16 research outputs found

    Cell wall structure and function in lactic acid bacteria

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    House dust mites possess a polymorphic, single domain putative peptidoglycan d,l endopeptidase belonging to the NlpC/P60 Superfamily

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    AbstractA 14kDa protein homologous to the Îł-d-glutamyl-l-diamino acid endopeptidase members of the NlpC/P60 Superfamily has been described in Dermatophagoides pteronyssinus and Dermatophagoides farinae but it is not clear whether other species produce homologues. Bioinformatics revealed homologous genes in other Sarcopteformes mite species (Psoroptes ovis and Blomia tropicalis) but not in Tetranychus urticae and Metaseiulus occidentalis. The degrees of identity (similarity) between the D. pteronyssinus mature protein and those from D. farinae, P. ovis and B. tropicalis were 82% (96%), 77% (93%) and 61% (82%), respectively. Phylogenetic studies showed the mite proteins were monophyletic and shared a common ancestor with both actinomycetes and ascomycetes. The gene encoding the D. pteronyssinus protein was polymorphic and intronless in contrast to that reported for D. farinae. Homology studies suggest that the mite, ascomycete and actinomycete proteins are involved in the catalysis of stem peptide attached to peptidoglycan. The finding of a gene encoding a P60 family member in the D. pteronyssinus genome together with the presence of a bacterial promotor suggests an evolutionary link to one or more prokaryotic endosymbionts

    Bacteriophages and other mobile genetic elements in microbial host adaptation, physiopathology and potential therapy

