- Volume 147, Issue 3, 2001
Volume 147, Issue 3, 2001
- Review Article
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- Microbiology Comment
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- Biochemistry
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NAD(P)H:menadione oxidoreductase of the amitochondriate eukaryote Giardia lamblia: a simpler homologue of the vertebrate enzyme
More LessThe GenBank accession number for the glQR1-encoding sequence reported in this paper is AF321405.
The amitochondriate eukaryote Giardia lamblia contains an NAD(P)H:menadione oxidoreductase (EC 1.6.99.2) (glQR) that catalyses the two-electron transfer oxidation of NAD(P)H with a quinone as acceptor. The gene encoding this protein in G. lamblia was expressed in Escherichia coli. The purified recombinant protein had an NAD(P)H oxidoreductase activity, with NADPH being a more efficient electron donor than NADH. Menadione, naphthoquinone and several artificial electron acceptors served as substrate for the enzyme. glQR shows high amino acid similarity to its homologues in vertebrates and also to a series of hypothetical proteins from bacteria. Although glQR is considerably smaller than the mammalian enzymes, three-dimensional modelling shows similar arrangement of the secondary structural elements. Most amino acid residues of the mammalian enzymes that participate in substrate binding or catalysis are conserved. Conservation of these features and the similarity in substrate specificity and in susceptibility to inhibitors establish glQR as an authentic member of this protein family.
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Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics
More LessThe GenBank accession number for the sequence reported in this paper is AY007251.
Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwα (comprising 29 residues) and Plwβ (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1:1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwα, and Plwβ contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.
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An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by ‘Pseudomonas butanovora’
More LessButane-grown ‘Pseudomonas butanovora’ expressed two soluble alcohol dehydrogenases (ADHs), an NAD+-dependent secondary ADH and an NAD+-independent primary ADH. Two additional NAD+-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD+-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1·0 mol pyrroloquinoline quinone (PQQ) and 0·25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C4 diols and aromatic alcohols. It exhibited the lowest K m (7±1 μM) and highest k cat/K m (72×104 M−1 s−1) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown ‘P. butanovora’.
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- Development And Structure
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The electrophoretic softness of the surface of Staphylococcus epidermidis cells grown in a liquid medium and on a solid agar
More LessMany Staphylococcus epidermidis strains possess capsule or slime layers and consequently the staphylococcal cell surface should be regarded as a soft, polyelectrolyte layer allowing electrophoretic fluid flow through a layer of fixed charges. The presence of such a soft layer decreases the energy barrier due to electrostatic repulsion in the interaction of the organisms with negatively charged substrata [Morisaki, H., Nagai, S., Ohshima, H., Ikemoto, E. & Kogure, K. (1999), Microbiology 145, 2797–2802] and hence plays an important role in their adhesion. In this paper, the authors compare the electrophoretic softness and amount of fixed charge in the outer cell surface layers of 20 S. epidermidis strains, grown in a liquid medium or on a solid agar, as determined from the dependencies of their electrophoretic mobilities upon the ionic strength of a suspending fluid. Most of the staphylococcal cell surfaces were relatively soft, with a mean cell surface softness (1/λ) for strains grown in liquid medium of 1·7±0·6 nm (standard deviation over all 20 strains) which is soft by comparison with a completely bald, peptidoglycan-rich streptococcal cell surface (1/λ=0·7 nm). When the staphylococcal strains were grown on solid agar, the cell surface softness of 17 of the 20 strains increased, sometimes by a factor of two. On average for 20 strains, the cell surface softness increased significantly (P<0·05, Student’s t-test) to 2·8±1·8 nm. The amount of fixed charge in the outer cell surface layer was −28±9 mM for bacteria grown in liquid medium and −24±12 mM for bacteria grown on agar. A soft, highly negatively charged polyelectrolyte layer was inferred by microelectrophoresis for all the staphylococcal cell surfaces, regardless of whether staining had indicated the presence of a capsule or slime layer.
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- Environmental Microbiology
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Sequence variation in dichloromethane dehalogenases/glutathione S-transferases
More LessThe GenBank accession numbers for the sequences determined in this work are AJ271131–38 (see text for details).
