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J Bacteriol, February 1998, p. 749-752, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The lytE Gene of Bacillus subtilis 168 Encodes a Cell Wall Hydrolase
Philippe
Margot,1
Michael
Wahlen,2
Ahmad
Gholamhuseinian,2,
Patrick
Piggot,2 and
Dimitri
Karamata1,*
Institut de Génétique et de
Biologie Microbiennes, CH-1005 Lausanne,
Switzerland,1 and
Department of
Microbiology and Immunology, Temple University School of Medicine,
Philadelphia, Pennsylvania 191402
Received 2 September 1997/Accepted 1 December 1997
 |
ABSTRACT |
Bacillus subtilis cell wall-bound protein CWBP33 is
encoded by lytE, a gene expressed during the exponential
growth phase. Sequence analysis of LytE, a 33-kDa protein, reveals two
domains. The N-terminal domain contains a threefold-repeated motif
common to several peptidoglycan binding proteins, while the C-terminal domain, probably carrying the catalytic activity, has homology with
certain exoproteins. Zymographs unambiguously reveal that the absence
of CWBP33, due to inactivation of lytE, is accompanied by
the loss of a lytic activity. In lytE mutants, the cell
autolysis rate is significantly decreased, although autolysis of
corresponding, purified cell walls does not seem to be
affected.
| |
TEXT |
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of
proteins contained in cell wall preparations has revealed that 12 major
proteins, the so-called cell wall-bound proteins (CWBP), are
associated with the cell wall of exponentially growing cells
(23). Four of the CWBP, attached by electrostatic forces, are peptidoglycan (PG) hydrolases (9, 20). They
comprise LytC, an N-acetylmuramyl-L-alanine
amidase, and LytD (21), an N-acetylglucosaminidase,
corresponding to CWBP49 (16) and CWBP90 (18),
respectively. The absence of any effect of lytC and
lytD inactivation on cell growth (18) prompted us
to search for novel PG hydrolases. We report the identification and
characterization of the gene lytE, whose product, a 33-kDa
polypeptide endowed with lytic activity, is involved in cell
autolysis.
Identification of the DNA sequence encoding CWBP35 and its
analysis.
CWBP were separated by SDS-PAGE and transferred onto a
membrane, and their N-terminal amino acid sequences were
determined (17). QSIKVKKGDTL, the sequence corresponding to
CWBP33, matched perfectly residues 26 to 36 of a putative protein
encoded by a gene called papQ (GenBank accession no.
U38819), subsequently renamed lytE (see below).
The N-terminal sequence of the mature LytE protein starts with amino
acid 26. The first 25 residues, corresponding to a signal peptide,
according to von Heijne (26), were cleaved. Amino acid sequence similarity analyses of LytE (1, 8, 19)
revealed two domains. The N-terminal domain contains three repeats of a 44-amino-acid motif (Fig. 1). It has been
proposed (12) that this motif found in a series of cell wall
lytic enzymes is required for their binding to PG. The C-terminal
domain of LytE has sequence homology with several classes of proteins
that are found outside the cytoplasmic membrane (Fig.
2) and, surprisingly, are endowed with rather different enzymatic activities, such as endopeptidase (Bacillus sphaericus) or amylase (Clostridium
acetobutylicum). None of these motifs were similar to those
characteristic of the main cell wall hydrolases of Bacillus
subtilis, i.e., LytC (14) and LytD (18),
suggesting that LytE is a novel type of PG hydrolase. The sequence
homologies between the C-terminal domains of LytE and a
well-characterized
-D-Glu-LD-(meso)-diaminopimelate
peptidase of B. sphaericus (11) suggests
that LytE may also encode such a peptidase. In addition, LytE has a
cysteine residue in a well-conserved motif which may be compatible with
an endopeptidase activity, since it has been proposed that the B. sphaericus peptidase is a cysteine enzyme (3).
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FIG. 1.
N-terminal domain of LytE. Repeats and their sequence
homology to the following cell wall-associated proteins are shown:
muramidase-2 of Enterococcus hirae (6),
muramidase of Enterococcus faecium (2),
invasion-associated protein P60 of Listeria ivanovii and
Listeria monocytogenes (4), muramidase of
Lactococcus lactis (5), B. subtilis
phage  29 lysozyme (25), and amidases of the B. subtilis defective phage PBSX (7, 15). The consensus
sequence is presented at the bottom of the figure (uppercase letters
represent amino acids conserved in all examined proteins; lowercase
letters represent conserved amino acids belonging to the same
family).
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FIG. 2.
C-terminal domain of LytE. Its homology to the following
exoproteins is shown: invasion-associated protein P60 of Listeria
ivanovii and Listeria monocytogenes (4),
amylase of Clostridium acetobutylicum (24), P54
of Enterococcus faecium (10), NlpC of
Escherichia coli (13), and
D-Glu-LD-(meso)-diaminopimelate
endopeptidase of B. sphaericus (13). The
consensus sequence is presented at the bottom of the figure (uppercase
letters represent amino acids conserved in all examined proteins;
lowercase letters represent conserved amino acids belonging to the same
family).
