- Volume 151, Issue 6, 2005
Volume 151, Issue 6, 2005
- Review
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Transcriptional takeover by σ appropriation: remodelling of the σ 70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA
Activation of bacteriophage T4 middle promoters, which occurs about 1 min after infection, uses two phage-encoded factors that change the promoter specificity of the host RNA polymerase. These phage factors, the MotA activator and the AsiA co-activator, interact with the σ 70 specificity subunit of Escherichia coli RNA polymerase, which normally contacts the −10 and −35 regions of host promoter DNA. Like host promoters, T4 middle promoters have a good match to the canonical σ 70 DNA element located in the −10 region. However, instead of the σ 70 DNA recognition element in the promoter's −35 region, they have a 9 bp sequence (a MotA box) centred at −30, which is bound by MotA. Recent work has begun to provide information about the MotA/AsiA system at a detailed molecular level. Accumulated evidence suggests that the presence of MotA and AsiA reconfigures protein–DNA contacts in the upstream promoter sequences, without significantly affecting the contacts of σ 70 with the −10 region. This type of activation, which is called ‘σ appropriation’, is fundamentally different from other well-characterized models of prokaryotic activation in which an activator frequently serves to force σ 70 to contact a less than ideal −35 DNA element. This review summarizes the interactions of AsiA and MotA with σ 70, and discusses how these interactions accomplish the switch to T4 middle promoters by inhibiting the typical contacts of the C-terminal region of σ 70, region 4, with the host −35 DNA element and with other subunits of polymerase.
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- Microbiology Comment
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- Biochemistry And Molecular Biology
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Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli
More LessEscherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA ‘suppressor’. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA1 fMet, and a distantly located metY gene encodes a variant, tRNA2 fMet. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA2 fMet with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA + host background. The mutations were observed when the plasmid-borne gene encoded tRNA2 fMet (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA1 fMet genes in E. coli in safeguarding their intactness, are discussed.
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The DNA-binding specificity of the Bacillus anthracis AbrB protein
More LessThe Bacillus subtilis AbrB protein is a DNA-binding global regulator of a plethora of functions that are expressed during the transition from exponential growth to stationary phase and under suboptimal growth conditions. AbrB orthologues have been identified in a variety of prokaryotic organisms, notably in all species of Bacillus, Clostridium and Listeria that have been examined. Based on amino acid sequence identity in the N-terminal domains of the orthologues from B. subtilis and Bacillus anthracis, it was predicted that the proteins might display identical DNA-binding specificities. The binding of purified B. anthracis AbrB (AbrBBA) and purified B. subtilis AbrB (AbrBBS) at DNA targets of B. subtilis, B. anthracis and a synthetic origin was compared. In all cases examined, DNA-binding specificity was identical as judged by DNase I footprinting. In B. subtilis cells, the B. anthracis promoters from the atxA and abrB genes were regulated by AbrBBS, and the B. subtilis promoter from the yxbB operon was regulated by AbrBBA.
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Regulation of two highly similar genes, omcB and omcC, in a 10 kb chromosomal duplication in Geobacter sulfurreducens
More LessThe Fe(III)-reducing micro-organism Geobacter sulfurreducens requires an outer-membrane c-type cytochrome, OmcB, for Fe(III) reduction, but a related cytochrome, OmcC, which is 73 % identical to OmcB, is not required. The omcB and omcC genes are part of a tandem chromosomal duplication consisting of two repeated clusters of four genes. The 2·7 kb sequences preceding omcB and omcC are identical with the exception of a single base pair change. Studies that combined genetic, Northern blotting and primer extension analyses demonstrated that both omcB and omcC are transcribed as monocistronic and polycistronic (orf1-orf2-omcB/omcC) transcripts. All of the promoters for the various transcripts were found to be located within the 2·7 kb identical region upstream of omcB and omcC. The sequences of the promoter regions for the two monocistronic transcripts are identical and equidistant from the omcB or omcC start codons. The promoters for the two polycistronic transcripts, in contrast, are distinct. One is specific for transcription of orf1-orf2-omcB and the other is associated with transcription of orf1-orf2-omcC. Studies with an RpoS-deficient mutant suggested that transcription from all four promoters is RpoS dependent under one or more growth conditions. Deletion of orfR, a gene immediately upstream of orf1-orf2-omcB that encodes a putative transcriptional regulator, significantly lowered the omcB transcription when Fe(III) was the electron acceptor and partially inhibited Fe(III) reduction. In contrast, levels of omcC transcripts were unaffected in the orfR mutant. These results indicate that omcB and omcC operons represent a rare instance in which duplicated operons, located in tandem on the chromosome, have different transcriptional regulation.
