- Volume 155, Issue 2, 2009
Volume 155, Issue 2, 2009
- Review
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Glycosylation and biogenesis of a family of serine-rich bacterial adhesins
Meixian Zhou and Hui WuGlycosylation of bacterial proteins is an important process for bacterial physiology and pathophysiology. Both O- and N-linked glycan moieties have been identified in bacterial glycoproteins. The N-linked glycosylation pathways are well established in Gram-negative bacteria. However, the O-linked glycosylation pathways are not well defined due to the complex nature of known O-linked glycoproteins in bacteria. In this review, we examine a new family of serine-rich O-linked glycoproteins which are represented by fimbriae-associated adhesin Fap1 of Streptococcus parasanguinis and human platelet-binding protein GspB of Streptococcus gordonii. This family of glycoproteins is conserved in streptococcal and staphylococcal species. A gene cluster coding for glycosyltransferases and accessory Sec proteins has been implicated in the protein glycosylation. A two-step glycosylation model is proposed. Two glycosyltransferases interact with each other and catalyse the first step of the protein glycosylation in the cytoplasm; the cross-talk between glycosylation-associated proteins and accessory Sec components mediates the second step of the protein glycosylation, an emerging mechanism for bacterial O-linked protein glycosylation. Dissecting the molecular mechanism of this conserved biosynthetic pathway offers opportunities to develop new therapeutic strategies targeting this previously unrecognized pathway, as serine-rich glycoproteins have been shown to play a role in bacterial pathogenesis.
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- Cell And Molecular Biology Of Microbes
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Microarray studies reveal a ‘differential response’ to moderate or severe heat shock of the HrcA- and HspR-dependent systems in Corynebacterium glutamicum
Genome-wide transcription profile analysis of the heat-shocked wild-type strain under moderate (40 °C) and severe heat stress (50 °C) revealed that a large number of genes are differentially expressed after heat shock. Of these, 358 genes were upregulated and 420 were downregulated in response to moderate heat shock (40 °C) in Corynebacterium glutamicum. Our results confirmed the HrcA/controlling inverted repeat of chaperone expression (CIRCE)-dependent and HspR/HspR-associated inverted repeat (HAIR)-dependent upregulation of chaperones following heat shock. Other genes, including clusters of orthologous groups (COG) related to macromolecule biosynthesis and several transcriptional regulators (COG class K), were upregulated, explaining the large number of genes affected by heat shock. Mutants having deletions in the hrcA or hspR regulators were constructed, which allowed the complete identification of the genes controlled by those systems. The up- or downregulation of several genes observed in the microarray experiments was validated by Northern blot analyses and quantitative (real-time) reverse-transcription PCR. These analyses showed a heat-shock intensity-dependent response (‘differential response’) in the HspR/HAIR system, in contrast to the non-differential response shown by the HrcA/CIRCE-regulated genes.
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The Rhizobium etli RpoH1 and RpoH2 sigma factors are involved in different stress responses
The physiological role and transcriptional expression of Rhizobium etli sigma factors rpoH1 and rpoH2 are reported in this work. Both rpoH1 and rpoH2 were able to complement the temperature-sensitive phenotype of an Escherichia coli rpoH mutant. The R. etli rpoH1 mutant was sensitive to heat shock, sodium hypochlorite and hydrogen peroxide, whereas the rpoH2 mutant was sensitive to NaCl and sucrose. The rpoH2 rpoH1 double mutant had increased sensitivity to heat shock and oxidative stress when compared with the rpoH1 single mutant. This suggests that in R. etli, RpoH1 is the main heat-shock sigma factor, but a more complete protective response could be achieved with the participation of RpoH2. Conversely, RpoH2 is involved in osmotic tolerance. In symbiosis with bean plants, the R. etli rpoH1 and rpoH2 rpoH1 mutants still elicited nodule formation, but exhibited reduced nitrogenase activity and bacterial viability in early and late symbiosis compared with nodules produced by rpoH2 mutants and wild-type strains. In addition, nodules formed by R. etli rpoH1 and rpoH2 rpoH1 mutants showed premature senescence. It was also determined that fixNf and fixKf expression was affected in rpoH1 mutants. Both rpoH genes were induced under microaerobic conditions and in the stationary growth phase, but not in response to heat shock. Analysis of the upstream region of rpoH1 revealed a σ 70 and a probable σ E promoter, whereas in rpoH2, one probable σ E-dependent promoter was detected. In conclusion, the two RpoH proteins operate under different stress conditions, RpoH1 in heat-shock and oxidative responses, and RpoH2 in osmotic tolerance.