Dichloromethane dehalogenase/glutathione S-transferase allows methylotrophic bacteria to grow with dichloromethane (DCM), a predominantly man-made compound. Bacteria growing with DCM by virtue of this enzyme have been readily isolated in the past. So far, the sequence of the dcmA gene encoding DCM dehalogenase has been determined for Methylobacterium dichloromethanicum DM4 and Methylophilus sp. DM11. DCM dehalogenase genes closely related to that of strain DM4 were amplified by PCR and cloned from total DNA from 14 different DCM-degrading strains, enrichment cultures and sludge samples from wastewater treatment plants. In total, eight different sequences encoding seven different protein sequences were obtained. Sequences of different origin were identical in several instances. Sequence variation was limited to base substitutions; strikingly, 16 of the 19 substitutions in the dcmA gene itself encoded amino acids that were different from those of the DM4 sequence. The kinetic parameters k cat and K m, the pH optimum and the stability of representative DCM dehalogenase variants were investigated, revealing minor differences between the properties of DCM dehalogenases related to that from strain DM4.
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- Genetics And Molecular Biology
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Regulation of the switch from early to late bacteriophage λ DNA replication
There are two modes of bacteriophage λ DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (θ or circle-to-circle) mode, and is later switched to the sigma (σ or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wild-type cells the replication starting from oriλ proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from oriλ is predominantly unidirectional. The regulation of directionality of replication from oriλ is mediated by positive control of λ p R promoter activity by DnaA, since the mode of replication of an artificial λ replicon bearing the p tet promoter instead of p R was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with λ, phage DNA replication proceeds predominantly according to the unidirectional θ mechanism and is switched early after infection to the σ mode. It is proposed that in wild-type E. coli cells infected with λ, phage DNA replication proceeds according to a bidirectional θ mechanism early after infection due to efficient transcriptional activation of oriλ, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of λ genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in λ DNA. It is proposed that this may lead to inefficient transcriptional activation of oriλ resulting in unidirectional θ replication followed by σ type replication.
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A unipolarly located, cell-surface-associated agglutinin, RapA, belongs to a family of Rhizobium-adhering proteins (Rap) in Rhizobium leguminosarum bv. trifolii
More LessThe GenBank accession numbers for the sequences reported in this paper are AF265222, AF265223, AF315809 and AF315810.
The phage-display cloning technique was used to find rhizobial proteins that bind to receptors located on the bacterial cell surface. The aim was to clone the gene(s) encoding rhicadhesin, a universal rhizobial adhesion protein, and/or other cell-surface-binding proteins. Four such R hizobium-adhering proteins (Rap) were revealed in Rhizobium leguminosarum bv. trifolii strain R200. The binding is mediated by homologous Ra domains in these proteins. One member of the Rap protein family, named RapA1, is a secreted calcium-binding protein, which are also properties expected for rhicadhesin. However, the size of the protein (24 kDa instead of 14 kDa) and its distribution among different rhizobia (present in only Rhizobium leguminosarum biovars and R. etli instead of all members of Rhizobiaceae) argue against RapA1 being rhicadhesin. Protein RapA1 consists of two homologous Ra domains and agglutinates R200 cells by binding to specific receptors located at one cell pole during exponential growth. Expression of these cell-surface receptors was detected only in rhizobia that produce the RapA proteins. The authors propose that the homologous Ra domains, found to be present also in other proteins with different structure, represent lectin domains, which confer upon these proteins the ability to recognize their cognate carbohydrate structures.
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Development of a genetic system for the transfer of DNA into Flavobacterium heparinum
More LessThe GenBank accession number for the sequence reported in this paper is AF221716.
Flavobacterium heparinum (now Pedobacter heparinus) is a Gram-negative soil bacterium which can produce yellow pigments. It synthesizes five enzymes that degrade glycosoaminoglycan molecules. The study of this unique bacterium has been limited by the absence of a genetic manipulation system. In this paper, the construction of a conjugation/integration plasmid system and a broad-host-range plasmid, both of which contain a F. heparinum functional selective marker created by placing the trimethoprim resistance gene, dhfrII, under the control of the hepA regulatory region is described. Both plasmids were introduced into F. heparinum by conjugation and/or electroporation, and trimethoprim resistant colonies were obtained. Fifty electroporants were obtained per microgram covalently closed circular plasmid DNA. The existence of integrated plasmid DNA was confirmed by Southern hybridization and PCR. The existence of a derivative of the broad-host-range plasmid pBBR1 in F. heparinum was demonstrated by plasmid digestion and Southern hybridization, and by transformation of Escherichia coli.
