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Volume 149,
Issue 2,
2003
Volume 149, Issue 2, 2003
- Reviews
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Prokaryotic motility structures
More LessProkaryotes use a wide variety of structures to facilitate motility. The majority of research to date has focused on swimming motility and the molecular architecture of the bacterial flagellum. While intriguing questions remain, especially concerning the specialized export system involved in flagellum assembly, for the most part the structural components and their location within the flagellum and function are now known. The same cannot be said of the other apparati including archaeal flagella, type IV pili, the junctional pore, ratchet structure and the contractile cytoskeleton used by a variety of organisms for motility. In these cases, many of the structural components have yet to be identified and the mechanism of action that results in motility is often still poorly understood. Research on the bacterial flagellum has greatly aided our understanding of not only motility but also protein secretion and genetic regulation systems. Continued study and understanding of all prokaryotic motility structures will provide a wealth of knowledge that is sure to extend beyond the bounds of prokaryotic movement.
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- Sgm Special Lecture
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Are dental diseases examples of ecological catastrophes?
More LessDental diseases are among the most prevalent and costly diseases affecting industrialized societies, and yet are highly preventable. The microflora of dental plaque biofilms from diseased sites is distinct from that found in health, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift towards community dominance by acidogenic and acid-tolerant Gram-positive bacteria (e.g. mutans streptococci and lactobacilli) at the expense of the acid-sensitive species associated with sound enamel. In contrast, the numbers and proportions of obligately anaerobic bacteria, including Gram-negative proteolytic species, increase in periodontal diseases. Modelling studies using defined consortia of oral bacteria grown in planktonic and biofilm systems have been undertaken to identify environmental factors responsible for driving these deleterious shifts in the plaque microflora. Repeated conditions of low pH (rather than sugar availability per se) selected for mutans streptococci and lactobacilli, while the introduction of novel host proteins and glycoproteins (as occurs during the inflammatory response to plaque), and the concomitant rise in local pH, enriched for Gram-negative anaerobic and asaccharolytic species. These studies emphasized (a) significant properties of dental plaque as both a biofilm and a microbial community, and (b) the dynamic relationship existing between the environment and the composition of the oral microflora. This research resulted in a novel hypothesis (the ‘ecological plaque hypothesis’) to better describe the relationship between plaque bacteria and the host in health and disease. Implicit in this hypothesis is the concept that disease can be prevented not only by directly inhibiting the putative pathogens, but also by interfering with the environmental factors driving the selection and enrichment of these bacteria. Thus, a more holistic approach can be taken in disease control and management strategies.
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- Cell And Developmental Biology
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Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB
More LessThe Dictyostelium spore is surrounded by a 220 μm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.
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- Biochemistry And Molecular Biology
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Cell-type-dependent repression of yeast a-specific genes requires Itc1p, a subunit of the Isw2p–Itc1p chromatin remodelling complex
More LessIn Saccharomyces cerevisiae MAT a haploid cells, the a-specific genes are expressed, whereas in the MATα haploid and MAT a/α diploid cell types their transcription is repressed. It is shown in this report that Itc1p, a component of the ATP-dependent Isw2p–Itc1p chromatin remodelling complex, is required for the repression of a-specific genes. It has previously been reported that disruption of the ITC1 gene leads, in MATα cells, to an aberrant cell morphology resembling the polarized mating projection of cells responding to pheromone. The activation of the pheromone signalling pathway in itc1 mutants of both mating types was examined and found to be constitutively active in MATα itc1 but not in MAT a itc1 cells. Furthermore, unlike the wild-type, MATα itc1 and MAT a/α itc1/itc1 cells secrete a-factor and express significant levels of other a-specific genes. The results indicate that the inappropriate a-factor production in a MATα context, due to the derepression of the a-specific genes, produces an autocrine signalling loop that leads to the aberrant morphology displayed by MATα itc1 cells. It is suggested that the Isw2p–Itc1p complex contributes to maintain the repressive chromatin structure described for the asg operator present in the promoters of a-specific genes.
