- Volume 153, Issue 11, 2007
Volume 153, Issue 11, 2007
- Cell And Developmental Biology
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Programmed cell death in Entamoeba histolytica induced by the aminoglycoside G418
This study presents morphological and biochemical evidence of programmed cell death (PCD) in Entamoeba histolytica induced by exposure of trophozoites to the aminoglycoside antibiotic G418. Morphological characteristics of PCD, including cell shrinkage, reduced cellular volume, nuclear condensation, DNA fragmentation and vacuolization were observed, with preservation of trophozoite membrane integrity. PCD is orchestrated biochemically by alterations in intracellular ion fluxes. In G418-treated trophozoites, overproduction of reactive oxygen species (ROS), decreased intracellular K+, increased cytosolic calcium, and decreased intracellular pH levels were observed. However, externalization of phosphatidylserine was not detected. These results suggest that amoebae can undergo PCD under stress conditions, and that this PCD shares several properties with PCD reported in mammals and in a variety of unicellular organisms.
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Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride
More LessLight is an environmental factor that regulates pivotal processes in living organisms, and appropriate perception is key to adaptation to the environment. Blue light activates asexual reproduction in Trichoderma atroviride through transcription factors BLR-1 and BLR-2 which regulate light-responsive genes. Here, we show that blr-2 expression is a limiting factor for photo-perception and photo-transduction. Overexpression of blr-2 resulted in increased photoconidiation and stronger expression of light-induced genes. In contrast, overexpression of blr-1 resulted in reduced photoconidiaton and weaker expression of light-induced genes. blr-2 overexpression caused a marked reduction of growth when the fungus was grown under defined photoperiods, including a period of strong sensitivity to light, followed by a period of insensitivity. Long periods of incubation under this condition permitted recovery of a rhythmic growth similar to that of the wild-type. In addition, blr-2 expression is apparently regulated at the post-transcriptional level through the BLR proteins and its expression level is BLR-1-dependent even in the dark. Finally, we demonstrated that blr-2 overexpression caused higher sensitivity to blue light and we therefore propose that the preformation of BLR-1/BLR-2 complexes is key to adequate light perception in T. atroviride.
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- Biochemistry And Molecular Biology
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Structure–function relationship of inducer peptide pheromones involved in bacteriocin production in Carnobacterium maltaromaticum and Enterococcus faecium
More LessThe production of several bacteriocins in lactic acid bacteria is regulated by inducer peptide pheromones that specifically interact with their cognate bacterial receptor. These peptide pheromones are between 19 and 27 aa long and contain a conserved (V/I)-X-X-X-F sequence followed by positively charged residues in the C-terminal domain. CbaX and EntF are peptide pheromones that share similarity and are involved in the production of carnobacteriocin A in Carnobacterium maltaromaticum LV17A and enterocins A and B in Enterococcus faecium CTC492, respectively. CbaX, EntF and two hybrids, CbaX : : EntF and EntF : : CbaX, were tested for pheromone activity in LV17A and CTC492. EntF and EntF : : CbaX only induced bacteriocin production in CTC492, whereas CbaX and CbaX : : EntF induced carnobacteriocin A production in LV17A and, at high concentrations, also cross-induced enterocin production in CTC492. Various peptide fragments of CbaX and EntF were made for further structure–function analysis. The C-terminal fragments, but not the N-terminal fragments, were able to effect bacteriocin induction. The 10-mer EntF(16–25), derived from the C-terminal domain of EntF, showed pheromone activity in LV17A. In contrast, the C-terminal 9-mer of CbaX, CbaX(16–24), inhibited pheromone activity in both LV17A and CTC492. EntF(16–25) and CbaX(16–24) differ by two amino acids. Changing either one of these abolished pheromone activity as well as the ability to inhibit pheromone activity. These results indicate that the C-terminal domain of these peptide pheromones interacts relatively non-specifically with the receptor, and that induction is greatly facilitated by the N-terminal domain that recognizes specifically its cognate receptor.
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Chronological and replicative life-span extension in Saccharomyces cerevisiae by increased dosage of alcohol dehydrogenase 1
Alcohol dehydrogenase 1 (Adh1)p catalyses the conversion of acetaldehyde to ethanol, regenerating NAD+. In Saccharomyces cerevisiae, Adh1p is oxidatively modified during ageing and, consequently, its activity becomes reduced. To analyse whether maintaining this activity is advantageous for the cell, a yeast strain with an extra copy of the ADH1 gene (2×ADH1) was constructed, and the effects on chronological and replicative ageing were analysed. The strain showed increased survival in stationary phase (chronological ageing) due to induction of antioxidant enzymes such as catalase and superoxide dismutases. In addition, 2×ADH1 cells displayed an increased activity of silent information regulator 2 (Sir2)p, an NAD+-dependent histone deacetylase, due to a higher NAD+/NADH ratio. As a consequence, a 30 % extension in replicative life span was observed. Taken together, these results suggest that the maintenance of enzymes that participate in NAD+/NADH balancing is important to chronological and replicative life-span parameters.
