Molecular Cloning, Sequence Analysis, and Characterization of a Penicillin-Resistant dd-Carboxypeptidase of Myxococcus xanthus

Abstract

We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistant dd-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity to N-acetylmuramoyl-l-alanine amidase and penicillin-resistant dd-carboxypeptidase, respectively. The pdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar to those of dd-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in pdcA deletion mutants.

Myxococcus xanthus is a gram-negative bacterium which lives in soil (7, 17, 35). It feeds upon other microorganisms by secreting bacteriolytic enzymes and antibiotics (14, 33, 37). The bacterium responds to nutrient starvation by forming a multicellular aggregate and fruiting body. M. xanthus cells coordinate fruiting-body formation by transmitting intercellular signals (18, 22, 34, 38). During the formation of the fruiting body, a morphological change from rod-shaped to spherical cells occurs, and the cells differentiate to form myxospores.

Although low-molecular-weight penicillin-binding proteins (PBPs) of Escherichia coli are dispensable for bacterial growth and division (3, 25), the morphological change during stationary phase requires the PBPs dd-transpeptidase and dd-carboxypeptidase (24, 39). In Bacillus species, many cell wall hydrolases, such as N-acetylmuramoyl-l-alanine amidases (20, 21) and endopeptidases (15), and dd-carboxypeptidases (36) contribute to sporulation and germination. On the other hand, dd-dipeptidase (VanX) and dd-carboxypeptidase (VanY) of vancomycin-resistant enterococci regulate the synthesis of new resistant peptidoglycan precursors and the elimination of wild-type sensitive peptidoglycan precursors (12, 32). There have been very few investigations dealing with cell morphological enzymes of M. xanthus, and the results that have been reported are inconclusive. Recently, we reported that M. xanthus produces dd-carboxypeptidases during development (19). In this paper, we report the cloning and sequencing of a penicillin-resistant dd-carboxypeptidase gene, pdcA, from M. xanthus, comparison of the amino acid sequence of PdcA with those of other penicillin-resistant dd-carboxypeptidases, and the characterization of a pdcA-deficient mutant.

Cloning of dd-carboxypeptidase gene from M. xanthus.

To examine whether M. xanthus IFO13542 (ATCC 25232) produces dd-carboxypeptidase, we attempted to clone the dd-carboxypeptidase gene with appropriate oligonucleotide probes designed from conserved sequences in the dd-carboxypeptidases of PBPs in E. coli or penicillin-resistant dd-carboxypeptidases of vancomycin-resistant Enterococcus. One positive phage was cloned by hybridization with an oligonucleotide probe (van Y_B). The sequence of van Y_B is 5′-CTGGTGCTCC(G)GAC(G)GTGCCCGG-3′ (the nucleotides in parentheses are degenerate), which was designed according to the conserved motifs (PGTSEHQ at amino acid positions 183 to 189) of dd-carboxypeptidase (VanY_B) of Enterococcus faecalis V583 (8). The 3.5-kb PstI fragment of the phage DNA was hybridized with the probe and then subcloned into the PstI site of pBluescript II SK(−) (Stratagene, La Jolla, Calif.) (Fig. 1).

FIG. 1.

Restriction map of the cloned PstI fragment harboring the pdcA gene of M. xanthus. (Top) Restriction map of a 3.5-kb PstI chromosomal fragment. Arrows indicate the positions of ORFs for pdcA and orf1. (Bottom) Insertion of a kanamycin resistance (Km^r) gene into the StuI site of pdcA.

Characterization of pdcA gene.

