New Complementation Constructs for Inducible and Constitutive Gene Expression in Neisseria gonorrhoeae and Neisseria meningitidis

Ramsey, Meghan E.; Hackett, Kathleen T.; Kotha, Chaitra; Dillard, Joseph P.

doi:10.1128/aem.07871-11

Cited by 45 publications

(54 citation statements)

References 66 publications

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“…For these experiments, E. coli strains carried aTc-inducible, C-terminally HA-tagged versions of N. gonorrhoeae amiC or arginase (argJ) as a negative control (37). Along with wildtype amiC and amiC Q316K , we included amiC E229D as an additional negative control, as the cell separation and fragment FIGURE 4. amiC Q316K mutation has increased enzymatic and lytic activity, whereas amiC E229D mutation protects E. coli from lytic activity.…”

Section: Resultsmentioning

confidence: 99%

“…amiC Q316K was amplified using overlap extension by first amplifying a 5Ј portion of amiC with JLDNA14 and JLDNA37, and a 3Ј portion of amiC with JLDNA36 and JLDNA12 and then joining an insert with JLDNA14 and JLDNA12. Each of the above inserts (amiC, amiC E229D , and amiC Q316K ) was digested with SacI and SpeI and cloned into pMR68 (37) to generate pJDL7, pJDL39, and pJDL40, respectively. The resulting plasmids were transformed into chemically competent TAM1 E. coli; Kan r colonies were selected, plasmids prepared, and inserts sequenced to confirm proper inframe assembly and the addition of the C-terminal HA tag.…”

Section: Methodsmentioning

confidence: 99%

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Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Lenz

Stohl

Robertson

et al. 2016

Journal of Biological Chemistry

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The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-L-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc (pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release. Peptidoglycan (PG)2 is a critical structural macromolecule that makes up the cell wall surrounding the cytoplasmic membrane of nearly all bacteria. This cage-like structure is made up of long polysaccharide strands that are composed of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with short peptide stems (3-5 amino acids) covalently attached to the MurNAc moiety (1). Depending on the organism, some proportion of peptide stems are crosslinked to peptides on adjacent strands, whereas others remain non-cross-linked. Cross-linking provides structural integrity for the macromolecule and contributes to the determination of bacterial shape.Neisseria gonorrhoeae (the gonococcus or GC) is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea. Gonorrhea is the second most common reportable bacterial infection in the United States with an estimated 820,000 cases annually (2). A robust inflammatory response is the cause of much of the damage observed during GC infection and is triggered by the release of lipo-oligosaccharide, porin proteins, and fragments of PG (3-5). In particular, N. gonorrhoeae release a greater abundance of PG fragments than Escherichia co...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Lenz

Stohl

Robertson

et al. 2016

Journal of Biological Chemistry

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“…Previously, we showed that the anhydrotetracycline (ATc)-inducible P tet promoter exhibited a broad dose-response curve in response to inducer in gonococci (47). We expressed traK-FLAG3 under the control of P tet at the iga-trpB chromosomal site and grew the resulting strain (MR675) in the presence of increasing amounts of ATc (Fig.…”

Section: Resultsmentioning

confidence: 99%

TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

et al. 2014

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b Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.

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“…The pbpG gene was amplified by PCR using oligonucleotides PBP4-SacI-F (5=-TGA GCT CCA TCC AAA CCG ACA CAC GAC GG) and PBP4-HindIII-R (5=-TTT AAG CTT TCA GGA GCG TTG CTG CAG C) and was digested with SacI and HindIII. The insert was ligated into pMR68 (43); the resulting colonies were selected from plates containing 500 g per ml erythromycin; and the construct was named pRS149 following sequencing.…”

Section: Methodsmentioning

confidence: 99%

Neisseria gonorrhoeae PBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

et al. 2019

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Neisseria gonorrhoeae releases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, Ͼ40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in either dacB, encoding PBP3, or pbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation in dacB caused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminal D-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of both dacB and pbpG eliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by the dacB pbpG mutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth. KEYWORDS NOD1, NOD1 agonist, carboxypeptidase, endopeptidase, penicillinbinding proteins, peptidoglycan Citation Schaub RE, Perez-Medina KM, Hackett KT, Garcia DL, Dillard JP. 2019. Neisseria gonorrhoeae PBP3 and PBP4 facilitate NOD1 agonist peptidoglycan fragment release and survival in stationary phase. Infect Immun

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New Complementation Constructs for Inducible and Constitutive Gene Expression in Neisseria gonorrhoeae and Neisseria meningitidis

Cited by 45 publications

References 66 publications

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

Neisseria gonorrhoeae PBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

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