Listeria monocytogenes est une bactérie à Gram+, ubiquiste, pathogène intracellulaire d'origine alimentaire, responsable chez l'homme, de nombreuses infections telles que les infections foeto-maternelles, des méningo-encéphalites et des septicémies. La bactérie utilise préférentiellement le glucose qui est transporté via le système phosphoenolpyruvate:sucre phsosphotransferase (PTS) et des perméases non-PTS. Les deux principaux transporteurs de glucose chez L. monocytogenes seraient des PTS de la classe mannose. Le premier est codé par l'opéron manLMN (man) et le deuxième, par l'opéron mpoABCD (mpo). Nous avons, dans un premier temps, mis en évidence le transport de glucose par ces PTS chez L. monocytogenes et aussi identifier d'autres transporteurs non-PTS de glucose. Des tests de croissance en milieu minimum (MM) additionné de glucose et des tests de consommation de glucose ont permis de montrer que les mutants ΔmanL (manL code pour l'EIIABMan) et ΔmanM (manM code pour l'EIICMan) utilisent moins vite le glucose que la souche sauvage AML73 ou EGDe (3 à 4 fois moins vite). Le mutant ΔmpoA (mpoA code pour l'EIIAMpo) montre un phénotype similaire à la souche sauvage tandis que le mutant ΔmpoB (mpoB code pour l'EIIBMpo) utilise 4 à 5 fois moins vite le glucose que la souche sauvage. Des tests de qRT-PCR ont par ailleurs permis de montrer que la délétion du gène mpoA permet une expression constitutive de l'opéron man tandis que la délétion du gène mpoB entraîne une inhibition de l'expression de cet opéron. Nous avons aussi montré que l'opéron man est induit par le glucose et l'opéron mpo est exprimé constitutivement. Le PTSMan est le principal système de transport de glucose chez L. monocytogenes et le PTSMpo pourrait fonctionner comme un senseur de glucose qui en présence de ce sucre stimule l'expression de l'opéron man en régulant l'activité de ManR. Le mutant ΔptsI (ptsI code pour la protéine générale EI du PTS) utilise 8 à 10 fois moins vite le glucose que la souche sauvage et présente une très faible expression de l'opéron man. L'utilisation du glucose (bien que faible) par le mutant ΔptsI permet d'affirmer qu'il existerait des transporteurs non-PTS qui permettraient à ce mutant d'utiliser le glucose. Des tests de complémentation hétérologue dans la souche E. coli LJ140 (incapable de transporter le glucose) ont permis de montrer que les trois protéines GlcU (GlcU1, GlcU2 et GlcU3, identifiées par homologie de séquences aux GlcU d'autres firmicutes) permettent le transport de glucose chez L. monocytogenes mais avec une très faible affinité. Un rôle potentiel du PTS et des transporteurs non-PTS dans la régulation de PrfA a également été mis en évidence par des tests de dosage β-D-glucuronidase à partir de cultures bactériennes réalisées en milieux liquides ou sur géloses et aussi par des tests de qRT-PCR (pour l'expression des gènes actA et hly). Ces tests ont été réalisés à partir de la souche L. monocytogenes AML73 (portant la fusion Phly-gus) et des mutants ΔmanL, ΔmanM, ΔmpoB, ΔmpoA, ΔptsI et glcU (construits dans cette souche). Les mutations manL, manM, mpoB, ptsI entraînent une augmentation de l'activité de PrfA (de 2 à 14 fois) et une augmentation de l'expression des gènes de virulence PrfA-dépendants (hly et actA) est également observée dans les mutants ΔmanL, ΔmanM et ΔmpoB. Les mutations glcU et mpoA ne montrent aucun effet sur l'activité de PrfA. Les mutants montrant une forte activité de PrfA contiennent peu ou pas de protéine EIIABMan qui est supposée jouer un rôle dans la régulation de l'activité de PrfA par le glucose. L'effet des mutations PTS observé sur l'expression des gènes de virulence dépend de PrfA car cet effet disparaît quand le gène prfA est délété dans les mutants ΔmanL, ΔmanM et ΔmpoB. Les mutations montrant un effet sur l'activité de PrfA ont également été étudiées in vitro par des infections des cellules épithéliales (Caco-2 et Jeg-3) avec les différents mutants et également in vivo dans la souris. La délétion du gène ptsI montre un effet dans l'infection plus particulièrement dans l'entrée des bactéries dans les cellules

Bacteriocin leader pepides are currently receiving much attention due to their possible functions. It is predicted that these leaders prevent cytoplasmic toxicity within the producer organism by rendering the bacteriocin inactive. Leucocin A, a class IIa bacteriocin produced by Leuconostoc gelidum UAL187-22 is synthesized with a 24 amino acid leader pepide which is cleaved during extracellular translocation. The antimicrobial activity of the leucocin A precursor, pre-leucocin A, was determined to gain insight into whether, the presence of a leader peptide has an impact on anti-listerial activity. The leucocin A and pre-leucocin A genes were generated by PCR of L. gelidum UAL187-22 plasmid DNA. Recombinant plasmids, pLcaA and pPreLcaA were isolated by cloning the amplified genes into the Escherichia coli pMAL.c2 vector, and by screening transformant colonies using blue white selection methods. The malE-LcaA and malE-preLcaA fusion genes were expressed, and resulting maltose binding fusion proteins, were purified using amylose affinity chromatography. Fractions collected, contained partially pure forms of MBP-LcaA (46.433 kDa) and MBP-preLcaA (49.088 kDa) fusion proteins. Following Factor Xa digestion, the MBP affinity tag was removed; and recombinant peptides, leucocin A and pre-leucocin A were further purified by reverese phase high performance liquid chromatography. It was determined that leucocin A was eluted with a retention time of 24.893, while pre-leucocin A was eluted with a retention time of 31.447. Fractions of pure leucocin A and pre-leucocin A were thereafter assayed for activity using a deferred antagonism assay, with Listeria monocytogenes being the indicator strain. Pre-leucocin A tested positive for antimicrobial activity. However, when compared to leucocin A it was found that the leucocin A precursor inhibits Listeria to a lesser degree than leucocin A. The relative bactericidal activities of leucocin A and pre-leucocin A was calculated at 6.0 x 10⁵ AU and 4.0 x 10⁵ AU. Taking this into consideration, it was estimated that the leucocin A precursor is ~66.667 % active as mature leucocin A. Hence the presence of a leader peptide does not have an influence on leucocin A antimicrobial activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407 / Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407 / Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [80]-104).

Freie Universiẗat, Diss.--Berlin, 2000.

Techn. University, Habil.-Schr., 2006--München.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references.