Download Novel surface layer protein genes in Bacillus

Microbiology (2005), 151, 2961–2973
DOI 10.1099/mic.0.28201-0
Novel surface layer protein genes in Bacillus
sphaericus associated with unusual insertion
elements
Katrin Pollmann, Johannes Raff, Michaela Schnorpfeil, Galina Radeva3
and Sonja Selenska-Pobell
Correspondence
Institute of Radiochemistry, Forschungszentrum Rossendorf, D-01314 Dresden, Germany
Katrin Pollmann
[email protected]
Received 17 May 2005
Revised
20 June 2005
Accepted 23 June 2005
The surface layer (S-layer) protein genes of the uranium mining waste pile isolate Bacillus
sphaericus JG-A12 and of its relative B. sphaericus NCTC 9602 were analysed. The almost
identical N-termini of the two S-layer proteins possess a unique structure, comprising three
N-terminal S-layer homologous (SLH) domains. The central parts of the proteins share a high
homology and are related to the S-layer proteins of B. sphaericus CCM 2177 and P-1. In contrast,
the C-terminal parts of the S-layer proteins of JG-A12 and NCTC 9602 differ significantly
between each other. Surprisingly, the C-terminal part of the S-layer protein of JG-A12 shares a
high identity with that of the S-layer protein of B. sphaericus CCM 2177. In both JG-A12 and
NCTC 9602 the chromosomal S-layer protein genes are followed by a newly identified putative
insertion element comprising three ORFs, which encode a putative transposase, a putative
integrase/recombinase and a putative protein containing a DNA binding helix–turn–helix motif, and
the S-layer-protein-like gene copies sllA (9602) or sllB (JG-A12). Interestingly, both B. sphaericus
strains studied were found to contain an additional, plasmid-located and silent S-layer protein
gene with the same sequence as sllA and sllB. The primary structures of the corresponding putative
proteins are almost identical in both strains. The N-terminal and central parts of these S-layer
proteins share a high identity with those of the chromosomally encoded functional S-layer proteins.
Their C-terminal parts, however, differ significantly. These results strongly suggest that the
S-layer protein genes have evolved via horizontal transfer of genetic information followed by DNA
rearrangements mediated by mobile elements.
INTRODUCTION
Regularly structured paracrystalline surface layers (S-layers)
are one of the most common surface structures found in
bacteria and archaea. Most of them are composed of protein
or glycoprotein monomers with the ability to self-assemble
in two-dimensional arrays (Sára & Sleytr, 2000; Sidhu &
Olsen, 1997). In most cases, they are the major protein
3Present address: Bulgarian Academy of Sciences, Institute of
Molecular Biology, Acad. G. Bonchev Str., Bl. 21, Sofia 1113, Bulgaria.
Abbreviations: S-layer, surface layer; SLH, S-layer homologous.
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are AJ866974 (B. sphaericus NCTC 9602),
AJ866975 (B. sphaericus JG-A12), AJ849547 (slfA), AJ849548 (sllA),
AJ849549 (slfB), and AJ849550 (sllB). The IS elements ISBsph1 and
ISBsph2 have been deposited in the ISFinder database (http://www-is.
biotoul.fr).
A table of all the oligonucleotides used for sequencing and
amplification is available as supplementary data with the online version
of this paper.
0002-8201 G 2005 SGM
species, constituting up to 15 % of the total protein produced by the cells (Kuen et al., 1994). As the outermost cell
structure the S-layers serve as an interface between the
bacterial cell and the environment. Several functions have
been ascribed to S-layers: a molecular sieve (Sára & Sleytr,
1987), virulence factors in several pathogenic bacteria
(Ishiguro et al., 1981; Lewis et al., 1987; Mesnage et al.,
2001; Mignot et al., 2001), an attachment structure for
high-molecular-mass extracellular proteins such as amylases (Matuschek et al., 1994; Egelseer et al., 1995, 1996), an
adhesin with affinity for human epithelial cells and fibronectin (Hynönen et al., 2002), and sorption of toxic heavy
metal ions (Raff, 2002; Merroun et al., 2005).
It is essential for micro-organisms to be able to respond
rapidly to changes in the environment. One way in which
they can do this is to alter their surface properties by varying
protein expression through programmed DNA rearrangements (Borst & Greaves, 1987). Such DNA rearrangements,
which can be mediated by a variety of mechanisms, affect
the phenotypic characteristics of surface structures, e.g. for
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K. Pollmann and others
evasion of host defences, antigenic variation or fine tuning of surface structures (Dybvig, 1993). Recombinational
events have also been described for the regulation of the
expression of S-layer protein genes from various bacterial
strains. It is assumed that bacteria carrying an S-layer use Slayer variation to adapt to different stress factors (JakavaViljanen et al., 2002; Kuen et al., 1997; Mignot et al., 2001,
2002). Chromosomal rearrangements include the inversion
of promoters located between two oppositely orientated Slayer gene cassettes (Dworkin & Blaser, 1996), recombination of S-layer gene segments (Blaser et al., 1994), and
inversion of chromosomal segments containing two variants
of S-layer-encoding genes orientated in opposite directions,
resulting in the placement of the formerly silent gene behind
the promoter (Boot et al., 1996a, b; Boot & Pouwels, 1996).
In the case of Geobacillus stearothermophilus PV72, S-layer
protein variation is mediated by the replacement of the
chromosomally encoded active S-layer protein gene sbsA
with the plasmid-encoded formerly silent S-layer protein
gene sbsB, resulting in the activation of sbsB (Scholz et al.,
2001).
The uranium mining waste pile isolate B. sphaericus JG-A12
is enveloped by an S-layer with a square symmetry, which
is composed of identical protein monomers (Raff, 2002).
The cells of B. sphaericus JG-A12, like those of the closely
related B. sphaericus NCTC 9602, are able to bind selectively
and reversibly high amounts of metals such as uranium,
lead, copper, aluminium, gallium and cadmium from drain
waters of uranium wastes (Selenska-Pobell et al., 1999).
Further analyses showed that the purified and recrystallized
S-layers of both strains bind high amounts of uranium
in a strain-specific way (Raff, 2002; Raff & Selenska-Pobell,
2004). This indicates that the S-layers play a key role in
interactions of the bacteria with metals (Raff, 2002).
