q resp (LC+δL)/(K L+LC+δL) [Q resp is maximal respiration rate (29.1 mmol O2 h-1per g biomass C), K L = 12.14 mg C per g biomass C], where LC is the absolute value of the pool size before amendment. Direct chemical determination of LC in cold TCA extracts revealed two fractions. The first fraction was mobile and showed a close correlation with both respiration and L. The second, ‘stable’, fraction did not correlate with respiration dynamics and was interpreted as material formed artifactually by acid degradation of polymeric cell components.
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Characterization of polar-flagellar-length mutants in Vibrio alginolyticus
Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof+Laf-), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5.0.9 μm, 2.5.0.6 μm and 11.2.3.6 μm, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. In the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 μm and 0.062 μm, respectively, whereas those of the wild-type flagella were 1.5 μm and 0.16 μm. The relative amounts of the various molecular components of the long Pof were different from those of the wild-type or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.
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Hydrogenosomes of Metopus contortus physiologically resemble mitochondria
The anaerobic free-living ciliated protozoon Metopus contortus is a grazer in anoxic marine sediments. It does not possess mitochondria, but it does have specialized organelles termed hydrogenosomes which release hydrogen gas. The cationic lipophilic cyanine dye DiOC7(3) is an indicator of transmembrane electrochemical potential. With the aid of confocal laser scanning microscopy (CLSM), the association of this dye with hydrogenosomes in situ was followed. Flow cytometric measurements showed that fluorescence of the membrane potential dye decreased in response to an elevated pH2 in the cell. CLSM also revealed localization of fluorescence of the calcium probe Fluo 3-AM, and of the transmembrane pH gradient probe BCECF-AM, within the lumen of the hydrogenosomes. In addition, hydrogenosomal inclusions were detected. X-ray microanalysis of these electron-dense granules revealed high levels of calcium, phosphate and magnesium. It is concluded that M. contortus hydrogenosomes are calcium stores, have a membrane potential, and an alkaline lumen. These physiological features resemble those of mitochondria in aerobic protozoa.
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Stimulation of sexual reproduction in Phytophthora cactorum by phospholipids is not due to sterol contamination
More LessPhytophthora cactorum did not form oospores on basal medium unless phosphatidylcholine (lecithin) or phosphatidylethanolamine (cephalin) was added. After removal of putative sterols by aminopropyl column chromatography, the activities of lecithin and cephalin were increased 47- and 2.8-fold, respectively, thus confirming the previous reports that sterols are not essential for sexual reproduction in this organism. Thin-layer chromatography (TLC) of the commercial lecithin revealed the presence of an unknown inhibitory substance which, when added to the purified lecithin, caused a 50% reduction of oospore formation. Commercial cephalin also showed a twofold increase in activity after removal of putative sterols and the existence of an unknown inhibitor when it was subjected to TLC. Addition of the inhibitor to the purified cephalin completely inhibited the growth of the test organism. One sample of lecithin tested was not stimulatory to oospore formation. However, after washing with deionized water or NaCl solution, it induced the production of 17300 and 24450 oospores (100 μg)-1, respectively. The ability of cephalin to induce oospore formation was increased 2⋅3-fold by washing with deionized water and 8⋅3-fold by washing with NaCl solution. Like sterols, the digitonin precipitable component (digitonide) of the non-phospholipid fraction of commercial lecithin or cephalin was stimulatory to oospore formation of P. cactorum but not Phytophthora parasitica. However, the non-digitonide component was not only more active than the digitonide component, but also stimulatory to P. parasitica. Gas chromatography and mass spectrometry (GC-MS) analysis of the digitonide component from lecithin failed to detect any putative sterol contaminant. The amount of the putative sterol contaminant in the digitonide component from cephalin was also below the detection limit of GC-MS. When 0.01-10 ng cholesterol was added to basal medium discs each containing 100 fig cephalin, the numbers of oospores produced by P. cactorum and P. parasitica were not significantly changed. It is concluded that, in the fungi tested, sterols did not play any significant role in the stimulation of sexual reproduction by highly purified phospholipids.
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- Genome Analysis
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A 12 kb nucleotide sequence containing the alanine dehydrogenase gene at 279° on the Bacillus subtilis chromosome
More LessIn the framework of the European project aimed at the sequencing of the Bacillus subtilis genome, a DNA fragment of 12315 bp was cloned and sequenced. The DNA fragment is located between rrnB (275°) and pai (284°). Twelve ORFs were predicted to encode putative proteins. Two of these (ald and yukl) coincided with known B. subtilis genes. The products of two other genes (yukK and yukL) showed significant similarity to known proteins present in databases, e.g. pyoverdine synthase of Pseudomonas aeruginosa and pristinamycine synthase D of Streptomyces pristinaespiralis.
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Physical mapping shows that the unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end of the linear chromosome
More LessA restriction map of the 8 Mb linear chromosome of Streptomyces rimosus R6-501 was constructed for the enzymes AseI (13 fragments) and DraI (7 fragments). Linking clones for all 12 AseI sites and 5 of the 6 DraI sites were isolated. The chromosome has terminal inverted repeats of 550 kb, which are the longest yet reported for a Streptomyces species. The oxytetracycline gene cluster lies about 600 kb from one end, which might account for its frequent spontaneous amplification and deletion. Several other markers were localized on the chromosome (dnaA and recA, the rrn operons, the attachment site for pSAM2 and prophages RP2 and RP3). Comparison of the conserved markers with the map of Streptomyces coelicolor A3(2) suggested there are differences in genome organization between the two species.
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- Corrigendum
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