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Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC
Highly branched dendritic swarming of B. subtilis on synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded by phrC) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genes sfp (activation of surfactin synthases), comA, abrB and codY (global regulators), hag (flagellin), mecA and yvzB (hag-like), and swrB (motility), to the different swarming stages. Surprisingly, mutations in genes comPX, comQ, comS, rapC and oppD, which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for driving srfABCD transcription. We showed that the phrC mutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated with phrC sfp+ and phrC+ sfp (GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormal phrC type, these were predominantly populated by sfp cells, which migrated faster than the phrC cells. This and other results indicated a specific migration defect in the phrC mutant that could not be trans-complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.
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Surface display of the receptor-binding domain of the F17a-G fimbrial adhesin through the autotransporter AIDA-I leads to permeability of bacterial cells
More LessSurface exposure of antigens on bacterial cells can be critical for eliciting an effective antibody response. Therefore, we investigated the cellular localization of the fimbrial F17a-G receptor-binding domain, fused to the translocator domain of the AIDA-I autotransporter. Synthesis of the fusion protein, under the control of the l-arabinose-inducible PBAD promoter, was shown to permeabilize Escherichia coli K-12 and Salmonella enterica serovar Typhimurium cells. The presence of permeable cells interfered with several methods that are typically used to determine surface exposure of proteins, such as protease treatment and whole-cell ELISA. Double immunofluorescence microscopy, using a second antibody directed against β-galactosidase, a bacterial protein expressed in the cytoplasm, allowed the simultaneous detection of antigen expression and permeability in individual cells.
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Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis
Yersinia pestis cells were grown in vitro at 26 and 37 °C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37 °C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small β-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37 °C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26 °C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26 °C, but not at 37 °C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658–y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a M r range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.
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Genes involved in the repression of mutacin I production in Streptococcus mutans
More LessStreptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibiotic mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high-phosphate brain heart infusion agar medium that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones for which we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups, including central metabolism, surface binding and sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when the gene is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.
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Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site
More LessThe important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the −10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.
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Stability and lability of circadian period of gene expression in the cyanobacterium Synechococcus elongatus
More LessMolecular aspects of the circadian clock in the cyanobacterium Synechococcus elongatus have been described in great detail. Three-dimensional structures have been determined for the three proteins, KaiA, KaiB and KaiC, that constitute a central oscillator of the clock. Moreover, a temperature-compensated circadian rhythm of KaiC phosphorylation can be reconstituted in vitro with the addition of KaiA, KaiB and ATP. These data suggest a relatively simple circadian system in which a single oscillator provides temporal information for all downstream processes. However, in vivo the situation is more complex, and additional components contribute to the maintenance of a normal period, the resetting of relative phases of circadian oscillations, and the control of rhythms of gene expression. We show here that two well-studied promoters in the S. elongatus genome report different circadian periods of expression under a given set of conditions in wild-type as well as mutant genetic backgrounds. Moreover, the period differs between these promoters with respect to modulation by light intensity, growth phase, and the presence or absence of a promoter-recognition subunit of RNA polymerase. These data contrast sharply with the current clock model in which a single Kai-based oscillator governs circadian period. Overall, these findings suggest that complex interactions among the circadian oscillator, perhaps other oscillators, and other cellular machinery result in a clock that is plastic and sensitive to the environment and to the physiological state of the cell.
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- Environmental And Evolutionary Microbiology
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Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomea
More LessA novel ligninolytic peroxidase gene (ACLnP) was cloned and characterized from a poroid brown-rot fungus, Antrodia cinnamomea. The genomic DNA of the fungus harboured two copies of ACLnP, with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of Pleurotus eryngii. ACLnP was cloned into vector pQE31, successfully expressed in Escherichia coli strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional ACLnP gene in A. cinnamomea in terms of wood degradation and colonization capacity in its unique niche is discussed.
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Plasmid diversity in Vibrio vulnificus biotypes
More LessVibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8 % of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65 % of these strains also had a putative conjugative plasmid with a molecular size of 52–56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48–56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.