Interestingly, the S-layers of the B. sphaericus strains JGA12 and NCTC 9602 both have square symmetry with
almost identical lattice constants, and their protein monomers have the same size and almost identical N-terminal Slayer homologous (SLH) domains (Raff, 2002). However,
they differ significantly in their pH and proteolytic stability
and in their kinetics of uranium binding (Raff, 2002; Raff
et al., 2004). Recently we demonstrated that the S-layer of
strain JG-A12 possesses a significantly higher binding capacity to uranium in solution: in contrast to strain NCTC
9602, the S-layer of strain JG-A12 is able to bind almost
all uranium dissolved in water in environmentally relevant
concentrations of 5 mg l21 and below (Raff & SelenskaPobell, 2004).
In order to elucidate the underlying causes of these differences, we studied the primary structures of the two S-layer
proteins. In the present work the chromosomally encoded
S-layer protein genes of B. sphaericus JG-A12 and B.
sphaericus NCTC 9602 and their upstream and downstream regions were sequenced. In addition to the functional
S-layer genes, a second silent, plasmid-located S-layerprotein-like gene was found and characterized in both the
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strains. The chromosomal S-layer protein genes are followed
by a putative mobile gene element carrying a truncated
S-layer-protein-like gene with the same sequence as the
plasmid-encoded silent gene. To our knowledge this is the
first report of an insertion element encoding an S-layerprotein-like gene as well as a transposase.
METHODS
Bacterial strains and culture conditions. B. sphaericus JG-A12
was recovered from a soil sample collected from the uranium
mining waste pile Haberland near the town of Johanngeorgenstadt,
Germany (Selenska-Pobell et al., 1999). B. sphaericus NCTC 9602
was purchased from the National Collection of Type Cultures,
London, UK. Both B. sphaericus strains were routinely grown in
nutrient broth (NB) medium consisting of 5 g peptone l21 and 3 g
meat extract l21. For isolation of RNA from bacterial cells treated
with Cd2+, the strains were grown in defined medium (Chan et al.,
1973). In the early exponential growth phase a solution of CdCl2
was added to give a final concentration of 50 mM Cd2+. The cells
were harvested after 3 h incubation at 30 uC.
DNA manipulations. For PCR amplification genomic DNA of B.
sphaericus NCTC 9602 and B. sphaericus JG-A12 was isolated and
purified using the NucleoSpin kit (Machery-Nagel) according to
the manufacturer’s instructions. For the construction of vectorette
libraries and for Southern hybridizations, genomic DNA was prepared as described by Ausubel et al. (1993). Digestion of DNA with
restriction endonucleases (Promega, Invitrogen) and separation of
DNA fragments by agarose gel electrophoresis were performed by
published methods (Sambrook et al., 1989).
PCR, oligonucleotides, vectorette libraries and sequence
analysis. PCR amplifications were carried out using a T3 Thermo-
cycler (Biometra). Oligonucleotide synthesis was done by MWGBiotech AG. The sequences of all oligonucleotides used are listed
in Supplementary Table S1 with the online version of this paper. All
amplification reactions were performed in a volume of 20 ml using
Taq DNA polymerase (Promega), and the conditions were optimized for each primer pair. The Universal Vectorette System UVS-1
(Sigma-Aldrich) was chosen to perform asymmetric PCR after hostindependent cloning. For the construction of different vectorette
libraries, 1 mg genomic DNA from each of the strains B. sphaericus
NCTC 9602 and JG-A12 was digested with the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HaeIII, HindIII, Sau3aI or TaqI.
The corresponding libraries were constructed according to the manufacturer’s instructions. PCR amplifications of the ligated fragments
were carried out in a 20 ml reaction volume containing 1 ml of
the vectorette library as template, 1 nmol each of dATP, dCTP,
dGTP and dTTP (Invitrogen), 2?5 mM MgCl2, 1?6 mg BSA, 20 pmol
Vectorette Primer, 20 pmol sequence-specific primer, and 1 unit
Taq polymerase in 16 reaction buffer.
For cloning of PCR products the TOPO-TA cloning kit (Invitrogen)
was used according to the manufacturer’s instructions. Plasmid
isolation was done using Wizard Plus SV Miniprep (Promega). The
plasmids were used as template for PCR amplification of S-layer gene
fragments using insert-specific primers.
The PCR products obtained were purified using the Quickstep 2 PCR
Purification Kit (Edge Bio Systems) and sequenced from each strand
using a Perkin-Elmer Applied Biosystems 377 instrument.
Isolation of RNA. Total RNA was isolated from exponentially
growing B. sphaericus NCTC 9602 and JG-A12 harvested at an
OD600 of 0?6 (NB medium) and from cells grown in defined
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Microbiology 151
Novel surface layer protein genes in B. sphaericus
medium treated with Cd2+ using the RNeasy Mini Kit (Qiagen)
according to the manufacturer’s instructions. The remaining DNA
was digested on-column by using the RNase free DNase Set
(Qiagen). RNA isolation and blotting was repeated three times.
blue and destaining. The N-terminal amino acid sequences of the
protein fragments were determined using an ABI 494A Procise HT
sequencer (Applied Biosystems).
Plasmid screening. Aliquots of 500 ml B. sphaericus overnight cul-
Northern and Southern hybridization. After digestion of total
DNA of B. sphaericus JG-A12 and NCTC 9602, DNA fragments were
separated on a 1 % agarose gel (SeakemLE). Southern blots were
performed on positively charged nylon membranes (Roche) using a
TurboBlotter (Schleicher & Schuell), following the manufacturer’s
instructions. Northern blotting of total RNA was performed according to published methods (Sambrook et al., 1989), by applying
2 mg total RNA of Cd2+-treated cells, and 5 mg total RNA of NBgrown cells of JG-A12 or 9602, respectively. For hybridization of
the Southern and Northern blots, DNA probes specific for S-layer
protein genes were labelled with DIG-dUTP by incorporation during
PCR using the PCR DIG Probe Synthesis Kit (Roche).