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New insights into the regulation of the Saccharomyces cerevisiae UGA4 gene: two parallel pathways participate in carbon-regulated transcription
More LessThe Saccharomyces cerevisiae UGA4 gene, which encodes the γ-aminobutyric acid (GABA) and δ-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation.
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Dynamic transcriptional changes in response to rehydration in Anabaena sp. PCC 7120
More LessGlobal transcriptional responses to dehydration and rehydration were determined in Anabaena sp. PCC 7120. Nearly 300 genes were up- or downregulated during both dehydration and rehydration. While as many as 133 genes showed dehydration-specific downregulation, only 29 genes showed dehydration-specific upregulation. In contrast, while only 13 genes showed rehydration-specific downregulation, as many as 259 genes showed rehydration-specific upregulation. The genes upregulated during rehydration responded rapidly and transiently, whereas those upregulated during dehydration did so gradually and persistently. The expression of various genes involved in DNA repair, protein folding and NAD synthesis, as well as genes responding to nitrogen depletion and CO2 limitation, was upregulated during rehydration. Although no genes for transcriptional regulators showed dehydration-specific upregulation, eight showed rehydration-specific upregulation. Among them, two genes, ancrpB and alr0618, encode putative transcriptional activators of the cAMP receptor protein (CRP) family. DNA microarray analysis using gene disruptants revealed that AnCrpB and Alr0618 regulate the genes induced by nitrogen depletion and by CO2 limitation, respectively. We conclude that rehydration is a complex process in which the expression of certain genes, particularly those for metabolism, is dramatically induced.
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- Biodiversity And Evolution
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Multilocus sequence typing (MLST) reveals high genetic diversity and clonal population structure of the toxic cyanobacterium Microcystis aeruginosa
More LessMicrocystis aeruginosa is one of the most prevalent bloom-forming cyanobacteria and has been the cause of increasing public health concern due to the production of hepatotoxins (microcystins). To investigate the genetic diversity, clonality and evolutionary genetic background with regard to the toxicity of M. aeruginosa, a multilocus sequence typing (MLST) scheme was developed, based on seven selected housekeeping loci (ftsZ, glnA, gltX, gyrB, pgi, recA and tpi). Analysis of a collection of 164 isolates from Japan and other countries identified 79 unique sequence types (STs), revealing a high level of genetic diversity (H=0.951). Although recombination between loci was indicated to be substantial by Shimodaira–Hasegawa (SH) tests, multilocus linkage disequilibrium analyses indicated that recombination between strains probably occurs at some frequency but not to the extent at which alleles are associated randomly, suggesting that the population structure of M. aeruginosa is clonal. Analysis of subsets of strains also indicated that the clonal population structure is maintained even in a local population. Phylogenetic analysis based on the concatenated sequences of seven MLST loci demonstrated that microcystin-producing genotypes are not monophyletic, providing further evidence for the gain and loss of toxicity during the intraspecific diversification of M. aeruginosa. However, toxic strains are genetically distinct from non-toxic strains in MLST allelic profiles, and it was also shown that non-toxic strains harbouring toxin genes fall into a single monophyletic clade, except for one case. These results suggest that the toxicity of M. aeruginosa is relatively stable in the short term, and therefore can be unequivocally characterized by MLST. The MLST scheme established here will be of great help for future detailed population genetic studies of M. aeruginosa.