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Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs
More LessIn order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon–Wiener (J′) indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D=0.83; J′=0.88) and Aphanizomenon flos-aquae the highest ones (D=J′=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.
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Amoeba co-culture of soil specimens recovered 33 different bacteria, including four new species and Streptococcus pneumoniae
More LessAmoeba-resistant bacteria (ARB), such as Legionella spp., are currently regarded as potential human pathogens that live in the natural environment, and thus their habitat is regarded as a reservoir of human pathogens. To detect ARB in human and environmental samples, co-culture with amoebae has been demonstrated to be an efficient tool. However, to date, only water samples from cooling towers and hospital water supplies have been investigated as possible reservoirs of ARB using this procedure. In the present study, we studied the ARB population of 11 diverse soil and sand sources in proximity to human environments; these sources included the university, the station, hospitals, the square, parks and public beaches in the city of Marseilles, France. As a result, a total of 33 different species of ARB were identified. The ability to grow within and/or lyse amoebae was demonstrated, for what is believed to be the first time, for several species; moreover, 20 of the isolates (61 %), including Streptococcus pneumoniae, have been described as human pathogens. However, Legionella spp. were not isolated. Four isolates are likely to be the members of new or uncharacterized genera or species, and their capability to be human pathogens needs to be determined. This preliminary work demonstrates that soils and sands in the vicinity of humans are reservoirs of human pathogenic ARB.
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- Genes And Genomes
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Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50
More LessBovicin HJ50 is a new lantibiotic containing a disulfide bridge produced by Streptococcus bovis HJ50; its encoding gene bovA was reported in our previous publication. To identify other genes involved in bovicin HJ50 production, DNA fragments flanking bovA were cloned and sequenced. The bovicin HJ50 biosynthesis gene locus was encoded by a 9.9 kb region of chromosomal DNA and consisted of at least nine genes in the following order: bovA, -M, -T, -E, -F, ORF1, ORF2, bovK and bovR. A thiol–disulfide oxidoreductase gene named sdb1 was located downstream of bovR. A knockout mutant of this gene retained antimicrobial activity and the molecular mass of bovicin HJ50 in the mutant was the same as that of bovicin HJ50 in S. bovis HJ50, implying that sdb1 is not involved in bovicin HJ50 production. Transcriptional analyses showed that bovA, bovM and bovT constituted an operon, and the transcription start site of the bovA promoter was located at a G residue 45 bp upstream of the translation start codon for bovA, while bovE through bovR were transcribed together and the transcription start site of the bovE promoter was located at a C residue 35 bp upstream of bovE. We also demonstrated successful heterologous expression of bovicin HJ50 in Lactococcus lactis MG1363, which lacks thiol–disulfide oxidoreductase genes; this showed that thiol–disulfide oxidoreductase genes other than sdb1 are not essential for bovicin HJ50 biosynthesis.
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- Microbial Pathogenicity
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Role of motility and chemotaxis in the pathogenesis of Dickeya dadantii 3937 (ex Erwinia chrysanthemi 3937)
Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94 %), cheY (80 %), cheW (74 %), cheB (54 %) and cheZ (48 %). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA<cheY<cheB=cheW<cheZ, which correlated with the degree to which swimming was affected. These results clearly indicate that motility plays an important role in the pathogenicity of this bacterium.
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The conserved portion of the putative virulence region contributes to virulence of avian pathogenic Escherichia coli
More LessColibacillosis is a common systemic disease of worldwide economic importance in poultry, caused by Escherichia coli. E. coli are normally found in the intestines of poultry, but some strains are able to cause extraintestinal disease. Plasmid pVM01 is essential for virulence in avian pathogenic Escherichia coli (APEC) strain E3 in chickens after aerosol exposure and contains the virulence-associated genes iucA, iss and tsh in distinct regions. The determination of the complete sequence of this plasmid identified many ORFs that were highly similar to genes found in the APEC O1 plasmid, as well as many hypothetical ORFs. Truncated versions of pVM01 were constructed and introduced into avirulent APEC strain E3/2.4 and the pathogenicity of these strains was assessed by aerosol exposure. The function of the region of pVM01 that contains the genes for conjugation was confirmed. Strains carrying the truncated plasmids appeared to be of intermediate virulence compared to the wild-type APEC strain E3. The conserved portion of the putative virulence region was found to contribute to the colonization of and generation of lesions in the air sacs. Both the conserved and variable portions of the putative virulence region were shown to contribute to the colonization of the trachea, but the variable portion of the putative virulence region was not required for the strain to confer a virulent phenotype. These results reveal that deletion of the conserved portion of the putative virulence region, but not the variable portion of the putative virulence region, is associated with a decrease in virulence of APEC.