Sequence determination of the 3.5-kb PstI fragment revealed that there was an open reading frame (ORF) encoding a protein with sequence similarity to dd-carboxypeptidases and N-acetylmuramoyl-l-alanine amidases (Fig. 1). The sequence of the 1.1-kb BamHI-ApaI fragment containing the ORF is displayed in Fig. 2, with some of the upstream sequence appended. In this ORF, designated the pdcA gene, 90% of the codons used C or G at the third base. The protein encoded by this ORF is 302 amino acids long and has an estimated molecular weight of 31,179 and a pI of 11.4. The putative initiation codon was preceded by a purine-rich Shine-Dalgarno-like sequence (AAGGGAAGAG at nucleotides 24 to 33). The 31-nucleotide sequence starting at position 63 downstream of the TGA stop codon has an inverted repeat of 14 bases and may function as a terminator. An incomplete ORF1, transcribed in the opposite direction of the pdcA gene, was present downstream of the pdcA gene (Fig. 1). A computer search with the BLAST program in the GenBank database indicated that the deduced amino acid sequence for ORF1 is similar to that of an antibiotic ATP-binding cassette transporter (26) (data not shown).

FIG. 2.

Nucleotide and deduced amino acid sequences of pdcA. A putative ribosome-binding site is double underlined. Boxed and shaded amino acid sequences represent postulated recognition sites for repeated units of peptidoglycan. The motifs [SxxK, S(Y)xN, and K(H/R)T(S)G] of the penicillin-interactive proteins are boxed. Arrows indicate the position of the palindrome sequence. The sequence corresponding to the probe is underlined.

The PdcA protein contained all the consensus motifs found in the penicillin-interactive proteins, PBPs, and β-lactamases (11, 16). The motifs SxxK (amino acids 133 to 136), S(Y)xN (amino acids 181 to 183, 241 to 243, and 254 to 256), and K(H/R)T(S)G (amino acids 24 to 26 and 119 to 121) were found in the PdcA product, but the order of the motifs was different from the typical order of penicillin-interactive proteins.

Based on the sequence homology, the PdcA protein was divided into two regions. The amino-terminal half of the PdcA protein (positions 1 to 178) exhibited sequence similarity to the carboxy-terminal half of the N-acetylmuramoyl-l-alanine amidase (CwlL) of Bacillus licheniformis (29) (28% identity with positions 176 to 360 of CwlL) and the amino-terminal half of the Zn²⁺-dd-carboxypeptidases (Zn-DD) of Streptomyces albus G (6) (23% identity with positions 1 to 140 of Zn-DD) (Fig. 3). The PdcA product contained four short repeated sequences [DGxF(V)GPKTQ(W)S(D)A(K)V(L) at positions 50 to 61, 73 to 84, 124 to 135, and 147 to 158], and the direct repeats are probably involved in the recognition of repeated units of peptidoglycan of the cell wall (27). Such imperfect direct repeats have been found in noncatalytic regions of various peptidoglycan hydrolases of bacilli (27).

FIG. 3.

Similarity of the deduced amino acid sequence of PdcA to an N-acetylmuramoyl-l-alanine amidase and penicillin-resistant dd-carboxypeptidases: CwlL (29); Zn-DD (6); VanY_B, E. faecalis V583 dd-carboxypeptidase (8); and VanY, E. faecium BM4147 dd-carboxypeptidase (1). The conserved motifs of the dd-carboxypeptidases are boxed; identical residues are shaded. Dashes indicate spaces introduced to maximize alignment.

The amino acid sequence of the carboxy-terminal half of the PdcA protein (positions 179 to 302) was similar to those of the dd-carboxypeptidases (VanY and VanY_B) of vancomycin-resistant enterococci (1, 8) (21 and 36% identities with positions 71 to 221 and 96 to 246, respectively). Motifs [SxHxxGxA(S)xD and EP(W)WH] conserved in dd-dipeptidases and dd-carboxypeptidases of vancomycin-resistant enterococci (32) were present in the carboxy-terminal half of PdcA (Fig. 3). The PdcA protein did not reveal significant similarity to E. coli dd-carboxypeptidases PBP5 and PBP6 (2), and the PdcA protein contained no hydrophobic transmembrane regions.

dd-carboxypeptidase activity of PdcA.