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Oxidative-stress-inducible qorA encodes an NADPH-dependent quinone oxidoreductase catalysing a one-electron reduction in Staphylococcus aureus
More LessThis work characterized the putative quinone oxidoreductase gene (qorA) from Staphylococcus aureus. The deduced amino acid sequence indicated that the 333 aa protein contains an NAD(P)H-binding motif. A Northern blot analysis revealed that 2·6 kb and 1·4 kb signals were detected by using a qorA probe. Both the signals were enhanced under the presence of a redox-cycling agent, 9,10-phenanthrenequinone (PQ). It was also revealed that the expression of three genes, SA1988, SA1989 (qorA) and SA1990, was enhanced at the transcriptional level by PQ exposure. The results suggested that the 2·6 kb signal detected by the qorA probe was in two co-transcripts, i.e. SA1990–qorA and qorA–SA1988 were transcribed. Besides, primer extension analyses confirmed the enhancement of qorA and SA1990 transcripts. The GST (glutathione S-transferase)-tagged QorA protein was expressed in Escherichia coli and purified using a glutathione affinity column. In purification steps, a 36 kDa band co-purified with the GST–QorA, and it was detected even in the thrombin-cleaved fraction. N-terminal amino acid sequences for the 36 kDa protein revealed that it was an intact QorA. They showed that QorA formed a multimer under physiological conditions. The purified recombinant GST–QorA catalysed NADPH consumption in the presence of PQ as a substrate, but not NADH. To characterize the catalytic activity of QorA, superoxide anion that was generated through one-electron reduction of PQ and hydroquinone that was produced by two-electron reduction of PQ were measured. During reduction of PQ by GST–QorA, superoxide anion was generated, whereas a small amount of 9,10-dihydroxyphenanthrene (hydroquinone of PQ) was produced. These results suggest that the activity of QorA is similar to ζ-Crystallin, catalysing an NADPH-dependent one-electron reduction of quinone.
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An amino acid change near the carboxyl terminus of the Streptococcus gordonii regulatory protein Rgg affects its abilities to bind DNA and influence expression of the glucosyltransferase gene gtfG
More LessThe Streptococcus gordonii glucosyltransferase structural gene, gtfG, is located immediately downstream from its positive transcriptional regulatory determinant, rgg. Recent genetic studies have indicated that the 3′ end of rgg is involved either directly as a binding site or indirectly, e.g. by playing a role in secondary structure, in the interaction of Rgg with the gtfG promoter. A previously identified spontaneous mutant with a point mutation near the 3′ end of rgg had only ∼25 % of the parental level of glucosyltransferase activity. To determine if this decreased activity was due to a change in the DNA binding site of trans-acting Rgg, or due to a change in the Rgg protein itself, complementation analyses and DNA-binding studies were performed. In Rgg-deficient strains, the chromosomal rgg point mutation did not influence the ability of plasmid-borne rgg to increase glucosyltransferase expression. However, plasmids carrying parental rgg were able to increase glucosyltransferase activity and expression of a gtfG promoter fusion to a greater extent than plasmids carrying the mutant allele, indicating that the mutant Rgg protein had decreased activity. The ability of NH2-terminal (hexahistidine) tagged proteins to bind to a 107 bp dsDNA fragment corresponding to the region immediately upstream of gtfG was demonstrated by surface plasmon resonance. Despite their differences in activity, both mutant and parental recombinant Rgg proteins bound to this dsDNA, albeit with different strengths. These studies provide insights into functional domains of S. gordonii Rgg which influence glucosyltransferase expression, and may have implications for Rgg-like regulatory proteins in related bacteria.