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GFP plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression
More LessWe have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (CmR). Using a different antibiotic resistance marker, kanamycin (KmR), did not impair invasiveness. Despite the effect of plasmid-encoded CmR, the strain containing chromosomally encoded GFP, also carrying a CmR gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH : : gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating CmR or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying CmR , should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.
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A novel PhoP-regulated locus encoding the cytolysin ClyA and the secreted invasin TaiA of Salmonella enterica serovar Typhi is involved in virulence
More LessSalmonella enterica serovar Typhi causes a human-restricted systemic infection called typhoid fever. We have identified a Typhi genomic region encoding two ORFs, STY1498 and STY1499, that are expressed during infection of human macrophages and organized in an operon. STY1498 corresponds to clyA, which encodes a pore-forming cytolysin, and STY1499 encodes a 27 kDa protein, without any attributed function, which we have named TaiA (Typhi-associated invasin A). In order to evaluate the roles of these genes in Typhi pathogenesis, isogenic Typhi strains harbouring a non-polar mutation of either clyA or taiA were constructed. In macrophages, taiA was involved in increasing phagocytosis, as taiA deletion reduced bacterial uptake, whereas clyA reduced or controlled bacterial growth, as clyA deletion enhanced Typhi survival within macrophages without affecting cytotoxicity. In epithelial cells, deletion of taiA had no effect on invasion, whereas deletion of clyA enhanced the Typhi invasion rate, and reduced cytotoxicity. Overexpression of taiA in Typhi or in Escherichia coli resulted in a higher invasion rate of epithelial cells. We have demonstrated that TaiA is secreted independently of both the Salmonella pathogenicity island (SPI)-1 and the SPI-2 type three secretion systems. We have shown that this operon is regulated by the virulence-associated regulator PhoP. Moreover, our results revealed that products of this operon might be involved in promoting the use of macrophages as a sheltered reservoir for Typhi and allowing long-term persistence inside the host.
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Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli
More LessEnterohaemorrhagic Escherichia coli (EHEC) colonizes and proliferates at the mucosal surface, inducing severe diarrhoea. Short-chain fatty acids (SCFAs) are abundant in the intestine owing to the metabolic activity of microflora, and are important for colonic health. We found that, although a high concentration of SCFAs inhibited the growth of EHEC, at low concentrations, the SCFAs markedly enhanced the expression of the virulence genes required for cell adherence and the induction of attaching and effacing (A/E) lesions. Of the SCFAs tested, butyrate markedly enhanced the expression of these virulence-associated genes, even at the low concentration of 1.25 mM, but acetate and propionate showed only a small effect at concentrations higher than 40 mM. Butyrate enhanced the promoter activity of the LEE1 operon, which encodes a global regulator of the LEE genes, Ler. This enhancement was dependent on a regulator, PchA. Butyrate sensing was completely abrogated by the deletion of lrp, the gene for the leucine-responsive regulatory protein, Lrp. Expression of a constitutively active mutant of Lrp enhanced the expression of the LEE genes in the absence of butyrate, and a response-defective Lrp derivative reduced the response to butyrate. Thus, upon entering the distal ileum, EHEC may respond to the higher butyrate level via Lrp by increasing its virulence expression, leading to efficient colonization of the target niche.