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The binding pattern of two carbohydrate-binding modules of laminarinase Lam16A from Thermotoga neapolitana: differences in β-glucan binding within family CBM4
More LessCarbohydrate-binding modules (CBMs) are often part of the complex hydrolytic extracellular enzymes from bacteria and may modulate their catalytic activity. The thermostable catalytic domain of laminarinase Lam16A from Thermotoga neapolitana (glycosyl hydrolase family 16) is flanked by two CBMs, 148 and 161 aa long. They share a sequence identity of 30%, are homologous to family CBM4 and are thus called CBM4-1 and CBM4-2 respectively. Recombinant Lam16A proteins deleted for one or both binding modules and the isolated module CBM4-1 were characterized. Proteins containing the N-terminal module CBM4-1 bound to the soluble polysaccharides laminarin (1,3-β-glucan) and barley 1,3/1,4-β-glucan, and proteins containing the C-terminal module CBM4-2 bound additionally to curdlan (1,3-β-glucan) and pustulan (1,6-β-glucan), and to insoluble yeast cell wall β-glucan. The activity of the catalytic domain on soluble 1,3-β-glucans was stimulated by the presence of CBM4-1, whereas the presence of CBM4-2 enhanced the Lam16A activity towards gelatinized and insoluble or mixed-linkage 1,3-β-glucan. Thermostability of the catalytic domain was not affected by the truncations. Members of family CBM4 can be divided into four subfamilies, members of which show different polysaccharide-binding specificities corresponding to the catalytic specificities of the associated hydrolytic domains.
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A multifunctional polyketide–peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans
More LessThe GenBank accession number for the sequence determined in this work is AF239749.
Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted M r 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS). At 4801 amino acids in length, XabB is the largest reported PKS–NRPS. Twelve catalytic domains in this multifunctional enzyme are arranged in the order N terminus–acyl-CoA ligase (AL)–acyl carrier protein (ACP)–β-ketoacyl synthase (KS)–β-ketoacyl reductase (KR)–ACP–ACP–KS–peptidyl carrier protein (PCP)–condensation (C)–adenylation–PCP–C. The modular architecture of XabB indicates likely steps in albicidin biosynthesis and approaches to enhance antibiotic yield. The novel pattern of domains, in comparison with known PKS–NRPS enzymes for antibiotic production, also contributes to the knowledge base for rational design of enzymes producing novel antibiotics.
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Contribution of the phosphoenolpyruvate:mannose phosphotransferase system to carbon catabolite repression in Lactobacillus pentosus
More LessThe role of the Lactobacillus pentosus phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) in sugar transport and control of sugar utilization was investigated. Growth experiments and measurements of PEP-dependent phosphorylation of sugars, of sugar transport and of catabolic enzyme activity were performed, to compare a wild-type strain with an EIIBMan mutant, LPE6, and a ccpA mutant, LPE4. Fructose uptake in wild-type bacteria demonstrated the presence of two fructose-specific PTSs: a high-affinity system, EIIFru (K m=52 μM) which is inducible by fructose, and a low-affinity system (K m=300 μM). The latter system was lacking in LPE6 and therefore corresponds to EIIMan. LPE6 was unable to phosphorylate glucose, mannose, N-acetylglucosamine and 2-deoxyglucose in a PEP-dependent reaction, indicating that these sugars are substrates of EIIMan. Transport and phosphorylation of these compounds was the same in LPE4 and in wild-type bacteria, although growth of LPE4 on these sugars was impaired. In wild-type bacteria and in LPE4 the activity of EIIFru was lowered by the presence of EIIMan substrates in the growth medium, but this decrease was not observed in LPE6. These results indicate that EIIMan but not CcpA regulates the synthesis of EIIFru. Mutations in EIIMan or CcpA resulted in a relief of catabolite repression exerted by EIIMan substrates on the activity of β-galactosidase and β-glucosidase, indicating that EIIMan and CcpA are important components in catabolite repression in L. pentosus. Fructose-mediated repression of these two enzymes appeared to be correlated with the activity of EIIFru.
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A Corynebacterium glutamicum mutant with a defined deletion within the rplK gene is impaired in (p)ppGpp accumulation upon amino acid starvation
The GenBank accession number for the 5·25 kb rplK region reported in this paper is AF130462.
