Peptidoglycan Remodeling in Mycobacterium tuberculosis: Comparison of Structures and Catalytic Activities of RipA and RipB

Both, D.; Schneider, Günter; Schnell, R.

doi:10.1016/j.jmb.2011.08.014

Cited by 55 publications

(66 citation statements)

References 38 publications

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“…Therefore, we designated the sagA alleles in HLL9 and HLL28 sagA2 and sagA3, respectively. In other Gram-positive bacteria, the SagA protein has been shown to function as a peptidoglycan endopeptidase that plays a role in septum formation and maintaining cell shape (17)(18)(19)(20). It cleaves the bond between D-Glu and meso-diaminopimelic acid in the peptide cross-link between the polysaccharide chains ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The FUGUE structural prediction software (http://tardis.nibio .go.jp/fugue/prfsearch.html) found that the closest structural homolog available is the C-terminal domain of the rv1477 protein (also designated RipA) from Mycobacterium tuberculosis (17). Originally identified as a gene product necessary for invasion and intracellular replication (19), rv1477 was later found to be a cell wall hydrolase similar in function to SagA homologs in the Firmicutes (25).…”

Section: Discussionmentioning

confidence: 99%

“…S3 in the supplemental material). This region contains the NLP/P60 cell wall hydrolase domain which catalyzes cleavage of the peptide bond between meso-DAP and D-glutamate in the peptide cross-link between glycan strands (17). In Mycobacterium smegmatis, a ripA mutant forms long chains and branches (25).…”

Section: Discussionmentioning

confidence: 99%

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Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

Liu¹,

Bouillaut²,

Sonenshein³

et al. 2012

Journal of Bacteriology

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bClostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFPassociated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility. Clostridium perfringens is a Gram-positive anaerobic bacteria that causes a wide variety of diseases in humans and animals, including acute food poisoning and gas gangrene (1). We have discovered that C. perfringens and other clostridia have the ability to produce type IV pili (TFP), a feature that was previously thought to be confined to Gram-negative bacteria (2). C. perfringens lacks flagella and flagellum-mediated swimming but can move across an agar medium with a unique type of gliding motility, in which curvilinear flares of densely packed cells move away from a colony (2). The flares are themselves composed of filaments of individual bacteria lined up in an end-to-end orientation. This orientation is essential for gliding motility; cells that are randomly oriented do not form the filaments and flares seen in motile cells (2; see Video S1 in the supplemental material). The bacteria within a filament can clearly be seen growing and dividing as the filament extends, suggesting that growth and division of individual bacteria provide at least some of the force necessary for movement across the surface of the agar (2; see Video S1). We also noted that the curvilinear flares move away from the colony in the direction of the long axis of the cells lined up within the filaments but eventually, due to their curvilinear nature, collide, leaving a region surrounded by C. perfringens cells...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

Liu¹,

Bouillaut²,

Sonenshein³

et al. 2012

Journal of Bacteriology

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“…30 Likewise, a separate study showed that mutation of glutamic acid to alanine in Rv1477 inactivates catalysis by changing the spatial configuration of Cys383. 31 However, no one has examined Rv1478 or its counterpart, MAP_1204, using mutational substitutions.…”

Section: Discussionmentioning

confidence: 99%

“…The binding assay was performed essentially as described previously. 30 Briefly, insoluble trichloroacetic acid treated peptidoglycan (1 mg/mL) from Staphylococcus aureus (Sigma chemicals) or Bacillus subtilis (Sigma chemicals) was suspended in binding buffer (0.15M NaCl and 25mM Na-Hepes, pH 7.0) and incubated with 0.2 mg/mL of each recombinant protein or the positive and negative controls, hen egg white lysozyme (HEWL) and bovine serum albumin (BSA), all of which were dialyzed in binding buffer. The proteinpeptidoglycan mixture was incubated for 5 min at 228C to allow binding.…”

Section: Peptidoglycan Binding Assaymentioning

confidence: 99%

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

et al. 2016

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A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases. Keywords: Mycobacterium; Johne's disease; crystal structure; peptidoglycan; proteins; antigens; paratuberculosis Additional Supporting Information may be found in the online version of this article.Importance: Johne's disease in ruminant livestock is caused by the bacterium Mycobacterium avium subspecies paratuberculosis. This research describes the functional aspects of two proteins that show promise in a subunit vaccine for Johne's disease. Through crystal structure determination and amino acid modification, we demonstrate that although both proteins have a similar structure, one of them lacked hydrolytic activity on peptidoglycan. We show that a specific amino acid is likely responsible for this lack of hydrolytic activity.

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Dermatophagoides pteronyssinus lytFM encoding an NlpC/P60 endopeptidase is also present in mite‐associated bacteria that express LytFM variants

2017

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The bodies and faecal pellets of the house dust mite ( HDM ), Dermatophagoides pteronyssinus , are the source of many allergenic and nonallergenic proteins. One of these, the 14‐ kD a bacteriolytic enzyme Lyt FM , originally isolated from the spent HDM growth medium, may contribute to bacteriolytic activity previously detected by zymography at 14 kD a in the culture supernatants of some bacterial species isolated from surface‐sterilised HDM . Based on previously reported findings of lateral gene transfer between microbes and their eukaryotic hosts, we investigated the presence of lyt FM in the genomes of nine Gram‐positive bacteria from surface‐sterilised HDM , and the expression by these isolates of Lyt FM and its variants Lyt FM 1/Lyt FM 2. The lyt FM gene was detected by PCR in the genomes of three of the isolates: Bacillus licheniformis strain 1, B. licheniformis strain 2 and Staphylococcus aureus . Expression of the variant Lyt FM 1 was detected in culture supernatants of these bacteria by mass spectrometry ( MS ) and ELISA , and the bacterial Lyt FM proteins were shown by zymography to be able to hydrolyse peptidoglycan. Our previous reports of Lyt FM homologues in other mite species and their phylogenetic analysis had suggested that they originated from a common mite ancestor. The phylogenetic analysis reported herein and the detection of other D. pteronyssinus proteins by MS in the culture supernatants of the three isolates that secreted Lyt FM 1 further support the hypothesis of lateral gene transfer to the bacterial endosymbionts from their HDM host. The complete sequence homology observed between the genes amplified from the microbes and those in their eukaryotic host indicated that the lateral gene transfer was an event that occurred recently.

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Peptidoglycan Remodeling in Mycobacterium tuberculosis: Comparison of Structures and Catalytic Activities of RipA and RipB

Cited by 55 publications

References 38 publications

Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

Dermatophagoides pteronyssinus lytFM encoding an NlpC/P60 endopeptidase is also present in mite‐associated bacteria that express LytFM variants

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