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Journal of Bacteriology, August 1999, p. 4696-4699, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Cloning, Sequence Analysis, and
Characterization of a Penicillin-Resistant
DD-Carboxypeptidase of Myxococcus
xanthus
Yoshio
Kimura,*
Yukie
Takashima,
Yushi
Tokumasu, and
Masayuki
Sato
Department of Life Sciences, Faculty of
Agriculture, Kagawa University, Kagawa 761-0795, Japan
Received 11 March 1999/Accepted 20 May 1999
 |
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.
| |
TEXT |
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
YB). The sequence of van YB 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 (VanYB) 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).
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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
(Kmr) gene into the StuI site of
pdcA.
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|
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).
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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.
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|
The PdcA protein contained all the consensus motifs found in the
penicillin-interactive proteins, PBPs, and
-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
Zn
2+-
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).
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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); VanYB, 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.
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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--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.
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% MgSO
4 · 7H
2O)
(
41) (data not shown),
this molecule may have a role similar
to that of VanY of vancomycin-resistant
enterococci. On the other hand,
since
-lactamase of
M. xanthus is induced by
-lactams
(
28), PdcA may also play a role in multiple
mechanisms to
resist
-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.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant-in-aid for scientific
research (no. 09760305) from the Ministry of Education, Science and
Culture of Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Life Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho,
Kagawa 761-0795, Japan. Phone: 81-87-891-3118. Fax: 81-87-891-3021. E-mail: kimura{at}ag.kagawa-u.ac.jp.
| |
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Journal of Bacteriology, August 1999, p. 4696-4699, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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