The filters were hybridized with a 922 bp PCR-generated probe
obtained with primer pair Mobil_F/Mobil_R (59-ATCGCCAATTTTGGTGTCGTGGA-39/59-ATGTACCGTTACCCGAAATACTCGA-39),
comprising a part of the putative transposase gene of JGA12 and 9602,
with a PCR-generated probe obtained with primer pair V83/R71 (59ATGCTTTATCTCCAATAATATC-39/59-AAATGTGTATGTACCGTTACCCGAA-39), comprising the putative recombinase genes of 9602
and JG-A12, with an 857 bp PCR-generated probe obtained by using
primer pair V8/R30 (59-AAAGAAATCGCAGGCGTAG-39/59-TTTRTAAGTAACTTTGTAAGTTTT-39) comprising the central region of
the S-layer protein gene of JG-A12, or with PCR-generated probes
obtained with primer pair V37/R64 (59-GGTTCCGTACTAAAAGATGCGGTG-39/59-TGGAGTTGGCTTTACTGTAATAG-39), comprising the 39-region of the sll genes. Hybridization temperature was
38 uC. Northern and Southern blots were developed using the DIG
Nucleic Acid Detection Kit (Roche). The size of the DNA fragments
was estimated using the DIG-dUTP-labelled DNA Molecular Weight
Marker II (Roche). The size of the S-layer-specific mRNA was
estimated using the DIG-dUTP-labelled RNA Molecular Weight
Marker II (Roche).
RT-PCR. For reverse transcription of the S-layer mRNA followed by
PCR the GeneAmp Reverse Transcriptase RNA PCR Kit (Applied
Biosystems) was used. Reverse transcription and PCR were performed according to the manufacturer’s instructions. For amplification of the upstream regions of the genes, primer pair UR2F/
9602_1R (59-TGATTGACAACTTTTAGGGATACAA-39/59-CGCTGTTACCTGGGTTGAAGTGA-39) was used. For amplification of the
39-regions of slf and sll, primer pairs R(V)87/V29 [59-ATCAGCATCTGATGCTACATC-39/59-CGCWCCAAACCGCGTGCATACA(GT)T-39] (slfA), R(V)77/V32 [59-GAACCCACTGGGCAATAATTTA-39/59-CGCGATGGTGGCGAAGTTGCGGC-39] (slfB) and R64/
V37 [59-TGGAGTTGGCTTTACTGTAATAG/59-GGTTCCGTACTAAAAGATGCGGTG-39] (sll) were used.
Western blotting. S-layer proteins of B. sphaericus NCTC 9602
and JG-A12 were purified as described previously (Raff, 2002). For
digestion with the endoproteinase Glu-C (Sigma-Aldrich), 200 mg of
purified S-layer proteins was resuspended in 32 ml of a 50 mM
KH2PO4/Na2HPO4 buffer (pH 7?8). After addition of 2 mg of the
endoproteinase Glu-C, the solution was incubated for 24 h at 37 uC.
The resulting fragments were separated on a 12?5 % SDS-PAGE
(Laemmli, 1970) using a Mini-PROTEAN II (Bio-Rad) and transferred onto a PVDF membrane using a Trans-Blot SD Semi-Dry
Transfer Cell (Bio-Rad), using 12 mM Tris base/8 mM CAPS/15 %
(v/v) methanol as cathode buffer and 12 mM Tris base/8 mM
CAPS/1?14 % (w/v) thioglycolate as anode buffer. The gel was blotted
for 80 min at a current of 1?5 mA cm22. Visible protein bands were
cut after staining the blotting membrane with Coomassie Brilliant
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tures grown in NB were inoculated in 10 ml fresh NB and shaken at
30 uC for about 1 h to reach an OD600 of 0?3. One millilitre of the
fresh culture was pelleted by centrifugation at 6000 g. The cells were
washed with TE buffer (10 mM Tris base, 1 mM EDTA, pH 8?0)
and resuspended in 20 ml 25 % sucrose in TE. Afterwards 20 ml
of a lysis solution containing lysozyme (4 mg ml21) and RNase A
(2 mg ml21) was carefully added. Lysis of the cells was achieved
directly in the wells of a low-percentage agarose gel and visualization
of the plasmids was performed as described previously (SelenskaTrajkowa et al., 1990).
Bioinformatic
analysis
of
S-layer
genes
and
proteins.
Sequence assembling was performed using AutoAssembler 2.0
(Applied Biosystems). For ORF predictions and restriction analyses
BioEdit was used. Isoelectric point and molecular mass predictions
were performed with the ExPASy Molecular Biology Server (http://
www.expasy.org). Multiple alignments of protein sequences were
performed using CLUSTALW. Predictions of secondary structures
were done using the mfold program (Zuker et al., 1999; http://www.
bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi).
Computer
searches and sequence comparisons were done using BLAST (Altschul
et al., 1990).
RESULTS
Characterization of the S-layer genes of
B. sphaericus NCTC 9602 and JG-A12
The entire sequence of the S-layer protein gene of B.
sphaericus NCTC 9602 (slfA) (accession no. AJ849547)
indicated one ORF of 3684 bp encoding a protein of
1228 aa. The ORF starts with ATG and is preceded by a
typical ribosome-binding site (GGAGG) with a distance of
13 bp between the middle A of this sequence and the start
codon. Two putative promoter sequences were identified
at 106 nt (TTGACA, 235) and 83 nt (TATACT, 210) upstream of the start codon, which are characteristic for strong
prokaryotic promoters. An inverted repeat of 34 nt was
found at positions 57–90 downstream from the stop codon
TAA of the ORF. This repeat forms the characteristic stem–
loop of a terminating transcription signal, followed by an
AT-rich region (Fig. 1a).
The entire S-layer protein gene sequence of the uranium
mining waste pile isolate B. sphaericus JG-A12 (slfB)
(accession no. AJ849549) indicated an ORF of 3714 bp,
encoding a protein of 1238 aa. A putative terminating signal
is found 29 nt downstream from the stop codon TAA,
forming a stem–loop of 42 nt followed by an AT-rich region
(Fig. 1a).
The region upstream of the slfB gene is identical to that of
the slfA gene of strain 9602, comprising the same ribosomebinding site and the same putative promoters (Fig. 1a).
Similarly, the first 630 nt of the two ORFs are almost
identical, whereas the central regions of the genes from
position 630 to position 2550 share a decreasing identity
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K. Pollmann and others
Fig. 1. Organization of the functional S-layer protein genes of B. sphaericus JG-A12 and NCTC 9602 (a) and of their silent
plasmid-encoded S-layer protein genes (b). RBS, ribosome-binding site; Pos., position on the transcript from the start codon.
