Recombinant Acinetobacter sp. UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA)

What is the role of MurA in bacterial cell wall biosynthesis?

UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) catalyzes the first committed step in the biosynthesis of peptidoglycan in bacterial cell walls. The enzyme transfers an enolpyruvyl group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDPAG) to form UDP-N-acetylglucosamine enolpyruvate, which is a precursor to UDP-N-acetylmuramate, a requisite building block of bacterial cell wall .

Methodological approach: To investigate MurA function, researchers should employ enzymatic assays that monitor the transfer of the enolpyruvyl group. This can be detected through HPLC analysis of reaction products or through colorimetric assays that measure phosphate release. The basic reaction can be set up with purified recombinant MurA, UDP-N-acetylglucosamine, and phosphoenolpyruvate in an appropriate buffer system, with activity monitored spectrophotometrically .

How is the murA gene organized in Acinetobacter species compared to other bacteria?

In Acinetobacter calcoaceticus, the murA gene (historically also referred to as murZ) is located downstream of the rpoN gene, which encodes the alternative sigma factor σ54. This represents a unique genetic organization, as A. calcoaceticus is identified as "the first exception from a conserved genetic context of rpoN observed in several other Gram-negative bacteria" .

Methodological approach: Researchers investigating murA genomic organization should perform comparative genomic analyses using bioinformatics tools. Southern blot analysis can confirm gene arrangement, while RT-PCR can be used to determine if murA is cotranscribed with rpoN, as suggested by the polar effect of rpoN mutations on phosphomycin resistance levels .

What are the optimal conditions for expressing recombinant Acinetobacter MurA?

While specific conditions for Acinetobacter MurA expression are not directly addressed in the search results, general approaches for recombinant MurA expression can be extrapolated from studies on related organisms.

Methodological approach: Expression systems using E. coli BL21(DE3) with pET-based vectors have been successful for MurA from various bacterial species. For purification, affinity chromatography using His-tagged constructs is commonly employed, followed by size exclusion chromatography to ensure high purity. Expression conditions typically include induction with 0.5-1 mM IPTG at 25-30°C for 4-6 hours to maximize soluble protein yield while minimizing inclusion body formation .

How can researchers assess the purity and activity of recombinant Acinetobacter MurA?

Methodological approach: Purity assessment should include SDS-PAGE analysis, with expected molecular weight around 45-50 kDa based on MurA proteins from related species. Activity can be measured using enzymatic assays that monitor the transfer of the enolpyruvyl group from PEP to UDPAG. For kinetic characterization, researchers should determine apparent affinity constants (Km) for both substrates under varying conditions. For example, in studies with Wolbachia MurA, the Km values were 0.03149 mM for UDP-N-acetylglucosamine and 0.009198 mM for phosphoenolpyruvate .

How does the genetic context of murA affect antimicrobial resistance in Acinetobacter species?

Studies have shown that multicopy plasmids encoding MurA conferred phosphomycin resistance to A. calcoaceticus, suggesting that increased expression of MurA contributes to antibiotic resistance. Furthermore, the polar effect of a rpoN mutation on phosphomycin resistance levels suggests that murA is, in part, cotranscribed with rpoN in A. calcoaceticus .

Methodological approach: To investigate this relationship, researchers should:

• Construct strains with varying murA copy numbers

• Perform minimum inhibitory concentration (MIC) assays with phosphomycin

• Create transcriptional fusions to measure murA expression under different conditions

• Analyze the impact of rpoN mutations on murA expression and phosphomycin resistance

What methodologies are effective for studying MurA inhibition in Acinetobacter species?

Methodological approach: A comprehensive inhibition study should include:

• In vitro enzyme inhibition assays: Using purified recombinant MurA, researchers can measure enzyme activity in the presence of potential inhibitors like fosfomycin, which has been shown to inhibit MurA activity approximately 2-fold in related bacterial species .

• Structural analysis: Homology modeling of Acinetobacter MurA based on crystal structures from related species can provide insights into inhibitor binding sites. For example, superimposition of a MurA homology model with the structural model of Haemophilus influenzae MurA has suggested similar binding sites for fosfomycin .