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Role of the Streptococcus agalactiae ClpP serine protease in heat-induced stress defence and growth arrest
The main causes of microbial death after heat exposure are not well understood. Here, it is shown that the heat-shock protein ClpP plays a major role in heat-induced growth arrest in Streptococcus agalactiae. A mutant lacking the ClpP protease was more sensitive to the inhibitory effects of heat, salt and oxidative stress than the isogenic wild-type strain. During growth arrest, this mutant displayed important modifications of its total protein content, including a decreased level of essential metabolic enzymes such as the alcohol dehydrogenase. Analysis of protein carbonylation demonstrated that the ClpP protease plays a role in preventing accelerated protein oxidation. Higher levels of oxidized DnaK, a key modulator of the heat-shock regulon, were observed in the ClpP mutant and these were increased following heat shock. Accumulation of oxidized/inactivated DnaK might explain why the ClpP mutant was unable to properly synthesize DNA and proteins, and why it exhibited an aberrant cell morphology. Even though ClpP plays a minor role in the virulence of S. agalactiae in a murine infection model, the data presented here point to the importance of ClpP in oxidative stress defence in preventing heat-induced cell alterations.
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Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp. strain 12S
Methylobacillus sp. strain 12S produces an exopolysaccharide (EPS), methanolan, composed of glucose, mannose and galactose. Twenty-four ORFs flanking a Tn5 insertion site in an EPS-deficient mutant were identified, and 21 genes (epsCBAKLDEFGHIJMNOPQRSTU) were predicted to participate in methanolan synthesis on the basis of the features of the primary sequence. Gene disruption analyses revealed that epsABCEFGIJNOP and epsR are required for methanolan synthesis, whereas epsKD and epsH are not essential. EpsFG and EpsE showed homology with Wzc (chain length regulator) and Wza (export protein) of group 1 capsule-producing Escherichia coli, suggesting that methanolan was synthesized via a Wzy-like biosynthesis system. This possibility was supported by the fact that the putative hydropathy profiles of EpsH and EpsM were similar to those of Wzx and Wzy, which are also involved in the flipping of the repeating unit in the cytoplasmic membrane and the polymerization of the capsule in the Wzy-dependent system. EpsBJNOP and EpsR are probably glycosyltransferases involved in the synthesis of the repeating unit onto the lipid carrier. In particular, EpsB appeared to catalyse the initial transfer of the glucose moiety. On the basis of their predicted location in the cells, it is proposed that EpsI and EpsL are involved in methanolan export to the cell surface. E. coli strains expressing EpsQ, EpsS and EpsT showed enhanced activities of GDP-mannose pyrophosphorylase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase, respectively, revealing that they were responsible for the production of the activated compositional sugars of methanolan. EpsU contains a conserved a lytic transglycosylase motif, indicating that it could participate in the degradation of polysaccharides. EpsA and EpsK, which have conserved DNA-binding and cAMP-binding motifs, respectively, were deduced to be transcriptional regulators. In particular, EpsA seems to positively regulate the transcription of methanolan synthesis genes, since the constitutive expression of epsA in strain 12S increased the EPS production. Interestingly, EpsD showed homology with peptidyl prolyl cis–trans isomerases that catalyse the folding of proteins following translocation across the cytoplasmic membrane.
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Catalytic properties of an endogenous β-lactamase responsible for the resistance of Azospirillum lipoferum to β-lactam antibiotics
More LessAzospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins. 6-β-Bromopenicillanic acid, an irreversible inhibitor of serine-β-lactamases, completely abolished this resistance. A β-lactamase was purified 518-fold from a cell-free extract of A. lipoferum RG20. A single band on SDS-PAGE (apparent molecular mass 31 000 Da) and on isoelectric focussing (pI9·35) was observed with the purified protein. The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable k cat values and catalytic efficiencies. However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower. The purified β-lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam. Its substrate and inhibitor profiles are consistent with those of the broad-spectrum β-lactamases inhibited by clavulanic acid (group 2b of the Bush–Jacoby–Medeiros scheme). The effect of pH on k cat and K m values for benzylpenicillin hydrolysis was studied. The dependence of k cat on pH suggests that the enzyme–substrate (ES) complex must be in at least three protonation states: two with k cat values equal to 2800 and 1450 s−1 and a third inactive one [pK 1(ES) 4·7 and pK 2(ES) 7·9]. Similarly, the dependence of k cat/K m on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with k cat/K m values equal to 2·7×106 and 3·7×108 M−1 s−1 and a third one unable to productively bind substrate. Interestingly, the dependence of k cat/K m on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK 1(E) 8·5 and pK 2(E) 7·2].