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The Bacillus subtilis YdfHI two-component system regulates the transcription of ydfJ, a member of the RND superfamily
More LessThe ydfHI genes encode a sensor kinase and a response regulator forming a two-component system. ydfJ is located downstream of ydfHI, and belongs to the RND (resistance-nodulation-cell division) superfamily, which is present in most major organisms. Four genes (secDF, yerP, ydfJ and ydgH) in Bacillus subtilis belong to this family. This study revealed that the YdfHI two-component system regulates ydfJ transcription. A gel shift assay using histidine-tagged YdfI (h-YdfI) showed that it directly binds to the ydfJ promoter region. Moreover, DNase I footprinting analysis revealed a tandem repeat sequence consisting of two conserved 12-mer sequences (GCCCRAAYGTAC) within the h-YdfI-binding site.
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Protein–protein interactions in the chemotaxis signalling pathway of Treponema denticola
More LessMotile bacteria employ sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioural responses. The proteins involved in this signalling pathway have been extensively studied on a molecular level in various model organisms, including enterobacteria and Bacillus subtilis, and specific protein–protein interactions have been identified. The chemotaxis operon of spirochaetes encodes a novel chemotaxis protein, CheX, in addition to homologues to the central components of established chemotaxis systems. Interestingly, the closest functionally characterized homologue of CheX is CheC of the complex B. subtilis chemotaxis pathway. In this study, the yeast two-hybrid system was applied to investigate protein–protein interactions within the chemotaxis signalling pathway of Treponema denticola, with special focus on CheX. CheX was found to interact with CheA and with itself. The other chemotaxis proteins exhibited interactions comparable to their homologues in known chemotaxis systems. Based on these findings, a model integrating CheX in the chemotaxis signal transduction pathway of T. denticola is proposed.
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Photo-oxidative stress in Rhodobacter sphaeroides: protective role of carotenoids and expression of selected genes
More LessIn Rhodobacter sphaeroides, carotenoids are essential constituents of the photosynthetic apparatus and are assumed to prevent the formation of singlet oxygen by quenching of triplet bacteriochlorophyll a (BChl a) in vivo. It was shown that small amounts of singlet oxygen are generated in vivo by incubation of R. sphaeroides under high light conditions. However, growth and survival rates were not affected. Higher amounts of singlet oxygen were generated by BChl a in a carotenoid-deficient strain and led to a decrease in growth and survival rates. The data support earlier results on the pivotal role of carotenoids in the defence against stress caused by singlet oxygen. Results obtained under photo-oxidative stress conditions with strains impaired in carotenoid synthesis suggest that sphaeroidene and neurosporene provide less protection against methylene-blue-generated singlet oxygen than sphaeroidenone in vivo. Despite their protective function against singlet oxygen, relative amounts of carotenoids did not accumulate in R. sphaeroides wild-type cultures under photo-oxidative stress, and relative mRNA levels of phytoene dehydrogenase and sphaeroidene monooxygenase did not increase. In contrast, singlet oxygen specifically induced the expression of glutathione peroxidase and a putative Zn-dependent hydrolase, but mRNA levels of hydrogen-peroxide-degrading catalase E were not significantly affected by photo-oxidative stress. Based on these results, it is suggested that singlet oxygen acts as a specific signal for gene expression in R. sphaeroides. Presumably transcriptional regulators exist to specifically induce the expression of genes involved in the response to stress caused by singlet oxygen.
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Oxidative protein damage causes chromium toxicity in yeast
Oxidative damage in microbial cells occurs during exposure to the toxic metal chromium, but it is not certain whether such oxidation accounts for the toxicity of Cr. Here, a Saccharomyces cerevisiae sod1Δ mutant (defective for the Cu,Zn-superoxide dismutase) was found to be hypersensitive to Cr(VI) toxicity under aerobic conditions, but this phenotype was suppressed under anaerobic conditions. Studies with cells expressing a Sod1p variant (Sod1H46C) showed that the superoxide dismutase activity rather than the metal-binding function of Sod1p was required for Cr resistance. To help identify the macromolecular target(s) of Cr-dependent oxidative damage, cells deficient for the reduction of phospholipid hydroperoxides (gpx3Δ and gpx1Δ/gpx2Δ/gpx3Δ) and for the repair of DNA oxidation (ogg1Δ and rad30Δ/ogg1Δ) were tested, but were found not to be Cr-sensitive. In contrast, S. cerevisiae msraΔ (mxr1Δ) and msrbΔ (ycl033cΔ) mutants defective for peptide methionine sulfoxide reductase (MSR) activity exhibited a Cr sensitivity phenotype, and cells overexpressing these enzymes were Cr-resistant. Overexpression of MSRs also suppressed the Cr sensitivity of sod1Δ cells. The inference that protein oxidation is a primary mechanism of Cr toxicity was corroborated by an observed ∼20-fold increase in the cellular levels of protein carbonyls within 30 min of Cr exposure. Carbonylation was not distributed evenly among the expressed proteins of the cells; certain glycolytic enzymes and heat-shock proteins were specifically targeted by Cr-dependent oxidative damage. This study establishes an oxidative mode of Cr toxicity in S. cerevisiae, which primarily involves oxidative damage to cellular proteins.
