- Volume 151, Issue 8, 2005
Volume 151, Issue 8, 2005
- Review
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Acclimation of unicellular cyanobacteria to macronutrient deficiency: emergence of a complex network of cellular responses
More LessCyanobacteria are equipped with numerous mechanisms that allow them to survive under conditions of nutrient starvation, some of which are unique to these organisms. This review surveys the molecular mechanisms underlying acclimation responses to nitrogen and phosphorus deprivation, with an emphasis on non-diazotrophic freshwater cyanobacteria. As documented for other micro-organisms, nutrient limitation of cyanobacteria elicits both general and specific responses. The general responses occur under any starvation condition and are the result of the stresses imposed by arrested anabolism. In contrast, the specific responses are acclimation processes that occur as a result of limitation for a particular nutrient; they lead to modification of metabolic and physiological routes to compensate for the restriction. First, the general acclimation processes are discussed, with an emphasis on modifications of the photosynthetic apparatus. The molecular mechanisms underlying specific responses to phosphorus and nitrogen-limitation are then outlined, and finally the cross-talk between pathways modulating specific and general responses is described.
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- Microbiology Comment
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- Cell And Developmental Biology
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Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869
More LessCorynebacterium glutamicum is a Gram-positive bacterium that lacks the cell division FtsA protein and actin-like MreB proteins responsible for determining cylindrical cell shape. When the cell division ftsZ gene from C. glutamicum (ftsZCg ) was cloned in different multicopy plasmids, the resulting constructions could not be introduced into C. glutamicum; it was assumed that elevated levels of FtsZ Cg result in lethality. The presence of a truncated ftsZCg and a complete ftsZCg under the control of Plac led to a fourfold reduction in the intracellular levels of FtsZ, generating aberrant cells displaying buds, branches and knots, but no filaments. A 20-fold reduction of the FtsZ level by transformation with a plasmid carrying the Escherichia coli lacI gene dramatically reduced the growth rate of C. glutamicum, and the cells were larger and club-shaped. Immunofluorescence microscopy of FtsZ Cg or visualization of FtsZ Cg –GFP in C. glutamicum revealed that most cells showed one fluorescent band, most likely a ring, at the mid-cell, and some cells showed two fluorescent bands (septa of future daughter cells). When FtsZ Cg –GFP was expressed from Plac, FtsZ rings at mid-cell, or spirals, were also clearly visible in the aberrant cells; however, this morphology was not entirely due to GFP but also to the reduced levels of FtsZ expressed from Plac. Localization of FtsZ at the septum is not negatively regulated by the nucleoid, and therefore the well-known occlusion mechanism seems not to operate in C. glutamicum.
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Mass flow and pressure-driven hyphal extension in Neurospora crassa
More LessMass flow of cytoplasm in Neurospora crassa trunk hyphae was directly confirmed by injecting oil droplets into the hyphae. The droplets move in a manner similar to cytoplasmic particles and vacuoles within the hyphae. The direction of mass flow is towards the growing hyphal tips at the colony edge. Based on flow velocities (about 5 μm s−1), hyphal radius and estimates of cytoplasm viscosity, the Reynolds number is about 10−4, indicating that mass flow is laminar. Therefore, the Poiseulle equation can be used to calculate the pressure gradient required for mass flow: 0·0005–0·1 bar cm−1 (depending on the values used for septal pore radius and cytoplasmic viscosity). These values are very small compared to the normal hydrostatic pressure of the hyphae (4–5 bar). Mass flow stops after respiratory inhibition with cyanide, or creation of an extracellular osmotic gradient. The flow is probably caused by internal osmotic gradients created by differential ion transport along the hyphae. Apical cytoplasm migrates at the same rate as tip extension, as do oil droplets injected near the tip. Thus, in addition to organelle positioning mediated by molecular motors, pressure-driven mass flow may be an integral part of hyphal extension.
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- Biochemistry And Molecular Biology
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Identification of tailoring genes involved in the modification of the polyketide backbone of rifamycin B by Amycolatopsis mediterranei S699
More LessRifamycin B biosynthesis by Amycolatopsis mediterranei S699 involves a number of unusual modification reactions in the formation of the unique polyketide backbone and decoration of the molecule. A number of genes believed to be involved in the tailoring of rifamycin B were investigated and the results confirmed that the formation of the naphthalene ring moiety of rifamycin takes place during the polyketide chain extension and is catalysed by Rif-Orf19, a 3-(3-hydroxyphenyl)propionate hydroxylase-like protein. The cytochrome P450-dependent monooxygenase encoded by rif-orf5 is required for the conversion of the Δ12, 29 olefinic bond in the polyketide backbone of rifamycin W into the ketal moiety of rifamycin B. Furthermore, Rif-Orf3 may be involved in the regulation of rifamycin B production, as its knock-out mutant produced about 40 % more rifamycin B than the wild-type. The work also revealed that many of the genes located in the cluster are not involved in rifamycin biosynthesis, but might be evolutionary remnants carried over from an ancestral lineage.
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Modulation of quorum sensing in Pseudomonas aeruginosa through alteration of membrane properties
Changes in the cellular envelope are major physiological adaptations that occur when micro-organisms encounter extreme environmental conditions. An appropriate degree of membrane fluidity is crucial for survival, and alteration of membrane lipids is an essential adaptive response. Emerging data suggest that microbial cells may recognize alterations in their membrane viscosity resulting from certain environmental changes as a trigger for adaptive cellular responses. In Pseudomonas aeruginosa, the quorum-sensing (QS) system involves a complex regulatory circuitry that coordinates the expression of genes according to a critical population density. Interestingly, it has been shown that the QS system of P. aeruginosa can also be activated by nutritional stress, independently of the cell density, and therefore may be part of a more general adaptive response to stressful environmental conditions. In order to examine the proposed link between membrane properties and stress signalling, the effects of genetically engineered alterations of the membrane phospholipid composition of P. aeruginosa PAO1 on the activation of the stringent response and the QS system were examined. The lptA gene encoding a functional homologue of PlsC, an Escherichia coli enzyme that catalyses the second step of the phospholipid biosynthesis pathway, was identified and disrupted. Inactivation of lptA altered the fatty acid profile of phospholipids and the membrane properties, resulting in decreased membrane fluidity. This resulted in a premature production of the QS signals N-butanoyl- and N-hexanoyl-homoserine lactone (C4-HSL and C6-HSL) and a repression of 2-heptyl-3-hydroxy-4-quinolone (PQS) synthesis at later growth phases. The effects on C4- and C6-HSL depended upon the expression of relA, encoding the (p)ppGpp alarmone synthase, which was increased in the lptA mutant. Together, the findings support the concept that alterations in membrane properties can act as a trigger for stress-related gene expression.
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The gene stlA encodes a phenylalanine ammonia-lyase that is involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01
More LessPhotorhabdus is a genus of Gram-negative bacteria from the family Enterobacteriaceae. Members of Photorhabdus have a complex life cycle during which the bacterium has a pathogenic interaction with insect larvae whilst also maintaining a mutualistic relationship with nematodes from the family Heterorhabditidae. During growth in the insect, Photorhabdus bacteria produce a broad-spectrum antibiotic identified as 3,5-dihydroxy-4-isopropylstilbene (ST). The biochemical pathway responsible for the production of this antibiotic has not been characterized. In this report, a mutant strain of Photorhabdus luminescens subsp. laumondii TT01, BMM901, has been isolated, by transposon mutagenesis, that is unable to produce the ST antibiotic. Using in silico studies, feeding experiments and biochemical analyses, it is shown that the gene mutated in this strain, stlA, encodes phenylalanine ammonia-lyase (PAL). PAL catalyses the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid and the enzyme is ubiquitous in plants, where it is involved in the production of phenylpropanoids such as lignin and phytoalexins. However, this is the first report of PAL activity in a member of the Proteobacteria.
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The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans
More LessThe secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Δppk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.
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Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system
More LessCloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450mor) and the Fe3S4 ferredoxin (Fdmor), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450mor system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1′, encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdRmor was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K m values of FdRmor for the substrate NADH (37·7±4·1 μM) and the artificial electron acceptors potassium ferricyanide (14·2±1·1 μM) and cytochrome c (28·0±3·6 μM) were measured. FdRmor was shown to interact functionally with its natural redox partner, the Fe3S4 protein Fdmor, and with the Fe2S2 protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fdmor in activity assays. These results indicated that FdRmor can utilize different ferredoxins, but that Fdmor requires the specific NADH : ferredoxin oxidoreductase FdRmor from the P450mor system for efficient catalytic function.
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Stability of the Synechococcus elongatus PCC 7942 circadian clock under directed anti-phase expression of the kai genes
More LessThe kaiA, kaiB and kaiC genes encode the core components of the cyanobacterial circadian clock in Synechococcus elongatus PCC 7942. Rhythmic expression patterns of kaiA and of the kaiBC operon normally peak in synchrony. In some mutants the relative timing of peaks (phase relationship) between these transcription units is altered, but circadian rhythms persist robustly. In this study, the importance of the transcriptional timing of kai genes was examined. Expressing either kaiA or kaiBC from a heterologous promoter whose peak expression occurs 12 h out of phase from the norm, and thus 12 h out of phase from the other kai locus, did not affect the time required for one cycle (period) or phase of the circadian rhythm, as measured by bioluminescence reporters. Furthermore, the data confirm that specific cis elements within the promoters of the kai genes are not necessary to sustain clock function.
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Adaptation and acclimatization to formaldehyde in methylotrophs capable of high-concentration formaldehyde detoxification
More LessFormaldehyde is a highly toxic chemical common in industrial effluents, and it is also an intermediate in bacterial metabolism of one-carbon growth substrates, although its role as a bacterial growth substrate per se has not been extensively reported. This study investigated two highly formaldehyde-resistant formaldehyde utilizers, strains BIP and ROS1; the former strain has been used for industrial remediation of formaldehyde-containing effluents. The two strains were shown by means of 16S rRNA characterization to be closely related members of the genus Methylobacterium. Both strains were able to use formaldehyde, methanol and a range of multicarbon compounds as their principal growth substrate. Growth on formaldehyde was possible up to a concentration of at least 58 mM, and survival at up to 100 mM was possible after stepwise acclimatization by growth at increasing concentrations of formaldehyde. At such high concentrations of formaldehyde, the cultures underwent a period of formaldehyde removal without growth before the formaldehyde concentration fell below 60 mM, and growth could resume. Two-dimensional electrophoresis and MS characterization of formaldehyde-induced proteins in strain BIP revealed that the pathways of formaldehyde metabolism, and adaptations to methylotrophic growth, were very similar to those seen in the well-characterized methanol-utilizing methylotroph Methylobacterium extorquens AM1. Thus, it appears that many of the changes in protein expression that allow strain BIP to grow using high formaldehyde concentrations are associated with expression of the same enzymes used by M. extorquens AM1 to process formaldehyde as a metabolic intermediate during growth on methanol.
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Candida albicans strains deficient in CHS7, a key regulator of chitin synthase III, exhibit morphogenetic alterations and attenuated virulence
Chitin is a structural polysaccharide present in most fungal cell walls, whose synthesis depends on a family of enzymic activities named chitin synthases (CSs). The specific role of each of them, as well as of their regulatory proteins, in cell morphogenesis and virulence is not well understood. Here, it is shown that most chitin synthesis in Candida albicans, one of the fungi most commonly isolated from opportunistic mycoses and infections, depends on CHS7. Thus, C. albicans chs7Δ null mutants showed reduced levels of chitin and CS activity, and were resistant to Calcofluor. Despite the sequence similarity and functional relationship with ScChs7p, CaChs7p was unable to restore CSIII activity in a Saccharomyces cerevisiae chs7Δ null mutant, because it was unable to direct ScChs3p export from the endoplasmic reticulum. C. albicans chs7Δ null mutants did not show any defect in growth rate, but yeast cells displayed minor morphogenetic defects affecting septum formation, and showed an increased tendency to form filaments. CaChs7p was not required for germ-tube emission, and null mutant strains underwent the dimorphic transition correctly. However, colony morphology appeared distinctively affected. chs7Δ hyphae were very curved and had irregular lateral walls, resulting in very compact colonies that seemed unable to spread out radially on the surface, unlike the wild-type. This growth pattern may be associated with the reduced virulence and high clearance rate observed when the chs7Δ strain was used in a murine model of infection. Therefore, CaChs7p is required for normal hyphal morphogenesis, suggesting that in C. albicans CSIII plays an important role in maintaining cell wall integrity, being essential when invading surrounding tissues.
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Molecular insight into extreme copper resistance in the extremophilic archaeon ‘Ferroplasma acidarmanus’ Fer1
More Less‘Ferroplasma acidarmanus’ strain Fer1 is an extremely acidophilic archaeon involved in the genesis of acid mine drainage, and was isolated from copper-contaminated mine solutions at Iron Mountain, CA, USA. Here, the initial proteomic and molecular investigation of Cu2+ resistance in this archaeon is presented. Analysis of Cu2+ toxicity via batch growth experiments and inhibition of oxygen uptake in the presence of ferrous iron demonstrated that Fer1 can grow and respire in the presence of 20 g Cu2+ l−1. The Fer1 copper resistance (cop) loci [originally detected by Ettema, T. J. G., Huynen, M. A., de Vos, W. M. & van der Oost, J. Trends Biochem Sci 28, 170–173 (2003)] include genes encoding a putative transcriptional regulator (copY), a putative metal-binding chaperone (copZ) and a putative copper-transporting P-type ATPase (copB). Transcription analyses demonstrated that copZ and copB are co-transcribed, and transcript levels were increased significantly in response to exposure to high levels of Cu2+, suggesting that the transport system is operating for copper efflux. Proteomic analysis of Fer1 cells exposed to Cu2+ revealed the induction of stress proteins associated with protein folding and DNA repair (including RadA, thermosome and DnaK homologues), suggesting that ‘Ferroplasma acidarmanus’ Fer1 uses multiple mechanisms for resistance to high levels of copper.
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- Biodiversity And Evolution
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Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin
More LessNumerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25–50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
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Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification
More LessAll known chlamydiae are either proven human or animal pathogens or possess such potential. Due to increasing reports of chlamydiae diversity in the environment, it is important to develop reliable means for identifying and characterizing Chlamydiales species. The identification of environmental chlamydiae at present relies on their branching pattern in 16S rRNA trees, as well as 16S/23S consensus motifs which display variability. At present, no reliable molecular signatures are known which are unique to all Chlamydiales species. Besides the rRNAs, sequence information for different Chlamydiales is not available for any other gene sequence. In this report, a number of molecular signatures are described that consist of conserved inserts and deletions (indels), in widely distributed proteins [RNA polymerase α subunit (RpoA), elongation factor (EF)-Tu, EF-P, DNA gyrase B subunit (GyrB) and lysyl-tRNA synthetase (LysRS)], that are distinctive characteristics of all available chlamydiae homologues (from Chlamydiaeceae species and Parachlamydiae sp. UWE25) and not found in any other bacteria. Using PCR primers for highly conserved regions in these proteins, the corresponding fragments of these genes from Simkania negevensis, Waddlia chondrophila, and in a number of cases for Neochlamydia hartmanellae, covering all families within the phylum Chlamydiae, have been cloned and sequenced. The shared presence of the identified signatures in these species provides strong evidence that these molecular signatures are distinctive characteristics of the entire order Chlamydiales and can be used to reliably determine the presence of chlamydiae or chlamydia-related organisms in environmental samples. The sequence information for these protein fragments was also used to determine the interrelationships among chlamydiae species. In a phylogenetic tree based on a combined dataset of sequences from RpoA, EF-Tu, EF-P and GyrB, the environmental chlamydiae (i.e. Simkania, Waddlia and Parachlamydia) and the traditional Chlamydiaceae (i.e. Chlamydophila and Chlamydia) formed two distinct clades. Similar relationships were also observed in individual protein phylogenies, as well as in a 16S rRNA tree for the same species. These results provide evidence that the divergence between the traditional Chlamydiaceae species and the other chlamydiae families occurred very early in the evolution of this group of bacteria.