The ribosome-binding sites are indicated in bold letters; the predicted promoters are underlined; the coding regions are
marked with boxes; the degree of identity of the coding sequences is indicated by shading; different sequences are marked
with different symbols.
from 90 % to 70 %, and after position 2550 the genes differ
significantly.
The sizes of the mRNAs transcribed from the two Slayer protein genes were determined by Nothern blotting
using an S-layer-protein-gene-specific DIG-labelled PCRgenerated DNA probe (primer pair V8/R30: see Methods).
Transcripts of about 3900 bp were detected in both strains,
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which is in accordance with the sizes of the two abovedescribed ORFs. The expression of the slfA and slfB genes
was confirmed by RT-PCR using primer pairs specific to
the upstream regions and the 59-terminal sequences and
by N-terminal sequencing of five (strain JG-A12) and nine
(strain 9602) protein fragments resulting from a digestion of
purified S-layer proteins by endoproteinase Glu-C (data not
shown).
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Novel surface layer protein genes in B. sphaericus
The S-layer protein genes of both strains are preceded by an
ORF of 714 bp that shows an identity of 42 % to the Bacillus
cereus ATCC 14579 N-acetylglucosaminyldiphosphoundecaprenol N-acetyl-b-D-mannosaminyltransferase (accession
no. NP_835080), which is involved in the biosynthesis of
teichoic acid linkage units in bacterial cell walls, and also
42 % identity to the teichoic acid synthesis protein A (accession no. AAS09508) of Lactobacillus johnsonii NCC 533.
Interestingly, similar to the upstream promoter-carrying
regions of the S-layer protein genes, the nucleotide sequences of the putative teichoic acid synthesis protein genes of
the strains JGA12 and 9602 share 100 % identity. To our
knowledge this is the first report of an S-layer protein gene
located downstream of a gene encoding an enzyme which is
involved in cell wall synthesis. Whether this localization is
connected to the regulation of cell wall synthesis has to be
elucidated in future.
The expressed S-layer proteins SlfA and SlfB have identical
signal peptides of 31 aa that are responsible for the protein
secretion to the cell surface. The predicted site for the
cleavage of the signal peptides is located between the alanine
residues 31 and 32. The theoretical molecular masses of the
mature proteins are 126 624 Da for SlfA and 126 254 Da for
SlfB, which are in accordance with the masses previously
determined by SDS-gel electrophoresis (Raff, 2002). The
calculated pI values of the mature proteins are 5?2 (SlfA)
and 5?1 (SlfB). Analyses of the hydrophobicity profiles of
the functional proteins (Kyte & Doolittle, 1982) show in the
central regions of the proteins – between residues 320–696
(SlfA) and 444–889 (SlfB) – a remarkably low content of
hydrophobic amino acids.
The primary structures of the two functional S-layer proteins studied demonstrate, similar to many other S-layers
(Bahl et al., 1997; Engelhardt & Peters, 1998; Ilk et al., 2002;
Kuen et al., 1997; Mesnage et al., 2001), a high content of
acidic amino acids (aspartate and glutamate) and of serine
and threonine residues. The low content of histidine, tryptophan and methionine, as well as the absence of cysteine, is
characteristic not only of the studied S-layer proteins but
also of all other described S-layer proteins of Bacillaceae
(Sára & Sleytr, 2000; Sidhu & Olsen, 1997). The N-terminal
regions of SlfA and SlfB were found to be identical, containing three acidic S-layer homologous (SLH) domains,
starting at positions 3, 68 and 135 after the end of the signal
peptide. These SLH domains are larger than those of the
other known S-layer proteins of B. sphaericus and their
primary structures are completely different. They are probably involved in anchoring the S-layer subunits to the cell
wall components as demonstrated for the SLH domains of
other S-layer proteins (Engelhardt & Peters, 1998; Ilk et al.,
1999; Sára & Sleytr, 2000).
The two proteins share a very high, slowly decreasing
identity from 96 % to about 60 % in their central region up
to positions 873 of SlfA and 869 of SlfB. In contrast, the
following C-terminal parts of the proteins show no similarity between each other (Fig. 2). The amount of glutamic
acid and aspartic acid residues, carrying carboxylic groups,
and of serine and threonine residues, carrying hydroxyl
groups, is strikingly high in these regions. However, the
number of serine and threonine residues is significantly
higher in SlfB than in SlfA. Stretches of these amino acids are
found throughout the central and C-terminal parts of both
Fig. 2. Comparison of the primary structures of the B. sphaericus S-layer proteins studied to date. The percentage amino
acid identity between the known S-layer proteins of B. sphaericus strains is shown. NTD, N-terminal domain; CD, central
domain; CTD, C-terminal domain; Pos., position; trunc., truncated; IS, putative IS element. Symmetry data are according to
Raff (2002).
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K. Pollmann and others
proteins. However, they are differently organized and distributed in the two proteins. A remarkable feature is the
occurrence of such accumulations in the variable regions of
the more conserved central parts: e.g. an insert with the
sequence GAVTTTSYT in SlfB, an insert with the sequence
TTVDKD in SlfA, and the sequence DTTETDKFT in SlfB.
Localization and analysis of a second S-layerprotein-like gene copy on large plasmids
The abrupt change to low identity of the S-layer protein
genes of strains 9602 and JG-A12 (Fig. 1a) may indicate a
horizontal gene transfer between different organisms and/or
DNA rearrangements. Bearing in mind that plasmids play an
important role in horizontal gene transfer and that they are
widely distributed within the genus Bacillus, we studied the
plasmid contents of B. sphaericus NCTC 9602 and JG-A12
and checked for the presence of S-layer protein gene copies
on them. After electrophoretic separation of cell lysates of
strains 9602 and JG-A12 on low-percentage agarose gels,
large plasmids were visualized in both strains. Southern
hybridization using an S-layer-protein-gene-specific probe
generated by primer pair V8/R30 (see Methods) showed that
a second S-layer gene copy is located on large plasmids in
both strains (data not shown).