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    La prĂ©sente thĂšse comprend trois chapitres qui Ă©tudient la maniĂšre dont les Ă©lĂ©ments gĂ©nĂ©tiques mobiles (EGMs), y compris les bactĂ©riophages (phages), peuvent ĂȘtre impliquĂ©s dans (i) l’adaptation des bactĂ©ries Ă  diffĂ©rents mammifĂšres, (ii) le parasitage des bactĂ©ries (par les phages) tout en prĂ©servant l’hĂŽte bactĂ©rien, et (iii) le dĂ©tournement des phages Ă  des fins thĂ©rapeutiques (phagothĂ©rapie) pour traiter les infections bactĂ©riennes. Le premier chapitre s’intĂ©resse au rĂ©cent saut d’espĂšces de la souche humaine de Staphylococcus aureus du Complexe Clonal 8 (CC8) aux bovins, chez qui elle est responsable de mastites. Nous dĂ©montrons que le premier Ă©vĂ©nement responsable de ce saut est l’acquisition d’une cassette chromosomique staphylococcique appelĂ©e SCCbov. SCCbov est un EGM codant pour une nouvelle protĂ©ine de surface appelĂ©e « Adherence-Like Bovine protein » (ADLB) impliquĂ©e dans la pathogenĂšse des mastites. En effet, les souches de CC8 bovines envahissent et tuent les cellules mammaires bovines en culture. Au contraire, l’excision de SCCbov ou l’inactivation d’ADLB diminuent la virulence dans ces conditions. Le second Ă©vĂ©nement du saut est la perte d’un phage insĂ©rĂ© dans le chromosome (ou prophage) qui interrompt le gĂšne de la lipase. La restauration de l’activitĂ© lipase est susceptible de faciliter la croissance des staphylocoques dans le milieu riche en lipides du lait. Le troisiĂšme Ă©vĂ©nement est la perte d’un prophage interrompant le gĂšne de la -hĂ©molysine. La restauration de la -hĂ©molysine promeut la colonisation Ă©pithĂ©liale. Enfin, nous avons construit des souches isogĂ©niques de CC8 comportant diffĂ©rentes combinaisons de ces EGMs, permettant d’étudier le saut d’espĂšce dans une approche « d’évolution inverse ». En conclusion, c’est ce trafic d’EGMs qui est Ă  l’origine du saut des CC8 humains aux bovins. Le second chapitre Ă©tudie la relation structure-fonction et la rĂ©gulation de l’activitĂ© de la lysine du phage PlySK1249 de Streptococcus dysgalactiae. Les phages produisent des lysines en fin de rĂ©plication pour lyser les bactĂ©ries hĂŽtes et libĂ©rer leur progĂ©niture. Cependant, libĂ©rer les lysines dans l’environnement risque de lyser des bactĂ©ries voisines qui portent le mĂȘme phage, ou des bactĂ©ries constituant de nouvelles proies. PlySK1249 a une structure multi- modulaire intrigante, constituĂ©e d’un domaine central de liaison Ă  la paroi bactĂ©rienne, encadrĂ© par un domaine amidase (bactĂ©riolytique) et domaine endopeptidase (CHAP; non- bactĂ©riolytique). Nous dĂ©montrons que cette multi-modularitĂ© remplit trois fonctions. (A) la combinaison des deux domaines enzymatiques et du domaine de liaison est hautement synergique en termes de dĂ©gradation de la paroi et de lyse bactĂ©rienne. (B) le domaine de liaison prĂ©vient la diffusion de la lysine en la gardant liĂ©e aux dĂ©bris de paroi cellulaire aprĂšs la lyse bactĂ©rienne. (C) en prĂ©sence de paroi bactĂ©rienne PlySK1249 est progressivement clivĂ©e et par une protĂ©ase de la paroi bactĂ©rienne (encore non-identifiĂ©e), qui dĂ©samorce son activitĂ© bactĂ©ricide. En rĂ©sumĂ©, la structure multi-modulaire de PlySK1249 implique une rĂ©gulation inter-domaines sophistiquĂ©e, permettant de circonscrire la lyse Ă  la cellule infectĂ©e afin de ne pas compromettre l’intĂ©gritĂ© des cellules avoisinantes. Le troisiĂšme chapitre est une preuve de concept de la phagothĂ©rapie, utilisant un cocktail de 12 phages anti-Pseudomonas aeruginosa et un modĂšle d’endocardite expĂ©rimentale (EE) chez le rat. Les phages se sont rĂ©vĂ©lĂ©s hautement bactĂ©ricides in vitro (perte de >6 logs CFU/ml en 6 h), mais sĂ©lectionnaient des pseudomonas rĂ©sistants. Les phages Ă©taient aussi bactĂ©ricides in vivo (perte de >2 logs CFU/g de vĂ©gĂ©tations en 6 h), mais ne sĂ©lectionnaient pas de rĂ©sistance chez les animaux. L’absence de rĂ©sistance in vivo Ă©tait due au coĂ»t d’adaptation chez l’animal, car elle affectait la synthĂšse des pili et du LPS, deux facteurs de virulence critiques de pseudomonas. Enfin, la combinaison de phages et d’antibiotiques (dans ce cas la ciprofloxacine) s’est rĂ©vĂ©lĂ©e hautement synergique, prĂ©venant la rĂ©sistance in vitro, et dĂ©montrant une frĂ©quence de stĂ©rilisation des vĂ©gĂ©tations cardiaques (>50% en 6 h) sans prĂ©cĂ©dent dans l‘EE Ă  P. aeruginosa. En conclusion, la phagothĂ©rapie combinĂ©e ou non