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NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis
More LessThe biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix–turn–helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These ΔnovG strains produced only 2 % of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His6-tagged NovG showed specific DNA-binding activity for the novG–novH and the cloG–cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)11CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.
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An acyl-CoA dehydrogenase is involved in the formation of the Δcis3 double bond in the acyl residue of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis
The lipopeptide antibiotic friulimicin, produced by Actinoplanes friuliensis, is an effective drug against Gram-positive bacteria, such as methicillin-resistant Staphylococcus epidermidis and Staphylococcus aureus strains. Friulimicin consists of a cyclic peptide core of ten amino acids and an acyl residue linked to an exocyclic amino acid. The acyl residue is essential for antibiotic activity, varies in length from C13 to C15, and carries a characteristic double bond at position Δcis3. Sequencing of a DNA fragment adjacent to a previously described fragment encoding some of the friulimicin biosynthetic genes revealed several genes whose gene products resemble enzymes of lipid metabolism. One of these genes, lipB, encodes an acyl-CoA dehydrogenase homologue. To elucidate the function of the LipB protein, a lipB insertion mutant was generated and the friulimicin derivative (FR242) produced by the mutant was purified. FR242 had antibiotic activity lower than friulimicin in a bioassay. Gas chromatography showed that the acyl residue of wild-type friulimicin contains a double bond, whereas a saturated bond was present in FR242. These results were confirmed by the heterologous expression of lipB in Streptomyces lividans T7, which led to the production of unsaturated fatty acids not found in the S. lividans T7 parent strain. These results indicate that the acyl-CoA dehydrogenase LipB is involved in the introduction of the unusual Δcis3 double bond into the acyl residue of friulimicin.
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A minor catalase/peroxidase from Burkholderia cenocepacia is required for normal aconitase activity
More LessThe opportunistic bacterium Burkholderia cenocepacia C5424 contains two catalase/peroxidase genes, katA and katB. To investigate the functions of these genes, katA and katB mutants were generated by targeted integration of suicide plasmids into the katA and katB genes. The catalase/peroxidase activity of the katA mutant was not affected as compared with that of the parental strain, while no catalase/peroxidase activity was detected in the katB mutant. However, the katA mutant displayed reduced resistance to hydrogen peroxide under iron limitation, while the katB mutant showed hypersensitivity to hydrogen peroxide, and reduced growth under all conditions tested. The katA mutant displayed reduced growth only in the presence of carbon sources that are metabolized through the tricarboxylic acid (TCA) cycle, as the growth defect was abrogated in cultures supplemented with glucose or glycerol. This phenotype was also correlated with a marked reduction in aconitase activity. In contrast, aconitase activity was not reduced in the katB mutant and parental strains. The authors conclude that the KatA protein is a specialized catalase/peroxidase that has a novel function by contributing to maintain the normal activity of the TCA cycle, while KatB is a classical catalase/peroxidase that plays a global role in cellular protection against oxidative stress.
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The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans
More LessAcidithiobacillus ferrooxidans is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous and ferric iron, making it an interesting model for understanding the biological mechanisms of bacterial iron uptake and homeostasis in extremely acid conditions. A candidate fur AF (Ferric Uptake Regulator) gene was identified in the A. ferrooxidans ATCC 23270 genome. FurAF has significant sequence similarity, including conservation of functional motifs, to known Fur orthologues and exhibits cross-reactivity to Escherichia coli Fur antiserum. The fur AF gene is able to complement fur deficiency in E. coli in an iron-responsive manner. FurAF is also able to bind specifically to E. coli Fur regulatory regions (Fur boxes) and to a candidate Fur box from A. ferrooxidans, as judged by electrophoretic mobility shift assays. FurAF represses gene expression from E. coli Fur-responsive promoters fiu and fhuF when expressed at high protein levels. However, it increases gene expression from these promoters at low concentrations and possibly from other Fur-regulated promoters involved in iron-responsive oxidative stress responses.