Both S-layer protein gene copies were analysed by PCR
amplification of the different vectorette libraries using Slayer-protein-gene-specific primers and by cloning and
sequencing of PCR products. The entire sequences of the
plasmid-located S-layer protein-like gene copy sllA (accession no. AJ849548) of strain 9602 and of the gene copy sllB
(accession no. AJ849550) of strain JG-A12 indicate ORFs
of 3297 bp and 3303 bp, encoding a protein of 1099 and
1101 aa, respectively. Thus, the two putative S-layer proteins should be significantly smaller than the functional
proteins (Fig. 1b). Remarkably, the sequences of the two sll
genes are almost identical. Furthermore, the 59-sequence
regions of the ORF of the two sll genes are identical to those
of the slf genes, whereas their 39-terminal regions differ
significantly from those of the functional genes (Fig. 2).
Moreover, the regions of the ribosome-binding sites of
the sll copies were found to be identical to those of the
chromosomally encoded S-layer genes. A direct repeat is
found at position 46–97 downstream from the stop codon
TAA, forming a deduced stem–loop of 51 nt followed by an
AT-rich region. Thus, the predicted transcripts should have
a size of about 3500 nt, which is significantly smaller than
those of the functional slf genes. However, neither Northern
blot analysis nor RT-PCR could prove expression of the Slayer-protein-like sll genes under the conditions described
in this work. The exclusive synthesis of the Slf proteins
was further confirmed by N-terminal sequencing of fragments of isolated S-layer proteins after digestion with the
endoproteinase Glu-C.
The N-terminal parts of the predicted silent proteins SllA
and SllB are similar to those of the functional proteins SlfA
and SlfB, possessing almost identical signal peptides with
the exception of one amino acid in SllA. The theoretical
molecular masses of the mature putative S-layer proteins
are 113 313 Da with a pI of 5?3 for SllA, and 111 990 Da with
a pI of 5?3 for SllB. Both proteins show all typical features of
S-layer proteins. The central regions of the putative proteins
SllA and SllB are very similar to those of SlfA and especially
SlfB. Interesting are several conserved regions shared by the
putative proteins and SllB. The almost identical C-terminal
regions of SllA and SllB differ significantly from those of
SlfA and SlfB and are shorter. Similar to the functional
proteins, accumulations of serine, threonine, aspartate, and
glutamate are found throughout the central and C-terminal
parts, but with a different distribution and a significantly
lower amount compared to SlfA and SlfB. However, since
no expression of these proteins has yet been detected, their
probable symmetries or interactions with metals remain
unknown.
Identification and organization of a novel
putative insertion element
The nucleotide sequences of the regions following the Slayer protein genes slfA and slfB were determined. Following the highly divergent 39-terminal parts of slfA and slfB,
large fragments of 7157 bp (strain 9602, accession number
AJ866974) and 7139 bp (strain JG-A12, accession number
AJ866975) were sequenced, and they showed a surprisingly
high identity to each other. Sequence analyses predicted the
presence of an ORF designated ORFA, followed by two
divergently transcribed ORFs, designated ORFB and ORFC,
and a sequence identical to the plasmid-encoded S-layer
protein genes sllA and sllB of strains JG-A12 and 9602
(Fig. 3). Analysis of the ORFA gene sequence showed a high
Fig. 3. Comparison of the organization of the putative IS elements ISBsph1 and ISBsph2, found on the chromosome of B.
sphaericus NCTC 9602 and JG-A12, respectively, with IS657 of B. halodurans. Inverted repeats are shown in capitals and
direct repeats are underlined.
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Microbiology 151
Novel surface layer protein genes in B. sphaericus
Putative
recombinase
An almost identical ORF was identified in B. sphaericus
NCTC 9602 starting 615 nt downstream from the stop
codon of the S-layer protein gene slfA. Sequence comparisons of the primary structure of the predicted protein
encoded by the ORF showed 97 % identity to the putative
protein encoded by the ORFA of strain JG-A12. Hence, the
S-layer protein genes of both analysed strains are followed by
a putative bacterial transposase-encoding gene.
In order to determine the number of putative transposase
gene copies distributed within the genomes of the two
strains, chromosomal DNA was digested using the restriction endonucleases EcoRV, ClaI and SpeI. After electrophoretic separation, the fragments were transferred to a
nylon membrane and hybridized with a DNA probe specific
to the putative transposase gene. Southern blots of all digests
gave one signal in each lane (Fig. 4), indicating the presence
of only one gene copy on the chromosome of each strain.
However, whether transposase genes are also associated with
the sll genes found on the plasmids remained unclear.
Characterization of ORFB
Following the putative transposase gene of strain JG-A12, an
ORF of 525 bp was identified at position 217 downstream
from the stop codon TAA of ORFA, encoding a putative
protein of 175 aa with a theoretical molecular mass of
19 902 Da and a calculated pI of 9?66. Interestingly, the
predicted gene is orientated in the opposite direction to slfB
and ORFA (Fig. 3). BLAST analysis of the protein sequence
showed an identity of 32 % to a putative integrase of Vibrio
vulnificus YJ016 and of 31 % to the integrase/recombinase
http://mic.sgmjournals.org
Putative
transposase
21
9.416
6.557
4.361
EcoRI
ClaI
SpeI
Marker
Marker
EcoRV
ClaI
2.322
2.027
Characterization of ORFA
ORFA, consisting of 450 bp, starts at position 317 downstream from the stop codon TAA of the S-layer protein gene
slfB of strain JG-A12. It encodes a putative protein of 150 aa
with a theoretical molecular mass of 17 716 Da and a
calculated pI of 9?59. BLAST analysis of the primary structure
of the protein showed 72 % identity to an IS605/IS200-like
transposase of Clostridium acetobutylicum ATCC 824, 71 %
to a transposase of Clostridium thermocellum ATCC 27405,
70 % to an IS605/IS200-like transposase of Staphylococcus
epidermidis ATCC 12228, and 67 % to the IS657 transposase
of the strain Bacillus halodurans C-125.
kbp
(a)
SpeI
identity of 68 % with the IS605/IS200-like transposase of
Clostridium acetobutylicum ATCC 824. In the case of strain
9602, the 6560 bp fragment comprising ORFA, ORFB,
ORFC and an intact copy of sllA is flanked by a direct repeat
of 2 bp (TT) and an imperfect inverted repeat of 14 bp. In
the case of strain JG-A12, the 6313 bp fragment containing
ORFA, ORFB, ORFC and a truncated sllB copy is flanked
by a direct repeat of 7 bp (TTGTGTG) and an imperfect
inverted repeat of 8 bp (Fig. 3). On the basis of these results
we suggest the presence of a new insertion element in each
strain, designated ISBsph1 (strain 9602) and ISBsph2 (strain
JG-A12), respectively.