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Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics
More LessThe albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30 %, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182T to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His78, His125, His141 and His189) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240–244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His125 might be the residue required for albicidin binding. Mutation of His125 to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His125 totally abolished binding activity. Mutation of His125 to arginine and tyrosine had no effect. These results indicate that His125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.
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- Biodiversity And Evolution
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Quantitative variation of biofilms among strains in natural populations of Candida albicans
More LessThis study examined the quantitative variation of biofilm formation and its relationship to multilocus genotypes in 115 strains of the human pathogenic fungus Candida albicans. These strains were isolated from three sources: 47 from oral cavities of healthy volunteers, 31 from the environment and 37 from the vaginas of patients with candidiasis. For each strain, biofilm formation was quantified as the ability to adhere to and grow on polystyrene plastic surfaces. Confocal laser scanning microscopy was used to visualize and confirm biofilm formation. Two methods were used to quantify biofilm formation abilities: (i) the XTT reduction assay, and (ii) absorbance following staining by crystal violet dye. Results obtained by the two methods were significantly correlated. Furthermore, biofilm formation ability was positively correlated with cell surface hydrophobicity. The analyses indicated that strains from each of the three sources varied widely in biofilm formation abilities. However, little correlation was observed between biofilm formation and multilocus genotypes as determined by PCR-RFLP at 16 polymorphic loci, regardless of source of strain. Strains with the same or similar multilocus genotypes often showed very different biofilm formation abilities. The results demonstrated that natural clones and clonal lineages of C. albicans exhibited extensive quantitative variation in biofilm formation.
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- Environmental Microbiology
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Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community
The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 16S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16S rDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like ‘Dehalococcoides ethenogenes’ or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56·6 %) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18·5 %), the Chloroflexi phylum (16·4 %), the Bacteroidetes phylum (6·3 %), the Synergistes genus (1·1 %) and a lineage that could not be affiliated with existing phyla (1·1 %). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured micro-organisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.
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Quorum-sensing-directed protein expression in Serratia proteamaculans B5a
N-Acyl-l-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.
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- Genes And Genomes
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Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus
More LessThioredoxins are small ubiquitous proteins that display different functions mainly via redox-mediated processes. The facultatively photosynthetic bacterium Rhodobacter capsulatus harbours at least two genes for thioredoxin 1 and 2, trxA and trxC. It is demonstrated that thioredoxin 2 of R. capsulatus can partially replace the thioredoxin 1 function as a hydrogen donor for methionine sulfoxide reductase but cannot replace thioredoxin 1 as a subunit of phage T7 DNA polymerase. By inactivating the trxC gene in R. capsulatus, it is shown that thioredoxin 2 is involved in resistance against oxidative stress. As thioredoxin 1 of Rhodobacter sphaeroides, R. capsulatus thioredoxin 2 affects the oxygen-dependent expression of photosynthesis genes, albeit in an opposite way. The trxC mutant of R. capsulatus shows a stronger increase in photosynthesis gene expression after a decrease in oxygen tension than the isogenic wild-type strain. The expression of the trxC gene is downregulated by oxygen.
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Spontaneous sequence duplications within capsule genes cap8E and tts control phase variation in Streptococcus pneumoniae serotypes 8 and 37
More LessCapsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes – cap8E (type 8) and tts (type 37) – were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF–ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [ Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001) . Mol Microbiol 42, 1223–1232]. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates.