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The leucyl aminopeptidase from Helicobacter pylori is an allosteric enzyme
More LessThis study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from Helicobacter pylori. A gene encoding LAP was cloned from H. pylori and the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity against l-leucine-p-nitroanilide. Optimal activity was observed at pH 8·0 and 45 °C, with V max of 232 μmol min−1 (mg protein)−1 and S 0·5 of 0·65 mM. The data suggest that LAP could be allosteric (n H=2·27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC50=49·9 nM) and H. pylori growth in vitro. The results point to the potential use of LAP as a drug target to develop novel anti-H. pylori agents.
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The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain?
Mycobacterium smegmatis is often used as a surrogate host for pathogenic mycobacteria, especially since the isolation of the transformable smooth morphotype strain mc2155 from the isogenic rough wild-type strain ATCC 607. Biochemical analysis of the cell envelope components revealed a lack of polar glycolipids, namely the lipooligosaccharides and the polar subfamilies of glycopeptidolipids, in the mc2155 strain. In addition, the latter strain differs from its parent by the distribution of various species of glycolipids and phospholipids between the outermost and deeper layers of the cell envelope. The presence of filamentous and rope-like structures at the cell surface of mc2155 cells grown in complex media further supported an ultrastructural change in the cell envelope of the mutant. Importantly, a significantly more rapid uptake of the hydrophobic chenodeoxycholate was observed for the mutant compared to wild-type cells. Taken together, these data indicate that the nature of the surface-exposed and envelope constituents is crucial for the surface properties, cell wall permeability and bacterial phenotype, and suggest that the transformable character of the mc2155 strain may be in part explained by these profound modifications of its cell envelope.
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Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole
More LessThe azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium–intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus.
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- Biodiversity And Evolution
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Population structure of group B streptococcus from a low-incidence region for invasive neonatal disease
The population structure of group B streptococcus (GBS) from a low-incidence region for invasive neonatal disease (Israel) was investigated using multilocus genotype data. The strain collection consisted of isolates from maternal carriage (n=104) and invasive neonatal disease (n=50), resolving into 46 sequence types. The most prevalent sequence types were ST-1 (17·5 %), ST-19 (10·4 %), ST-17 (9·7 %), ST-22 (8·4 %) and ST-23 (6·5 %). Serotype III was the most common, accounting for 29·2 % of the isolates. None of the serotypes was significantly associated with invasive neonatal disease. burst analysis resolved the 46 sequence types into seven lineages (clonal complexes), from which only lineage ST-17, expressing serotype III only, was significantly associated with invasive neonatal disease. Lineage ST-22 expressed mainly serotype II, and was significantly associated with carriage. The distribution of the various sequence types and lineages, and the association of lineage ST-17 with invasive disease, are consistent with the results of analyses from a global GBS isolate collection. These findings could imply that the global variation in disease incidence is independent of the circulating GBS populations, and may be more affected by other risk factors for invasive GBS disease, or by different prevention strategies.
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- Environmental Microbiology
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Structural and replicative diversity of large plasmids from sphingomonads that degrade polycyclic aromatic compounds and xenobiotics
More LessThe plasmids from 16 sphingomonads which degrade various xenobiotics and polycyclic aromatic compounds were compared with the previously sequenced plasmid pNL1 from Sphingomonas aromaticivorans F199. The replicase genes repAaAb from plasmid pNL1 were amplified by PCR and used as a gene probe for the identification of plasmids belonging to the same incompatibility group as plasmid pNL1. Plasmids were prepared from various sphingomonads and hybridized with the repA gene probe. Positive hybridization signals were obtained with plasmids of approximately 160–195 kb from Sphingomonas subterranea and S. aromaticivorans B0695, which had been isolated from the same subsurface location as S. aromaticivorans F199. The repA probe also hybridized with plasmids from Sphingomonas xenophaga BN6, Sphingomonas sp. HH69 and Sphingomonas macrogoltabidus, which had been isolated from different continents and which utilize different organic compounds than S. aromaticivorans F199 and the other subsurface strains. The results of the hybridization experiments were confirmed by PCR experiments using primers deduced from the repAaAb region of plasmid pNL1. Nucleotide sequence comparisons suggested that three gene clusters were conserved between plasmid pNL1 and plasmid pBN6 from the naphthalenesulfonate- degrading strain S. xenophaga BN6. From these sequence comparisons, PCR primers were derived in order to detect the respective gene clusters in the other strains and to deduce their position relative to each other. These experiments demonstrated that all analysed subsurface strains harboured the same three gene clusters, but that the position and distance from each other of the clusters varied considerably among the different strains.