aux antibiotiques ouvre des promesses nouvelles pour le traitement des infections bactĂ©riennes systĂ©miques sĂ©vĂšres. -- The present thesis comes in three chapters studying how mobile genetic elements (MGEs), including bacterial viruses called bacteriophages (phages), may be involved in (i) adaptation of bacteria to different mammalian hosts, (ii) parasiting bacteria (in the case of prophages) while preserving their bacterial hosts, and (iii) new therapeutic applications. The first chapter studied the recent human-to-bovine host jump of typical human Staphylococcus aureus Clonal Complex 8 (CC8) to cowherds, where it causes invasive mastitis. We show that the first event driving the human-to-bovine jump was the acquisition, by human CC8 strains, of a new “staphylococcal cassette chromosome” called SCCbov. SCCbov is a new MGE encoding for a new bacterial surface protein called Adherence-Like Bovine protein (or ADLB), which is implicated in mastitis pathogenesis. Indeed, while parent bovine CC8 readily invaded mammary cells lines, then escaped endosomes, and eventually lysed cultured cells, excision of SCCbov or knocking out ADLB decreased invasiveness and mammary cell death. We then show that the second event of the host-jump was the loss of a phage inserted in the bacterial chromosome (called a prophage), which interrupted the lipase- encoding gene. The loss of this prophage restored S. aureus lipase activity, which is likely to facilitate staphylococcal growth in the lipid-rich milk milieu. The third event was the loss of a ÎČ- hemolysin-interrupting prophage, restoring ÎČ-hemolysin activity, which was shown to promote epithelial colonization. Finally, we constructed isogenic bovine CC8 strains carrying these MGEs in various combinations, which will help determine the host-jump dynamics in a “reverse evolution” approach. Thus, MGE trafficking was the driving force behind the adaptation of human CC8 staphylococci to cows. The second chapter dissected the molecular structure-function activity and regulation of phage lysin PlySK1249 from Streptococcus dysgalactiae. Phages produce lysins at the end of their replication cycle to lyse host bacteria and release their progeny. However, uncontrolled diffusion of lysins in the surrounding might also lyse neighboring bacteria carrying sibling phages or potential new bacterial hosts. PlySK1249 has an intriguing multimodular structure with a central cell wall-binding domain (CWBD) bracketed by a bacteriolytic amidase domain and a non-bacteriolytic endopeptidase (CHAP) domain. We show that this multi-modularity serves a triple purpose. On the one hand, combining the two enzymatic and the CWBD domains led to a synergistic activity in cell wall degradation and bacterial lysis. On other hand, the CWBD prevented lysin diffusion after bacterial lysis by keeping lysin attached to cell wall debris. Finally, we found that in the presence (but not in the absence) of cell wall protein extract, PlySK1249 was subject to proteolytic cleavage by an as yet unknown bacterial wall protease, further introducing posttranscriptional modification in order to control its lytic activity. Thus, the multimodular structure of PlySK1249 implicates sophisticated inter-domain interactions promoting synergistic – yet cell-restricted – lysis, in order to release the phage progeny without jeopardizing neighboring host cells. The third chapter was a proof of concept study of phage therapy, using a cocktail of 12 phages directed against Pseudomonas aeruginosa and a model of P. aeruginosa experimental endocarditis (EE). Phages were highly bactericidal in vitro (loss of >6 logs CFU/ml in 6 h), but selected for phage resistance. Phages were also bactericidal in vivo (loss of >2 logs CFU/g of vegetations in 6 h), but did not select for resistance in animals. The absence of in vivo resistance selection was due to the fitness cost of resistance, which affected synthesis of bacterial pilus or LPS, two P. aeruginosa virulence factors that are critical for in vivo infection. Finally, combining phages with antibiotics (namely ciprofloxacin) was highly synergistic, prevented resistance selection in vitro, and cured notoriously difficult-to-treat EE due to P. aeruginosa at an unprecedented rate, healing >50% of the animals within 6 h of therapy. Thus, thoughtful utilization of phage therapy combined or not with antibiotics might become an alternative to treat severe systemic bacterial infections