• Antimicrobial susceptibility testing: Minimum inhibitory concentration (MIC) assays against different Acinetobacter strains can evaluate the antibacterial activity of potential MurA inhibitors.

How do different environmental conditions affect MurA expression and activity in Acinetobacter species?

While the search results don't provide specific information on MurA expression under different conditions in Acinetobacter, studies on related organisms can provide methodological guidance.

Methodological approach: Researchers should investigate:

• Carbon source effects: Using defined media with different carbon sources (e.g., acetate, citrate, pyruvate, succinate) to evaluate MurA expression and activity. Studies on A. baylyi ADP1 have shown that carbon sources significantly influence protein expression patterns in response to various stresses .

• Proteomic analysis: Employing techniques such as iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC/MS/MS to compare MurA expression levels under different growth conditions .

• Transcriptional analysis: Using qRT-PCR or RNA-seq to measure murA transcript levels under various environmental conditions.

What is the potential of MurA as a target for developing new antimicrobials against multidrug-resistant Acinetobacter infections?

Acinetobacter species, particularly A. baumannii, have garnered significant attention due to their remarkable ability to acquire resistance to multiple antibiotics. Common antimicrobial resistance genes in Acinetobacter include class D oxacillinases (OXA-23, OXA-24/40, OXA-58), multiple drug resistance efflux pumps (AdeABC, AdeIJK, AdeFGH), and various aminoglycoside-modifying enzymes .

Methodological approach: To evaluate MurA as a drug target, researchers should:

• Target validation: Create conditional murA knockout strains in Acinetobacter to confirm essentiality. Similar studies in other bacteria have demonstrated that disruption of murA leads to loss of viability .

• Structural analysis: Determine the crystal structure of Acinetobacter MurA to identify unique features that could be exploited for selective inhibitor design.

• High-throughput screening: Develop assays suitable for screening compound libraries for novel MurA inhibitors. Considerations should include:

  • Selection of appropriate enzyme concentration and substrate concentrations near Km values

  • Development of a robust readout system (fluorescence, colorimetric, etc.)

  • Controls for non-specific inhibition

• Selectivity profiling: Evaluate promising inhibitors against human cell lines to ensure safety and specificity.

How does MurA contribute to the pathogenicity of clinical Acinetobacter isolates?

Methodological approach: To investigate the role of MurA in pathogenicity:

• Virulence studies: Compare wildtype and MurA-attenuated strains in appropriate infection models.

• Gene expression analysis: Analyze murA expression during infection processes using techniques like qRT-PCR or RNA-seq.

• Host-pathogen interaction studies: Investigate how MurA activity affects recognition by the host immune system.

• Combination studies: Evaluate potential synergistic effects between MurA inhibitors and other antibiotics against clinical isolates of Acinetobacter, particularly those with defined resistance mechanisms .

What site-directed mutagenesis approaches are most informative for studying the catalytic mechanism of Acinetobacter MurA?

Methodological approach: Based on studies of MurA from other bacterial species, researchers should:

• Target conserved residues: Focus on the Cys115 equivalent (numbering based on E. coli MurA), which is the target of fosfomycin in many bacterial species.

• Structure-guided mutagenesis: Use homology models to identify residues involved in substrate binding and catalysis.

• Kinetic analysis of mutants: Perform detailed kinetic studies on mutant enzymes to determine effects on:

  • Substrate binding (Km values)

  • Catalytic efficiency (kcat)

  • Inhibitor binding

• Thermal stability studies: Employ techniques like differential scanning fluorimetry to assess the impact of mutations on protein stability.

How can researchers develop MurA-targeted inhibitors specific for Acinetobacter species?

Methodological approach:

• Virtual screening approaches: Use homology models of Acinetobacter MurA to perform in silico screening of compound libraries.

• Fragment-based drug design: Screen fragment libraries against purified MurA using techniques like thermal shift assays, NMR, or X-ray crystallography.

• Structure-activity relationship studies: Synthesize and test structural analogs of known MurA inhibitors like fosfomycin.

• Evaluation criteria for potential inhibitors: