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Optimization Of Udp N Acetylmuramic Acid Synthesis Essay

Introduction

Bacteria belonging to the Verrucomicrobia phylum are Gram-negative heterotrophic organisms that are generally found in soil and fresh water environments. The phylum is considered to have two sister phyla, Chlamydiae and Lentisphaerae (Cho et al., 2004). Members of the Verrucomicrobia are of interest due to their close evolutionary relationship to bacteria from the genus Chlamydia in addition to their unusual morphology of possessing wart-like and tube-like appendages that protrude from the cell membrane, commonly referred to as prosthecae (Wagner and Horn, 2006; McGroty et al., 2013). Most of the research that has been done with bacteria from this phylum has been done using Verrucomicrobium spinosum as the model organism. The bacterium was found to employ the type III secretion system and is pathogenic toward Drosophila melanogaster and Caenorhabditis elegans (Sait et al., 2011). In addition, research from our group recently demonstrated that the bacterium employs the L,L-diaminopimelate aminotransferase (DapL) pathway for the synthesis of meso-diaminopimelate involved both in the cross-linking of peptidoglycan (PG) and in lysine anabolism (Nachar et al., 2012; McGroty et al., 2013). Due to the morphological complexity and unusual cellular plan of V. spinosum, the synthesis of PG is of interest to our group given its close relationship to the pathogenic organisms from the genus Chlamydia. In addition, the recent discovery of PG in Chlamydia has made this project more intriguing, given the fact that even though β-lactam antibiotics are effective against Chlamydia, definitive evidence of PG in Chlamydial species has been lacking until this recent discovery (Pilhofer et al., 2013; Packiam et al., 2015).

Cell wall PG (also named murein) is ubiquitous in the bacterial domain. The PG of bacteria is composed of tandem repeats of the sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by a short peptide stem containing usually L-lysine or meso-diaminopimelate at the third position (Park, 1987; Vollmer et al., 2008). Due to its rigid structure and tensile strength, the PG has several overarching roles such as protecting the osmotic integrity of the cell in addition to maintaining the shape of the bacteria.

The synthesis of PG in bacteria occurs via a pathway that has three distinct steps: the cytoplasmic, membrane, and periplasmic steps. In the cytoplasmic steps, the nucleotide sugar-linked precursor UDP-MurNAc-pentapeptide is synthesized in a series of reactions catalyzed by the enzymes MurA, MurB, MurC, MurD, MurE, MurF, and Ddl (Figure 1) (Barreteau et al., 2008). The next steps in PG formation are the synthesis of the lipid precursor intermediates, undecaprenyl-diphospho-MurNAc-pentapeptide (lipid I) and undecaprenyl-diphospho-MurNAc-(pentapeptide)-GlcNAc (lipid II), by the enzymes MraY and MurG, respectively; these reactions occur at the level of the cytoplasmic membrane (Bouhss et al., 2008). The ultimate steps in the pathway are the transglycosylation and transpeptidation reactions, characterized by the polymerization of the sugar-peptide units and their incorporation into the growing PG; these reactions take place in the extra-cytoplasmic space and are catalyzed by the penicillin-binding proteins (PBPs) (Figure 1) (Scheffers and Pinho, 2005).

Figure 1. Schematic representation depicting the three stages of PG biosynthesis in bacteria. The cytoplasmic, membrane, and periplasmic sgteps are shown. The abbreviations of the enzymes are as follows: MurA, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; MurB, UDP-N-acetylenolpyruvoylglucosamine reductase; MurC, UDP-N-acetylmuramate:L-alanine ligase; MurD, UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase; MurE, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:2,6-diaminopimelate ligase or UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase; MurF, UDP-N-acetylmuramoyl-tripeptide:D-alanyl-D-alanine ligase; Ddl, D-alanine:D-alanine ligase; MraY, phospho-N-acetylmuramoyl-pentapeptide transferase; MurG, undecaprenyl-diphospho-N-acetylmuramoyl-pentapeptide β-N-acetylglucosaminyl transferase; and PBPs, penicillin-binding proteins. The enzymatic activities theoretically carried by the MurB/C fusion enzyme from V. spinosum are shaded in black. UDP-GlcNAc-EP stands for UDP-N-acetylenolpyruvoylglucosamine.

