Applied and Environmental Microbiology, Feb 1, 2002
Through the analysis of spontaneous insertion mutants of Lactobacillus sakei L45, a second operon... more Through the analysis of spontaneous insertion mutants of Lactobacillus sakei L45, a second operon involved in lactocin S production was identified and characterized. The new, bicistronic unit, termed lasXY, is situated immediately upstream of the previously characterized nine-open reading frame (ORF) lactocin S operon (lasA-W) and is transcribed in the opposite direction. The proximal of the two newly identified genes, lasX, specifies a 285-residue protein that is similar to a group of proteins with reported gene regulation functions in gram-positive bacteria. It was demonstrated that the lasX mutants have a strongly reduced level of lasA and lasA-W mRNA, thus indicating the likely cause of the Bac ؊ phenotype of these mutants. The second ORF in the operon, lasY, specifies a 300-residue ABC transporter homolog, the function of which is currently obscure. Transcription initiation mapping of the lasXY operon demonstrates that the two lactocin S promoters overlap such that both transcripts initiate within the ؊35 region of the oppositely oriented promoter. This organization of promoters is unique among this group of regulons and may constitute a modulatory site in the proposed LasX-dependent expression of lasA and downstream genes.
Antimicrobial Agents and Chemotherapy, Jun 1, 2005
Bacterial strain and growth conditions. The bacterium studied was E. faecalis V583 (V583), which ... more Bacterial strain and growth conditions. The bacterium studied was E. faecalis V583 (V583), which was the first vancomycin-resistant clinical isolate reported in the United States (24) and whose genome sequence has been completed (20). V583 samples were grown overnight in flasks with brain heart infusion (BHI) medium (Difco) at 37°C on a rotary shaker (300 rpm). Cultures were then diluted 50ϫ and grown in BHI, as described above, for 1 h. Cultures were split in two, and erythromycin (Sigma) was added to one of the cultures. The final concentration of erythromycin was 50 g/ml. The two cultures (BHI and BHI plus erythromycin) were then incubated further, and 3-ml samples of each culture were collected immediately after the addition of erythromycin (t0), and after 15 (t15), 30 (t30), 60 (t60), and 90 (t90) min. Growth of the cultures was monitored spectrophotometrically (optical density at 600 nm [OD 600 ]). Samples were centrifuged for 5 min at 4°C, and pellets were flash frozen in liquid N 2 prior to RNA extraction. Construction of V583 microarrays. PCR primers for all V583 open reading frames (The Institute for Genomic Research, May 2002 update) were designed using Primer3 (freeware; .cgi), which has commonly been used for primer design during microarray construction (3, 13). Primers were designed to amplify full-length or ϳ500-bp amplicons from genomic DNA. PCRs (50-l reaction volume) were run with 50 mM KCl; 10 mM Tris-HCl (pH 9.0); 0.1% Triton X-100; 2.5 mM MgCl 2 ; 2 M of each primer; 0.2 mM of each dATP, dCTP, dGTP, and dTTP; 10 to 20 ng genomic DNA; and 1.25 U Taq polymerase (Promega). Amplifications were performed with Perkin-Elmer 9600 thermocyclers with an initial denaturation at 94°C for 3 min and then 35 cycles of denaturation at 94°C for 1 min, annealing at 54 to 56°C for 1 min (the annealing temperature varied depending on the melting temperatures of the primers), and an extension at 74°C for 1 min. The PCR was finalized with incubation at 74°C for 10 min. The quality of the PCR products was assessed by agarose gel electrophoresis. The PCR products were cleaned up using a QIAquick 96 PCR purification kit (QIAGEN). Of a total of 3,337 predicted open reading frames in the V583 genome, 3,160 PCR products were obtained. The purified PCR products were eluted in 3ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.01% sodium dodecyl sulfate (SDS) and printed on Corning UltraGaps slides. All PCR products, representing the predicted V583 open reading frames, were printed in five copies on the slides. In addition, negative controls (three genes from Arabidopsis thaliana, buffer [3ϫ SSC, 0.01% SDS], and empty spots) and digested genomic DNA from V583
Applied and Environmental Microbiology, Apr 1, 2007
We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic ... more We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the "test" strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.
Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage fLC3 (32, 172 b... more Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage fLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs). Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function. fLC3 contained a unique replication module and orf201 was identified as the putative replication initiator proteinencoding gene. fLC3 was closely related to the L. lactis r1t phage (73% DNA identity). Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively. fLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange. Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages. These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination. The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.
HindIlI restriction fragment containing the structural gene of plantaricin A was cloned and seque... more HindIlI restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed p1nA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, pinC and pinD) downstream ofp1nA. The DNA sequences ofplnB, plnC, and plnD revealed that their products closely resemble
Journal of Industrial Microbiology & Biotechnology, Sep 1, 2013
Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA... more Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36cderived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZaA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.
SCIENTIFIC OPINION Applications (EFSA-GMO-RX-MON810) for renewal of authorisation for the continu... more SCIENTIFIC OPINION Applications (EFSA-GMO-RX-MON810) for renewal of authorisation for the continued marketing of (1) existing food and food ingredients produced from genetically modified insect resistant maize MON810; (2) feed consisting of and/or containing maize MON810, including the use of seed for cultivation; and of (3) food and feed additives, and feed materials produced from maize MON810, all under Regulation (EC) No 1829/2003
Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedne... more Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published for Staphylococcus aureus (A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672-1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer.
Applied and Environmental Microbiology, Oct 1, 2000
A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named... more A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.
Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL5... more Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63: 4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above.
Antimicrobial Agents and Chemotherapy, Sep 1, 2005
A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is prese... more A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is presented. This is the first study describing a complete transcriptional profile of Enterococcus. E. faecalis is a common and nonvirulent bacterium in many natural environments, but also an important cause of nosocomial infections. We have used a genome-wide microarray based on the genome sequence of V583 to study gene expression in cells exposed to erythromycin. V583 is resistant to relatively high concentrations of erythromycin, but growth is retarded by the treatment. The effect of erythromycin treatment on V583 was studied by a time course experiment; samples were extracted at five time points over a period of 90 min. A drastic change in gene transcription was seen with the erythromycin-treated cells compared to the untreated cells. Altogether, 260 genes were down-regulated at one or more time points, while 340 genes were up-regulated. Genes encoding hypothetical proteins and genes encoding transport and binding proteins were the two most dominating groups of differentially expressed genes. The gene encoding ermB (EFA0007) was expressed, but not differentially, which indicated that other genes are important for the survival and growth maintenance of V583 treated with erythromycin. One of these genes is a putative MsrC-like protein, which was up-regulated at all time points studied. Other specific genes that were found to be up-regulated were genes encoding ABC transporters and two-component regulatory systems, and these may be genes that are important for the specific response of V583 to erythromycin.
Applied and Environmental Microbiology, Sep 15, 2007
Resistance to bile is a prerequisite property of the gastrointestinal bacterial flora. Bile acids... more Resistance to bile is a prerequisite property of the gastrointestinal bacterial flora. Bile acids are powerful detergents, and resistance to sodium dodecyl sulfate (SDS) has therefore often been considered relevant to studies of bile resistance. We have studied the effects of bovine bile (BB) and SDS on Enterococcus faecalis V583 by traditional growth studies and microarrays. Transcriptional responses were studied by time course experiments. In the presence of BB (V583-BB) or SDS (V583-SDS), 308 and 209 genes were identified as differentially expressed at one or more time points, respectively. In V583 treated with both BB and SDS (V583-BB-SDS), 254 genes showed differential expression. Detergents exert their toxic effects primarily on the microbial membrane. The enrichment of differentially transcribed genes that encode proteins with membrane-associated functions and/or locations indicates a major impact of all three treatments on the integrity and functionality of the cell membrane. Two gene clusters involved in fatty acid biosynthesis were repressed in V583-BB and V583-BB-SDS and partly induced in V583-SDS. Furthermore, two EmrB/QacA family drug resistance transporters and a vacuolar-type ATPase were induced in V583-BB and V583-BB-SDS. None of the putative bile salt hydrolase homologs in V583 showed differential expression during the bile treatments. The transcriptional profile of V583-BB-SDS was qualitatively more similar to the response in V583-BB than to that in V583-SDS, suggesting that the presence of bile suppresses the effects of SDS in V583-BB-SDS. The overall results presented here indicate that different mechanisms are involved in detergent resistance in E. faecalis.