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Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence
More LessPlasmid SCP2* is a 31 kb, circular, low-copy-number plasmid originally identified in Streptomyces coelicolor A3(2) as a fertility factor. The plasmid was completely sequenced. The analysis of the 31 317 bp sequence revealed 34 ORFs encoding putative proteins from 31 to 710 aa long, most of them lacking similarity to known proteins. Three functional regions had been identified previously: the replication region, the transfer and spreading region, and the stability region. Three genes were identified in the stability region which contribute to the stability of SCP2 as shown by plasmid stability testing. The first gene, mrpA, encodes a new member of the λ integrase family of site-specific recombinases. The two genes downstream of mrpA were called parA and parB. The gene product, ParA, shows similarity to a family of ATPases involved in plasmid partition. An increase of plasmid stability could be seen only when both genes were present. By deletion analysis, the replication region could be narrowed down to a 1·6 kb region, consisting of a 650 bp non-coding region and two genes, repI and repII, encoding proteins of 161 and 131 aa. Only RepI exhibits similarities to DNA binding elements and contains a putative helix–turn–helix motif. The traA gene that is essential for DNA transfer and pock formation was identified previously. Upstream of traA, 10 ORFs were found in the same orientation as traA which might be involved in conjugation and DNA spreading, together with one gene in the opposite orientation with similarities to transcriptional regulators of DNA transfer. Two transposable elements were found on SCP2*. IS1648 belongs to the IS3 family of insertion sequences. The second element, Tn5417, shows the highest similarity to the Tn4811 element located in the terminal inverted repeats of the Streptomyces lividans chromosome.
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Two new cellulosome components encoded downstream of celI in the genome of Clostridium thermocellum: the non-processive endoglucanase CelN and the possibly structural protein CseP
More LessClostridium thermocellum produces a great number of extracellular cellulases which are free or cellulosome-bound. The nucleotide sequence of a gene cluster containing the genes celI, celN and cseP was determined from C. thermocellum strain F7. Gene products Cel9I and Cel9N are structurally related enzymes having a glycosyl hydrolase family 9 and a carbohydrate-binding module (CBM3c), but show characteristic differences: Cel9I is a non-cellulosomal protein with an additional CBM (CBM3b), whereas Cel9N contains a cellulosomal dockerin module and no additional CBM. Although Cel9I is a processive endoglucanase, Cel9N is non-processive. Both enzymes hydrolyse phosphoric acid swollen cellulose, but the products of hydrolysis are different. The CseP protein encoded in the gene cluster is the first component attached to the cellulosomal scaffoldin for which no catalytic activity could be detected. It was shown to be present in the cellulosome. Its sequence is homologous to the spore-coat assembly protein CotH of Bacillus subtilis, suggesting a structural role of CseP in the cellulosome.
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CsgD, a regulator of curli and cellulose synthesis, also regulates serine hydroxymethyltransferase synthesis in Escherichia coli K-12
More LessThe homologous CsgD and AgfD proteins are members of the FixJ/UhpA/LuxR family and are proposed to regulate curli (thin aggregative fibres) and cellulose production by Escherichia coli and Salmonella enterica serovar Typhimurium, respectively. A plasmid containing part of the csgD gene was isolated during a screen for multicopy suppressors of glycine auxotrophy caused by deleting the folA gene in E. coli. The sequence of the plasmid suggests it encodes a chimaeric protein. Plasmids containing the intact csgD or agfD gene also caused suppression. Cells transformed with the recombinant plasmids contained higher serine hydroxymethyltransferase (SHMT) activity than controls. The increase could also be monitored by assaying β-galactosidase activity from a reporter strain with part of the SHMT gene, glyA, fused to lacZ. The increase in SHMT activity was sufficient to correct the glycine auxotrophy of strains lacking folA. The recombinant plasmids also enabled K-12 strains that are not curli-proficient to make curli. Curlin, the major component of curli, contains more glycine than normal E. coli proteins. It is proposed that CsgD upregulates glyA to facilitate synthesis of curli. It is suggested that recombinant plasmids produce enough CsgD or chimaeric protein to titrate out a ligand that switches CsgD into its inactive form. As a result, sufficient active CsgD is present to activate genes in its regulon. It is concluded that CsgD increases expression of the glyA gene either directly or indirectly.