The first three cytoplasmic steps of the PG synthesis pathway, which are the topic of this paper, are as follows. First, UDP-N-acetylglucosamine-1-carboxyvinyltransferase (MurA, EC 2.5.1.7) catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate to the 3′-hydroxyl end of UDP-GlcNAc to produce UDP-N-acetylenolpyruvoylglucosamine (UDP-GlcNAc-EP) (Figure 1) (Marquardt et al., 1992; Wanke et al., 1992). Then, UDP-N-acetylenolpyruvoylglucosamine reductase (MurB, EC 1.3.1.98) catalyzes the reduction of the enolpyruvyl moiety of UDP-GlcNAc-EP to lactyl ether to produce UDP-N-acetylmuramic acid (UDP-MurNAc) (Figure 2A) (Benson et al., 1993; Tayeh et al., 1995). Finally, UDP-N-acetylmuramate:L-alanine ligase (MurC, EC 6.3.2.8) catalyzes the third reaction, which consists in the addition of L-Ala to the carboxyl group of UDP-MurNAc to produce UDP-MurNAc-L-Ala (Figure 2B) (Liger et al., 1995; Falk et al., 1996; Gubler et al., 1996).

Figure 2. Enzymatic reactions catalyzed by the MurB/C fusion enzyme. (A) Reaction catalyzed by UDP-N-acetylenolpyruvoylglucosamine reductase (MurB). (B) Reaction catalyzed by UDP-N-acetylmuramate: L-alanine ligase (MurC).

Here we report the identification and biochemical partial characterization of a novel MurB/C fusion enzyme from V. spinosum. While in vitro assays demonstrate that the enzyme is able to catalyze the ligase (MurC) reaction, attempts to demonstrate the reductase (MurB) activity in vitro were unsuccessful. Nevertheless, in vivo analyses demonstrated that the fusion gene is able to functionally complement two Escherichia coli strains that harbor mutations in the murB and murC genes. Given the facts that (i) the MurB and MurC enzymes are not normally fused and are encoded by separate ORFs as is the case of the two E. coli proteins (Pucci et al., 1992; Liger et al., 1995), and (ii) the PG biosynthesis pathway is essential and is only present in the bacterial domain, the identification and characterization of this unusual fusion enzyme involved in PG biosynthesis in V. spinosum, a close relative of the pathogenic organism Chlamydia, is intriguing. This study has the potential to contribute to the further understanding of the kinetic, physical and structural properties of enzymes involved in the synthesis of PG in order to facilitate the development and/or discovery of antibacterial compounds that are able to combat current and emerging bacterial infections and diseases, especially those that are deemed to be resistant to antibiotics that are currently used in a clinical setting.

Materials and Methods

Materials

L-[14C]Ala (5.99 GBq.mmol−1) and L-[14C]Ser (6.07 GBq.mmol−1) were purchased from Perkin Elmer, [14C]Gly (3.88 GBq.mmol−1) from CEA, and UDP-[14C]GlcNAc (2 GBq.mmol−1) from ARC Isobio. UDP-[14C]MurNAc was prepared according to published procedure (Bouhss et al., 2002). UDP-GlcNAc-EP was purchased from the BaCWAN facility.

V. spinosum Growth Conditions/Plasmids and Strains used in this Study

V. spinosum DSM 4136T was cultured in R2A medium supplemented with 5% (w/v) artificial sea water at 26°C (Schlesner, 1987). The plasmids and strains used in this study are listed in Table 1.

Table 1. Plasmids and strains used in this study.

Domain Mapping of MurB/CVs Identification

The protein family (Pfam) domains of MurB/CVs were identified using the Pfam server (http://pfam.sanger.ac.uk/) and (http://pfam.janelia.org/) (Finn et al., 2014). The conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) was used to identify the domains of MurB/CVs (Marchler-Bauer et al., 2015).

Identification of Lineages Containing Fused MurB/C Protein and Phylogenetic Analysis of MurB and MurC Proteins