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Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs
More LessIn this study, the Enterobacteriaceae microbiota, including their diversity as well as the distribution of haemolytic and virulence gene-harbouring Escherichia coli of 56-day-old healthy piglets, was characterized. Both the composition and the diversity of Enterobacteriaceae populations varied considerably between individual pigs and intestinal sections. E. coli, Enterobacter cloacae, Citrobacter freundii and Klebsiella pneumoniae dominated the Enterobacteriaceae microbiota. However, mucosa-associated Enterobacteriaceae were scarce or in some cases undetectable. The majority of E. coli clones from the jejunum were also found in the colon, with up to 10 different E. coli clones in one intestinal section. Other Enterobacteriaceae species were represented by only one clone localized to one intestinal section. While several piglets did not harbour virulence gene-positive or haemolytic E. coli, such strains dominated intestinal sections of other animals. This study reveals that the diversity of intestinal Enterobacteriaceae is clearly individual. In general, Enterobacteriaceae do not appear to be a consistent fraction of the microbiota of the jejunum. High numbers of adherent bacteria do not appear to be essential for successful intestinal colonization, and E. coli clones do not necessarily colonize distinct intestinal sections based on the particular phylogenetic affiliation. Furthermore, dominance of haemolytic or virulence gene-positive E. coli does not correlate with disease. Finally, probiotic Enterococcus faecium feed supplementation does not affect the Enterobacteriaceae microbiota.
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- Environmental Microbiology
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Ammonium ions inhibit nitrogen fixation but do not affect heterocyst frequency in the bloom-forming cyanobacterium Nodularia spumigena strain AV1
More LessIn the presence of ammonium ion, Nodularia spumigena strain AV1, a filamentous, heterocystous cyanobacterium isolated from the Baltic Sea, lost aerobic nitrogen-fixation activity while maintaining heterocyst frequency along the filaments. Real-time RT-PCR showed that the expression of nifH (encoding the dinitrogenase reductase component of the nitrogenase enzyme) was suppressed and the levels of NifH protein decreased dramatically in response to treatment with ammonium. On the other hand, ntcA (encoding the global nitrogen regulator in cyanobacteria) and hetR (the key regulatory gene in heterocyst differentiation) were expressed and their expression patterns were not affected by the treatment with ammonium. These data demonstrate that N. spumigena strain AV1 maintains heterocyst frequency along the filaments in the presence of ammonium and in the absence of detectable N2-fixation activity.
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Responses to arsenate stress by Comamonas sp. strain CNB-1 at genetic and proteomic levels
Comamonas sp. strain CNB-1, a chloronitrobenzene-degrading bacterium, was demonstrated to possess higher arsenate tolerance as compared with the mutant strain CNB-2. pCNB1, a plasmid harboured by CNB-1 but not CNB-2, contained the genetic cluster ars(RPBC)Com , which putatively encodes arsenate-resistance regulator, family II arsenate reductase, arsenite efflux pump and family I arsenate reductase, respectively, in Comamonas strain CNB-1. The arsC-negative Escherichia coli could gain arsenate resistance by transformation with arsPCom or arsCCom , indicating that these two genes might express functional forms of arsenate reductases. Intriguingly, when CNB-1 cells were exposed to arsenate, the transcription of arsPCom and arsCCom was measurable by RT-PCR, but only ArsP Com was detectable at protein level. To explore the proteins responding to arsenate stress, CNB-1 cells were cultured with and without arsenate and differential proteomics was carried out by two-dimensional PAGE (2-DE) and MALDI-TOF MS. A total of 31 differential 2-DE spots were defined upon image analysis and 23 proteins were identified to be responsive specifically to arsenate. Of these spots, 18 were unique proteins. These proteins were identified to be phosphate transporters, heat-shock proteins involved in protein refolding, and enzymes participating in carbon and energy metabolism.
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- Genes And Genomes
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A genome-wide survey of short coding sequences in streptococci
Identification of short genes that encode peptides of fewer than 60 aa is challenging, both experimentally and in silico. As a consequence, the universe of these short coding sequences (CDSs) remains largely unknown, although some are acknowledged to play important roles in cell–cell communication, particularly in Gram-positive bacteria. This paper reports a thorough search for short CDSs across streptococcal genomes. Our bioinformatic approach relied on a combination of advanced intrinsic and extrinsic methods. In the first step, intrinsic sequence information (nucleotide composition and presence of RBSs) served to identify new short putative CDSs (spCDSs) and to eliminate the differences between annotation policies. In the second step, pseudogene fragments and false predictions were filtered out. The last step consisted of screening the remaining spCDSs for lines of extrinsic evidence involving sequence and gene-context comparisons. A total of 789 spCDSs across 20 complete genomes (19 Streptococcus and one Enterococcus) received the support of at least one line of extrinsic evidence, which corresponds to an average of 20 short CDSs per million base pairs. Most of these had no known function, and a significant fraction (31 %) are not even annotated as hypothetical genes in GenBank records. As an illustration of the value of this list, we describe a new family of CDSs, encoding very short hydrophobic peptides (20–23 aa) situated just upstream of some of the positive transcriptional regulators of the Rgg family. The expression of seven other short CDSs from Streptococcus thermophilus CNRZ1066 that encode peptides ranging in length from 41 to 56 aa was confirmed by real-time quantitative RT-PCR and revealed a variety of expression patterns. Finally, one peptide from this list, encoded by a gene that is not annotated in GenBank, was identified in a cell-envelope-enriched fraction of S. thermophilus CNRZ1066.