    Distinct and Specific Role of NlpC/P60 Endopeptidases LytA and LytB in Cell Elongation and Division of Lactobacillus plantarum

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    Peptidoglycan (PG) is an essential lattice of the bacterial cell wall that needs to be continuously remodeled to allow growth. This task is ensured by the concerted action of PG synthases that insert new material in the pre-existing structure and PG hydrolases (PGHs) that cleave the PG meshwork at critical sites for its processing. Contrasting with Bacillus subtilis that contains more than 35 PGHs, Lactobacillus plantarum is a non-sporulating rod-shaped bacterium that is predicted to possess a minimal set of 12 PGHs. Their role in morphogenesis and cell cycle remains mostly unexplored, except for the involvement of the glucosaminidase Acm2 in cell separation and the NlpC/P60 D, L-endopeptidase LytA in cell shape maintenance. Besides LytA, L. plantarum encodes three additional NlpC/P60 endopeptidases (i.e., LytB, LytC and LytD). The in silico analysis of these four endopeptidases suggests that they could have redundant functions based on their modular organization, forming two pairs of paralogous enzymes. In this work, we investigate the role of each Lyt endopeptidase in cell morphogenesis in order to evaluate their distinct or redundant functions, and eventually their synthetic lethality. We show that the paralogous LytC and LytD enzymes are not required for cell shape maintenance, which may indicate an accessory role such as in PG recycling. In contrast, LytA and LytB appear to be key players of the cell cycle. We show here that LytA is required for cell elongation while LytB is involved in the spatio-temporal regulation of cell division. In addition, both PGHs are involved in the proper positioning of the division site. The absence of LytA activity is responsible for the asymmetrical positioning of septa in round cells while the lack of LytB results in a lateral misplacement of division planes in rod-shaped cells. Finally, we show that the co-inactivation of LytA and LytB is synthetically affecting cell growth, which confirms the key roles played by both enzymes in PG remodeling during the cell cycle of L. plantarum. Based on the large distribution of NlpC/P60 endopeptidases in low-GC Gram-positive bacteria, these enzymes are attractive targets for the discovery of novel antimicrobial compounds

    Determining upper limb kinematics and dynamics during everyday tasks

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    PhD ThesisIn planning orthopaedic procedures or designing joint replacements for the upper limb, detailed knowledge on the kinematic and dynamic behaviour of the shoulder, elbow and wrist joints during the performance of everyday tasks is essential. Previous studies have included kinematic analyses of everyday activities involved in feeding and personal hygiene though none have included both the kinematic and dynamic analyses of these tasks. This study has involved the development, validation and application of experimental methods and analysis techniques, enabling the measurement and modelling of upper limb kinematics and dynamics. A four camera video-based motion analysis system was used to track reflective spheres attached at specific locations on the upper limb and trunk. Novel methods for the definition of the embedded trunk frame and glenohumeral rotation centre were incorporated. Joint attitudes, cadences, angular velocities and angular accelerations were calculated prior to the determination of external forces and moments through the dynamic modelling of the upper limb. The procedures developed have been validated against known measurements and the results of previous studies. These have been applied to obtain kinematic and dynamic data from unimpaired subjects and subjects with shoulder impairment during performance of ten everyday tasks involved in feeding, personal hygiene and the use of everyday objects. Elbow and shoulder flexion were found to be the primary components for the successful completion of the selected tasks. Reaching to the opposite side of the neck was identified as being the most complex of the activities tested in terms of rotation at the shoulder and elbow. Characteristic patterns of motion at the joints of the upper limb were identified during anterior targeted lifting. Differences in performance between the unimpaired and impaired subjects were identified, particularly in the results for cadence and the individual joint velocities and accelerations.Engineering and Physical Sciences Research Council, DePuy Internationa

    Phenotype comparison between wild type <i>L. casei</i> BL23, Lc-p75-negative mutant and complemented Lc-p75 mutant.

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    <p>Pictures of wild type <i>L. casei</i> (A, C, F, H), Lc-p75-negative mutant (B, D, I) and complemented Lc-p75 mutant (E). Colony morphology (A, B), phase contrast microscopy (C, D, E) fluorescence microscopy with merged FM-4-64 (red) and DAPI (blue) staining (F, G) and transmission electron microscopy (H, I).</p

    Influence du métabolisme du peptidoglycane sur les propriétés immunomodulatrices de Lactobacillus casei

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    Le peptidoglycane (PG) est le composant majeur de la paroi des bactĂ©ries Ă  Gram positif. Il assure la forme et l’intĂ©gritĂ© de la cellule bactĂ©rienne. Le PG ou des fragments dĂ©rivĂ©s sont connus pour ĂȘtre des inducteurs du systĂšme d’immunitĂ© innĂ©e de l’hĂŽte, en particulier au travers des rĂ©cepteurs Nod2. Au cours de ce travail, nous avons Ă©tudiĂ© l’influence du mĂ©tabolisme du PG sur les propriĂ©tĂ©s immunomodulatrices de Lactobacillus casei BL23, en Ă©tudiant principalement sa capacitĂ© Ă  moduler la rĂ©ponse des cellules dendritiques humaines. Nous avons tout d’abord caractĂ©risĂ© les hydrolases du PG (PGHs) majeures de L. casei BL23. Une recherche in silico a rĂ©vĂ©lĂ© que L. casei possĂšde un systĂšme de PGHs relativement complexe comprenant treize enzymes putatives avec des domaines catalytiques variĂ©s. Une analyse protĂ©omique d’extraits de paroi de L. casei BL23 a permis de dĂ©tecter la production de sept d’entre elles pendant la croissance bactĂ©rienne. Quatre d’entre elles ont Ă©tĂ© Ă©tudiĂ© plus en dĂ©tails. La PGH la plus fortement exprimĂ©e, Lc-p75, a une activitĂ© de -D-glutamyl-L-lysyl-endopeptidase et est responsable de la sĂ©paration des cellules aprĂšs division. De plus, Lc-p75 associĂ©e Ă  la paroi est localisĂ©e au niveau des septa cellulaires. Il s’agit Ă©galement de l’une des protĂ©ines majeures secrĂ©tĂ©e dans le surnageant de culture de L. casei BL23. Lc-p75 possĂšde la particularitĂ© d’ĂȘtre une glycoprotĂ©ine. La PGH Lc-p40 possĂšde un domaine CHAP douĂ© d’une activitĂ© endopeptidase avec un site de clivage situĂ© au niveau des ponts interpeptidiques du PG. Lc-p40 parait localisĂ©e au niveau de la paroi latĂ©rale des cellules de L. casei. Lc-p45 est une deuxiĂšme -D-glutamyl-L-lysyl-endopeptidase avec un rĂŽle dans le maintien de la forme de la bactĂ©rie. Enfin nous avons caractĂ©risĂ© deux enzymes de prophages, Lc-Lys et Lc-Lys2, codĂ©e par le gĂ©nome de L. casei BL23, qui possĂšde toute deux un domaine de liaison au PG d’un nouveau type qui possĂšde la particularitĂ© de lier spĂ©cifiquement le D-Asp amidĂ© prĂ©sent dans les ponts interpeptidiques du PG de L. casei BL23. La dĂ©lĂ©tion des deux gĂšnes qui codent pour les endopeptidases Lc-p75 et Lc-p45 chez L. casei BL23 conduit Ă  l’absence de disaccharide dipeptide dans la structure du PG du mutant, tandis que la dĂ©lĂ©tion de Lc-p75 seulement conduit Ă  une rĂ©duction de la quantitĂ© du disaccharide-dipeptide. Ce disaccharide dipeptide est un ligand des rĂ©cepteurs Nod2. Les deux mutants obtenus par dĂ©lĂ©tion de Lc-p75 ou bien par dĂ©lĂ©tion des deux endopeptidases ont Ă©tĂ© comparĂ©s avec la souche sauvage BL23 pour leur capacitĂ© Ă  activer in vitro des cellules dendritiques humaines dĂ©rivĂ©es de monocytes sanguins. Suite Ă  l’activation des cellules dendritiques par les souches de L. casei, quatre cytokines pro-inflammatoires, les interleukines IL-6, IL-8, IL-12 et le TNF- ont Ă©tĂ© produites. La quantitĂ© de chaque cytokine sĂ©crĂ©tĂ©e en rĂ©ponse aux mutants simple Lc-p75 et double Lc-p75/Lc-p45 Ă©tait diminuĂ©e par rapport Ă  celle induite par la souche sauvage L. casei BL23.En conclusion, L. casei BL23 est dotĂ© d’un Ă©quipement complexe en PGHs. Les PGHs caractĂ©risĂ©es au cours de ce travail prĂ©sentent des caractĂ©ristiques uniques et jouent un rĂŽle important dans la division des bactĂ©ries ainsi que dans le maintien de leur morphologie. Nos rĂ©sultats indiquent que la souche sauvage de L. casei Bl23 et les mutants dĂ©rivĂ©s obtenus par inactivation d’enzymes Ă  activitĂ© endopeptidase, qui diffĂšrent Ă  la fois au niveau de leur contenu enzymatique ainsi qu’au niveau de la structure de leur PG, ont des effets diffĂ©rents sur les cellules dendritiques humaines, avec un caractĂšre anti-inflammatoire plus Ă©levĂ© pour les mutantsPeptidoglycan (PG) is the major component of the Gram-positive bacteria cell wall. It ensures bacterial cell shape and integrity. PG or PG-derived fragments have been shown to stimulate the host innate immune system, through Nod-2 receptors. In this work, we studied the influence of PG metabolism on immunomodulatory properties of Lactobacillus casei BL23, mainly its ability to modulate the response of human dendritic cells (DCs).We have first characterized the main peptidoglycan hydrolases (PGHs) of L. casei BL23. In silico search revealed that L. casei BL23 has a rather complex PGH complement including thirteen predicted PGHs with various catalytic domains. Proteomic analysis of bacterial cell wall extracts revealed the expression of seven of them during bacterial growth. We characterized four of them in details. Lc-p75 is the major PGH with a Îł-D-glutamyl-L-lysyl-endopeptidase specificity and is responsible for daughter cell separation. Lc-p75 associated to the cell wall localizes at the cell septa. It is also one of the major secreted proteins of L. casei found in culture supernatant. Besides, we showed that L. casei Lc-p75 is a glycosylated protein. Lc-p40 is a PGH with a CHAP-domain endowed with endopeptidase hydrolytic specificity toward peptidoglycan cross-bridges and appears to localize on lateral cell wall. Lc-p45 is a second Îł-D-glutamyl-L-lysyl endopeptidase with a role in cell shape maintenance. We further demonstrated that two prophage endolysins Lc-Lys and Lc-Lys2, encoded in L. casei BL23 genome, share a common novel type peptidoglycan-binding domain that recognizes specifically D-Asn cross-bridge, present in L. casei BL23 peptidoglycan.Deletion of the two endopeptidases, Lc-75 and Lc-p45, resulted in a complete loss ofdisaccharide-dipeptide, which is a ligand of Nod-2 receptor, in the muropeptide structure of L. casei BL23, whereas deletion of Lc-p75 gene led only to a reduction of disaccharide dipeptide. The two PGH-mutants, obtained by deletion of Lc-p75 gene alone or both Lc-p75 and Lc-p45 endopeptidase genes were compared with wild type L. casei BL23 for their capacity to stimulate signaling pathways in vitro in DCs derived from human monocytes. As a consequence of DC activation by L. casei strains, four pro-inflammatory cytokines IL-6, IL-8, IL-12 and TNF-α were produced. The concentrations of secreted cytokines in response to the single Lc-p75 and Lc-p75/p45 double mutant were lower than those induced by wild type L. casei BL23.In conclusion, L. casei BL23 has a complex PGH complement. The PGHs described in this work present unique features and play important role in cell division and morphology of L. casei. Our results indicate that wild type L. casei and endopeptidase-negative mutants, which differ in their PGH content and in their PG structure, have distinct effects on human DCs, with a higher anti-inflammatory character of the endopeptidase-negative mutants

    Analysis of the peptidoglycan hydrolase complement of **Lactobacillus casei** and characterization of the major <tex>\gamma$</tex>-D-glutamyl-L-lysyl-endopeptidase

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    Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties. The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75) was identified as the major one. This protein is the homolog of p75 (Msp1) major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a c-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. A

    Indirect immunofluorescence localization of Strep-tagged Lc-p75 in <i>L. casei</i>.

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    <p>Strep-tagged Lc-p75 was localized in overexpressing strain (A) and in complemented negative mutant (B) with monoclonal antibody directed against Strep-tag as first antibody.</p
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