Publicly available draft and complete genome sequences belonging to members of the phylum Planctomycetes, Verrucomicrobia, Chlamydiae, and Lentisphaerae (PVC) were downloaded from NCBI. The proteomes were re-predicted with Prodigal version 2.6 (Hyatt et al., 2010) and then used for phylogenomic tree construction with PhyloPhlAN (Segata et al., 2013). The manually curated database of protein families known as TIGFAMs was used to identify MurB (TIGR00179) and MurC (TIGR00182) proteins (Haft et al., 2003). The predicted whole proteomes were subjected to similarity search based on the hidden Markov model (HMM) profiles TIGR00179 and TIGR00182 using HMMsearch (–cut_tc option) (Eddy, 2011), and species containing unusual composition of MurB and MurC, i.e., fused MurB/C, missing MurB, and/or missing MurC, were annotated in the constructed species tree. Additionally, the gene organization of contigs containing the fused murB/C open reading frame (ORF) was further analyzed with EasyFig version 2.1 (Sullivan et al., 2011). Additional MurB and MurC proteins were also mined from the UniProt (http://www.uniprot.org/) (The UniProt Consortium) to be included for phylogenetic analysis. Briefly, the proteins were aligned and trimmed using mafft-linsi and trimal (-gappyout setting) (Capella-Gutiérrez et al., 2009; Katoh and Standley, 2013), and phylogenetic inference was performed using IQ-TREE version 1.3.10 (Nguyen et al., 2014) and visualized using FigTree version 1.42 (http://tree.bio.ed.ac.uk/software/figtree/).

PCR Amplification and Cloning of the V. spinosum murB/C Open Reading Frame (ORF)

The ORF annotated by the locus tag (VspiD_010100018130) UDP-N-acetylenolpyruvoylglucosamine reductase /UDP-N-acetylmuramate:L-alanine ligase was amplified by PCR using the primers murB/CVs-forward (5′-C ACCATGAATCACGCCGTCGTCAGTTTGTTGAA G-3′) and murB/CVs-reverse (5′-GTCGACCTATAGCGGAAG CGGTTCCTCTTCGCCAAT-3′). The underlined sequence represents the restriction enzyme site used to facilitate sub-cloning of the ORF while the bold sequences represent initiation and termination codons. The PCR reaction contained 12 pmol of forward and reverse primers, 1 mM MgSO4, 0.5 mM of each of the four deoxynucleotide triphosphates, 0.5 ng of genomic DNA and 1 unit of Platinum Pfx DNA polymerase (Invitrogen Corporation, Carlsbad, CA, USA). PCR conditions were: 1 cycle at 94°C for 2 min, followed by 30 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 3 min. The murB/C PCR fragment was ligated into the plasmid pET100D/topo (Invitrogen Corporation, Carlsbad, CA, USA) to produce the plasmid pET100D::murB/CVs. The recombinant protein encoded by this plasmid carries an N-terminal MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFT additional sequence containing a hexa-histidine tag (bold) derived from the pET100D plasmid. To confirm the fidelity of the PCR reaction, the ORF was sequenced from pET100D using the T7 promoter primer, 5′-TAATACGACTCACTATAGGG-3′ and the T7 reverse primer, 5′-TAGTTATTGCTCAGCGGTGG-3′. The cloned murB/C ORF was 100% identical to the sequence deposited in the Integrated Microbial Genomes public database (http://img.jgi.doe.gov/cgi-bin/w/main.cgi).

Cloning of murC and murB Domains

The murC and murB domains of the MurB/CVs fusion protein were also cloned separately in expression vectors, as follows. The murC domain sequence (two different end points were chosen) was amplified by PCR by using murCVs-forward (5′-GCGCTCATGAATCACGCCGT CGTCAGTTTGTTGAAG-3′) as the forward primer and either murCVs-reverse-1 (5′-GCGCAGATCTGCCTTCGCG ATTGAGCACCGTAGTGAG-3′) or murCVs-reverse-2 (5′-GCGCAGATCTGACCGTGCC ACCGCCTTCGCGATTGAG-3′) as the reverse primer, designed to end the MurC domain at the Gly477 and Val481 residues, respectively. The underlined sequences correspond to introduced BspHI and BglII restriction sites and the murC gene initiation codon is indicated in bold. The two PCR products were digested by BspHI and BglII and inserted between the compatible NcoI and BglII sites of the expression vector pTrcHis60 that allows expression of proteins with a C-terminal hexa-histidine tag under the control of the IPTG-inducible trc promoter (Pompeo et al., 1998). The two plasmids thus generated, pTrcHis60::murCVs-1 and pTrcHis60::murCVs-2, directed expression of Met1-Gly477 and Met1-Val481 fragments from the MurB/CVs fusion protein (770 residues in total), respectively, fused to a C-terminal tag extension consisting in Arg-Ser-His6.