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Subtractive hybridization reveals a high genetic diversity in the fish pathogen Photobacterium damselae subsp. piscicida: evidence of a SXT-like element
More LessPhotobacterium damselae subsp. piscicida is the causative agent of fish pasteurellosis, a severe disease affecting cultured marine fish worldwide. In this study, suppression subtractive hybridization was used to identify DNA fragments present in the virulent strain PC554.2, but absent in the avirulent strain EPOY 8803-II. Twenty-one genomic regions of this type (that included twenty-six distinct putative ORFs) were analysed by DNA sequencing. Twenty ORFs encoded proteins with homology to proteins in other bacteria, including four homologues involved in siderophore biosynthesis, and four homologues related to mobile elements; three of these were putative transposases and one was a putative conjugative transposon related to the Vibrio cholerae SXT element. This sequence was shown to be integrated into a prfC gene homologue. Six ORFs showed no significant homology to known bacterial proteins. Among the 21 DNA fragments specific to strain PC554.2, 5 DNA fragments (representing 7 ORFs) were also absent in the avirulent strain ATCC 29690. The analysis of these differential regions, as well as the screening of their presence in a collection of strains, demonstrated the high genetic heterogeneity of this pathogen.
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Structure and distribution of the phosphoprotein phosphatase genes, prpA and prpB, among Shigella subgroups
More LessPhosphoprotein phosphatases encoded by the prpA and prpB genes function in signal transduction pathways for degradation of misfolded proteins in the extracytoplasmic compartments of Escherichia coli. In order to trace the evolution of prp genes and assess their roles in other enteric pathogens, the structure and distribution of these genes among closely related Shigella subgroups were studied. PCR amplification, probe hybridization studies and DNA sequencing were used to determine the prp genotypes of 58 strains from the four Shigella subgroups, Dysenteriae, Boydii, Sonnei and Flexneri. It was found that the prp alleles among Shigella subgroups were extremely susceptible to gene inactivation and that the mutations involved in prp allele inactivation were varied. They included IS insertions, gene replacement by an IS element, a small deletion within the gene or large deletion engulfing the entire gene region, and base substitutions that generated premature termination codons. As a result, of 58 strains studied, only eight (14 %) possessed intact prpA and prpB genes. Of the Shigella strains examined, 76 % (44/58) showed at least one of the prp alleles inactivated by one or more IS elements, including IS1, IS4, IS600 and IS629. Phylogenetic analysis revealed that IS elements have been independently acquired in multiple lineages of Shigella, suggesting that loss of functional alleles has been advantageous during Shigella strain evolution.