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The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker
More LessA new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copper-potassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis- (repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG1363 and L. lactis IL1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/counterselection marker in many different organisms.
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Quantitative proteomics and transcriptomics of anaerobic and aerobic yeast cultures reveals post-transcriptional regulation of key cellular processes
Saccharomyces cerevisiae is unique among yeasts in its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of S. cerevisiae to anaerobiosis was investigated using metabolic stable-isotope labelling in aerobic and anaerobic glucose-limited chemostat cultures, followed by relative quantification of protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many of the responses of S. cerevisiae to oxygen availability were, to our knowledge, previously unreported. Comparison of transcriptomes and proteomes from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl-tRNA synthesis, purine nucleotide synthesis and amino acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights into the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the mRNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology.
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Direct evidence for redundant segmental replacement between multiple 18S rRNA genes in a single Prototheca strain
More LessInformational genes such as those encoding rRNAs are related to transcription and translation, and are thus considered to be rarely subject to lateral gene transfer (LGT) between different organisms, compared to operational genes having metabolic functions. However, several lines of evidence have suggested or confirmed the occurrence of LGT of DNA segments encoding evolutionarily variable regions of rRNA genes between different organisms. In the present paper, we show, for the first time to our knowledge, that variable regions of the 18S rRNA gene are segmentally replaced by multiple copies of different sequences in a single strain of the green microalga Prototheca wickerhamii, resulting in at least 17 genotypes, nine of which were actually transcribed. Recombination between different 18S rRNA genes occurred in seven out of eight variable regions (V1–V5 and V7–V9) of eukaryotic small subunit (SSU) rRNAs. While no recombination was observed in V1, one to three different recombination loci were demonstrated for the other regions. Such segmental replacement was also implicated for helix H37, which is defined as V6 of prokaryotic SSU rRNAs. Our observations provide direct evidence for redundant recombination of an informational gene, which encodes a component of mature ribosomes, in a single strain of one organism.
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Exploring the evolution of the Bacillus cereus group repeat element bcr1 by comparative genome analysis of closely related strains
More Lessbcr1 is a chromosomal ∼155 bp repeated element found uniquely and ubiquitously in the Bacillus cereus group of Gram-positive bacteria; it exhibits several features characteristic of mobile elements, including a variable distribution pattern between strains. Here, highly similar bcr1 elements in non-conserved genomic loci are identified in a set of closely related B. cereus and Bacillus thuringiensis strains near the Bacillus anthracis phylogenetic cluster. It is also shown that bcr1 may be present on small RNA transcripts in the 100–400 bp size range. In silico folding of bcr1 at the RNA level indicated that transcripts may form a double-hairpin-like structure predicted to have high structural stability. A functional role of bcr1 at the RNA level is supported by multiple cases of G–U base-pairing, and compensatory mutations maintaining structural stability of the RNA fold. In silico folding at the DNA level produced similar predicted structures, with the potential to form a cruciform structure at open DNA complexes. The predicted structural stability was greater for bcr1 elements showing high sequence identities to bcr1 elements in non-conserved chromosomal loci in other strains, relative to other bcr1 copies. bcr1 mobility could thus be dependent on the formation of a stable DNA or RNA intermediate. Furthermore, bcr1 elements potentially encoding structurally stable and less stable transcripts were phylogenetically intermixed, indicating that loss of bcr1 mobility may have occurred multiple times during evolution. Repeated elements with similar features in other bacteria have been shown to provide functions such as mRNA stabilization, transcription termination and/or promoter function. Similarly, bcr1 may constitute a mobile element which occasionally gains a function when it enters an appropriate chromosomal locus.