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A PCR-based specific assay reveals a population of bacteria within the Chloroflexi associated with the reductive dehalogenation of polychlorinated biphenyls
More LessPolychlorinated biphenyls (PCBs) accumulate and persist in sediments posing a risk to human health and the environment. Highly chlorinated PCBs are reductively dechlorinated in anaerobic sediments and two bacteria, designated o-17 and DF-1, from a novel phylogenetic group that reductively dechlorinate PCBs have recently been identified. However, there is a paucity of knowledge about the distribution, diversity and ecology of PCB-dechlorinating bacteria due to difficulty in obtaining pure cultures and the lack of detection by universal PCR 16S rRNA gene primer sets in sediments. A specific PCR primer was developed and optimized for detection of o-17/DF-1 and other closely related bacteria in the environment. Using this primer set it was determined that bacteria of this group were enriched in sediment microcosms from Baltimore Harbour concurrent with active dechlorination of 2,2′,3,4,4′,5′-hexachlorobiphenyl. Additional 16S rRNA gene sequences that had high levels of similarity to described PCB dechlorinators were detected in sediments from the Elizabeth River tributary of Chesapeake Bay, which had confirmed PCB-dechlorinating activities. Phylogenetic comparison of these detected 16S rRNA gene sequences revealed a relatively diverse group of organisms within the dehalogenating Chloroflexi that are distinct from the Dehalococcoides spp. Results from this study indicate that reductive PCB dechlorination activity may be catalysed by a previously undescribed group of micro-organisms that appear to be prevalent in PCB-impacted sites.
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Pectin utilization by the methylotrophic yeast Pichia methanolica
More LessThe methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Δ and mod1Δmod2Δ strains showed a severe defect in the growth yield, although the mod2Δ strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.
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- Genes And Genomes
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Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2
More LessThe gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD2C2. To assess the extent to which FlhD2C2 directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42–44 bp inverted repeat FlhD2C2 binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD2C2 binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD2C2 over the K D range 38–356 nM, and of the nine corresponding chromosomal promoter–lacZ fusions, those of the four genes b1904, b2446, wzz fepE and gltI showed up to 50-fold dependence on FlhD2C2 in vivo. In comparison, four representative flagella class II promoters bound FlhD2C2 in the K D range 12–43 nM and were upregulated in vivo 30- to 990-fold. The FlhD2C2-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted −35 motif of the FlhD2C2-activated σ 70 promoters, as is the case with FlhD2C2-dependent class II flagellar promoters. The data indicate a wider FlhD2C2 regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.
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sae is essential for expression of the staphylococcal adhesins Eap and Emp
Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.
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Comparative analysis of the exopolysaccharide biosynthesis gene clusters from four strains of Lactobacillus rhamnosus
B. Péant, G. LaPointe, C. Gilbert, D. Atlan, P. Ward and D. RoyThe exopolysaccharide (EPS) biosynthesis gene clusters of four Lactobacillus rhamnosus strains consist of chromosomal DNA regions of 18·5 kb encoding 17 ORFs that are highly similar among the strains. However, under identical conditions, EPS production varies considerably among these strains, from 61 to 1611 mg l−1. Fifteen genes are co-transcribed starting from the first promoter upstream of wzd. Nevertheless, five transcription start sites were identified by 5′-RACE PCR analysis, and these were associated with promoter sequences upstream of wzd, rmlA, welE, wzr and wzb. Six potential glycosyltransferase genes were identified that account for the assembly of the heptasaccharide repeat unit containing an unusually high proportion of rhamnose. Four genes involved in the biosynthesis of the sugar nucleotide precursor dTDP-l-rhamnose were identified in the EPS biosynthesis locus, which is unusual for lactic acid bacteria. These four genes are expressed from their own promoter (P2), as well as co-transcribed with the upstream EPS genes, resulting in coordinated production of the rhamnose precursor with the enzymes involved in EPS biosynthesis. This is believed to be the first report demonstrating that the sequence, original organization and transcription of genes encoding EPS production are highly similar among four strains of Lb. rhamnosus, and do not vary with the amount of EPS produced.
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The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes
More LessThe integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded GmR green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attI was detected by PCR in 8 % of GmR colonies and the frequency of attI integration was estimated as 2·0×10−8 per P. stutzeri Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter (Pc) was localized to the 5′ end of the intI gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the attI site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attI and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.