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The mid genes of Rhizobium sp. strain TAL1145 are required for degradation of mimosine into 3-hydroxy-4-pyridone and are inducible by mimosine
More LessMimosine is a toxin present in the tree-legume leucaena (Leucaena leucocephala), including its root nodules and the root exudates. The leucaena-nodulating Rhizobium sp. strain TAL1145 degrades mimosine (Mid+) and utilizes it as a source of carbon and nitrogen. Twelve TAL1145 mutants defective in mimosine degradation (Mid−) were made through Tn3Hogus, TnphoA or kanamycin-resistance-cassette insertions. A 5·0 kb PstI fragment of TAL1145, subcloned from a cosmid clone containing mid genes for mimosine degradation, complemented most of the Mid− mutants. Sequencing this fragment and the adjacent 0·9 kb PstI fragment identified five genes, midA, midB, midC, midD and midR, of which the first three genes encode ABC transporter proteins involved in mimosine uptake, while midD encodes an aminotransferase required for degrading mimosine into 3-hydroxy-4-pyridone, and midR is a regulatory gene encoding a LysR-type transcriptional activator. The location of MidA in the periplasm was shown by making two midA : : phoA fusions, which made active alkaline phosphatase in the periplasm. The various mid : : gus and midA : : phoA fusions were inducible by mimosine, and a midD : : gus fusion mutant showed β-glucuronidase activity in the leucaena nodules, indicating that midD is expressed in the nodules. Similarly, a midA : : phoA fusion expressed alkaline phosphatase activity in the leucaena nodules, indicating that mimosine induces midA transcription in the bacteroids. mid genes are specific for the Mid+ strains of leucaena Rhizobium and are absent in strains of other Rhizobium, Sinorhizobium and Bradyrhizobium spp.
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- Pathogens And Pathogenicity
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Cell line differences in bacterially translocated ExoS ADP-ribosyltransferase substrate specificity
More LessExoenzyme S (ExoS) is an ADP-ribosyltransferase (ADPRT) directly translocated into eukaryotic cells by the type III secretory (TTS) process of Pseudomonas aeruginosa. Comparisons of the functional effects of ExoS on human epithelial and murine fibroblastic cells showed that human epithelial cells exhibited an overall increased sensitivity to the effects of bacterially translocated ExoS on cell proliferation, morphology and re-adherence. ExoS was also found to ADP-ribosylate a greater number of low-molecular-mass G (LMMG) proteins in human epithelial cells, as compared to murine fibroblasts. Examination of the cellular mechanism for differences in ExoS ADPRT substrate modification found that the more restricted pattern of substrate modification in murine fibroblasts was not linked to the efficiency of bacterial adherence nor to the efficiency of ExoS internalization by the TTS process. In exploring the cellular nature of patterns of substrate modification, more extensive substrate modification was detected in human and simian cell lines, while rodent cell lines, including rat, mouse and hamster lines, consistently exhibited the more limited pattern of LMMG protein ADP-ribosylation. Patterns of substrate modification were not altered by cellular transformation and occurred independently of cell type. These studies suggest that eukaryotic cell properties, as recognized through studies of cells of different animal origins, affect the substrate targeting of ExoS ADPRT activity, and that this in turn can influence the severity of effects of ExoS on host-cell function.
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Volumes and issues
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Volume 169 (2023)
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Volume 168 (2022)
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Volume 167 (2021)
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Volume 166 (2020)
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Volume 165 (2019)
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Volume 164 (2018)
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Volume 163 (2017)
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Volume 161 (2015)
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Volume 143 (1997)
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Volume 123 (1981)
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Volume 122 (1981)
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Volume 121 (1980)
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Volume 120 (1980)
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Volume 119 (1980)
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Volume 118 (1980)
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Volume 117 (1980)
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Volume 116 (1980)
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Volume 115 (1979)
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Volume 114 (1979)
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Volume 113 (1979)
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Volume 112 (1979)
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Volume 111 (1979)
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Volume 110 (1979)
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Volume 109 (1978)
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Volume 108 (1978)
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Volume 107 (1978)
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Volume 105 (1978)
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Volume 104 (1978)
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Volume 91 (1975)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 76 (1973)
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Volume 68 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 60 (1970)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 49 (1967)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 19 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)
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