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Probiotics prevent enterohaemorrhagic Escherichia coli O157 : H7-mediated inhibition of interferon-γ-induced tyrosine phosphorylation of STAT-1
More LessEnterohaemorrhagic Escherichia coli (EHEC) O157 : H7 inhibits interferon (IFN)-γ-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-1 in epithelial cells. We determined the effects of probiotics on EHEC-mediated disruption of IFN-γ-stimulated STAT-1 activation in epithelial cell lines. Confluent Intestine 407, HEp-2 and Caco-2 epithelial cells were pre-treated (3 h) with either probiotics or surface-layer proteins derived from Lactobacillus helveticus R0052 prior to infection with EHEC O157 : H7 strain CL56 (m.o.i. 100 : 1, 6 h, 37 °C in 5 % CO2). Subsequently, cells were washed and stimulated with human recombinant IFN-γ (50 ng ml−1, 0.5 h, 37 °C) followed by whole-cell protein extraction and immunoblotting for tyrosine-phosphorylated STAT-1. Relative to uninfected cells, STAT-1-activation was reduced after EHEC O157 : H7 infection. Pre-incubation with the probiotic L. helveticus R0052 followed by EHEC infection abrogated pathogen-mediated disruption of IFN-γ–STAT-1 signalling. As determined using Transwell inserts, probiotic-mediated protection was independent of epithelial cell contact. In contrast, pre-incubation with boiled L. helveticus R0052, an equal concentration of viable Lactobacillus rhamnosus R0011, or surface-layer proteins (0.14 mg ml−1) did not restore STAT-1 signalling in EHEC-infected cells. The viable probiotic agent L. helveticus R0052 prevented EHEC O157 : H7-mediated subversion of epithelial cell signal transduction responses.
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Secreted indole serves as a signal for expression of type III secretion system translocators in enterohaemorrhagic Escherichia coli O157 : H7
More LessIndole is produced by tryptophanase during growth of enteric bacteria and accumulates in the culture medium. The physiological role of indole production is poorly understood. We discovered that enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 with a tnaA deletion has decreased secretion of EspA and EspB via the type III secretion system and as a result there is reduced formation of attaching and effacing (A/E) lesions in HeLa cells. Addition of indole restored and enhanced secretion of EspA and EspB and formation of A/E lesions by the tnaA deletion mutant EHEC. Indole addition moderately increased the promoter activity of LEE4 genes, including espA and espB, in the locus of enterocyte effacement. Thus in EHEC indole can serve to signal EspA and EspB expression and secretion and stimulate the ability of EHEC to form A/E lesions on human cells.
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Kinase activity of the dgk gene product is involved in the virulence of Streptococcus mutans
C-terminal deletion of the diacylglycerol kinase (Dgk) homologue of the cariogenic oral bacterium Streptococcus mutans resulted in loss of aciduricity. To confirm the role of the C terminus of the Dgk homologue in aciduricity, various mutants of S. mutans UA159 with a C-terminally truncated Dgk homologue were constructed. The deletion of one or two amino acid residues at the C terminus had no effect on the acid-tolerance properties of mutants. When further amino acid residues at the C terminus were removed, mutants became more acid-sensitive. The mutant with deletion of eight amino acid residues at the C terminus did not grow at pH 5.5, suggesting that the C-terminal tail of the Dgk homologue was indispensable for tolerance to acid stress in S. mutans. Kinase activity assays revealed that deletion of the C-terminal amino acids of Dgk led to a reduction of kinase activity for undecaprenol. A truncated mutant that had completely lost kinase activity was unable to grow at pH 5.5. These results suggest that the acid tolerance of S. mutans is closely related to kinase activity of the Dgk homologue. Additionally, the dgk deletion mutant exhibited markedly reduced levels of smooth-surface carious lesions in pathogen-free rats, despite there being no difference between the mutant and the parental organism in the extent of total smooth surface plaque. The results suggest that Dgk activity may play a direct role in the virulence of S. mutans.
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Production of a unique pneumococcal capsule serotype belonging to serogroup 6
More LessSerogroup 6 of Streptococcus pneumoniae contains three serotypes, named 6A, 6B and 6C, with highly homologous capsule gene loci. The 6A and 6B capsule gene loci consistently differ from each other by only one nucleotide in the wciP gene. The 6A capsule gene locus has a galactosyltransferase, which has been replaced with a glucosyltransferase in the 6C capsule gene locus. We considered that a new serotype named ‘6X1’ would be possible if the galactosyltransferase of the 6B capsule gene locus is replaced with the glucosyltransferase of 6C. We demonstrate that this gene transfer yields a viable pneumococcal strain and that the capsular polysaccharide (PS) from this strain has the predicted chemical structure and serological similarity to the capsular PS of the 6B serotype. The new strain (i.e. serotype 6X1) is typed as 6B by the quellung reaction, but it can be distinguished from 6B strains with mAbs to 6B PS. Reexamination of 264 pneumococcal isolates that had been previously typed as 6B with classical typing methods revealed no isolates expressing serotype 6X1. Nevertheless, this study shows that this capsular PS is biochemically possible and could exist/emerge in nature.