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Mutations affecting predation ability of the soil bacterium Myxococcus xanthus
More LessMyxococcus xanthus genetic mutants with characterized phenotypes were analysed for the ability to prey on susceptible bacteria. Quantification of predatory ability was scored by a newly developed method under conditions in which prey bacteria provided the only source of nutrients. These results were corroborated by data derived using a previously published protocol that measures predation in the presence of limited external nutrients. First, early developmental regulatory mutants were examined, because their likely functions in assessing the local nutrient status were predicted to be also important for predation. The results showed that predation efficiency is reduced by 64–80 % for mutants of three A-signalling components, AsgA, AsgC and AsgE, but not for AsgB. This suggests that an Asg regulon function that is separate from A-signal production is needed for predation. Besides the Asg components, mutations in the early developmental genes sdeK and csgA were also consistently observed to reduce predatory efficacy by 36 and 33 %, respectively. In contrast, later developmental components, such as DevRS, 4406 and PhoP4, did not appear to play significant roles in predation. The predatory abilities of mutants defective for motility were also tested. The data showed that adventurous, but not social, motility is required for predation in the assay. Also, mutants for components in the chemotaxis-like Frz system were found to be reduced in predation efficiency by between 62 and 85 %. In sum, it was demonstrated here that defects in development and development-related processes affect the ability of M. xanthus to prey on other bacteria.
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Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies
A total of 67 Mycoplasma gallisepticum field isolates from the USA, Israel and Australia, and 10 reference strains, were characterized by gene-targeted sequencing (GTS) analysis of portions of the putative cytadhesin pvpA gene, the cytadhesin gapA gene, the cytadhesin mgc2 gene, and an uncharacterized hypothetical surface lipoprotein-encoding gene designated genome coding DNA sequence (CDS) MGA_0319. The regions of the surface-protein-encoding genes targeted in this analysis were found to be stable within a strain, after sequencing different in vitro passages of M. gallisepticum reference strains. Gene sequences were first analysed on the basis of gene size polymorphism. The pvpA and mgc2 genes are characterized by the presence of different nucleotide insertions/deletions. However, differentiation of isolates based solely on pvpA/mgc2 PCR size polymorphism was not found to be a reliable method to differentiate among M. gallisepticum isolates. On the other hand, GTS analysis based on the nucleotide sequence identities of individual and multiple genes correlated with epidemiologically linked isolates and with random amplified polymorphic DNA (RAPD) analysis. GTS analysis of individual genes, gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, respectively. GTS analysis using multiple gene sequences mgc2/pvpa and gapA/MGA_0319/mgc2/pvpA identified 38 and 40 sequence types, respectively. GTS of multiple surface-protein-encoding genes showed better discriminatory power than RAPD analysis, which identified 36 pattern types from the same panel of M. gallisepticum strains. These results are believed to provide the first evidence that typing of M. gallisepticum isolates by GTS analysis of surface-protein genes is a sensitive and reproducible typing method and will allow rapid global comparisons between laboratories.
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ftsZ mutations affecting cell division frequency, placement and morphology in Bacillus subtilis
More LessA key event in cytokinesis in bacteria is the assembly of the essential division protein FtsZ into ring-like structures at the nascent division site. FtsZ is the prokaryotic homologue of tubulin, and is found in nearly all bacteria. In vitro, FtsZ polymerizes in the presence of GTP to form higher-ordered polymers. FtsZ consists of two domains, with the GTP-binding site located in the N-terminal domain. The less-conserved C-terminal domain contains residues important for GTP hydrolysis, but its overall function is still unclear. This paper reports the development of a simple strategy to generate mutations in the essential division gene ftsZ. Nine novel and viable ftsZ mutants of Bacillus subtilis are described. Eight of the mutations would affect the C-terminus of FtsZ. The collection of mutants exhibits a range of morphological phenotypes, ranging from normal to highly filamentous cells; some produce minicells, or divide in a twisted configuration; one mutation has a temperature-sensitive effect specifically impairing sporulation. The sites of the amino acid changes generated by the mutations could be informative about FtsZ function and its protein–protein interactions.