The rplK gene of Corynebacterium glutamicum ATCC13032 comprises 438 nucleotides and encodes a protein of 145 amino acids with a molecular mass of 15·3 kDa. The amino acid sequence revealed extensive similarities to the large ribosomal subunit protein L11 from several Gram-positive and Gram-negative bacteria. The C. glutamicum rplK gene is located downstream of secE, representing part of the protein export apparatus, and of nusG, encoding a transcription antiterminator protein. The rplK gene is followed by an ORF homologous to rplA encoding the 50S ribosomal protein L1. Northern analysis revealed that transcription of the rplK–rplA cluster resulted in two different transcripts of 1·5 and 0·6 kb. The 1·5 kb transcript corresponds to the entire rplK–rplA cluster and the short transcript originates from the rplK gene. A C. glutamicum rplK mutant strain carrying a 12 bp in-frame deletion within rplK, which resulted in the loss of the tetrapeptide Pro-Ala-Leu-Gly in the L11 protein, was constructed. The mutant failed to accumulate (p)ppGpp in response to amino acid starvation and exhibited an increased tolerance to the antibiotic thiostrepton. Evidently, the C. glutamicum rplK gene is required for (p)ppGpp accumulation upon nutritional starvation.
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- Pathogenicity And Medical Microbiology
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Phenotypic consequences of red–white colony type variation in Mycobacterium avium
More LessMycobacterium avium undergoes reversible morphotypic switching between the virulent transparent colony type and the less virulent opaque colony type. A new morphotypic switch in M. avium, termed red–white, that becomes visible when opaque colonies of clinical isolates are grown on agar media containing Congo red, was recently described. White opaque (WO) variants were found to be more resistant to multiple antibiotics than were red opaque (RO) variants. The present paper reports that transparent derivatives of RO and WO clones retain the differential Congo red binding properties of their opaque parents, indicating that the opaque–transparent switch operates independently of the red–white switch. White transparent variants were more resistant to clarithromycin and rifampin in vitro, and better able to survive within human macrophages, than their red transparent counterparts. Neither red nor white variants were markedly favoured during growth in vitro; however, red variants were better able to spread on soft agar (sliding motility), a potential selective advantage under some environmental circumstances. White-to-red switching was frequently observed in vitro and was accompanied by decreased antibiotic resistance and increased motility. Red-to-white switching has yet to be observed in vitro, indicating that the red morphotype is very stable. Significantly, some widely studied laboratory reference strains of M. avium, including strain 2151 and the genome sequence strain 104, are stable red clones. These strains are intrinsically antibiotic resistant and virulent in animal models, but they may not express genes encoding the elevated levels of antibiotic resistance and intracellular survival observed in white variants.
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Salmonella typhimurium thyA mutants fail to grow intracellularly in vitro and are attenuated in mice
More LessSalmonella typhimurium ATCC14028 readily multiplies in professional phagocytes in vitro and is highly virulent in mice. Mutants lacking thymidylate synthase activity (thyA) were isolated and shown to be strictly dependent on thymidine monophosphate precursors in the growth medium. The thyA mutants were found to be virtually incapable of intracellular growth and survival in vitro, both in macrophage-like cell line P338D1 and in the human epithelial cell line Hep-2, and their virulence was impaired in BALB/c mice. Intraperitoneal immunization of mice with two doses of live S. typhimurium thyA provided protection against a challenge with 103 times the 50% lethal dose of the virulent parent strain.
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Campylobacter upsaliensis exerts a cytolethal distending toxin effect on HeLa cells and T lymphocytes
More LessCampylobacter upsaliensis is an emerging human enteropathogen. However, little is known about the pathogenesis of C. upsaliensis infection. In this study the authors demonstrate that C. upsaliensis whole-cell preparations and extracts produce a cytolethal distending toxin (CDT)-like effect on HeLa cells characterized by progressive distension and nuclear fragmentation culminating in cell death over 5 d. To further delineate the nature of this toxic effect in relation to CDT from other pathogens, the effect of C. upsaliensis on cellular events in epithelial cells and immunocytes was investigated. C. upsaliensis lysate-treated HeLa cells subjected to FACScan analysis using carboxyfluorescein diacetete succinimidyl ester (CFDA-SE) as a cell tracer demonstrated cell division arrest. Propidium iodide (PI) staining of HeLa cells revealed that cell cycle arrest occurred in G2/M. Human T lymphocytes exposed to C. upsaliensis lysates also showed cell cycle arrest in G2/M. Using a combination of Annexin V/PI staining and TUNEL assay, cytodistended HeLa cells were shown to undergo apoptotic cell death. These data provide the first insights into the virulence mechanisms of this novel enteropathogen.