To investigate the biological function of PdcA, expression plasmid pPDC-T was constructed by subcloning a 2.2-kb NcoI fragment containing the pdcA gene into a region downstream of the thioredoxin gene (encoding TrxA) in pET-32a(+) (Novagen, Madison, Wis.) and then transferred to E. coli BL21(DE3) (Novagen). Formation of the TrxA-PdcA fusion product (48 kDa) was induced by 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for 2 h, and the protein was produced in soluble fractions in E. coli. While the cells transformed with pET-32a(+) showed low levels of dd-carboxypeptidase activity, cells transformed with pPDC-T produced a large amount of dd-carboxypeptidase (Table 1). The dd-carboxypeptidase activity was impervious to penicillin at concentrations of 5 to 10 mM. The enzyme activity was also not affected by the addition of 5 mM EDTA or Mg²⁺. Zn-DD (metalloprotease) of S. albus G (5) has been reported to be penicillin resistant. Since the PdcA product was not inhibited by 5 mM EDTA, it was not a metalloprotease. The fusion product did not show dd-dipeptidase activity (data not shown). These results indicate that the characteristics of PdcA are similar to those of penicillin-resistant dd-carboxypeptidases of vancomycin-resistant enterococci (40). We are not aware of any reports on penicillin-resistant dd-carboxypeptidases of gram-negative bacteria.

TABLE 1.

dd-Carboxypeptidase activity of enzyme extracts of E. coli harboring pET-32a or pPDC-T^a

Plasmid	Fusion product induction	Total activity (mU)	Sp act (mU/mg)
pET-32a(+)	−	3.0	0.6
	+	3.6	0.6
pPDC-T (pET-32aΩpdcA)	−	5.0	1.2
	+	55.7	16.6

^aThe dd-carboxypeptidase activity was measured by incubating enzyme extract with 5 mM diacetyl-l-Lys-d-Ala-d-Ala in a final volume of 50 μl for 30 min at 30°C and by estimating the amount of C-terminal d-alanine liberated enzymatically (9). One unit of enzyme catalyzed the hydrolysis of 1 μmol of the substrate per min. 

Characterization of the pdcA mutant.

A kanamycin resistance gene of pTF1 (10) was inserted into the StuI site of the pdcA gene. The insertion mutation was moved into the chromosome of M. xanthus by the electroporation method of Plamann et al. (31). Using Southern hybridization and PCR analyses, we confirmed that the kanamycin resistance gene was inserted into the pdcA gene on the chromosome of the mutant. To investigate its biological function in M. xanthus, the cell morphology, sporulation, and germination of a pdcA deletion mutant were examined. The pdcA deletion mutant produced fruiting bodies of normal size and spore morphology on clone fruiting (CF) agar (13) (data not shown). Differences in cell morphology or germination between the wild type and a pdcA deletion mutant were not observed when vegetative cells or spores were incubated in Casitone-yeast extract (CYE) medium (4).

In vancomycin-resistant enterococci, the vanY gene is a member of the vancomycin resistance van gene cluster (23). In VanA-type enterococci, VanY is nonessential for resistance and has been reported to control the abundance of peptidoglycan precursors (1). M. xanthus produces the antibiotic TA, which inhibits the polymerization step in cell wall formation, leading to an accumulation of lipid intermediates (41), and its mode of action is similar to that of vancomycin (30). Although no significant differences in growth were also observed between the wild type and pdcA mutants grown in antibiotic TA production medium, 0.5 CT (0.5% Casitone and 0.2% MgSO₄ · 7H₂O) (41) (data not shown), this molecule may have a role similar to that of VanY of vancomycin-resistant enterococci. On the other hand, since β-lactamase of M. xanthus is induced by β-lactams (28), PdcA may also play a role in multiple mechanisms to resist β-lactams it encounters in soil. Future work will provide insight into the roles of PdcA in this bacterium.

Nucleotide sequence accession number.

The nucleotide sequence data reported here will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession no. AB023893.