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- Pathogens And Pathogenicity
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Identification and regulation of cold-inducible factors of Bordetella bronchiseptica
More LessThe expression of bacterial cold-shock proteins (CSPs) is highly induced in response to cold shock, and some CSPs are essential for cells to resume growth at low temperature. Bordetella bronchiseptica encodes five CSPs (named CspA to CspE) with significant amino acid homology to CspA of Escherichia coli. In contrast to E. coli, the insertional knock-out of a single csp gene (cspB) strongly affected growth of B. bronchiseptica independent of temperature. In the case of three of the csp genes (cspA, cspB, cspC) more than one specific transcript could be detected. The net amount of cspA, cspB and cspC transcripts increased strongly after cold shock, while no such effect could be observed for cspD and cspE. The exposure to other stress conditions, including translation inhibitors, heat shock, osmotic stress and nutrient deprivation in the stationary phase, indicated that the csp genes are also responsive to these conditions. The coding regions of all of the cold-shock genes are preceded by a long non-translated upstream region (5′-UTR). In the case of the cspB gene, a deletion of parts of this region led to a significant reduction of translation of the resulting truncated transcript, indicating a role of the 5′-UTR in translational control. The cold-shock stimulon was investigated by 2D-PAGE and mass spectrometric characterization, leading to the identification of additional cold-inducible proteins (CIPs). Interestingly, two cold-shock genes (cspC and cspD) were found to be under the negative control of the BvgAS system, the main transcriptional regulator of Bordetella virulence genes. Moreover, a negative effect of slight overexpression of CspB, but not of the other CSPs, on the transcription of the adenylate cyclase toxin CyaA of Bordetella pertussis was observed, suggesting cross-talk between the CSP-mediated stress response stimulon and the Bordetella virulence regulon.
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Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae
More LessThe capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0·04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS− mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS− phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS− mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.
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Oral immunization with a dam mutant of Yersinia pseudotuberculosis protects against plague
More LessInactivation of the gene encoding DNA adenine methylase (dam) has been shown to attenuate some pathogens such as Salmonella enterica serovar Typhimurium and is a lethal mutation in others such as Yersinia pseudotuberculosis strain YPIII. In this study the dam methylase gene in Yersinia pseudotuberculosis strain IP32953 was inactivated. Unlike the wild-type, DNA isolated from the mutant could be digested with MboI, which is consistent with an altered pattern of DNA methylation. The mutant was sensitive to bile salts but not to 2-aminopurine. The effect of dam inactivation on gene expression was examined using a DNA microarray. In BALB/c mice inoculated orally or intravenously with the dam mutant, the median lethal dose (MLD) was at least 106-fold higher than the MLD of the wild-type. BALB/c mice inoculated with the mutant were protected against a subcutaneous challenge with 100 MLDs of Yersinia pestis strain GB and an intravenous challenge with 300 MLDs of Y. pseudotuberculosis IP32953.
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Diverse humoral immune responses and changes in IgG antibody levels against mycobacterial lipid antigens in active tuberculosis
More LessHumoral immune responses of active TB patients against six mycobacterial lipid antigens [trehalose 6,6′-dimycolate (TDM) from Mycobacterium bovis BCG (TDM-T) and Mycobacterium avium complex (TDM-M), trehalose 6-monomycolate (TMM) from M. bovis BCG (TMM-T) and M. avium complex (TMM-M), triacyl (PL-2) and tetraacyl (PL-1) phosphatidylinositol dimannosides] were examined by ELISA. IgG antibodies of TB patients with active disease reacted against the six lipid antigens distinctively, but heterogeneously. If tests were combined and an overall positive was scored cumulatively when any one of the six tests was positive, a good discrimination between patient and normal subject was obtained. A positive result in any one of the six tests was obtained in 91·5 % of all 924 hospitalized patients and 93·3 % of 210 patients at their first visit to the outpatient clinic. The IgG antibody response differed considerably from patient to patient, and the response patterns were grouped into several types. IgG antibody levels paralleled the bacterial burden; however, the smear-negative (culture-positive) patient group also showed high positive rates and mean ELISA ΔA values against the six lipid antigens. There were also marked differences in positive rate and mean ΔA values between cavity-positive and -negative groups, the former being higher than the latter. After anti-TB chemotherapy was initiated, IgG antibody levels decreased dramatically, paralleling the decrease in the amount of excretion of bacteria. Since multiple-antigen ELISA using particular lipid antigens was highly sensitive, and IgG antibody levels vary greatly at different stages of the disease, this technique is applicable for early diagnosis of smear-negative (and -positive) active TB and the prognosis for completion of anti-TB chemotherapy.
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Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis
Since avian pathogenic Escherichia coli (APEC) and human uropathogenic E. coli (UPEC) may encounter similar challenges when establishing infection in extraintestinal locations, they may share a similar content of virulence genes and capacity to cause disease. In the present study, 524 APEC and 200 UPEC isolates were compared by their content of virulence genes, phylogenetic group, and other traits. The two groups showed substantial overlap in terms of their serogroups, phylogenetic groups and virulence genotypes, including their possession of certain genes associated with large transmissible plasmids of APEC. Based on these results, the propensity of both groups to cause extraintestinal infections, and a well-documented ability of avian E. coli to spread to human beings, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. However, significant differences in the prevalence of the traits occurred across the two groups, suggesting that if APEC are involved in human urinary tract infections, they are not involved in all of them.