(b)
21
9.416
.
6 557
4.361
2.322
2.027
Fig. 4. Southern hybridization. Genomic DNA of Bacillus
sphaericus JG-A12 (a) and NCTC 9602 (b) was digested
using different restriction endonucleases. After electrophoretic
separation and blotting, the fragments were hybridized using a
DIG-labelled PCR probe specific to the putative recombinase
or the putative transposase of JG-A12 generated by the primer
pairs V83/R71 and Mobil_F/Mobil_R, respectively (see Methods
for primer sequences). Weak signals are indicated by arrows.
XerD of Vibrio parahaemolyticus RIMD 2210633. NCBI
conserved domain search (Marchler-Bauer et al., 2003)
showed high similarity to the domain INT-REC-C of the
tyrosine recombinase/integrase family. Similar results were
found for B. sphaericus NCTC 9602. A predicted ORF of
525 bp in the opposite orientation (Fig. 3) showed an identity of 97 % to the putative integrase/recombinase protein
encoded by the ORFB of B. sphaericus JG-A12.
To analyse the distribution of the putative integrase genes
within the genomes of strains 9602 and JG-A12, chromosomal DNA of the two strains was digested with the restriction endonucleases EcoRV, ClaI and SpeI. Southern
hybridization of all digests gave one signal in each lane
with a size corresponding to the signals detected using the
transposase-gene-specific probe (Fig. 4). However, in the
case of the ClaI digests, a second small fragment giving a
weak signal was detected, indicating the presence of a second
recombinase copy within the genomes of 9602 and JG-A12.
It remains to be determined whether this putative gene copy
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2967
K. Pollmann and others
is located on the chromosomes or on the large plasmids
harboured by the strains.
Characterization of ORFC
After the putative recombinase of B. sphaericus JG-A12 an
ORF of 849 bp was found at position 385 after the stop
codon of ORFB, in the opposite orientation (Fig. 3), encoding a protein of 283 bp with a theoretical molecular mass
of 33 419 Da and a calculated pI of 5?68. NCBI Conserved
Domain Search of the entire protein sequence found a
high identity to helix–turn–helix XRE family-like proteins
(HTH_XRE), which are prokaryotic DNA-binding proteins
belonging to the xenobiotic response element family of
transcriptional regulators (Luscombe et al., 2000; Wintjens
& Rooman, 1996; Wood et al., 1990). A similar organization was found for strain 9602: after the putative recombinase gene an ORF of 843 bp is found, encoding a protein
of 281 aa with 90 % identity to that of the predicted
HTH_XRE-like protein of JG-A12.
Identification of an additional silent and
truncated S-layer protein gene copy
Surprisingly, following the ORF of the HTH-like protein
in both strains a gene sequence was found in the opposite
orientation with a high identity to the functional, chromosomally encoded S-layer protein genes slfA and slfB, and
especially to the silent plasmid-located S-layer-protein-like
genes sllA and sllB (Fig. 3). However, some sequence differences were found. The entire sequence of the inverted Slayer-protein-like gene of strain 9602 determined in this
study indicates an ORF of 3297 bp, encoding a protein of
1099 aa with a theoretical molecular mass of 116 472 Da,
showing 100 % identity to the plasmid-encoded sllA. The
potential ribosome-binding site preceding the ORF at 13 nt
upstream of the start codon is incomplete (TGAGG), suggesting that this gene is silent. Further, the upstream region
shows no identity to that of the functional gene slfA, and
putative promoters are found at a far distance from the
start codon at 2452 (235, TTGAAT) and 2426 (210,
TATAGA).
The corresponding sequence of the inverted S-layerprotein-like gene of strain JG-A12 represents a truncated
form of the plasmid-encoded sllB possessing a large deletion of 391 bp of the 59-terminal region (Figs 3 and 5).
Interestingly, the upstream region of this copy shows a high
identity to the upstream region of the chromsomally located
sllA copy of strain 9602, although a sequence of 65 bp
containing the modified ribosome-binding site is truncated
(Fig. 5). Additionally, a large part of the upstream region
identical to that in 9602 was found to be repeated (Fig. 5).
Characterization of the region following the
silent S-layer protein gene sequences
Following the truncated sllB copy of JG-A12, the nucleotide sequence of a 1407 bp fragment was determined. BLAST
analysis of the translated sequence showed a high identity of
50 % of the last 513 bp to the 59-terminal region (starting at
aa 896) of a putative extracellular nuclease of Deinococcus
radiodurans R1 with a size of 1067 aa. These results indicate
the presence of a truncated ORF, probably disrupted by the
insertion of ISBsph2.
Comparative analysis of B. sphaericus S-layer
proteins
S-layer proteins of several B. sphaericus strains have been
described previously: B. sphaericus CCM 2177 (Ilk et al.,
2002), P-1 (Deblaere et al., 1995) and WHO 2362 (Bowditch
et al., 1989; Deblaere et al., 1995). These strains and those of
the present study belong to different DNA homology groups
(Miteva et al., 1999). The mosquito-pathogenic strain WHO
2362 belongs to DNA homology group II and possesses, like
other pathogenic strains of the genus Bacillus (Lewis et al.,
1987; Mesnage et al., 2001; Sidhu & Olsen, 1997), an oblique
structured S-layer (Raff, 2002; Sidhu & Olsen, 1997), which
is believed to contribute to their pathogenity (Lewis et al.,
1987; Mesnage et al., 2001). In contrast, the S-layers of the
non-pathogenic strains B. sphaericus CCM 2177 and P-1,
which belong to DNA homology group III, possess square
symmetry (Ilk et al., 2002; Lepault et al., 1986; Ohnesorge
et al., 1992). The S-layers of B. sphaericus NCTC 9602 and
JG-A12 have square symmetry as well (Raff, 2002), although
these strains belong to a different DNA homology group
(group I) (Selenska-Pobell et al., 1999).
