Journal of Bacteriology, December 2000, p. 6824-6826, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
B Is Involved in
Regulation of Biofilm Expression in a Staphylococcus aureus
Mucosal Isolate
Institut für Molekulare Infektionsbiologie, D-97070 Würzburg,1 and Institut für Mikrobiologie und Molekularbiologie, 15, D-17487 Greifswald,2 Germany
Received 26 June 2000/Accepted 12 September 2000
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ABSTRACT |
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Osmotic stress was found to induce biofilm formation in a
Staphylococcus aureus mucosal isolate. Inactivation of a
global regulator of the bacterial stress response, the alternative
transcription factor
B, resulted in a biofilm-negative
phenotype and loss of salt-induced biofilm production. Complementation
of the mutant strain with an expression plasmid encoding
B completely restored the wild-type phenotype. The
combined data suggest a critical role of
B in S. aureus biofilm regulation under environmental stress conditions.
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TEXT |
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For numerous pathogenic bacteria,
biofilms represent a source of persisting and relapsing infections and
thus contribute significantly to pathogenesis (4). Biofilms
seem to protect bacteria from unfavorable external conditions, and, in
some bacterial ecosystems, the conversion of planktonic cells into a
biofilm-producing community is triggered by environmental stress
factors (6, 12, 13). In the human pathogen
Staphylococcus aureus, biofilm formation is mediated by the
production of the extracellular polysaccharide adhesin PIA, whose
synthesis depends on the expression of the icaADBC-encoded
enzymes (5, 15). The regulation of biofilm expression in
this organism is poorly understood. All S. aureus strains
analyzed so far contain the entire ica gene cluster, but only a few express the operon and produce biofilms in vitro
(5). In this study, we investigated whether the alternative
transcription factor
B is involved in the regulation of
ica expression.
B is known to be a global
regulator of the stress response in S. aureus and also
influences various virulence-associated genes (7, 11, 16,
20). To elucidate the possible role of
B in
biofilm formation, we used a genetic approach and constructed an
S. aureus sigB::ermB insertion mutant
of the biofilm-forming, methicillin-sensitive mucosal isolate S. aureus MA12 (18). Biofilm formation and ica
expression of the mutant were compared with the phenotypes of the
corresponding wild-type strain and a complemented strain that carried a
sigB copy on an expression vector.
Construction of a sigB insertion mutant and
complementation of the mutation.
The inactivation of
sigB was done by insertion of an erythromycin resistance
cassette into the sigB gene of S. aureus MA12 by
double-crossover integration. For this purpose, the
temperature-sensitive shuttle vector pSK8, which carries a
sigB::ermB mutation, was constructed. A
937-bp fragment containing the entire sigB gene was
amplified by PCR from S. aureus MA12 by using the primers 5'
CGG GAT CCG GTG TGA CAA TCA GTA TGA C 3' and 5' CGG AAT TCG CGA CAT TTA
TGT GGA TAC AC 3'. The DNA fragment was inserted into the shuttle
vector pBT1 (17), resulting in pSK7. Then the
ermB cassette of pEC1 (1) was excised by
XbaI-HindIII digestion, treated with the
Klenow fragment of Escherichia coli DNA polymerase, and
ligated with EcoRV-digested pSK7, resulting in plasmid pSK8. Following passage through the restriction-negative strain S. aureus RN4220, pSK8 was reisolated and transformed into S. aureus MA12 by electroporation (19). Replacement of the
chromosomal S. aureus MA12 sigB wild-type gene
was achieved by double-crossover integration of the
sigB::ermB insert of pSK8 following a
temperature shift to the nonpermissive temperature of the shuttle
vector (42°C). Erythromycin-resistant and chloramphenicol-sensitive
colonies were isolated, and the
sigB::ermB integrations were confirmed by Southern hybridization, PCR, and nucleotide sequencing (data not
shown). From these experiments, the
sigB::ermB insertion mutant S. aureus MA12.2 was selected for further analysis. To restore the
B function, the S. aureus MA12.2
sigB mutant strain was complemented with plasmid pSK9.
Plasmid pSK9 was constructed by inserting the sigB PCR
fragment into the expression shuttle vector pHPS9 (8). Prior
to transformation into S. aureus MA12.2, the vector was transformed into the restriction-negative cloning host S. aureus RN4220. The plasmid was reisolated and transferred into
S. aureus MA12.2, resulting in the complemented strain
S. aureus MA12.2-1(pSK9).
Analysis of the
B function.
Inactivation of the
sigB gene in S. aureus MA12.2 and restoration of
its function in the complemented strain S. aureus
MA12.2-1(pSK9) was investigated by Northern analysis of the
asp23-specific gene expression. asp23 encodes an
alkaline shock protein, and the gene was shown to be preceded by a
B-dependent promoter (7, 11). Recent studies
have shown that asp23 transcription is absent in S. aureus sigB mutants. It has therefore been concluded that
asp23 transcription reliably reflects the activity of the
B factor (7, 11, 16).
B-negative strain (2, 11), and in contrast to
both the wild-type and complemented strains, the mutant strain had lost
its yellow pigmentation and showed enhanced hemolysis on blood agar
plates (data not shown). Taken together, these data indicate that
S. aureus MA12.2 is a sigB mutant and that the
sigB mutation is restored in the complemented strain
S. aureus MA12.2-1(pSK9).
Effect of the sigB mutation on biofilm production and
ica expression.