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Attenuated virulence of an Aeromonas salmonicida subsp. salmonicida type III secretion mutant in a rainbow trout model
More LessAeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a severe systemic disease affecting salmonid fish. This bacterium contains a type III protein secretion system that is responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of fish cells. This study showed that inactivation of the type III secretion system by marker-replacement mutagenesis of the gene ascV, which encodes an inner-membrane component of the type III secretion system, attenuated virulence in a rainbow trout model. The isogenic ascV deletion mutant was phagocytosed by peripheral blood leukocytes but the wild-type (wt) A. salmonicida subsp. salmonicida isolate was not. Histological examination of fish experimentally infected with the wt bacterium revealed extensive tissue necrosis and bacterial aggregates in all organs examined, including the heart, kidney and liver, indicating that the isolate established a systemic infection. Cumulative mortality of fish experimentally infected with the wt bacterium reached 88 %. In contrast, no mortality was observed among fish infected with the same dose of the ascV mutant, and histological examination of fish infected with this strain revealed healthy organs. The results indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required to establish systemic infection.
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- Physiology
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Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans
More LessOxylipins called psi factors have been shown to alter the ratio of asexual to sexual sporulation in the filamentous fungus Aspergillus nidulans. Analysis of the A. nidulans genome has led to the identification of three fatty acid oxygenases (PpoA, PpoB and PpoC) predicted to produce psi factors. Here, it is reported that deletion of ppoB (ΔppoB) reduced production of the oleic-acid-derived oxylipin psiBβ and increased the ratio of asexual to sexual spore development. Generation of the triple mutant ΔppoAΔppoBΔppoC resulted in a strain deficient in producing oleic- and linoleic-acid-derived 8′-hydroxy psi factor and caused increased and mis-scheduled activation of sexual development. Changes in asexual to sexual spore development were positively correlated to alterations in the expression of brlA and veA, respectively. PpoB and/or its products antagonistically mediate the expression levels of ppoA and ppoC, thus revealing regulatory feedback loops among these three genes. Phylogenetic analyses showed that ppo genes are present in both saprophytic and pathogenic Ascomycetes and Basidiomycetes, suggesting a conserved role for Ppo enzymes in the life cycle of fungi.
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Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms
Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0·050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.
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The specificity of oligopeptide transport by Streptococcus thermophilus resembles that of Lactococcus lactis and not that of pathogenic streptococci
More LessPeptide transport is a crucial step in the growth of Streptococcus thermophilus in protein- or peptide-containing media. The objective of the present work was to determine the specificity of peptide utilization by this widely used lactic acid bacterium. To reach that goal, complementary approaches were employed. The capability of a proteinase-negative S. thermophilus strain to grow in a chemically defined medium containing a mixture of peptides isolated from milk as the source of amino acids was analysed. Peptides were separated into three size classes by ultrafiltration. The strain was able to use peptides up to 3·5 kDa during growth, as revealed by liquid chromatography and mass spectrometry analyses. The same strain was grown in chemically defined medium containing a tryptic digest of casein, and the respective time-course consumption of the peptides during growth was estimated. The ability to consume large peptides (up to 23 residues) was confirmed, as long as they are cationic and hydrophobic. These results were confirmed by peptide transport studies. Extension of the study to 11 other strains revealed that they all shared these preferences.
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Plasma membrane H+ and K+ transporters are involved in the weak-acid preservative response of disparate food spoilage yeasts
More LessThe food spoilage yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae have been proposed to resist weak-acid preservative stress by different means; Z. bailii by limiting influx of preservative combined with its catabolism, S. cerevisiae by active extrusion of the preservative weak-acid anion and H+. Measurement of H+ extrusion by exponential-phase Z. bailii cells suggest that, in common with S. cerevisiae, this yeast uses a plasma membrane H+-ATPase to expel H+ when challenged by weak-acid preservative (benzoic acid). Simultaneous measurement of Z. bailii net H+ and K+ fluxes showed that net K+ influx accompanies net H+ efflux during acute benzoic acid stress. Such ionic coupling is known for S. cerevisiae in short-term preservative stress. Both yeasts significantly accumulated K+ on long-term exposure to benzoic acid. Analysis of S. cerevisiae K+ transporter mutants revealed that loss of the high affinity K+ uptake system Trk1 confers sensitivity to growth in preservative. The results suggest that cation accumulation is an important factor in adaptation to weak-acid preservatives by spoilage yeasts and that Z. bailii and S. cerevisiae share hitherto unsuspected adaptive responses at the level of plasma membrane ion transport.
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