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- Pathogens And Pathogenicity
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Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities
More LessHelicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, possesses a hydrogenase and a urease, both of which are nickel-containing enzymes. Analysis of the genome sequence of H. hepaticus revealed a full set of accessory genes which are required for the nickel maturation of each enzyme in other micro-organisms. Erythromycin-resistant mutants were constructed in four of these genes, hypA, hypB, ureE and ureG. Controls for polar effect were provided for hypA or hypB mutants by disrupting each gene located immediately downstream, i.e. hp0809 or hypC, respectively. Urease and hydrogenase activities were determined for each strain with or without supplemented nickel in the medium. As expected, the ureE and the ureG mutants had negligible urease activity, but they retained normal levels of hydrogenase activity. Urease levels could not be increased by the addition of nickel to the medium. The H. hepaticus hypA and hypB strains were deficient in both urease and hydrogenase activities, suggesting that both gene products act in a similar fashion as their counterparts in H. pylori. However, in contrast with the analogous mutants of H. pylori, the addition of nickel into the growth medium failed to restore either urease or hydrogenase enzyme levels in the H. hepaticus hypA or hypB mutants, indicating a probably unique role for these genes in the mouse liver pathogen.
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Legionella pneumophila exhibits plasminogen activator activity
More LessBased on their localization at the boundary of the bacterial cell and its environment, outer-membrane proteins (Omps) are important determinants for interaction of bacteria with their host cell. Therefore, they can be considered as important determinants for virulence. Looking for Legionella pneumophila Omps potentially involved in virulence, we identified a gene encoding a homologue of the plasminogen activator (Pla) of Yersinia pestis. Pla belongs to the class of omptins, a family of surface proteases/adhesins that exhibit different virulence-associated functions. In this report we describe the cloning and identification of the plasminogen activator homologue Lpa of L. pneumophila and demonstrate its outer-membrane localization. Transcriptional analysis of the Lpa region revealed expression of the gene in both exponential and stationary growth phase and showed that transcription of the lpa gene is directed by its own promoter. We also show, to our knowledge for the first time, that L. pneumophila has the capacity to convert plasminogen into plasmin by the action of the outer-membrane Lpa protein.
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The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens
More LessThe Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) mediates the transfer of single-stranded DNA and protein virulence factors into plant cells, and also determines the assembly of the T-pilus, which is believed to play a role in host recognition. The T-pilus is composed of the major component VirB2 and the minor component VirB5. Using immuno-electron microscopy we detected the major component VirB2 along the entire length of detached T-pili, but not on cell-bound T-pili or on the cell surface. In contrast, the minor T-pilus component VirB5 was detected on the tips of cell-bound T-pili as well as on the ends of detached T-pili and on the cell surface. To gain further insights into the role of VirB5 we introduced changes at its C terminus. C-terminal deletions of up to four amino acids and alanine replacements did not abolish T-pilus formation and incorporation of the VirB5 variants at the tip, although they did impact the length of T-pili. Also, these changes differentially affected the ability of the T4SS to transfer DNA into plant and bacterial recipients, suggesting differential effects on host-cell specificity. The data presented here suggest that VirB5 localizes at the T-pilus tip, and provide novel insights into its role during the type IV secretion process.
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Biochemical characterization of the enterotoxigenic Escherichia coli LeoA protein
More LessEnterotoxigenic Escherichia coli (ETEC) causes enterotoxin-induced diarrhoea and significant mortality. The molecular mechanisms underlying how the heat-labile enterotoxin (LT) is secreted during infection are poorly understood. ETEC produce outer-membrane vesicles (OMVs) containing LT that are endocytosed into host cells. Although OMV production and protein content may be a regulated component of ETEC pathogenesis, how LT loading into OMVs is regulated is unknown. The LeoA protein plays a role in secreting LT from the bacterial periplasm. To begin to understand the function of LeoA and its role in ETEC H10407 pathogenesis, a site-directed mutant lacking the putative GTP-binding domain was constructed. The ability of wild-type and mutant LeoA to hydrolyse GTP in vitro was quantified. This domain was found to be responsible for GTP binding; it is important to LeoA's function in LT secretion, and may play a modest role in the formation and protein content of OMVs. Deletion of leoA reduced the abundance of OmpX in outer-membrane protein preparations and of LT in OMVs. Immunoprecipitation experiments revealed that LeoA interacts directly with OmpA, but that the GTP-binding domain is non-essential for this interaction. Deletion of leoA rendered ETEC H10407 non-motile, through apparent periplasmic accumulation of FliC.
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Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to hydrogen peroxide
More LessMycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia, colonizes the cilia of swine lungs, causing ciliostasis and cell death. M. hyopneumoniae is a component of the porcine respiratory disease complex (PRDC) and is especially problematic for the finishing swine industry, causing the loss of hundreds of millions of dollars in farm revenues worldwide. For successful infection, M. hyopneumoniae must effectively resist oxidative stresses due to the release of oxidative compounds from neutrophils and macrophages during the host's immune response. However, the mechanism that M. hyopneumoniae uses to avert the host response is still unclear. To gain a better understanding of the transcriptional responses of M. hyopneumoniae under oxidative stress, cultures were grown to early exponential phase and exposed to 0.5 % hydrogen peroxide for 15 min. RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. This study revealed significant downregulation of important glycolytic pathway genes and gene transcription proteins, as well as a protein known to activate oxidative stressor cascades in neutrophils. Sixty-nine per cent of the upregulated genes were hypothetical with no known function. This study has also revealed significantly differentially expressed genes common to other environmental stress responses, indicating that further investigation of universal stress response genes of M. hyopneumoniae is merited.
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Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells
More LessThe human pathogen Mycoplasma pneumoniae can cause atypical pneumonia through adherence to epithelial cells in the respiratory tract. The major immunogenic protein, P1, participates in the attachment of the bacteria to the host cells. To investigate the adhesion properties of P1, a recombinant protein (rP1-II) covering amino acids 1107–1518 of the P1 protein was produced. This protein inhibited the adhesion of M. pneumoniae to human HEp-2 cells, as visualized in a competitive-binding assay using immunofluorescence microscopy. Previous studies have shown that mAbs that recognize two epitopes in this region of P1 also reduce M. pneumoniae adhesion. Therefore, peptides covering these epitopes, of 8 and 13 aa, respectively, were synthesized to further investigate the adhesion region. None of these synthetic peptides reduced the binding of M. pneumoniae to the receptors on the host cells. Instead, 10 overlapping synthetic peptides covering the whole of rP1-II were evaluated in the competitive-binding assay using immunofluorescence microscopy. A reduction in the number of M. pneumoniae microcolonies was seen, which was confirmed for five peptides using a POLARstar OPTIMA reader to measure fluorescence intensity. The number of M. pneumoniae microcolonies adhering to the host cells was significantly reduced by these five peptides. Further investigations showed that inhibiting peptide 7 (amino acids 1347–1396) of the major adhesin protein P1 bound directly to host receptors, suggesting that the observed M. pneumoniae-inhibiting peptides occupied HEp-2 receptors, which are otherwise available for P1-mediated M. pneumoniae adhesion.
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Protein FOG is a moderate inducer of MIG/CXCL9, and group G streptococci are more tolerant than group A streptococci to this chemokine's antibacterial effect
More LessStreptococcus dysgalactiae subsp. equisimilis (group G streptococci; GGS) cause disease in humans but are often regarded as commensals in comparison with Streptococcus pyogenes (group A streptococci; GAS). The current study investigated the degree and kinetics of the innate immune response elicited by the two species. This was assessed as expression of the chemokine MIG/CXCL9 and bacterial susceptibility to its bactericidal effect. No significant difference in MIG/CXCL9 expression from THP-1 or Detroit 562 cells was observed when comparing whole GGS or GAS as stimuli. The study demonstrates that protein FOG was released from the bacterial surface directly and by neutrophil elastase. Expression of MIG/CXCL9 following stimulation with soluble M proteins of the two species (the recently described protein FOG of GGS and protein M1 of GAS) was reduced for protein FOG in both the monocytic and the epithelial cell line. When the antibacterial effects of MIG/CXCL9 were examined in conditions of increased ionic strength, MIG/CXCL9 killed GAS more efficiently than GGS. Also in the absence of MIG/CXCL9, GGS were more tolerant to increased salt concentrations than GAS. In summary, both GGS and GAS evoke MIG/CXCL9 expression but they differ in susceptibility to its antibacterial effects. This may in part explain the success of GGS as a commensal and its potential as a pathogen.
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Uncultivated Tannerella BU045 and BU063 are slim segmented filamentous rods of high prevalence but low abundance in inflammatory disease-associated dental plaques
More LessUncultivated clones BU045 and BU063 and Tannerella forsythia, a ‘consensus periodontal pathogen’, are the closest known relatives within the genus Tannerella. They have been described to inhabit different ecological niches of the human oral cavity. In this study, fluorescent in situ hybridization (FISH) and immunofluorescence were combined to investigate the prevalence and abundance of BU045 and BU063 in comparison to T. forsythia in plaques from gingivitis, necrotizing ulcerative gingivitis (NUG) and chronic periodontitis. Phylotype-specific FISH probes identified BU045 and BU063 as elongated thin rods with a segmented structure. Two structurally similar and previously unknown, rare phylotypes (127+ and 997+) were also identified due to partial 16S rRNA sequence identity with T. forsythia. In gingivitis, NUG and periodontitis patients, BU045, BU063, 127+, 997+ and T. forsythia were detected with prevalences of 50/83/71/14 and 81 %, 100/100/86/17 and 53 %, and 100/100/12/0 and 100 %, respectively. Supragingivally, colonization density of all five organisms was generally low, rarely exceeding 0.1 % of the total biota. In periodontal pocket samples, however, cell numbers of T. forsythia, but not of the uncultivable phylotypes, were greatly elevated. Our data demonstrate that Tannerella phylotypes BU045, BU063, 127+ and 997+ consist of long slim rods with segments, which, with respect to FISH stainability, often behaved as independent units. The phylotypes are frequent but low-level colonizers of various periodontal disease-associated plaques. Their apparent inability to proliferate to high density seems to exclude any relevance for the pathogenesis of periodontal diseases.