Similarly, the murB domain sequence (two different starting points were chosen) was amplified by using either murBVs-forward-1 (5′-G AAGCCATGGGCACGGTCAAGCTCTATGAGCCGAT G-3′) or murBVs-forward-2 (5′-GCGCTCATGAAGCTCTATG AGCCGATGGCCAACCAC-3′) as the forward primer and murBVs-reverse (5′-GCGCAGATCTTAGCGGAAG CGGTTCCTCTTCGCCAATG-3′) as the reverse primer. NcoI, BspHI and BglII restriction sites introduced in these sequences are underlined and the murB gene initiation codons are indicated in bold. The two PCR products were digested by NcoI or BspHI and BglII and inserted between the compatible NcoI and BglII sites of the vector pTrcHis60. The two plasmids thus generated, pTrcHis60::murBVs-1 and pTrcHis60::murBVs-2, directed expression of the Gly479-Leu770 and Lys482-Leu770 fragments (preceded by a Met residue) from the MurB/CVs fusion protein, respectively, here too with a C-terminal tag consisting in Arg-Ser-His6.

Expression and Purification of Recombinant MurB/CVs

The E. coli Rosetta (DE3) pLysS strain (Novagen) was transformed with the plasmid pET100D::murB/CVs and grown at 37°C in 2YT medium containing 50 μg.mL−1 ampicillin and 25 μg.mL−1 chloramphenicol. An overnight pre-culture of the resulting strain was used to inoculate 2YT medium (2-liter cultures). The culture was incubated with shaking at 37°C. When the optical density reached 0.9, the temperature of the culture was decreased to 20°C and IPTG was added at a 1 mM final concentration. Growth was continued for 18 h at 20°C. Cells were harvested at 4°C and the pellet was washed with cold 20 mM phosphate buffer, pH 7.2, containing 1 mM dithiothreitol (buffer A). Bacteria were resuspended in buffer A (12 mL) and disrupted by sonication in the cold using a Bioblock Vibracell 72412 sonicator. The resulting suspension was centrifuged at 4°C for 30 min at 200,000 × g with a Beckman TL100 apparatus, and the pellet was discarded. The supernatant was kept at −20°C.

The His6-tagged protein was purified on Ni2+-nitrilotriacetate (Ni2+-NTA)-agarose following the manufacturer's recommendations (Qiagen). All procedures were performed at 4°C. The supernatant was mixed for 1 h with the polymer previously washed with buffer A containing 0.3 M KCl and 10 mM imidazole. Washing and elution steps were performed with a discontinuous gradient of imidazole (20–300 mM) in buffer A containing 0.3 M KCl. Protein contents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and relevant fractions were pooled and dialyzed against 100 volumes of buffer A. At this step, precipitation of a significant part of the protein was observed. The non-precipitated protein was concentrated on an Amicon Ultra 50,000 molecular mass cutoff filter. Glycerol (10% final concentration) was added for storage at −20°C. Protein concentrations were determined by quantitative amino acid analysis with a Hitachi L8800 analyzer (ScienceTec) after hydrolysis of samples at 105°C for 24 h in 6 M HCl containing 0.05% 2-mercaptoethanol. No attempts were made to remove the additional sequence containing the hexa-histidine tag after protein purification.

Construction of the Plasmid to Facilitate Functional Complementation

The plasmid used for functional complementation of the E. coli murB and murC mutants was produced by sub-cloning the XbaI and SalI fragment from the pET100D::murB/CVs plasmid into pBAD33 to produce the plasmid pBAD33::murB/CVs, which is under control of the arabinose inducible promoter (Guzman et al., 1995). The protein produced from the pBAD33 construct is identical to the protein produced from the pET100D construct. Note that there is an XbaI site 20 base pairs upstream of the ribosome binding site of the pET100D vector that was used to facilitate sub-cloning from pET100D into pBAD33.

Functional Complementation of the E. coli murB and murC Thermosensitive Mutants

The E. coli murB mutant (ST5-strain #6442) [thr-1, araC14, leuB6 (Am), secA216, fhuA61, lacY1, galT23, λ trp-84, his-215, thyA710, rpsL263 (strR), xylA5, mtl-1, murB1-(ts), thi-1] and murC mutant (ST222-strain #5988) [thr-1, araC14, leuB6 (Am), murC3(ts), secA216, fhuA61, lacY1, galT23, λ trp-84, his-215, thyA710, rpsL263 (strR), xylA5, mtl-1, thi-1] were both obtained from the Coli Genetic Stock Center (http://cgsc.biology.yale.edu/). These mutants were transformed with the pBAD33 vector or the pBAD33::murB/CVs plasmid and transformants were selected on LB agar medium supplemented with 50 μg.mL−1 thymine, 34 μg.mL−1 chloramphenicol, and 0.2% (w/v) arabinose at 30°C. Colonies were then replica-plated onto LB agar medium plus 0.2% (w/v) arabinose and 10 μg.mL−1 thymine. Liquid cultures were also performed in LB medium supplemented with arabinose and thymine. In both cases, the growth phenotype was assessed at 30 and 42°C for up to 24 h.

