MurA Protein Research Articles

In silico assessments of bioactive compounds from Dayak onion (Eleutherine bulbosa) as antibacterial agents targeting UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA)

Antibiotic resistance is a formidable global health challenge, a concern that also occurs in Indonesia. Dayak onion (Eleutherine bulbosa), a traditional medicinal plant, holds promising potential as a natural remedy. This study focused on in silico assessment on the antibacterial potential of Dayak onion metabolites as future antibiotics. MurA protein (PDB ID: 1UAE) was selected as the target protein. Eleuthoside C displayed the most favorable binding with a remarkably low ΔG value of -10.5 kcal/mol, denoting robust stability, followed by Eleutherinoside A (-10.1 kcal/mol) and 2,5-dimethyl-10-hydroxynaphtopyrone 8-O-β-glucopyranoside (-10.0 kcal/mol). In contrast, the standard drug fosfomycin showed a lower binding affinity (-4.6 kcal/mol). Eleuthoside C demonstrated 100% of binding site similarity (%BSS), closely followed by Eleuthoside B (91.30%), Eleutherinoside A (78.26%) and Eleutherinoside B (78.26%), with the main target to Cys115. The druglikeness criteria was only fulfilled by Eleutherinoside A, yet being proposed as the alternative lead compound for antibiotic treatment of pathogenic bacteria.

Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis.

We implemented a unique strategy to construct a recombinant attenuated Edwardsiella vaccine (RAEV) with a biological containment phenotype that causes regulated bacterial cell wall lysis. This process ensures that the vaccine strain is not able to persist in the environment. The murA gene is responsible for the catalysis of one of the first steps in the biosynthesis of muramic acid, which is a crucial component of the bacterial cell wall. The regulated lysis phenotype was achieved by inserting the tightly regulated araC ParaBAD cassette in place of the chromosomal murA promoter. Strains with this mutation require growth media supplemented with arabinose in order to survive. Without arabinose, they are unable to synthesize the peptidoglycan cell wall. Following the colonization of fish lymphoid tissues, the murA protein is no longer synthesized due to the lack of arabinose. Lysis is subsequently achieved in vivo, thus preventing the generation of disease symptoms and the spread of the strain into the environment. Vaccine strain χ16016 with the genotype ΔPmurA180::TT araC ParaBADmurA is attenuated and shows a higher LD50 value than that of the wild-type strain. Studies have demonstrated that χ16016 induced TLR4, TLR5, TLR8, TLR9, NOD1 and NOD2-mediated NF-κB pathways and upregulated the gene expression of various cytokines, such as il-8, il-1β, tnf-a, il-6 and ifn-γ in catfish. We observed significant upregulation of the expression profiles of cd4, cd8 and mhc-II genes in different organs of vaccinated catfish. Vaccine strain χ16016 induced systemic and mucosal IgM titers and conferred significant protection to catfish against E. piscicida wild-type challenge. Our lysis RAEV is the first live attenuated vaccine candidate designed to be used in the aquaculture industry that displays this biological containment property.

Asp50Glu mutation in MurA results in fosfomycin resistance in Enterococcus faecium

Enterococcus faecium is one of the important pathogens causing nosocomial infection, which can be resistant to fosfomycin by obtaining the plasmid-encoded fosfomycin resistance genes, and the mutation of MurA protein encoded by chromosome is a newly discovered fosfomycin resistance mechanism in recent years. In this study, we found a fosfomycin-resistant clinical isolate of E. faecium Efm_1415 with fosfomycin MIC of 512 mg/L, carrying Asp50Glu mutant of MurA protein, which was never reported before. To study the role and mechanism of this mutant protein in fosfomycin resistance, we used gene cloning, protein expression, and purification, steady-state kinetic, fosfomycin inhibition assay, and next-generation sequencing (NGS) to investigate the functions, characters, and enzymatic kinetic properties of MurA protein. The results revealed that the Asp50Glu MurA can mediate a 4-fold increase in the fosfomycin MIC of the host bacteria. Compared with the wild-type MurA, the affinity of the Asp50Glu MurA to the substrates was increased, and the enzyme activity cannot be inhibited by the concentration of fosfomycin less than 100 mg/L. The research on the mutant MurA had gained a new understanding of the fosfomycin resistance mechanisms and helped to find new antibiotics with MurA enzyme as the target of action.

Growth promotion in Corynebacterium glutamicum by overexpression of the NCgl2986 gene encoding a protein homologous to peptidoglycan amidases.