Quantitative biofilm measurement was
done in a microtiter assay as described previously (3, 22).
Bacteria were grown overnight in 96-well, flat-bottomed tissue culture
plates (Greiner, Nürtingen, Germany) at 37°C using either
tryptic soy broth (TSB) (Difco, BBL, Detroit, Mich.) or TSB
supplemented with 3% sodium chloride as growth medium. Based upon
the optical densities (OD) of the biofilms, the strains were classified
as nonadherent strains (OD
0.120), weak biofilm producers
(0.120 < OD
0.240), or strongly adherent strains (OD > 0.240) according to the scheme introduced by Christensen et al.
(3). As shown in Fig. 1, the biofilm formation of S. aureus MA12 was weak when the strain
was grown in TSB, but it could be significantly stimulated under
osmotic stress conditions (that is, TSB containing 3% sodium
chloride). In contrast, the S. aureus MA12.2 sigB
insertion mutant failed to produce any detectable biofilm, irrespective
of whether it was grown in TSB alone or in TSB containing 3% sodium
chloride. In the case of S. aureus MA12.2(pSK9), in which
the inactivated chromosomal copy of sigB was complemented by
an expression plasmid carrying sigB, both basal and the
osmotic stress-stimulated biofilm formation were restored.
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B. Obviously, this conclusion appears to
contradict previous results which demonstrated that S. aureus RN4220 exhibits a biofilm-positive phenotype when
the icaADBC genes are provided on a plasmid (5, 14). Therefore, we have done an additional set of experiments. We
have transformed plasmid pCN27 (9), which carries the entire ica operon of Staphylococcus epidermidis, into
S. aureus RN4220. Consistent with previous data
(14), the strain formed a biofilm when grown in TSB (Fig.
1). However, in obvious contrast to the pSK9 complementation
experiments described above, no induction of biofilm production was
observed under osmotic stress conditions. Finally, we found that
propagation of pCN27 in Staphylococcus carnosus TM300
(9) increased the biofilm production of this strain under
high-salt conditions. The combined data led us to hypothesize that the
biofilm-forming phenotype of S. aureus RN4220(pCN27) may
result from a basal, vector-driven ica expression, which is enhanced by the copy effect of the plasmid. The absence of biofilm induction, however, suggests that
B is required for the
activation of biofilm formation in response to osmotic stress.
Conclusions.
In this study, we provide evidence that the
biofilm-forming phenotype observed in a mucosal isolate of
S. aureus can be induced by changing environmental
conditions (in this case, osmotic stress). The results are consistent
with our recent data obtained for S. epidermidis (S. Rachid
and W. Ziebuhr, unpublished data), which demonstrated that the
expression of the biofilm-mediating ica operon is enhanced
by high osmolarity, high temperature, and subinhibitory concentrations
of certain antibiotics. In contrast to S. epidermidis, all
S. aureus strains analyzed so far carry the ica
gene cluster, but only a few spontaneously express biofilms in vitro
(5). It is thus tempting to speculate that, in these
biofilm-negative strains, the ica expression may be tightly
controlled. The data presented in this study strongly support the idea
that this suppression might be overcome by activation of
B in response to external stress. Another explanation
for the biofilm-negative phenotype in the majority of the S. aureus strains would be the presence of mutations in the
sigB gene itself or in its adjacent rsbU,
rsbV, and rsbW regulatory genes (11,
20). It is also conceivable that S. aureus biofilm
production may be influenced by mechanisms that have been shown to be
used by S. epidermidis in the control of the ica
expression (e.g., phase variation and gene rearrangements)
(21-23). All these hypotheses still need careful experimental evaluation. It should also be noted that, in this study,
we have characterized only the mucosal isolate S. aureus MA12 in detail. Even though we have obtained conclusive
evidence for the critical involvement of the
B factor in
biofilm formation in this isolate, the actual role of
B
in ica expression remains to be determined. The nucleotide
sequence immediately upstream of the ica operon does not
resemble any of the known
B-dependent promoter
structures, suggesting an indirect activation of the ica
expression by additional unknown regulatory factors. This idea is also
supported by the identification of S. aureus strains which
express the sigB gene but, nevertheless, are biofilm negative (Rachid and Ziebuhr, unpublished). To answer the question of
whether
B is directly or indirectly involved in S. aureus ica expression, more experimental work, including primer
extension analyses under different growth conditions, is needed.
Finally, a broad range of S. aureus strains has to be
analyzed to identify possible variations in biofilm regulation among
different strains.
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ACKNOWLEDGMENTS |
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This work was supported by the BMBF (grant no. 01KI9608), the Deutsche Forschungsgemeinschaft (Graduiertenkolleg Infektiologie), and the Fond der Chemischen Industrie.
We are grateful to Jürgen Kreft, Lehrstuhl für Mikrobiologie, Universität Würzburg, for providing plasmid pHSP9, and to Friedrich Götz, Mikrobielle Genetik, Universität Tübingen, for plasmid pCN27.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931- 31 2154. Fax: 49-931- 31 2578. E-mail: w.ziebuhr{at}mail.uni-wuerzburg.de.
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