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lytE regulation.
While a sequence corresponding to
the consensus 
10 domain of a 
A promoter is apparent
70-bp upstream of the start codon, there is no satisfactory match with
the corresponding 35 consensus sequence. Unambiguous evidence
that lytE is transcribed by a vegetative phase promoter are
(i) the presence of LytE in native cell walls obtained from
exponentially growing cells (Fig.
3) and (ii) the expression of a
lytE-lacZ transcriptional fusion during vegetative growth
(data not shown). Since cell wall preparations of parent strains
and of sigD- and sinR-bearing mutants contained
comparable amounts of CWBP33 (data not shown), it follows that
the alternate factor D does not play a role
in lytE transcription. Moreover, transfer of the
lytE-lacZ fusion into a sinR background did not
affect lytE expression (data not shown).
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FIG. 3.
(A) SDS-PAGE of CWBP extracted from L16601
lyt+ (lane 1) and L16638 lytE (lane
2). Standard Mr proteins (Bio-Rad broad range)
are also shown (lane 3). (B) Results of renaturation gel assay
performed with CWBP extracted from L16601 (lane 1) and L16638
lytE (lane 2). Pictures of the gels were digitally recorded
with a charge-coupled device camera (Vilbert Lourmat, Marme la
Vallée, France) and processed by computer. Molecular weights of
standard proteins are shown on the right (in thousands).
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lytE mutant behavior.
To construct strain L16638
carrying a 
lytE::Cam, a cat
cassette was inserted between the ClaI and PstI
restriction sites (bases 644 to 1184 of the sequence of GenBank
accession no. U38819) of a plasmid whose insert contains the
whole sequenced region. The resulting plasmid was used to transform
strain L16601 to chloramphenicol resistance (17). The
motility, growth rate, chain separation, and sporulation in the MTOa or
Schaeffer media (17, 22) did not differ between the
lytE-deficient and the parent strains. Analysis of native
cell walls of strain L16638 lytE by SDS-PAGE revealed the
absence of CWBP33, confirming that lytE does indeed encode
this protein (Fig. 3A). As expected, the renaturation gel assay
(9) of strain L16638 did not reveal the clearing zone corresponding to the CWBP33 lytic activity (Fig. 3B). LytE may correspond to CwlF, a 35-kDa protein (20), or to autolysin
A3, a 34-kDa protein (9). However, methods for
extracting the CwlF and autolysin A3 differ from that used for
CWBP33, making comparison difficult. Incidentally, the weak
clearing zone corresponding to a protein with a slightly higher
Mr (Fig. 3B) may be due to either CwlF or to
autolysin A3.
Native cell walls of strains L16601 lyt+ and
L16638 lytE exhibited similar autolysis rates (data not
shown), suggesting that the contribution of LytE to global cell wall
lytic activity is marginal, a behavior characteristic of LytD, the
glucosaminidase (18). However, autolysis of whole cells
(17) of the lytE-deficient mutant was
considerably decreased and delayed as compared to that of the parent
strain (Fig. 4), a behavior
resembling that of a strain deficient in LytC, the main
vegetative amidase (16, 21). However, while
lytC-deficient mutants are characterized by a very long
delay in their cell wall turnover kinetics (16), no such delay was observed in lytE-bearing strains (data not shown).
We hypothesize that LytE may be located at some specific points in the
cell wall and, through its action, could create points suitable for the
action of the LytC amidase; this, in turn, could rapidly perforate the
cell wall and accelerate the release of the cytoplasmic material. In
the absence of LytE, the main vegetative amidase, LytC, most likely
located at the outer wall surface, would have to gradually degrade the
entire surface of the cell wall, leading to delayed autolysis. This
interpretation is compatible with our observation that the absence of
LytE has no significant effect on the autolysis of isolated cell wall
preparations.
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FIG. 4.
Cell autolysis. The symbols along the solid line
(L16601) and the broken line (L16638 lytE) represent average
values from three different experiments. The vertical line for each
point corresponds to the standard deviation. Results were normalized to
nephelometric density (ND) at time 0 (ND0), i.e., percent
lysis at time t = (ND0 ND at time
t)/ND0.
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ACKNOWLEDGMENTS |
The expertise of Arthur Moir (University of Sheffield, Sheffield,
United Kingdom), who performed the protein sequencing, is gratefully
acknowledged.
This work was supported by grant 31-42322.94 from the Fonds National
Suisse de la Recherche Scientifique (to D.K.) and by Public Health
Service grant GM43577 from the National Institutes of Health (to P.P.).
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut de
Génétique et de Biologie Microbiennes, Rue César-Roux
19, CH-1005 Lausanne, Switzerland. Phone: 41 21 3206075. Fax: 41 21 3206078. E-mail: dimitri.karamata{at}igbm.unil.ch
Present address: Kerman University of Medical Sciences, Kerman,
Iran.
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J Bacteriol, February 1998, p. 749-752, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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