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- Physiology And Growth
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A role for DNA supercoiling in the regulation of the cytochrome bd oxidase of Escherichia coli
More LessThe cydAB operon of Escherichia coli encodes cytochrome bd, a terminal oxidase in the aerobic respiratory chain. The high oxygen affinity of this oxidase explains its increased synthesis under low-oxygen conditions. Expression of the cydAB operon is controlled by the ArcA/ArcB two-component system and the oxygen-sensing transcriptional regulator Fnr. However, cydAB expression is still induced upon entry into stationary phase or following a shift to anaerobic conditions in a mutant deleted for arcA and fnr [Cotter, P. A. & Gunsalus, R. P. (1992), FEMS Microbiol Lett 91, 31–36]. Indeed, such a mutant contains 60% of the wild-type levels of spectrally detectable cytochrome bd. A possible mechanism to account for this regulation is that changes in negative supercoiling, which occur during a shift to low-oxygen or anaerobic conditions, may contribute to the regulation of the cydAB operon. This paper reports several lines of evidence in support of this idea. Firstly, the expression of cydAB, and the final level of spectrally detectable cytochrome bd, is sensitive to inhibitors of DNA gyrase, the enzyme responsible for introducing negative supercoils into DNA. Both nalidixic acid and novobiocin reduce cydA–lacZ expression in a concentration-dependent manner. Secondly, in a gyrA mutant, defective in DNA gyrase activity, expression of cydAB is reduced to a basal level that is no longer sensitive to the oxygen status. Both gyrase inhibitors and the gyrA mutation reduce cydAB expression in a strain deleted for arcA and fnr, indicating that their effects are not mediated indirectly through ArcA or Fnr, but rather that they are likely to be direct effects on cydAB expression. In conclusion, the authors have shown that changes in DNA supercoiling play a role in the induction of cydAB expression and may provide a general way of increasing cytochrome bd levels in the cell in response to environmental stress.
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Characterization of a copper-transport operon, copYAZ, from Streptococcus mutans
More LessA copper-transport (copYAZ) operon was cloned from the oral bacterium Streptococcus mutans JH1005. DNA sequencing showed that the operon contained three genes (copY, copA and copZ), which were flanked by a single promoter and a factor-independent terminator. copY encoded a small protein of 147 aa with a heavy-metal-binding motif (CXCX4CXC) at the C-terminus. CopY shared extensive homology with other bacterial negative transcriptional regulators. copA encoded a 742 aa protein that shared extensive homology with P-type ATPases. copZ encoded a 67 aa protein that also contained a heavy-metal-binding motif (CXXC) at the N-terminus. Northern blotting showed that a 3·2 kb transcript was produced by Cu2+-induced Strep. mutans cells, suggesting that the genes were synthesized as a polycistronic message. The transcriptional start site of the cop operon was mapped and shown to lie within the inverted repeats of the promoter–operator region. Strep. mutans wild-type cells were resistant to 800 μM Cu2+, whereas cells of a cop knock-out mutant were killed by 200 μM Cu2+. Complementation of the cop knock-out mutant with the cop operon restored Cu2+ resistance to wild-type level. The wild-type and the mutant did not show any differences in susceptibility to other heavy metals, suggesting that the operon was specific for copper. By using a chloramphenicol acetyltransferase reporter gene fusion, the cop operon was shown to be negatively regulated by CopY and could be derepressed by Cu2+.
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Molecular characterization, enzyme properties and transcriptional regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in a ruminal bacterium, Selenomonas ruminantium
More LessThe GenBank accession numbers for the S. ruminantium pck and pyk sequences reported in this paper are AB016600 and AB037182, respectively.