As described above, the individual murC and murB domains from the murB/CVs fusion gene were also cloned separately in the pTrcHis60 vector that allows high-level gene expression under control of the strong IPTG-dependent trc promoter. The resulting plasmids pTrcHis60::murBVs and pTrcHis60::murCVs were then tested for functional complementation, using the E. coli murB mutant ST5 and the murC mutant strain H1119 (Wijsman, 1972), respectively. Transformants were selected on LB agar medium supplemented with 100 μg.mL−1 ampicillin and 50 μg.mL−1 thymine at 30°C and were subsequently replicated on similar plates incubated at 30°C or 42°C, in the presence or absence of 0.5 mM IPTG. Growth was observed after 24 h of incubation.

Determination of the Kinetic Constants of the MurC Activity

The standard MurC activity assay (Liger et al., 1995) measured the formation of UDP-MurNAc-L-Ala in a mixture (final volume, 40 μL) containing 100 mM Tris-HCl, pH 9.0, 10 mM MgCl2, 3 mM ATP, 10 mM ammonium sulfate, 0.5 mg.mL−1 bovine serum albumin (BSA), 0.9 mM UDP-MurNAc, 0.3 mM L-[14C]Ala (400 Bq), and enzyme (20 μL of an appropriate dilution in buffer A). For the determination of the Km values for UDP-MurNAc, UDP-[14C]MurNAc was used as the radiolabelled substrate.

In all cases, the mixtures were incubated for 30 min at 37°C and the reactions were stopped by the addition of glacial acetic acid (10 μL) followed by lyophilization. Radioactive substrate and product were then separated by HPLC on a Nucleosil 100C18 5 μm column (150 × 4.6 mm; Alltech France) using 50 mM ammonium formate, pH 3.2, at a flow rate of 0.6 mL.min−1. Radioactivity was detected with a flow detector (model LB506-C1, Berthold) using the Quicksafe Flow 2 scintillator (Zinsser Analytic) at 0.6 mL.min−1. Quantification was performed with the Radiostar software (Berthold).

For the determination of the kinetic constants, the same assay was used with various concentrations of one substrate and fixed concentrations of the others. In all cases, the enzyme concentration was chosen so that substrate consumption was < 20%, the linearity being ensured within this interval even at the lowest substrate concentration. Data were fitted to the equation v = VmaxS/(Km + S) by the Levenberg-Marquardt method (Press et al., 1986), where v is the initial velocity and S is the substrate concentration, and values ± standard deviation at 95% of confidence were calculated. The MDFitt software developed by M. Desmadril (I2BC, Orsay, France) was used for this purpose.

In vitro Spectrophotometric Assay of the MurB Activity

The MurB spectrophotometic assay was performed as described previously (Benson et al., 1993). The reaction mixture contained, in a final volume of 100 μL, 50 mM Tris-HCl, pH 8.0, 20 mM KCl, 0.5 mM dithiothreitol, 0.1 mM UDP-GlcNAc-EP, and 0.15 mM NADPH. The mixture was placed in a 1- cm path length cell and the reaction was started by the addition of the enzyme. The decrease in NADPH absorbance at 340 nm was monitored with a Jasco V-630 spectrophotometer.

In vitro Coupled Assay of the MurA/MurB Activities

The reaction mixture contained, in a final volume of 40 μL, 50 mM Tris-HCl, pH 7.6, 25 mM KCl, 0.1 mM NADPH, 55 μM UDP-[14C]GlcNAc (500 Bq), 75 μM phosphoenolpyruvate, E. coli MurA (1 μg), and enzyme. In some experiments, 5 mM ATP, 10 mM MgCl2, and 0.15 mM L-Ala were included. After 30 min at 37°C, the reaction was stopped by the addition of glacial acetic acid (8 μL) followed by lyophilization. The radioactive substrate and product were separated on a Nucleosil 100C18 5 μm column (150 × 4.6 mm; Alltech France) using 50 mM ammonium formate, pH 3.2, at a flow rate of 0.6 mL.min−1. Detection and quantification of the radioactivity were performed as described above. The retention times for UDP-GlcNAc, UDP-GlcNAc-EP, UDP-MurNAc, and UDP-MurNAc-L-Ala were 6, 10, 12, and 20 min, respectively.