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- Genes And Genomes
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Characterization of a regulatory gene essential for the production of the angucycline-like polyketide antibiotic auricin in Streptomyces aureofaciens CCM 3239
More LessA gene, aur1P, encoding a protein similar to the response regulators of bacterial two-component signal transduction systems, was identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. Expression of the gene was directed by a single promoter, aur1Pp, which was transcribed at low levels during the exponential phase and induced just before the stationary phase. A divergently transcribed gene, aur1R, has been identified upstream of aur1P, encoding a protein homologous to transcriptional repressors of the TetR family. The aur1P gene was disrupted in the S. aureofaciens CCM 3239 chromosome by homologous recombination. The mutation in the aur1P gene had no effect on growth and differentiation. However, biochromatographic analysis of culture extracts from the S. aureofaciens aur1P-disrupted strain revealed that auricin was not produced in the mutant. This indicated that aur1P is essential for auricin production. Transcription from the previously characterized aur1Ap promoter, directing expression of the first gene, aur1A, in the auricin gene cluster, was dramatically decreased in the S. aureofaciens CCM 3239 aur1P mutant strain. Moreover, the Aur1P protein, overproduced in Escherichia coli, was shown to bind specifically upstream of the aur1Ap promoter region. The results indicated that the Aur1P regulator activates expression of the auricin biosynthesis genes.
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Effects of growth phase and the developmentally significant bldA-specified tRNA on the membrane-associated proteome of Streptomyces coelicolor
More LessPrevious proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.
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Cell lysis directed by σ E in early stationary phase and effect of induction of the rpoE gene on global gene expression in Escherichia coli
It has been shown that Escherichia coli cells with increased expression of the rpoE gene encoding σ E exhibit enhanced cell lysis in early stationary phase. Further analysis of the lysis phenomenon was performed using a transient expression system of the rpoE gene and by DNA microarray. The former analysis revealed a σ E-directed cell lysis, specific for early stationary phase but not for the exponential phase. The microarray analysis with RNAs from exponential and early stationary phase cells revealed that a large number of genes were up- or down-regulated when the rpoE gene was induced, and that several genes were induced in a phase-specific manner. The upregulated genes include many previously identified σ E regulon genes, suggesting that a large number of genes are under the control of σ E in this organism. These genes are involved in various cellular activities, including the cell envelope, cellular processes, regulatory functions, transport and translation. Genes that are presumably related to phase-specific cell lysis in E. coli are discussed.
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- Pathogens And Pathogenicity
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Two glutathione peroxidases in the fungal pathogen Cryptococcus neoformans are expressed in the presence of specific substrates
More LessGlutathione peroxidases catalyse the reduction of peroxides by reduced glutathione. To determine if these enzymes are important for resistance to oxidative stress and evasion of the innate immune system by the fungal pathogen Cryptococcus neoformans, two glutathione peroxidase homologues, which share 38 % identity, were identified and investigated. In this study, these peroxidases, Gpx1 and Gpx2, their localization, their contribution to total glutathione peroxidase activity, and their importance to the oxidative and nitrosative stress resistance of C. neoformans are described. It is shown that the two glutathione peroxidase genes are differentially expressed in response to stress. While both GPX1 and GPX2 are induced during t-butylhydroperoxide or cumene hydroperoxide stress and repressed during nitric oxide stress, only GPX2 is induced in response to hydrogen peroxide stress. Deletion mutants of each and both of the glutathione peroxidases were generated, and it was found that they are sensitive to various peroxide stresses while showing wild-type resistance to other oxidant stresses, such as superoxide and nitric oxide. While the glutathione peroxidase mutants are slightly sensitive to oxidant killing by macrophages, they exhibit wild-type virulence in a mouse model of cryptococcosis.
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The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans
More LessCandida albicans is an opportunistic pathogen that has adapted to live and grow in the human body as its natural environment. Under these conditions, this fungus faces numerous challenges, including oxidative, osmotic and enzymic processes that may damage external and internal structures. In view of the key role of MAP kinase signalling pathways in the physiology of C. albicans, the effect of agents mimicking in vivo environmental conditions on the activation of the p42-44 MAP kinases has been analysed. It has been found that Mkc1p is phosphorylated in the presence of oxidative stress, changes in osmotic pressure, cell wall damage and a decrease in the growth temperature. This phosphorylation is dependent on Pkc1p, indicating that both proteins operate in the same signalling pathway in C. albicans. Under some stimuli, the phosphorylation of Mkc1p required the presence of Hog1p, the MAP kinase of the high osmolarity glycerol (HOG) pathway. This suggests the existence of a new regulatory role, at least under some conditions, for these MAP kinase pathways in yeast.