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Induction of morphological changes in Ustilago maydis cells by octyl gallate
More LessThe effects of octyl gallate on Ustilago maydis yeast cells were analysed in relation to its capacity to oxidize compounds (pro-oxidant actions). All phenolic compounds tested inhibited the alternative oxidase (AOX). However, only octyl gallate induced a morphological change in yeast cells and collapsed the mitochondrial membrane potential. In contrast to octyl gallate, propyl gallate and nordihydroguaiaretic acid caused only a negligible cell change and the membrane potential was not affected. Our findings show that structurally related phenolic compounds do not necessarily exert similar actions on target cells. Preincubation of U. maydis cells with trolox inhibited the change to pseudohyphal growth produced by octyl gallate. These results suggest that in addition to the inhibitory action of octyl gallate on the AOX, this compound induces a switch from yeast to a mycelium, probably through the formation of lipid peroxides.
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- Physiology And Biochemistry
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Verification of a topology model of PorT as an integral outer-membrane protein in Porphyromonas gingivalis
More LessPorT is a membrane-associated protein shown to be essential for the maturation and secretion of a class of cysteine proteinases, the gingipains, from the periodontal pathogen Porphyromonas gingivalis. It was previously reported that PorT is located on the periplasmic surface of the inner membrane to function as a chaperone for the maturing proteinases. Our modelling suggested it to be an integral outer-membrane protein with eight anti-parallel, membrane-traversing β-strands. In this report, the outer-membrane localization model was confirmed by the structural and functional tolerance of PorT to hexahistidine (6×His) tag insertions at selected locations within the protein using site-directed mutagenesis. Interestingly, those PorT mutations adversely affecting gingipain secretion enhanced expression of the porT gene but at the same time suppressed the transcription of the gingipain rgpB gene. Further, PorT mutants deficient in gingipain activities produced significantly more di- and triaminopeptidase activities. PorT homologues have been found in restricted members of the Bacteroidetes phylum where there is potential for PorT to participate in the maturation and secretion of proteins with characteristic C-terminal domains (CTDs). Knowledge of the cellular localization of PorT will enable analysis of the role of this protein in a new secretory pathway for the export of gingipains and other CTD-class proteins.
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Phagocytosis, germination and killing of Bacillus subtilis spores presenting heterologous antigens in human macrophages
More LessBacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5 %. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.
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Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum
More LessCorynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, α-glucan phosphorylases were assumed to be involved in glycogen degradation, forming α-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two α-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum ΔmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the ΔmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-α-glucanotransferase (MalQ), glucokinase (Glk) and α-phosphoglucomutase (α-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and β-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and α-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria.
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Cyclopropane fatty acyl synthase in Sinorhizobium meliloti
More LessCyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under Pi starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under Pi starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under Pi starvation. The cfa2 gene was also required for cyclopropanation of non-phosphorus-containing lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.
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The Hog1 MAP kinase controls respiratory metabolism in the fungal pathogen Candida albicans
More LessSignal transduction pathways mediated by mitogen-activated protein kinases (MAPKs) play crucial roles in eukaryotic cells. In the pathogenic fungus Candida albicans the HOG MAPK pathway regulates the response to external stresses (osmotic and oxidative among others) and is involved in morphogenesis and virulence. We show here that the lack of the Hog1 MAPK increases the sensitivity of this fungus to inhibitors of the respiratory chain. hog1 mutants also show an enhanced basal respiratory rate compared to parental strains, and higher levels of intracellular reactive oxygen species despite an increased expression of detoxifying enzymes. We also demonstrate that although oxidative phosphorylation is essentially unaffected, hog1 mutants have an altered mitochondrial membrane potential. Data indicate that hog1-defective mutants are more dependent on mitochondrial ATP synthesis, probably due to an increased cellular ATP demand. Our results therefore link a MAPK pathway with respiratory metabolism in pathogenic fungi.