We previously reported the extracellular production of antibody fragment Fab by Corynebacterium glutamicum. In the course of searching for genes which improve the secretion efficiency of Fab, we coincidentally found that the final growth increased significantly when the NCgl2986 gene encoding an amidase-like protein was overexpressed. This effect was observed when cells were grown on the production medium MMTG, which contains high concentrations of glucose and neutralizing agent CaCO3, but not on MMTG without CaCO3 or Lennox medium. Not only turbidity but also dry cell weight was increased by NCgl2986 overexpression, although the growth rate was not affected. It was recently reported that the Mycobacterium tuberculosis homolog Rv3915 functions as an activator of MurA protein, which catalyzes the initial step of peptidoglycan synthesis. Growth promotion was also observed when the MurA protein was overproduced. His-tagged NCgl2986 protein was purified, but its peptidoglycan hydrolyzing activity could not be detected. These results suggest that NCgl2986 promotes cell growth by activating the peptidoglycan synthetic pathway.

Characterization of mycobacterial UDP-N-acetylglucosamine enolpyruvyle transferase (MurA)

The mycobacterial peptidoglycan has structure and biosynthetic pathways to similar those of other bacteria. UDP-N-acetylglucosamine enolpyruvyle transferase (MurA) catalyzes the first reaction in the biosynthesis of peptidoglycan. The MurA enzyme has been identified from various bacterial species, but the in-depth biochemical properties of mycobacterial MurA have not been characterized. In this study, both Mycobacterium tuberculosis MurA protein and Mycobacterium smegmatis MurA protein were overexpressed in Escherichia coli and purified by affinity chromatography. MurA activity was detected by HPLC. A colorimetric assay of MurA activity was also developed and the kinetic properties of Mtb MurA and Msm MurA were determined using this colorimetric assay. A conditional murA gene knockout strain was constructed by DNA homologous recombination. The disruption of murA in the genome of M. smegmatis led to loss of viability at a non-permissive temperature. Drastic morphological and structural alterations in the M. smegmatis murA knockout strain were observed by scanning electron microscopy and transmission electron microscopy. These results demonstrated that murA was an essential gene for growth of M. smegmatis. Therefore, MurA is a potential target for developing new anti-tuberculosis drugs.

Cell hybridization technology produces monoclonal antibodies against MurA protein of Listeria monocytogenes (P3132)

Abstract The research aims to develop and characterize monoclonal antibodies(MAbs) with high specificity and affinity for surface antigens of all major serotypes of Listeria monocytogenes(L. monocytogenes). Hybridomas were produced using extraction of L. monocytogenes surface protein as the immunogen. Detected by enzyme-linked-immunosorbent assay (ELISA), MAb1B10 was capable of binding to cell-surface antigens of live L. monocytogenes. MAb1B10, 2C10 and 1G3 reacted with both formalin-inactivated and heat-killed L. monocytogenes without any cross reactions with other Listeria spp.; MAb1C10 and 3G8 highly reacted with L. monocytogenes, L. innocua and L. welshimeri, but seldom reacted with non-Listeria spp.. Immunoprecipitation-mass spectrometry (IP-MS) and western blot were applied to convince that the reactive epitope was on the MurA protein of 72kDa. Recombinant MurA protein produced in Escherichia coli was confirmed to be the antigen which was recognized by MAb1B10, 2C10 and 1G3. To sum up, MAbs reacting with live L. monocytogenes, formalin-inactivated and heat-killed L. monocytogenes were produced. The results indicated that MurA could be exploited as a marker for specifically identifying pathogenic L. monocytogenes in suspect food samples.

Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

Streptococcus mutans (S. mutans) is the primary etiological agent of human dental caries. A gene (SMU_1525) encoding MurA was identified in S. mutans UA159 (ATCC700610). The deduced amino-acid sequence has homology (51% identity) with E. coli MurA protein (AAC76221). In this study, we cloned S. mutans murA (SMU_1525) gene, expressed soluble recombinant MurA protein in E. coli BL21(DE3). The recombinant protein was purified by affinity chromatography. The molecular weight of expressed protein was about 45.6 kD. The activity of the protein was identified by high-pressure liquid chromatography (HPLC) and was shown to have UDP-N-acetylglucosamine enolpyruvyl transferase activity. Then one microtiter plate based colourimetric assay for S. mutans MurA activity was developed. The optimal temperature and pH of the purified enzyme were 37°C and 7.5, respectively. The Km for UDP-GlcNAc was 0.120 mM and for PEP was 0.086 mM. The Vmax for UDP-GlcNAc was 0.048 mM min-1 mg-1 and for PEP was 0.098 mM min-1 mg-1. The activity of S. mutans MurA was inhibited by the fosfomycin, known MurA inhibitor.