Applied and Environmental Microbiology, Feb 1, 2002
Through the analysis of spontaneous insertion mutants of Lactobacillus sakei L45, a second operon... more Through the analysis of spontaneous insertion mutants of Lactobacillus sakei L45, a second operon involved in lactocin S production was identified and characterized. The new, bicistronic unit, termed lasXY, is situated immediately upstream of the previously characterized nine-open reading frame (ORF) lactocin S operon (lasA-W) and is transcribed in the opposite direction. The proximal of the two newly identified genes, lasX, specifies a 285-residue protein that is similar to a group of proteins with reported gene regulation functions in gram-positive bacteria. It was demonstrated that the lasX mutants have a strongly reduced level of lasA and lasA-W mRNA, thus indicating the likely cause of the Bac ؊ phenotype of these mutants. The second ORF in the operon, lasY, specifies a 300-residue ABC transporter homolog, the function of which is currently obscure. Transcription initiation mapping of the lasXY operon demonstrates that the two lactocin S promoters overlap such that both transcripts initiate within the ؊35 region of the oppositely oriented promoter. This organization of promoters is unique among this group of regulons and may constitute a modulatory site in the proposed LasX-dependent expression of lasA and downstream genes.
Antimicrobial Agents and Chemotherapy, Jun 1, 2005
Bacterial strain and growth conditions. The bacterium studied was E. faecalis V583 (V583), which ... more Bacterial strain and growth conditions. The bacterium studied was E. faecalis V583 (V583), which was the first vancomycin-resistant clinical isolate reported in the United States (24) and whose genome sequence has been completed (20). V583 samples were grown overnight in flasks with brain heart infusion (BHI) medium (Difco) at 37°C on a rotary shaker (300 rpm). Cultures were then diluted 50ϫ and grown in BHI, as described above, for 1 h. Cultures were split in two, and erythromycin (Sigma) was added to one of the cultures. The final concentration of erythromycin was 50 g/ml. The two cultures (BHI and BHI plus erythromycin) were then incubated further, and 3-ml samples of each culture were collected immediately after the addition of erythromycin (t0), and after 15 (t15), 30 (t30), 60 (t60), and 90 (t90) min. Growth of the cultures was monitored spectrophotometrically (optical density at 600 nm [OD 600 ]). Samples were centrifuged for 5 min at 4°C, and pellets were flash frozen in liquid N 2 prior to RNA extraction. Construction of V583 microarrays. PCR primers for all V583 open reading frames (The Institute for Genomic Research, May 2002 update) were designed using Primer3 (freeware; .cgi), which has commonly been used for primer design during microarray construction (3, 13). Primers were designed to amplify full-length or ϳ500-bp amplicons from genomic DNA. PCRs (50-l reaction volume) were run with 50 mM KCl; 10 mM Tris-HCl (pH 9.0); 0.1% Triton X-100; 2.5 mM MgCl 2 ; 2 M of each primer; 0.2 mM of each dATP, dCTP, dGTP, and dTTP; 10 to 20 ng genomic DNA; and 1.25 U Taq polymerase (Promega). Amplifications were performed with Perkin-Elmer 9600 thermocyclers with an initial denaturation at 94°C for 3 min and then 35 cycles of denaturation at 94°C for 1 min, annealing at 54 to 56°C for 1 min (the annealing temperature varied depending on the melting temperatures of the primers), and an extension at 74°C for 1 min. The PCR was finalized with incubation at 74°C for 10 min. The quality of the PCR products was assessed by agarose gel electrophoresis. The PCR products were cleaned up using a QIAquick 96 PCR purification kit (QIAGEN). Of a total of 3,337 predicted open reading frames in the V583 genome, 3,160 PCR products were obtained. The purified PCR products were eluted in 3ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.01% sodium dodecyl sulfate (SDS) and printed on Corning UltraGaps slides. All PCR products, representing the predicted V583 open reading frames, were printed in five copies on the slides. In addition, negative controls (three genes from Arabidopsis thaliana, buffer [3ϫ SSC, 0.01% SDS], and empty spots) and digested genomic DNA from V583
Applied and Environmental Microbiology, Apr 1, 2007
We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic ... more We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the "test" strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.
Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage fLC3 (32, 172 b... more Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage fLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs). Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function. fLC3 contained a unique replication module and orf201 was identified as the putative replication initiator proteinencoding gene. fLC3 was closely related to the L. lactis r1t phage (73% DNA identity). Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively. fLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange. Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages. These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination. The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.
HindIlI restriction fragment containing the structural gene of plantaricin A was cloned and seque... more HindIlI restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed p1nA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, pinC and pinD) downstream ofp1nA. The DNA sequences ofplnB, plnC, and plnD revealed that their products closely resemble
Journal of Industrial Microbiology & Biotechnology, Sep 1, 2013
Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA... more Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36cderived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZaA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.
SCIENTIFIC OPINION Applications (EFSA-GMO-RX-MON810) for renewal of authorisation for the continu... more SCIENTIFIC OPINION Applications (EFSA-GMO-RX-MON810) for renewal of authorisation for the continued marketing of (1) existing food and food ingredients produced from genetically modified insect resistant maize MON810; (2) feed consisting of and/or containing maize MON810, including the use of seed for cultivation; and of (3) food and feed additives, and feed materials produced from maize MON810, all under Regulation (EC) No 1829/2003
Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedne... more Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published for Staphylococcus aureus (A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672-1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer.
Applied and Environmental Microbiology, Oct 1, 2000
A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named... more A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.
Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL5... more Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63: 4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above.
Antimicrobial Agents and Chemotherapy, Sep 1, 2005
A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is prese... more A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is presented. This is the first study describing a complete transcriptional profile of Enterococcus. E. faecalis is a common and nonvirulent bacterium in many natural environments, but also an important cause of nosocomial infections. We have used a genome-wide microarray based on the genome sequence of V583 to study gene expression in cells exposed to erythromycin. V583 is resistant to relatively high concentrations of erythromycin, but growth is retarded by the treatment. The effect of erythromycin treatment on V583 was studied by a time course experiment; samples were extracted at five time points over a period of 90 min. A drastic change in gene transcription was seen with the erythromycin-treated cells compared to the untreated cells. Altogether, 260 genes were down-regulated at one or more time points, while 340 genes were up-regulated. Genes encoding hypothetical proteins and genes encoding transport and binding proteins were the two most dominating groups of differentially expressed genes. The gene encoding ermB (EFA0007) was expressed, but not differentially, which indicated that other genes are important for the survival and growth maintenance of V583 treated with erythromycin. One of these genes is a putative MsrC-like protein, which was up-regulated at all time points studied. Other specific genes that were found to be up-regulated were genes encoding ABC transporters and two-component regulatory systems, and these may be genes that are important for the specific response of V583 to erythromycin.
Applied and Environmental Microbiology, Sep 15, 2007
Resistance to bile is a prerequisite property of the gastrointestinal bacterial flora. Bile acids... more Resistance to bile is a prerequisite property of the gastrointestinal bacterial flora. Bile acids are powerful detergents, and resistance to sodium dodecyl sulfate (SDS) has therefore often been considered relevant to studies of bile resistance. We have studied the effects of bovine bile (BB) and SDS on Enterococcus faecalis V583 by traditional growth studies and microarrays. Transcriptional responses were studied by time course experiments. In the presence of BB (V583-BB) or SDS (V583-SDS), 308 and 209 genes were identified as differentially expressed at one or more time points, respectively. In V583 treated with both BB and SDS (V583-BB-SDS), 254 genes showed differential expression. Detergents exert their toxic effects primarily on the microbial membrane. The enrichment of differentially transcribed genes that encode proteins with membrane-associated functions and/or locations indicates a major impact of all three treatments on the integrity and functionality of the cell membrane. Two gene clusters involved in fatty acid biosynthesis were repressed in V583-BB and V583-BB-SDS and partly induced in V583-SDS. Furthermore, two EmrB/QacA family drug resistance transporters and a vacuolar-type ATPase were induced in V583-BB and V583-BB-SDS. None of the putative bile salt hydrolase homologs in V583 showed differential expression during the bile treatments. The transcriptional profile of V583-BB-SDS was qualitatively more similar to the response in V583-BB than to that in V583-SDS, suggesting that the presence of bile suppresses the effects of SDS in V583-BB-SDS. The overall results presented here indicate that different mechanisms are involved in detergent resistance in E. faecalis.
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Papers by Ingolf Nes