Sequence comparisons of the different B. sphaericus S-layer
proteins demonstrate the unique structure of the S-layer
proteins of B. sphaericus NCTC 9602 and JG-A12. The
primary structures of the SLH domains studied in this work
differ significantly from those described for the S-layer
Fig. 5. Organization of the chromosomally encoded copies of sllA and sllB. mRBS, modified ribosome-binding site; SP,
signal peptide; NTD, N-terminal domain; CTD, C-terminal domain; DR, direct repeat.
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Microbiology 151
Novel surface layer protein genes in B. sphaericus
proteins of B. sphaericus CCM 2177, B. sphaericus P-1 and B.
sphaericus WHO 2362 (Fig. 2), whose N-terminal SLH
domains share a surprisingly high identity (Fig. 2). The
region following the N-terminal domains of the S-layer
proteins of B. sphaericus NCTC 9602 and JG-A12 shares a
striking high (61 %) and slowly decreasing identity with the
S-layer proteins of strains CCM 2177 and P-1, whereas no
significant homology was found to the S-layer protein of
strain 2362. The high identity of the N-terminal and central
parts of SlfA, SlfB, SllA and SllB changes abruptly in their
C-terminal parts (Fig. 2). Whereas the C-terminal part of
SlfA shows no similarity to any known S-layer protein, the
corresponding region of SlfB shares a surprisingly high
identity of 57 % (Fig. 2) with that of the S-layer protein of B.
sphaericus CCM 2177. This high identity is very interesting,
since the C-terminal regions of the Bacillus S-layer proteins
studied to date are highly variable. Further, sequence
comparisons of S-layer protein genes of even closely related
bacterial strains demonstrated a high diversity of these
proteins (Hansmeier et al., 2004). The almost identical
structure of the C-terminal regions of the putative S-layer
proteins SllA and SllB is therefore surprising.
DISCUSSION
Primary structure characteristics of the S-layer
proteins of B. sphaericus NCTC 9602 and
JG-A12
The amino acid compositions of the two functional S-layer
proteins studied demonstrate features typical of many other
S-layers (Bahl et al., 1997; Engelhardt & Peters, 1998; Ilk
et al., 2002; Kuen et al., 1997; Mesnage et al., 2001). A
remarkable feature is the stretches of aspartic and glutamic
acid residues as well as serine and threonine residues, which
were found especially in the C-terminal parts of SlfA and
SlfB as well as of the putative proteins SllA and SllB, with
different amounts and distribution. In this context, the
results of previous work in which the interactions of the
S-layers of B. sphaericus JG-A12 and NCTC 9602 with
uranium were studied (Merroun et al., 2005; Raff et al.,
2004) are interesting. The analyses showed that the S-layer
SlfB of strain JG-A12 is much more effective in uranium
binding than the S-layer of strain NCTC 9602 (Raff, 2002;
Raff & Selenska-Pobell, 2004). EXAFS (extended X-ray
absorption fine structure) demonstrated that the uranium
bound by the S-layers of both strains is coordinated mainly
to phosphate and carboxyl groups of the proteins (Merroun
et al., 2005; Raff et al., 2003, 2004). Analyses of posttranslational modifications confirmed the phosphorylation
of the proteins (Raff, 2002; Raff & Selenska-Pobell, 2004). In
addition, ICP-MS (inductively coupled plasma mass spectrometry) and colorimetric methods showed that the S-layer
protein SlfB of the uranium mining waste pile isolate JGA12 possesses six times more phosphorus than the S-layer
protein SlfA of strain NCTC 9602. Bearing in mind that
threonine and serine are the most frequently phosphorylated residues in prokaryotic proteins (Cozzone, 1988; Saier
et al., 1990; Thomas & Trust, 1995), the higher level of
phosphorylation of SlfB in comparison to SlfA may be
related to the differences in the content and distribution of
threonine and serine residues in the central and C-terminal
parts of the S-layer proteins, and to the accumulations of
serine and threonine stretches found in these regions. Since
phosphate groups possess extremely high metal complexing
activity, the high ability of strain JG-A12 to bind uranium
and other metals (Selenska-Pobell et al., 1999) may be
explained by the high content of phosphate residues of SlfB,
but also by the stretches of residues carrying carboxyl groups
found in the protein.
Identification of novel IS elements in
B. sphaericus
As well as the plasmid-encoded silent S-layer gene copies
sllA and sllB an additional truncated gene copy was found
downstream of slfA and slfB, showing the same sequence as
sllA and sllB (Fig. 6). Interestingly, the truncated S-layer
protein gene copies were found to be associated with genes
Fig. 6. Organization of the chromosomally encoded S-layer gene regions of B. sphaericus NCTC 9602 and JG-A12. Tas,
putative teichoic acid synthesis protein; UR, upstream region; SP, signal peptide; NTD, N-terminal domain; CD, central
domain; CTD, C-terminal domain, Tr, putative transposase; Rec, putative integrase/recombinase; HTH, protein with helix–
turn–helix motif; ENuc, putative extracellular recombinase. The arrows indicate the direction of transciption; the percentage
identity of the nucleotide sequences is shown.
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K. Pollmann and others
encoding proteins involved in genetic rearrangements, indicating the presence of two novel IS elements, designated
ISBsph1 and ISBsph2 (Fig. 6).
Although BLAST analyses of the putative transposase indicated the affiliation of ISBpsh1 and ISBsph2 to the IS605/
IS200-family, the IS elements show some significant differences from members of this family. IS200 contains only a
single ORF encoding a transposase and does not carry terminal inverted repeats (Beuzon & Casadesus, 1997; Lam &
Roth, 1983a, b). In the case of IS605, a second transposase is
associated with a second divergently transcribed ORF, which
is related to a putative transposase (Kersulyte et al., 1998,
2000; Murai et al., 1995). The putative transposase found in
the present study is closely related to an ORF found in IS657
of the alkaliphilic strain Bacillus halodurans C-125 (Takami
et al., 2001). IS657 contains only one ORF and no inverted
repeats were found (Fig. 3). Thus, the insertion elements
found in the present work are quite unusual. Their size and
organization are more indicative of a transposon rather than
an insertion element, although BLAST analysis showed no
similarities to a known transposon and no similar gene
element was found in any bacterial genome sequenced so far.