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The Streptococcus equi prophage-encoded protein SEQ2045 is a hyaluronan-specific hyaluronate lyase that is produced during equine infection
Streptococcus equi causes equine ‘strangles’. Hyaluronate lyases, which degrade connective tissue hyaluronan and chondroitins, are thought to facilitate streptococcal invasion of the host. However, prophage-encoded hyaluronate lyases are hyaluronan-specific and are thought to be primarily involved in the degradation of the hyaluronan capsule of streptococci during bacteriophage infection. To understand the role of prophage-encoded hyaluronate lyases further, we have biochemically characterized such a hyaluronate lyase, SEQ2045 from S. equi, and have shown that it is produced during equine infection. Prophage-encoded hyaluronan-specific hyaluronate lyases may therefore play a more direct role in disease pathogenesis than previously thought.
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Differences between human subjects in the composition of the faecal bacterial community and faecal metabolism of linoleic acid
Conjugated linoleic acid (CLA) is formed from linoleic acid (LA; cis-9,cis-12-18 : 2) by intestinal bacteria. Different CLA isomers have different implications for human health. The aim of this study was to investigate LA metabolism and the CLA isomers formed in two individuals (V1 and V2) with different faecal metabolic characteristics, and to compare fatty acid metabolism with the microbial community composition. LA incubated with faecal samples was metabolized at similar rates with both subjects, but the products were different. LA was metabolized extensively to stearic acid (SA; 18 : 0) in V1, with minor accumulation of CLA and more rapid accumulation of vaccenic acid (VA; trans-11-18 : 1). CLA accumulation at 4 h was almost tenfold higher with V2, and little SA was formed. At least 12 different isomers of CLA were produced from LA by the colonic bacteria from the two individuals. The predominant (>75 %) CLA isomer in V1 was rumenic acid (RA; cis-9,trans-11-18 : 2), whereas the concentrations of RA and trans-10,cis-12-18 : 2 were similar with V2. Propionate and butyrate proportions in short-chain fatty acids were higher in V1. A 16S rRNA clone library from V1 contained mainly Bacteroidetes (54 % of clones), whereas Firmicutes (66 % of clones) predominated in V2. Both samples were devoid of bacteria related to Clostridium proteoclasticum, the only gut bacterium known to metabolize VA to SA. Thus, the CLA formed in the intestine of different individuals may differ according to their resident microbiota, with possibly important implications with respect to gut health.
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Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5
More LessThe transcriptional regulation of three distinct alcohol oxidation systems, alcohol dehydrogenase (ADH)-I, ADH-IIB and ADH-IIG, in Pseudomonas putida HK5 was investigated under various induction conditions. The promoter activities of the genes involved in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC, encoding ADH-I and a cytochrome c, respectively. Primary and secondary C3 and C4 alcohols and butyraldehyde specifically induced the divergent promoters of qbdBA and aldA, encoding ADH-IIB and an NAD-dependent aldehyde dehydrogenase, respectively. The qgdA promoter of ADH-IIG responded well to (S)-(+)-1,2-propanediol induction. In addition, the roles of genes encoding the response regulators exaE and agmR, located downstream of qedA, were inferred from the properties of exaE- or agmR-disrupted mutants and gene complementation tests. The gene products of both exaE and agmR were strictly necessary for qedA transcription. The mutation and complementation studies also suggested a role for AgmR, but not ExaE, in the transcriptional regulation of qbdBA (ADH-IIB) and qgdA (AGH-IIG). A hypothetical scheme describing a regulatory network, which directs expression of the three distinct alcohol oxidation systems in P. putida HK5, was derived.
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Methionine sulphoxide reductases protect iron–sulphur clusters from oxidative inactivation in yeast
More LessMethionine residues and iron–sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of micro-organisms. Here, we show that methionine redox modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxrΔ double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild-type reproduced the Cu-resistance phenotype, and FeS-cluster functions were found to be defective in the mxrΔ mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. 55Fe-labelling studies showed that FeS clusters are turned over more rapidly in the mxrΔ mutant than the wild-type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes in helping to protect the essential function of FeS clusters in aerobic settings.
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PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis
More LessPichia membranifaciens CYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 °C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibit Brettanomyces bruxellensis while Saccharomyces cerevisiae was fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well as P. membranifaciens CYC 1086, was able to inhibit B. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions.
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