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Expression of Legionella pneumophila paralogous lipid A biosynthesis genes under different growth conditions
Legionella pneumophila is an opportunistic pathogen that in the environment colonizes biofilms and replicates within amoebae. The bacteria employ the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to grow intracellularly in a specific vacuole. Using Acanthamoeba castellanii as a host cell, we have previously identified lcsC (Legionella cytotoxic suppressor), a paralogue of the lipid A disaccharide synthase lpxB, as a cytotoxic factor of L. pneumophila. A bioinformatic analysis of the genome revealed that L. pneumophila is unique in harbouring two paralogues of lpxB and two and three paralogues of the lipid A biosynthesis acyltransferases lpxA and lpxD, respectively. LcsC (lpxB1) forms a transcriptional unit with gnnA, encoding a putative UDP-GlcNAc oxidase in the biosynthetic pathway leading to 3-aminoglucosamine analogues of lipid A. LpxB2 clusters with lpxD2, lpxA2 and lpxL paralogues, encoding secondary acyltransferases. LcsC/lpxB1 and lpxB2 were found to partially complement the growth defect of an Escherichia coli lpxB conditional mutant strain, indicating that both corresponding enzymes possess lipid A disaccharide synthase activity. The two L. pneumophila lpxB paralogues are not functionally equivalent, since expression of lcsC/lpxB1 but not lpxB2 in an L. pneumophila icmG mutant is cytotoxic for A. castellanii, and LPS purified from the two strains triggers CD14-dependent tumour necrosis factor (TNF)α production by macrophages with a different potency. The lpxB and lpxA paralogues are expressed under various growth conditions, including broth, biofilms and in A. castellanii. While the flagellar gene flaA is mainly expressed in late stationary phase, the lpxB and lpxA paralogues are preferentially expressed in the exponential and early stationary phases. Upon exposure to hypotonic stress and nutrient deprivation, lpxA1, and to a lesser extent lcsC/lpxB1, is upregulated. The differential regulation of lpxB or lpxA paralogues in response to changing environmental conditions might allow L. pneumophila to adapt its lipid A structure.
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- Physiology
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Propionate inactivation of butane monooxygenase activity in ‘Pseudomonas butanovora’: biochemical and physiological implications
More LessButane monooxygenase (BMO) catalyses the oxidation of alkanes to alcohols in the alkane-utilizing bacterium ‘Pseudomonas butanovora’. Incubation of alkane-grown ‘P. butanovora’ with butyrate or propionate led to irreversible time- and O2-dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane. Although both the time and O2 dependency of propionate inactivation of BMO imply that propionate might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14C labelling of BMO polypeptides by [2-14C]propionate during inactivation. Propionate-dependent BMO inactivation was also explored in mutant strains of ‘P. butanovora’ containing single amino acid substitutions in the α-subunit of the BMO hydroxylase. Propionate-dependent BMO inactivation in two mutant strains with amino acid substitutions close to the catalytic site differed from wild-type (one was more sensitive and the other less), providing further evidence that propionate-dependent inactivation involves interaction with the BMO catalytic site. A putative model is presented that might explain propionate-dependent inactivation of BMO when framed within the context of the catalytic cycle of the closely related enzyme, soluble methane monooxygenase.