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 microg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

Lateral Transfers of Serine Hydroxymethyltransferase (glyA) and UDP-N-Acetylglucosamine Enolpyruvyl Transferase (murA) Genes from Free-living Actinobacteria to the Parasitic Chlamydiae

The chlamydiae are important human and animal pathogens which form a phylogentically distinct lineage within the Bacteria. There is evidence that some genes in these obligate intracellular parasites have undergone lateral exchange with other free-living organisms. In the present work, we describe two interesting cases of lateral gene transfer between chlamydiae and actinobacteria, which have been identified based on the shared presence of conserved inserts in two important proteins. In the enzyme serine hydroxymethyltransferase (SHMT or GlyA protein), which links amino acid and nucleotide metabolisms by generating the key intermediate for one-carbon transfer reactions, two conserved inserts of 3 and 31 amino acids (aa) are uniquely present in various chlamydiae species as well as in a subset of Actinobacteria and in the Treponema species. Similarly, in the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which is involved in the synthesis of cell wall peptidoglycan, a 16-aa conserved insert is specifically present in various sequenced chlamydiae and a subset of actinobacteria (i.e., Streptomyces, Actinomyces, Tropheryma, Bifidobacterium, Leifsonia, Arthrobacter, and Brevibacterium). To determine the phylogenetic depths of the GlyA and MurA inserts, the fragments of these genes from two chlamydiae-like species, Simkania negevensis and Waddlia chondrophila, were PCR amplified and sequenced. The presence of the corresponding inserts in both these species strongly indicates that these inserts are distinctive characteristics of the Chlamydiales order. In phylogenetic trees based on GlyA and MurA protein sequences, the chlamydiae species (and also the Treponema species in the case of GlyA) branched with a high affinity with various insert-containing actinobacteria within a clade of other actinobacteria. These results provide strong evidence that the shared presence of these indels in these bacteria is very likely a consequence of ancient lateral gene transfers from actinobacteria to chlamydiae. Pairwise sequence identity and the branching pattern of the GlyA homologues in the phylogenetic tree indicates that the glyA gene was initially transferred from an actinobacteria to an ancestor of the Treponema genus and from there it was acquired by the common ancestor of the Chlamydiales.

Simultaneous Deficiency of both MurA and p60 Proteins Generates a Rough Phenotype inListeria monocytogenes

We examined eight spontaneously occurring rough mutants of Listeria monocytogenes for their ability to express two previously reported autolysins, p60 and MurA. All mutants lack MurA expression and show strongly reduced levels of extracellular p60. One rough strain harbors a variant of the p60 protein with a partially truncated catalytic domain. In seven cases there were shifts in the localization of p60 to the membrane fraction. Mutations within the secA2 gene, encoding an auxiliary protein secretion system paralog, were previously shown to be involved in the smooth-rough phenotypic variation seen with Listeria strains. An isogenic DeltasecA2 EGDe deletion strain displays a strong pleiotropic reduction of p60 and MurA, in addition to a large number of secreted and surface proteins. However, we observed no apparent SecA2 dysfunction in several of the investigated strains as determined by direct sequencing of the secA2 gene and complementation of the DeltasecA2 mutant with the respective allele cloned from the rough mutant. To determine the gene products required for the smooth-rough transition, we created mutants lacking the individual iap and murA genes as well as a Deltaiap DeltamurA double mutant. The double mutant displays a rough phenotype and exhibits many of the properties seen with the DeltasecA2 mutant. Our results implicate p60 and MurA as important determinants in controlling the cell shape of L. monocytogenes. We also identified homologous MurA and SecA2 proteins in other Listeria species. The muramidase in two species, L. innocua and L. welshimeri, shows activity similar to that of the MurA protein in L. monocytogenes.

Conditional lethal amber mutations in essential Escherichia coli genes.

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.

Jittery, a Mutator distant relative with a paradoxical mobile behavior: excision without reinsertion.

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709-amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator-Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements.

MDM-1 and MDM-2: two mutator-derived MITE families in rice.

Numerous miniature inverted repeat transposable elements (MITEs) are present in the rice genome but their transposition mechanisms are unknown. In this report, we present evidence that two novel MITE families may have arisen from Mutator-related transposable elements and thus may use a transposition mechanism similar to that of Mutator elements. Two families of novel MITEs, namely, MDM-1 and MDM-2, were identified by searching for MITEs nested with Kiddo, a previously identified MITE family. MDM-1 and MDM-2 bear hallmarks of Mutator elements, such as long terminal inverted repeats (LTIRs), 9-bp target-site duplications (TSDs), and putative transposase binding sites. Strikingly, the MDM-1 family has a 9-bp terminus identical to that of a rice Mutator-like element ( MULE-9) and the MDM-2 family has an 8-bp terminus identical to that of the maize autonomous Mutator element MuDR. A putative transposase homologous to MURA protein is identified for the MDM-2 family. Thus, these two novel MITE families, with a total copy number of several hundred in rice, are designated Mutator-derived MITEs ( MDMs). Interestingly, sequence decay analysis of MDM families revealed a number of insertion site duplications (ISDs) in the alignment gaps, and widespread historical nesting events are proposed to account for the existence of these ISDs. In addition to its value for discovering new MITEs, the nesting analysis approach used in this study simultaneously identifies MITE insertion polymorphisms.