To our knowledge, this is the first description of a mobile
element encoding an S-layer-protein-like gene.
S-layer protein genes of B. sphaericus JG-A12
and NCTC 9602 have probably evolved by
horizontal gene transfer and rearrangements
The sequence comparisons (Fig. 2) strongly suggest an
intragenic ‘patchwork’ evolution of the functional S-layer
genes in strains of B. sphaericus. It is obvious that the Nterminal parts of the S-layer proteins of B. sphaericus strains
9602 and JG-A12 have evolved from a common ancestor,
which is different from that of the other B. sphaericus strains
studied. Similarly, the high identity of the N-terminal
domains of the S-layer proteins of B. sphaericus strains CCM
2177, WHO 2362 and P-1 (Fig. 2) indicates a common
derivation of these domains, although the strains belong to
different DNA groups and possess different symmetries. On
the other hand, the sequence comparisons demonstrate that
the central parts of the S-layer proteins SlfA and SlfB and
those of strains CCM 2177 and P-1 share a common origin,
whereas the S-layer protein of strain WHO 2362 shows a
unique structure. Further, the results indicate that the Cterminal parts of the S-layer proteins of strains JG-A12 and
CCM 2177 have evolved from a common ancestor, whereas
the C-terminal parts of strains 9602, P-1, and WHO 2362
have evolved from three different, unrelated ancestors.
The presence of the IS elements, consisting of a truncated sll
copy, a putative transposase and a putative recombinase
(Fig. 6), supports the above suggestion. Whereas transposases are needed for efficient transposition of the insertion
sequence, functions of recombinases include the site-specific
insertion of the mobile element and, most interesting in
the present study, DNA inversions controlling expression
of surface proteins (Sadowski, 1986; Stark et al., 1992).
2970
Therefore, we may speculate that the insertion elements
found were involved in the evolution of the S-layer protein
genes in both strains studied. Parts of the S-layer protein
genes may have been exchanged between the different gene
copies by integrase/recombinase-mediated chromosomal
rearrangements.
Shuffling of functional protein domains as well as horizontal
gene transfer are well known and play an important role in
microbial evolution. These processes generate proteins of
a great variety, with similar functions but different specificities. Prominent examples of protein families produced
by domain shuffling and horizontal gene transfer are the
insecticidal crystal proteins of Bacillus thuringiensis (Bravo,
1997). Horizontal transfer and rearrangements of segments
of S-layer protein genes have also been reported. It is
assumed that the bacteria use S-layer variation for adaptation to different stress factors (Kuen et al., 1997; JakavaViljanen et al., 2002) or for their virulence (Blaser et al.,
1994; Dworkin & Blaser, 1996; Mesnage et al., 2001; Mignot
et al., 2003). Mechanisms of S-layer protein variation
include the inversion of promoters, recombination of Slayer gene segments (Dworkin & Blaser, 1996), and the
replacement of an active S-layer protein gene by a formerly
silent gene (Boot et al., 1996b). G. stearothermophilus PV72
expresses two different S-layer protein gene variants (sbsA
and sbsB) under different growth conditions. Whereas sbsA
is located on the bacterial chromosome, sbsB is carried on a
large plasmid harboured by the strain. For the expression of
sbsB, the gene is translocated from the plasmid to a specific
chromosomal expression site, whereas the coding region of
sbsA is irreversibly transferred to the plasmid (Scholz et al.,
2001). Due to the lack of significant sequence identity of
the sbsA and sbsB genes, a replacement of the two genes by
homologous recombination was excluded (Scholz et al.,
2001).
In contrast, the chromosomally located (slfA and slfB) and
the plasmid-located (sllA and sllB) genes of B. sphaericus
NCTC 9602 and JG-A12 share large regions with an
extremely high identity. Moreover, the two plasmid-located
genes sllA and sllB are almost identical. To our knowledge,
NCTC 9602 and JG-A12 are the first strains of B. sphaericus
found to contain large plasmids encoding silent S-layer
protein genes. However, to date we have not been able to
induce the expression of any of the sll genes described in this
work. Rearrangements of S-layer protein genes mediated by
insertion elements have not been reported so far. In the case
of Aeromonas salmonicida (Gustafson et al., 1994), G. stearothermophilus PV72 (Scholz et al., 2000) and G. stearothermophilus ATCC 12980 (Egelseer et al., 2000), transposable
elements have been described which affect the expression of
S-layer proteins by insertion. However, these elements were
much smaller than those described in the present study,
comprising only a transposase and not being associated
with an S-layer protein gene. For B. sphaericus NCTC 9602,
the occurrence of a spontaneous variant in a frozen stock
culture was reported in an earlier study, expressing a
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Microbiology 151
Novel surface layer protein genes in B. sphaericus
15–20 kDa smaller variant of the S-layer protein (Hastie &
Brinton, 1979a, b). Both variants, the wild-type and the
smaller variant, as well as several truncated forms were not
able to bind to the sacculi of the S-layer-possessing related
strain B. sphaericus P-1 (Hastie & Brinton, 1979b). From our
sequence analyses this finding can be explained by the
different N-termini of the wild-type S-layer protein of
NCTC 9602 and possibly also of the smaller variant in
comparison to the S-layer protein of P-1 (Deblaere et al.,
1995). It can be suggested that the spontaneous mutation
was caused by the replacement of slfA with sllA possibly
mediated by ISBsph1. However, a transposition or inversion
of ISBsph1 or ISBsph2 has not yet been observed, and if
and under what conditions the IS elements are mobilized
remains to be determined.
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ACKNOWLEDGEMENTS
This study was supported by grant DFG/SE 671/7-2 from DFG, Bonn,
Germany, by grant 7531.50-03.0370-01/5 from SMWK, Dresden,
Germany, and by EC grant GRD1-2001-40424. We thank R. Getzlaff,
I. Plumeier (GBF Braunschweig) and K. Flemming for technical
assistance.
Evidence that an N-terminal S-layer protein fragment triggers
the release of a cell-associated high-molecular-weight amylase in
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causing loss of S-layer-gene expression in Bacillus stearothermophilus
ATCC 12980. Microbiology 146, 2175–2183.
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