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Induction of the SOS regulon of Haemophilus influenzae does not affect phase variation rates at tetranucleotide or dinucleotide repeats
More LessHaemophilus influenzae has microsatellite repeat tracts in 5′ coding regions or promoters of several genes that are important for commensal and virulence behaviour. Changes in repeat number lead to switches in expression of these genes, a process referred to as phase variation. Hence, the virulence behaviour of this organism may be influenced by factors that alter the frequency of mutations in these repeat tracts. In Escherichia coli, induction of the SOS response destabilizes dinucleotide repeat tracts. H. influenzae encodes a homologue of the E. coli SOS repressor, LexA. The H. influenzae genome sequence was screened for the presence of the minimal consensus LexA-binding sequence from E. coli, CTG(N)10CAG, in order to identify genes with the potential to be SOS regulated. Twenty-five genes were identified that had LexA-binding sequences within 200 bp of the start codon. An H. influenzae non-inducible LexA mutant (lexA NI) was generated by site-directed mutagenesis. This mutant showed increased sensitivity, compared with wild-type (WT) cells, to both UV irradiation and mitomycin C (mitC) treatment. Semi-quantitative RT-PCR studies confirmed that H. influenzae mounts a LexA-regulated SOS response following DNA assault. Transcript levels of lexA, recA, recN, recX, ruvA and impA were increased in WT cells following DNA damage but not in lexA NI cells. Induction of the H. influenzae SOS response by UV irradiation or mitC treatment did not lead to any observable SOS-dependent changes in phase variation rates at either dinucleotide or tetranucleotide repeat tracts. Treatment with mitC caused a small increase in phase variation rates in both repeat tracts, independently of an SOS response. We suggest that the difference between H. influenzae and E. coli with regard to the effect of the SOS response on dinucleotide phase variation rates is due to the absence of any of the known trans-lesion synthesis DNA polymerases in H. influenzae.
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Campylobacter jejuni activates mitogen-activated protein kinases in Caco-2 cell monolayers and in vitro infected primary human colonic tissue
More LessThe mitogen-activated protein kinases (MAPKs) play a central role in many host signalling pathways. These signalling proteins are known to be involved in host responses against invasive bacteria including generation of chemotactic and inflammatory cytokines. It was hypothesized that Campylobacter jejuni may activate MAPKs, as intestinal infection may induce a clinical and pathological picture of acute colonic inflammation. Infection of Caco-2 cell monolayers (human colonic epithelial cell line) and human colonic tissue with C. jejuni in vitro demonstrated increased MAPK activity for ERK 1/2 (p44/42 MAPK), JNK and p38 MAPKs. Kinase activity and phosphorylated forms were increased in infected Caco-2 cells and human colonic explants, suggesting that these pathways are important in inflammatory responses induced by C. jejuni in man.
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Escherichia coli O157 : H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon
Enterohaemorrhagic Escherichia coli O157 : H7 is a human pathogen that causes no apparent disease in cattle, its primary reservoir host. Recent research has demonstrated that E. coli O157 : H7 predominately colonizes the distal few centimetres of the bovine rectum, and in this study, the LEE4 operon encoding a type III secretion system translocon and associated proteins was shown to be essential for colonization. A deletion mutant of LEE4 failed to colonize cattle, in contrast to a co-inoculated strain containing a chromosomal complement of the operon, therefore fulfilling ‘molecular’ Koch's postulates for this virulence determinant. In addition, attaching and effacing (A/E) lesions were detectable in E. coli O157 : H7 microcolonies from the terminal rectum of both naturally and experimentally colonized cattle when examined by transmission electron microscopy. This study proves that type III secretion is required for colonization of cattle by E. coli O157 : H7, and that A/E lesion formation occurs at the bovine terminal rectum within E. coli O157 : H7 microcolonies. The research confirms the value of using type III secreted proteins as vaccine candidates in cattle.