MurB/CVs Cleavage Assay

For the cleavage assay, V. spinosum was grown in liquid medium R2A medium supplemented with 5% (w/v) artificial sea water at 26°C for 5 days. Following centrifugation, the cells were lysed by sonication in the following buffer systems 50 mM Tris-HCl, pH 7.6, 50 mM Tris-HCl, pH 8.5, and 50 mM HEPES-KOH, pH 7.6. The purified recombinant enzyme (7.5 μg) was incubated with 15 μg of V. spinosum extract at 30°C. The proteins were resolved on a 10% (w/v) acrylamide gel and stained with Coomassie blue for visualization. Protein concentration was measured using the Bradford assay with BSA as the standard (Bradford, 1976).

Results

Identification of the MurB/C Fusion Enzyme from V. spinosum

The complete set of genes in V. spinosum required for the de novo synthesis of PG was initially identified from a comparative analysis of the V. spinosum proteome using the known PG biosynthesis proteins as queries (Nachar et al., 2012). The search revealed an anomaly when it was realized that both the MurB and MurC proteins were encoded by a single locus tag VspiD_010100018130 (Table 2).

Table 2. List of PG biosynthesis genes from V. spinosum.

Domain Mapping of the MurB/C Fusion Enzyme from V. spinosum

The length of the MurB and MurC E. coli proteins are 342 and 491 residues, respectively, and the lengths of the MurB/C fusion enzyme protein is 770 residues. As such, we were interested to assess if the fusion enzyme had the domains that are indicative of typical MurB and MurC enzymes. Domains were identified using the NCBI's CDD and the protein families database (Pfam) (Finn et al., 2014; Marchler-Bauer et al., 2015). The CDD and Pfam analyses resulted in the identification of the following domains: (1) the Mur ligase catalytic domain (Pfam01225), (2) the Mur ligase middle domain (Pfam08245), (3) the Mur ligase family amino acid-binding domain (Pfam02875), (4) the FAD-binding domain (Pfam01565), and (5) the UDP-N-acetylenolpyruvoylglucosamine reductase C-terminal domain (Pfam02873). This analysis also demonstrated that the residues responsible for the MurC activity were located toward the N-terminal end of the fusion enzyme, while those for the MurB activity are located toward the C-terminal end. MurC and MurB are separated by a linker region of ~100 residues as depicted in the schematic. For comparison, the figure also depicts the domain structures of the E. coli MurB and MurC enzymes (Figure 3).

Figure 3. Domain mapping of the MurB/CVs fusion enzyme of V. spinosum and the individual MurB and MurC enzymes from E. coli showing the predicted Pfam domains and residue locations of the individual domains.

The murB/CVs Gene is able to Functionally Complement the E. coli murB and murC Mutants

The E. coli strains ST5 and ST222 obtained from the Coli Genetic Stock Center harbor mutations in the murB and murC genes, respectively. These mutations result in a temperature-sensitive growth phenotype where the mutants are able to grow at the permissive temperature of 30°C, but not at the non-permissive temperature of 42°C (Matsuzawa et al., 1969; Miyakawa et al., 1972). To answer the question of whether the fusion gene is able to complement the murB and murC E. coli mutants, the mutant strains were transformed with an empty vector (pBAD33) or with a vector containing the murB/C gene (pBAD33::murB/CVs). Using replica-plating, the results from this analysis demonstrate that at the permissive temperature of 30°C, the mutant strains harboring the vector control (pBAD33) and the vector containing the recombinant gene (pBAD33::murB/CVs) were both able to grow. However, when exposed to the non-permissive temperature of 42°C, only the strains expressing the murB/C recombinant gene were able to grow (Figure 4A). This result was corroborated by assessing bacterial growth in liquid medium over a period of 24 h. At 30°C the mutants harboring the vector-only and the murB/C− expressing vector grew as expected. The growth phenotype at the non-permissive temperature of 42°C demonstrated that only the mutant strains expressing the murB/C gene were able to grow when compared to the vector-only controls (Figure 4B). The lack of growth based on the optical density of the vector-only controls at 42°C can be attributed to rapid lysis of the cells due to the lack of proper peptidoglycan synthesis (Figure 4B). The assessment of crude soluble protein extracts from the complementation experiment using SDS-PAGE analysis confirmed the production of the recombinant MurB/C fusion enzyme (~87.3 kDa) in the mutant backgrounds grown at 42°C that was not present in the extracts from the mutant harboring the vector-only control when both were under inducing conditions using arabinose (Figure 4C). Together, these analyses demonstrated that the recombinant MurB/C fusion enzyme from V. spinosum was endowed with the reductase (MurB) and ligase (MurC) activities, and that the amount produced was sufficient to sustain the growth of the E. coli mutants.

