Characterization of <i>Chlamydia</i> MurC‐Ddl, a fusion protein exhibiting D‐alanyl‐D‐alanine ligase activity involved in peptidoglycan synthesis and D‐cycloserine sensitivity

McCoy, Andrea J.; Maurelli, Anthony T.

doi:10.1111/j.1365-2958.2005.04661.x

Cited by 49 publications

(58 citation statements)

References 42 publications

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“…MurE Ct displays maximum activity at pH 8.6. The optimal pH values for the chlamydial enzymes of PG synthesis studied so far are somewhat diverse: 8 to 8.5 for MurC (23), 6 to 8 for Ddl (32), and 6 for MurA (33). In E. coli, the Mur ligases have optimal pH values between 8 and 9.2 (15,26,37,46).…”

Section: Discussionmentioning

confidence: 99%

“…The Chlamydia trachomatis genome contains most of the genes coding for proteins involved in, or associated with, PG synthesis (54). Chlamydial MurA, MurC-Ddl, and the MurC domain of the latter fusion protein are active in vitro and complement Escherichia coli mutants deficient in the respective enzymes (23,32,33). Furthermore, proteomic analysis reveals that the murE gene product, which was assigned as UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid ligase (UDP-MurNAc-L-Ala-D-Glu:meso-A 2 pm ligase), is expressed in RBs (52).…”

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confidence: 99%

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Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

et al. 2009

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Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A 2 pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A 2 pm ligase activity. Recombinant MurE from C. trachomatis (MurE Ct ) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE Ct demonstrated ATP-dependent meso-A 2 pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the LL and DD isomers of A 2 pm, D-lysine) were also recognized by MurE Ct. However, the activities for these amino acid substrates were weaker than that for meso-A 2 pm. The specificity of MurE Ct for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k cat /K m ratios for UDP-MurNAc-L-Ala-D-Glu, UDPMurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A 2 pm. However, due to the lack of specificity of MurE Ct for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

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Section: Discussionmentioning

confidence: 99%

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Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

et al. 2009

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“…The C. trachomatis genome contains other examples of fusion proteins made up of functionally related proteins. For example, MurC-Ddl is a fusion of two enzymes involved in peptidoglycan synthesis (27), and LcrE is a fusion of two type III secretion proteins (12). There is precedent for a membrane-associated transcription factor that can respond to changes in the external environment by altering gene expression.…”

Section: Vol 193 2011mentioning

confidence: 99%

Identification and Functional Analysis of CT069 as a Novel Transcriptional Regulator in Chlamydia

et al. 2011

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Only a small number of transcription factors have been predicted in Chlamydia spp., which are obligate intracellular bacteria that include a number of important human pathogens. We used a bioinformatics strategy to identify novel transcriptional regulators from the Chlamydia trachomatis genome by predicting proteins with the general structure and characteristic functional domains of a bacterial transcription factor. With this approach, we identified CT069 as a candidate transcription factor with sequence similarity at its C terminus to Treponema pallidum TroR. Like TroR, the gene for CT069 belongs to an operon that encodes components of a putative ABC transporter for importing divalent metal cations. However, CT069 has been annotated as YtgC because of sequence similarity at its N terminus to TroC, a transmembrane component of this metal ion transporter. Instead, CT069 appears to be a fusion protein composed of YtgC and a TroR ortholog that we have called YtgR. Although it has not been previously reported, a similar YtgC-YtgR fusion protein is predicted to be encoded by other Chlamydia spp. and several other bacteria, including Bacillus subtilis. We show that recombinant YtgR polypeptide bound specifically to an operator sequence upstream of the ytg operon and that binding was enhanced by Zn 2؉ . We also demonstrate that YtgR repressed transcription from the ytg promoter in a heterologous in vivo reporter assay. These results provide evidence that CT069 is a negative regulator of the ytg operon, which encodes a putative metal ion transporter in C. trachomatis.

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“…For example, the outer membrane of EB contains a poorly immunogenic truncated lipopolysaccharide (LPS) (14, 30) and immunodominant epitopes of the major outer membrane protein (MOMP) vary substantially among closely related chlamydial strains (13). EB also lack detectable peptidoglycan (2, 20, 60), although functional murein biosynthetic enzymes (2,5,16,21,32,43,45,46) are expressed in RB during productive and persistent infection (44). To compensate for the loss of murein, EB are enveloped by a protein P-layer, which lends osmotic stability to the infectious particle (29).…”

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Identification ofChlamydia trachomatisOuter Membrane Complex Proteins by Differential Proteomics

Liu¹,

Afrane

Clemmer³

et al. 2010

J Bacteriol

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The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra-and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosylsoluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.Bacteria in the phylum Chlamydiae are characterized by their complex intracellular developmental cycles. Chlamydiae must assume at least two functionally distinct morphotypes, the intracellular, replicative reticulate body (RB) and the extracellular, infectious elementary body (EB), to replicate and be transmitted to new hosts (50). The divergence of distinct RB and EB forms may have been driven by the different pressures these pathogens face inside host cells during replication and outside host cells during transmission. For example, the outer membrane of EB contains a poorly immunogenic truncated lipopolysaccharide (LPS) (14, 30) and immunodominant epitopes of the major outer membrane protein (MOMP) vary substantially among closely related chlamydial strains (13). EB also lack detectable peptidoglycan (2, 20, 60), although functional murein biosynthetic enzymes (2,5,16,21,32,43,45,46) are expressed in RB during productive and persistent infection (44). To compensate for the loss of murein, EB are enveloped by a protein P-layer, which lends osmotic stability to the infectious particle (29).Attempts to identify components of the P-layer and outer membrane proteins of Chlamydia were advanced by the observation that these layers can be separated from many soluble EB proteins using the detergent N-lauroyl sarcosine (Sarkosyl). Caldwell et al. dubbed the Sarkosyl-insoluble fraction the chlamydial outer membrane complex (COMC) and noted that purified COMC maintained the shape of intact EB and contained a complete outer membrane, and they reported that a single outer membrane protein, MOMP, accounted for more than 60% of total COMC protein content (15). Other studies revealed that the COMC is ...

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Characterization of Chlamydia MurC‐Ddl, a fusion protein exhibiting D‐alanyl‐D‐alanine ligase activity involved in peptidoglycan synthesis and D‐cycloserine sensitivity

Abstract: Summary Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the

Cited by 49 publications

References 42 publications

Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

Identification and Functional Analysis of CT069 as a Novel Transcriptional Regulator in Chlamydia

Identification ofChlamydia trachomatisOuter Membrane Complex Proteins by Differential Proteomics

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