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- Physiology
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Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli
Acid resistance (AR) in Escherichia coli is important for its survival in the human gastrointestinal tract and involves three systems. The first AR system is dependent on the sigma factor RpoS. The second system (the GAD system) requires the glutamate decarboxylase isoforms encoded by the gadA and gadB genes. The third system (the ARG system) requires the arginine decarboxylase encoded by adiA. Loss of topoisomerase I function from topA deletion or Tn10 insertion mutations lowered the resistance to killing by pH 2 or 2·5 treatment by 10-fold to >100-fold. The RpoS and GAD systems were both affected by the topA mutation, but the ARG system of AR was not affected. Northern blot analysis showed that induction of gadA and gadB transcription in stationary phase and at pH 5·5 was decreased in the topA mutant. Western blot analysis showed that the topA mutation did not affect accumulation of RpoS, GadX or GadW proteins. Topoisomerase I might have a direct influence on the transcription of AR genes. This influence does not involve R-loop formation as the overexpression of RNase H did not alleviate the decrease of AR caused by the topA mutation. The effect of the topA mutation could be suppressed by an hns mutation, so topoisomerase I might be required to counteract the effect of H-NS protein on gene expression, in addition to its influence on RpoS-dependent transcription.
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- Plant-Microbe Interactions
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Specific gene targeting in Spiroplasma citri: improved vectors and production of unmarked mutations using site-specific recombination
More LessIn Spiroplasma citri, where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, oriC plasmids. To improve the probability of selecting rare recombination events without fastidious, extensive passaging of the transformants, a new targeting vector was constructed, which was used to inactivate the crr gene encoding the IIA component of the glucose phosphotransferase system (PTS) permease. Selection of recombinants was based on a two-step strategy using two distinct selection markers, one of which could only be expressed once recombination had occurred through one single crossover at the target gene. According to this strategy, spiroplasmal transformants were screened and multiplied in the presence of gentamicin before the crr recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the crr-disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in S. citri the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon γδ TnpR/res recombination system to produce unmarked mutations in S. citri was demonstrated. In an arginine deiminase (arcA-disrupted) mutant, the tetM gene flanked by the res sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative oriC plasmid. Due to oriC incompatibility, plasmid loss occurred spontaneously when selection pressure was removed. This approach will be helpful for constructing unmarked mutations and generating multiple mutants with the same selection marker in S. citri. It should also be relevant to other species of mollicutes.
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Influence of fusaric acid on phenazine-1-carboxamide synthesis and gene expression of Pseudomonas chlororaphis strain PCL1391
More LessProduction of the antifungal metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis strain PCL1391 is essential for the suppression of tomato foot and root rot caused by the soil-borne fungus F. oxysporum f. sp. radicis-lycopersici. The authors have shown previously that fusaric acid (FA), a phytotoxin produced by Fusarium oxysporum, represses the production of PCN and of the quorum-sensing signal N-hexanoyl-l-homoserine lactone (C6-HSL). Here they report that PCN repression by FA is maintained even during PCN-stimulating environmental conditions such as additional phenylalanine, additional ferric iron and a low Mg2+ concentration. Constitutive expression of phzI or phzR increases the production of C6-HSL and abolishes the repression of PCN production by FA. Transcriptome analysis using P. chlororaphis PCL1391 microarrays showed that FA represses expression of the phenazine biosynthetic operon (phzABCDEFGH) and of the quorum-sensing regulatory genes phzI and phzR. FA does not alter expression of the PCN regulators gacS, rpoS and psrA. In conclusion, reduction of PCN levels by FA is due to direct or indirect repression of phzR and phzI. Microarray analyses identified genes of which the expression is strongly influenced by FA. Genes highly upregulated by FA are also upregulated by iron starvation in Pseudomonas aeruginosa. This remarkable overlap in the expression profile suggests an overlapping stress response to FA and iron starvation.
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Volumes and issues
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