To elucidate the regulatory mechanism for propionate production in Selenomonas ruminantium, the molecular properties and gene expression of phosphoenolpyruvate carboxykinase (Pck) and pyruvate kinase (Pyk) were investigated. The Pck was deduced to consist of 538 aa with a molecular mass of 59·6 kDa, and appeared to exist as a monomer. The Pyk was revealed to consist of four identical subunits consisting of 469 aa with a molecular mass of 51·3 kDa. Both Mg2+ and Mn2+ were required for the maximal activity of Pck, and Pck utilized ADP, not GDP or IDP, as a substrate. Either Mg2+ or Mn2+ was required for Pyk activity, and the enzyme was activated by phosphoenolpyruvate (PEP) and fructose 1,6-bisphosphate (FBP). Pyk activity was severely inhibited by Pi, but restored by the addition of FBP. The K m value of Pck for PEP (0·55 mM) was nearly equal to the K m value of Pyk for PEP, suggesting that the partition of the flow from PEP in the fermentation pathways is determined by the activity ratio of Pck to Pyk. Both pck and pyk genes were monocistronic, although two transcriptional start sites were found in pyk. The level of pyk mRNA was not different whether glucose or lactate was the energy substrate. However, the pck mRNA level was 12-fold higher when grown on lactate than on glucose. The level of pck mRNA was inversely related to the sufficiency of energy, suggesting that Pck synthesis is regulated at the transcriptional level when energy supply is altered. It was conceivable that the transcription of pck in S. ruminantium is triggered by PEP and suppressed by ATP.
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Transcription of arcA and rpoS during growth of Salmonella typhimurium under aerobic and microaerobic conditions
More LessPhysiology of the exponential and stationary phase of growth, under both aerobic and microaerobic conditions, of Salmonella typhimurium and its isogenic mutants nuoG::Km, cydA::TnphoA, ΔarcA and ΔrpoS was studied using luxAB transcriptional fusions with the rpoS and arcA genes. In the wild-type strain, rpoS transcription was greater under aerobic than under microaerobic conditions, whereas transcription of arcA was suppressed by aerobiosis. Under aerobic conditions, no interaction between NuoG, CydA, ArcA and RpoS was detected. Under microaerobic conditions, rpoS was suppressed in the nuoG mutant as compared with the wild-type strain, but it was overexpressed in the cydA and arcA mutants. A deletion in the rpoS gene, on the other hand, resulted in non-restricted, increased arcA expression in stationary-phase cultures under microaerobic conditions. Based on the rpoS transcription in the nuoG mutant the authors propose that the decrease in the NADH:NAD ratio that occurs when carbon sources become limiting serves as a signal for increased rpoS transcription, while active respiration catalysed by CydA and controlled by ArcA downregulates rpoS transcription. When, finally, the RpoS-controlled stationary phase of growth is reached, arcA is suppressed in an RpoS-dependent fashion. Transition into stationary phase under microaerobic conditions is thus controlled by coordinated action of the RpoS and ArcA regulators, depending on subtle changes in the environment.
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Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes
More LessAcid resistance is an important feature of both pathogenic and non-pathogenic Escherichia coli. It enables survival in the acidic regions of mammalian gastrointestinal tracts and is largely responsible for the small number of bacteria required for infection/colonization. Three systems of acid resistance have been identified, the most efficient of which requires glutamic acid during pH 2 acid challenge. Three proteins associated with glutamate-dependent acid resistance have been identified. They are glutamate decarboxylase (encompassing two isozymes encoded by gadA and gadB) and a putative glutamate:γ-amino butyric acid antiporter (encoded by gadC). The results confirm that the GadA and GadB proteins increase in response to stationary phase and low environmental pH. The levels of these proteins correspond to concomitant changes in gadA and gadBC mRNA levels. Fusions between lacZ and the gadA and gadBC operons indicate that this control occurs at the transcriptional level. Western blot, Northern blot and fusion analyses reveal that regulation of these genes is complex. Expression in rich media is restricted to stationary phase. However, in minimal media, acid pH alone can trigger induction in exponential or stationary phase cells. Despite this differential control, there is only one transcriptional start site for each gene. Expression in rich media is largely dependent on the alternate sigma factor σS and is repressed by the cAMP receptor protein (CRP). In contrast, σS has only a minor role in gad transcription in cells grown in minimal media. Deletions of the regulatory region upstream of gadA provided evidence that a 20 bp conserved region located 50 bp from the transcriptional start of both operons is required for expression.