Figure 4. Functional complementation analysis of the E. coli murB and murC mutants. (A) Replica-plating experiment of the murC and murB mutants transformed with pBAD33 and pBAD33::murB/CVs grown at 30 and 42°C. (B) Analysis of the complementation experiment at 30 and 42°C in liquid culture assessing the growth phenotype by measurement of the optical density (OD) at 600 nm for a 24 h period. The growth experiments were done four times giving the similar growth profiles. The graphs in (B) represent one of those growth curves. (C) SDS-PAGE analysis of proteins from the complementation experiment to assess the expression of MurB/C in the mutant backgrounds. Lane (1), protein ladder (kDa). Lane (2), 10 μg of protein extract from the E. coli murC mutant harboring pBAD33 grown at 30°C. Lane (3), 10 μg of protein extract from the E. coli murC mutant harboring pBAD33::murB/CVsgrown at 42°C. Lane (4), 10 μg of protein extract from the E. coli murB mutant harboring pBAD33 grown at 30°C. Lane (5), 10 μg of protein extract from the E. coli murB mutant harboring pBAD33::murB/CVsgrown at 42°C. Crude extracts were obtained via sonication after 24 h from the samples grown at 30°C and 42°C. The black arrows show expression of the MurB/C recombinant enzyme. The proteins were resolved on a 10% (w/v) acrylamide gel and stained with Coomassie blue for visualization.

Attempts to clone and assay the in vivo activity of the individual MurB and MurC domains of the MurB/CVs fusion protein were then made. Protein dissection was performed on the basis of the mapping experiments described above, i.e., alignment of the V. spinosum protein sequence with that of MurB and MurC ortholog proteins from E. coli. These two domains were cloned in the pTrcHis60 vector, in each case in two versions: with the MurC domain starting at the Met1 residue and terminating either at the Gly477 or the Val481 residue, and the MurB domain starting either at the Gly479 or the Lys482 residue (preceded by a Met residue) and terminating at the last residue (Leu770) of the fusion protein. The two pTrcHis60::murCVs constructs thus generated complemented the thermosensitive murC mutant strain H1119, indicating that the shortest version ending at Gly477 clearly exhibited MurC activity. IPTG was not required for complementation, indicating that basal expression of the murCVs domain from the pTrcHis60 vector was sufficient to sustain cell growth and viability of the murC mutant at the non-permissive temperature of 42°C (Supplemental Figure 1). However, the two other pTrcHis60::murBVs constructs failed to complement the growth defect of the murB mutant strain ST5 and induction of gene expression with 0.5 mM IPTG yielded the same result. The latter finding could be interpreted in several ways: inappropriate design of the murB domain initiation codon, physical instability of these truncated forms of the fusion protein, or inability of the MurB domain to function independently (absolute requirement for the presence of the MurC domain). Our data show that this is not the case for the MurC domain whose activity did not depend on the presence of the MurB domain.

Properties and Kinetic Parameters of MurC Activity from MurB/CVs

The in vitroL-alanine-ligase activity of the MurB/CVs fusion enzyme was revealed using a radioactive assay, which was also used to determine the properties and kinetic parameters (Table 3). The optimal pH and temperature for MurCVs were found to be 9.0 and 44–46°C, respectively. As it is the case for the other Mur ligases (Barreteau et al., 2008), magnesium ions were essential for the activity: the optimal concentration was 10 mM. It was shown that the addition of 10 mM ammonium sulfate and 0.5 mg.mL−1 BSA increased the activity by 35 and 55%, respectively. With L-alanine as the amino acid substrate, the apparent kcat of the enzyme was 480 min−1 (Vmax, ca. 5500 nmol.min−1.mg−1). Amino acids Gly and L-Ser, which are found at position 1 of the peptide stem in some bacteria (Schleifer and Kandler, 1972; Vollmer et al., 2008), were also tested as substrates (Table 4). L-Ala was the best substrate, which is in agreement with the amino acid composition of V. spinosum peptidoglycan (McGroty et al., 2013). However, the difference with the two others was not as important as for the E. coli MurC ortholog (Liger et al., 1995).