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Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2
More LessThis study was designed to characterize naphthalene metabolism in Polaromonas naphthalenivorans CJ2. Comparisons were completed using two archetypal naphthalene-degrading bacteria: Pseudomonas putida NCIB 9816-4 and Ralstonia sp. strain U2, representative of the catechol and gentisate pathways, respectively. Strain CJ2 carries naphthalene catabolic genes that are homologous to those in Ralstonia sp. strain U2. Here we show that strain CJ2 metabolizes naphthalene via gentisate using respirometry, metabolite detection by GC-MS and cell-free enzyme assays. Unlike P. putida NCIB 9816-4 or Ralstonia sp. strain U2, strain CJ2 did not grow in minimal medium saturated with naphthalene. Growth assays revealed that strain CJ2 is inhibited by naphthalene concentrations of 78 μM (10 p.p.m.) and higher, and the inhibition of growth is accompanied by the accumulation of orange-coloured, putative naphthalene metabolites in the culture medium. Loss of cell viability coincided with the appearance of the coloured metabolites, and analysis by HPLC suggested that the accumulated metabolites were 1,2-naphthoquinone and its unstable auto-oxidation products. The naphthoquinone breakdown products accumulated in inhibited, but not uninhibited, cultures of strain CJ2. Furthermore, naphthalene itself was shown to directly inhibit growth of a regulatory mutant of strain CJ2 that is unable to metabolize naphthalene. These results suggest that, despite being able to use naphthalene as a carbon and energy source, strain CJ2 must balance naphthalene utilization against two types of toxicity.
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Role of individual nap gene cluster products in NapC-independent nitrate respiration of Wolinella succinogenes
More LessBacterial nap gene clusters, encoding periplasmic nitrate reductase (NapA), are complex and diverse, and the composition of the electron transport chain donating electrons to NapA is poorly characterized in most organisms. Exceptionally, Wolinella succinogenes transfers electrons from formate via the menaquinone pool to NapA independently of a membrane-bound c-type cytochrome of the NapC family. The role of individual ORFs of the W. succinogenes napAGHBFLD gene cluster is assessed here by characterizing in-frame gene inactivation mutants. The ability of the mutants to grow by nitrate respiration was tested and their NapA content and specific nitrate reductase activity were determined. The napB and napD gene products proved to be essential for nitrate respiration, with NapD being required for the production of mature NapA. Inactivation of either subunit of the putative membrane-bound menaquinol dehydrogenase complex NapGH almost abolished growth by nitrate respiration. Substitution of the twin-arginine sequence of NapG had the same effect as absence of NapG. Phenotypes of mutants lacking either NapF or NapL suggest that both proteins function in NapA assembly and/or export. The data substantiate the current model of the composition of the NapC-independent electron transport chain as well as of NapA maturation, and indicate the presence of an alternative electron transport pathway to NapA.
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Strain-specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium
More LessPseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on mucous plugs in the lungs of cystic fibrosis (CF) patients, resulting in chronic infections. Pulmonary P. aeruginosa isolates often display a mucoid (alginate-producing) phenotype, whereas non-mucoid strains are generally associated with acute infections. We characterized the cytosolic proteomes of biofilm-associated and planktonic forms of a CF pulmonary isolate, P. aeruginosa FRD1, and a non-mucoid strain, PAO1. Since Ca2+ metabolism is altered in CF pulmonary fluids, we also analysed the effect of Ca2+ on the proteome responses of these strains. Both strains altered the abundances of 40–60 % of their proteins in response to biofilm growth and/or [Ca2+]. Differentially expressed proteins clustered into 12 groups, based on their abundance profiles. From these clusters, 146 proteins were identified by using MALDI-TOF/TOF mass spectrometry. Similarities as well as strain-specific differences were observed. Both strains altered the production of proteins involved in iron acquisition, pyocyanin biosynthesis, quinolone signalling and nitrogen metabolism, proteases, and proteins involved in oxidative and general stress responses. Individual proteins from these classes were highly represented in the biofilm proteomes of both strains. Strain-specific differences concerned the proteins within these functional groups, particularly for enzymes involved in iron acquisition and polysaccharide metabolism, and proteases. The results demonstrate that a mucoid CF isolate of P. aeruginosa responds to biofilm-associated growth and [Ca2+] in a fashion similar to strain PAO1, but that strain-specific differences may allow this CF isolate to successfully colonize the pulmonary environment.
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- Theoretical Microbiology
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Senescence can explain microbial persistence
More LessIt has been known for many years that small fractions of persister cells resist killing in many bacterial colony–antimicrobial confrontations. These persisters are not believed to be mutants. Rather it has been hypothesized that they are phenotypic variants. Current models allow cells to switch in and out of the persister phenotype. Here, a different explanation is suggested for persistence, namely senescence. Using a mathematical model including age structure, it is shown that senescence provides a natural explanation for persistence-related phenomena, including the observations that the persister fraction depends on growth phase in batch culture and dilution rate in continuous culture.
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