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Volume 147,
Issue 3,
2001
Volume 147, Issue 3, 2001
- Physiology And Growth
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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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