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Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum
More LessWhen grown in the presence of sunflower cell walls, Sclerotinia sclerotiorum, an ubiquitous necrotrophic fungus, secretes several acid proteases including a non-aspartyl protease. The gene acp1, encoding an acid protease, has been cloned and sequenced. The intronless ORF encodes a preproprotein of 252 aa and a mature protein of 200 residues. In vitro expression of acp1 is subject to several transcriptional regulatory mechanisms. Expression induced by plant cell-wall proteins is controlled by both carbon and nitrogen catabolite repression. Glucose on its own represses acp1 expression while ammonium repression requires the simultaneous presence of a carbon source. Ambient pH higher than pH 5 overrides induction resulting in full repression of acp1. These transcriptional regulatory mechanisms and the presence of several motifs in the promoter of acp1 that may encode binding sites for the regulators CREA, AREA and PacC suggest the involvement of these regulators in the control of acp1 expression. acp1 is expressed in planta during sunflower cotyledon infection. Expression is low at the beginning of infection but increases suddenly at the stage of necrosis spreading. Comparison of in vitro and in planta acp1 expression suggests that glucose and nitrogen starvation together with acidification can be considered as key factors controlling Scl. sclerotiorum gene expression during pathogenesis.
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- Plant-Microbe Interactions
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An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas
More LessThe GenBank accession number for the sequence reported in this work is AF225904.
Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2·5 kbp EcoRI–HindIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.
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Supply of O2 regulates demand for O2 and uptake of malate by N2-fixing bacteroids from soybean nodules
More LessBacteroids, prepared anaerobically from soybean root nodules by fractional centrifugation or by sucrose or Percoll density-gradient methods, were retained within a stirred, flow-through reaction chamber and used to determine rates of respiration and N2 fixation at various rates of O2 supply. Liquid reaction solutions containing malate, oxyleghaemoglobin, dissolved N2 and various levels of dissolved O2 were passed through the reaction chamber at measured rates of flow. The relative oxygenation of leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([O2 free]) and the rates of O2 consumption were calculated. N2 fixation was measured by analysis of fractions of effluent. The principal finding was that stepwise increases in the flow rate (increasing the supply of O2 and malate) induced an increase in O2 demand (respiration) resulting in a decrease in [O2 free] and increased N2 fixation. In some experiments, samples of bacteroids were withdrawn from the flow chamber during steady states and the rates of malate uptake were measured in standard, microaerobic assays. Progressive taking of samples from the flow chamber whilst maintaining constant flow rates (increasing the supply of O2 and malate per bacteroid) also resulted in increased O2 demand and declines in [O2 free]. With increased bacteroid respiration, transport of malate into bacteroids (linear with time between 1 and 5 min after starting each assay) increased proportionally. This suggests that the rate of malate transport is tightly coupled with bacteroid respiration. Thus, bacteroid respiration, coupled with malate uptake, must be regulated by the rate of O2 supply, rather than by the [O2 free] prevailing in the stirred chamber as found or assumed in previous work. These features are discussed in relation to N2 fixation by anaerobically isolated bacteroids.
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- Systematics And Evolution
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Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica
More LessThe GenBank accession numbers for the sequences reported in this paper are AY012160–AY012201.
The evolutionary history of the GDP-mannose pathway in Salmonella enterica was studied via sequencing manB and manC genes from 13 representative strains for O antigens containing mannose and/or sugar derivatives of GDP-D-mannose. In addition, colanic acid (CA) manB and manC genes were sequenced from selected strains, as the basis for a detailed comparison. Interestingly, including the eight previously characterized O antigen gene clusters, 12 of the 21 S. enterica strains studied in total (each representing a different O antigen structure) possess a manB gene which displays DNA identity, ranging from 93 to 99%, to the CA manB gene of S. enterica LT2. Furthermore, the CA-like manB genes (as well as the CA manB and manC genes) display subspecies specificity, and the CA and CA-like manB genes (for individual strains) appear to be evolving in concert via gene conversion events. In comparison, the manC genes were generally not CA-like, a situation also apparent in Escherichia coli,and therefore most strongly reflected the evolutionary history of the S. enterica Oantigen GDP-mannose pathway. It appears that, in relatively recent times, gene capture from a distant source has occurred infrequently, and that groups of manB and manC genes have been maintained and are continuing to evolve within S. enterica and more closely related species.
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