Table 3. Properties and apparent kinetic parameters of V. spinosum MurC in comparison with its orthologs from E. coli and C. trachomatis.

Table 4. Specificity of MurCVs for the amino acid substrate.

Attempts to Demonstrate the In vitro MurB Activity from MurB/CVs

Several attempts were made to measure the in vitro reductase activity of MurB/CVs with two assays: a spectrophotometric assay using commercial UDP-GlcNAc-EP and NADPH, and a coupled MurA/MurB assay using UDP-[14C]GlcNAc and E. coli MurA. However, neither decrease of absorbance at 340 nm nor appearance of labeled UDP-MurNAc occurred, even at high protein concentrations. It was checked that the expected reaction took place in both assays when MurB/CVs was replaced by E. coli MurB (data not shown). In order to ascertain that the absence of reaction in the coupled assay was not due to strong inhibition by the MurB product, L-alanine, ATP, and Mg2+ were added so that the MurCVs activity might displace the reaction toward the right. A new radioactive compound appeared, but its retention time (25 min) was not consistent with that of UDP-MurNAc-L-Ala (20 min) (data not shown). It was presumably the result of the direct addition of L-alanine to UDP-GlcNAc-EP, a reaction that has been shown to occur with E. coli MurC (Liger et al., 1995).

Is MurB/CVs Active as a Fusion Enzyme in vivo?

The SDS-PAGE showing the expression of the MurB/CVs in the murB and murC mutant backgrounds demonstrated that the recombinant enzyme was not cleaved in E. coli (Figure 4C). However, given the fact that there is a 100-residue linker region between the MurC and MurB domains (Figure 3), we were interested in answering the question of whether the fusion enzyme is cleaved in V. spinosum. This would indicate that after translation, the enzyme is processed by a protease to create two separate and distinct polypeptides of MurB and MurC. To answer this question, the purified recombinant enzyme (Figure 5) was used in a cleavage assay using crude soluble protein extract from V. spinosum. The assay showed that the recombinant enzyme was not cleaved when incubated with an extract from V. spinosum over a period of 120 min (Figure 6). It should be noted that the result was consistent when the assay was done using several concentrations of V. spinosum protein extract of up to 15 μg and several buffer systems with varying pH values (data not shown). Based on the complementation, and supported by the cleavage assay, it is probable that the enzyme is active as a fusion enzyme in V. spinosum. Fusion enzymes involved in PG biosynthesis are not a new phenomenon; a MurC/Ddl fusion enzyme from Chlamydia trachomatis has been characterized and it was shown that the D-Ala:D-Ala ligase activity of the Ddl domain is dependent on the fusion structure of the MurC/Ddl protein (McCoy and Maurelli, 2005). Our present data show that at least the MurC domain of the V. spinosum MurB/C fusion protein is functionally independent as its expression allowed complementation of a temperature-conditional murC defect in E. coli.

Figure 5. Purification of MurB/C by affinity chromatography. Lane (1), protein ladder (kDa). Lane (2), 0.5 μg of purified MurB/C. The proteins were resolved on a 10% (w/v) acrylamide gel and stained with Coomassie blue for visualization.

Figure 6. Assay to assess cleavage of MurB/C using a crude protein extract from V. spinosum. Lane (1), protein ladder (kDa). Lane (2), 15 μg of V. spinosum extract. Lane (3), 15 μg of V. spinosum extract plus 7.5 μg of purified MurB/C at time zero. Lane (4), 15 μg of V. spinosum extract plus 7.5 μg of purified MurB/C at 60 min. Lane (5), 15 μg of V. spinosum extract plus 7.5 μg of purified MurB/C at 120 min. Lane (6), 7.5 μg of purified MurB/C. The temperature of the assay was 30°C. The proteins were resolved on a 10% (w/v) acrylamide gel and stained with Coomassie blue for visualization.

Unusual MurB and MurC Composition is Prevalent in the Currently Sequenced Members of Verrucomicrobia

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