Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università degli Studi di Pisa, Via S. Zeno 35, Pisa 56127, Italy1
Food Research Institute, Department of Food Microbiology and Toxicology, University of Wisconsin-Madison, 1925 Willow Drive, Madison, WI 53706, USA2
Author for correspondence: Sonia Senesi. Tel: +39 050 836566. Fax: +39 050 836570. e-mail: senesi{at}biomed.unipi.it
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ABSTRACT |
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Keywords: flagellum, chemotaxis, haemolysin BL
Abbreviations: CI, capilliary index; HBL, haemolysin BL; RAPD, randomly amplified polymorphic DNA; TBS, tris-buffered saline
The EMBL accession number for the sequence reported in this paper is Y08031.
a Present address: FBI Academy, Range Road, Quantico, VA 22135, USA.
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INTRODUCTION |
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In contrast to other processes of differentiation in bacteria, swarming is not a starvation response and significantly varies depending on the organism and environmental growth conditions (Eberl et al., 1996 ; Harshey, 1994
; Harshey & Matsuyama, 1994
). The rapid migration of swarm cells and the active growth of dedifferentiated swimmer cells provide swarming communities with the remarkable ability to progressively colonize the available surface. Swarming, therefore, is thought to be a successful strategy developed by flagellated micro-organisms to ensure their rapid expansion in the natural environment, where microbial activities are often associated with solid surfaces. Swarming motility was also proven to play a role in the colonization of host mucosal surfaces by infectious agents (Allison et al., 1992a
; Belas & Colwell, 1982
). Moreover, the finding that some bacteria, such as uropathogenic strains of P. mirabilis, produce higher levels of specific virulence factors during their swarm-cell state (Allison et al., 1992b
, 1994
), is of intrinsic interest as well as of great medical relevance.
Swarming differentiation has been studied mostly in Gram-negative rods and much has been learned about the regulatory mechanisms of swarming in Serratia liquefaciens, Serratia marcescens, Salmonella typhimurium, Xenorhabdus nematophilus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Yersinia enterocolitica and Escherichia coli, as well as in P. mirabilis (Belas et al., 1991 ; Eberl et al., 1996
; Givaudan & Lanois, 2000
; Harshey & Matsuyama, 1994
; ORear et al., 1992
; Rashid & Kornberg, 2000
; Stewart et al., 1997
; Young et al., 1999
). Characterization of swarming-defective mutants demonstrated that molecular components of both the chemotaxis system and flagellar apparatus are essential for swarming differentiation in all the Gram-negative bacteria studied so far (Belas et al., 1991
, 1995
; Burkart et al., 1998
; Givaudan & Lanois, 2000
; Gygi et al., 1995
; ORear et al., 1992
; Young et al., 1999
). However, while the role of chemotaxis proteins in swarm-cell differentiation has not been clarified completely, the inability to swarm or to swarm properly exhibited by mutants defective in flagellar proteins (Belas et al., 1991
, 1995
; ORear et al., 1992
; Young et al., 1999
), in motor-switch proteins (Belas et al., 1995
; Burkart et al., 1998
) or in proteins involved in the assembly of flagellar filaments (Gygi et al., 1995
; Young et al., 1999
), is consistent with the essential role played by a functional flagellum-mediated motility for the differentiation of hyperflagellated swarm cells.
Almost nothing is known about the molecular components needed for swarming differentiation in Gram-positive bacteria, although this striking behaviour has been long recognized in some species of Clostridium and Bacillus (Henrichsen, 1972 ; Hoeniger & Taushel, 1974
). The ability to swarm has been never described in Bacillus cereus; the only flagellum-dependent surface motility recognized in this species is its ability to rapidly spread on the surface of solid culture media. Spreading of B. cereus, briefly discussed in a general review on bacterial surface translocation (Henrichsen, 1972
), has been reported to be due to swimming motility only, brought about by individual cells that do not exhibit hyperflagellation or elongation.
In this report, we demonstrate that B. cereus is capable of swarming differentiation and that the production of differentiated swarm cells requires the activity of fliY (Celandroni et al., 2000 ), the homologue of which in Bacillus subtilis encodes an essential component of the flagellar-switch complex (C-ring) controlling the direction of flagellum rotation (Bischoff & Ordal, 1992
). Complementation of a fliY deletion, identified in a motile but non-swarming and non-chemotactic spontaneous mutant of B. cereus, restores the ability to produce differentiated swarm cells as well as to respond to chemoattractants. Finally, evidence is produced showing that secretion of the L2 component of haemolysin BL, a tripartite pore-forming necrotizing toxin from B. cereus, exclusively occurs during differentiation of swarm cells on solid media.
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METHODS |
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Swarming motility.
Bacteria from stationary-phase TrB cultures were seeded by spot inoculation (5 µl, 2·0x108 cells ml-1) onto the centre of TrA (1%, w/v, agar) plates and colony development was allowed to proceed at 37 °C in humidified chambers. Swarming migration was followed microscopically (IMT-2, Olympus) at different time intervals during growth. Formation of swarm cells was evaluated by recording the length and hyperflagellation of bacteria recovered from different portions of growing colonies. Cell length was assessed by microscopic observations of Gram-stained preparations, while flagellar staining was performed as described by Harshey & Matsuyama (1994) . Since B. cereus flagella are very fragile, bacterial samples were taken by slide overlay of single agar blocks (5x5 mm) that contained different colony portions. Swarming of strain NCIB 8122 was also evaluated in TrA plates containing different agar concentrations (0·42·5%, w/v) and at temperature values ranging from 25 to 40 °C. The effect of mannitol on the swarming behaviour of B. cereus was evaluated on TrA (1%, w/v, agar) plates supplemented with mannitol (Sigma) at concentrations ranging from 0·2 to 2·0 mM.
Cell differentiation assay.
The extent of swarm-cell differentiation was quantified by monitoring the increase in both cell length and cell-surface flagellin at fixed time intervals during growth on solid media. Differentiation assays were initiated by spreading 200 µl stationary-phase TrB cultures (2·0x108 cells ml-1) onto TrA (1%, w/v, agar) plates (14 cm diameter) and incubating at 37 °C. Cells were harvested by washing the entire surface of duplicate plates with 3·0 ml cold Tris-buffered saline (pH 7·4; TBS) at 2 h intervals. Cell suspensions for each time point analysed were standardized to an OD600 of 6·0 in TBS and vortexed for 5 min. Cell length was estimated by phase-contrast microscopy (BH-2, Olympus) for a total of 100 cells fixed in 1% (v/v) formaldehyde, 0·9% (w/v) NaCl. Cell-surface flagellin was recovered from supernatants of vortexed cells by centrifugation at 5000 g for 15 min. Flagellar filaments were collected from supernatants by high-speed centrifugation at 100000 g for 1 h. Flagellin was assayed by densitometry of protein stained with Coomassie brilliant blue after separation by SDS-PAGE on 12·5% (w/v) polyacrylamide gels, as described by Gygi et al. (1995) .
Swimming and chemotaxis assays.
Swimming in liquid media was evaluated under a phase-contrast microscope (BH-2, Olympus) by observing the smooth swimming or tumbling phenotype exhibited by bacteria suspended in a drop of LB broth. Swimming motility was also evaluated by seeding stationary phase cells (5 µl, 2·0x108 cells ml-1) on the centre of motility TrA plates (swim plates, 0·25%, w/v, agar). Plates were incubated at 37 °C and the diameter of developing colonies, recorded 6 h post-inoculation, was taken as a measure of bacterial migration in semisolid agar.
Chemotaxis was evaluated by using capillaries filled with attractants, as described by Ordal & Goldman (1975) with modifications. Briefly, cells were grown overnight in TrB at 37 °C and diluted 1:50 in G medium (Hanson et al., 1961
). After a 4 h incubation (100 r.p.m.) at 37 °C, glycerol (0·05%) was added to the cell suspensions. Cells were then harvested, diluted to OD600 0·05 in chemotaxis buffer [10 mM potassium phosphate buffer (pH 7·0), 0·3 mM (NH4)2SO4, 0·14 mM CaCl2, 0·1 mM EDTA] and assayed for their ability to chemotax towards L-glutamine, L-alanine, L-cysteine, L-histidine or mannitol (50 mg each ml-1 in chemotaxis buffer). The mean number of c.f.u. entering the attractant-filled capillaries (CFUa) and that entering control capillaries (CFUc) filled with chemotaxis buffer only were determined on LB agar plates by repeating each assay five times on separate days. The capillary index (CI) for each strain and attractant was calculated as follows: CI=(CFUa-CFUc)/CFUc. The ability of bacteria to chemotax in semisolid media was also determined in swim TrA plates supplemented with either glutamine or mannitol, at concentrations ranging from 0·2 to 20 mM.
Immunoassays for haemolysin BL (HBL) and detection of hblA and hblD genes.
B. cereus HBL components (B, L1 and L2) were assayed in supernatants of late-exponential phase cultures grown in TrB at 30 °C by Western blotting (Beecher et al., 1995a ). Culture supernatants were concentrated 20-fold with Millipore Ultrafree centrifugal filters. Proteins from concentrated culture supernatants were separated by SDS-PAGE on 12% (w/v) polyacrylamide gels and transferred electrophoretically to PVDF membranes (Millipore). Detection of HBL components produced on solid media was performed by seeding a liquid inoculum (5 µl stationary-phase TrB cultures) on sterile disks of nitrocellulose membrane (Millipore) layered on TrA (1%, w/v, agar) plates. The liquid inoculum was allowed to dry and after 24, 48 or 72 h at 30 °C, cells were washed from the membrane with TBS. All the membranes were soaked for 1 h at 37 °C in TBS containing 3% (w/v) BSA and probed with rabbit antisera specific to the individual HBL components (Beecher et al., 1995b
). Bound antibody was detected by using goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) and the chromogenic substrate 4-chloro-1-naphthol (Sigma).
B. cereus was lysed and chromosomal DNA purified as previously described (Celandroni et al., 2000 ). PCRs for assessing the presence of the hblA (encoding component B) and hblD (encoding component L1) genes were performed using standard conditions and varying the annealing temperatures from 70 to 50 °C. PCR reactions were performed using the primers HblA1 (5'-GCTAATGTAGTTTCACCTGTAGCAAC-3')/HblA2 (5'-AATCATGCCACTGCGTGGACATATAA-3') (Prüb et al., 1999
) or DL1F (5'-GTAGGCAATTATGCATTAGGCC-3')/DL1R (5'-CAACAATAACTACCGCTCCTCC-3') for the amplification of an 883 bp and a 670 bp fragment of B. cereus hblA and hblD, respectively.
Randomly amplified polymorphic DNA (RAPD) analysis, Southern blotting and fliY amplification.
PCR mixtures (50 µl) for RAPD analysis contained a single oligonucleotide primer at a concentration ranging from 1·0 to 3·0 µM, 2·5 U Taq polymerase (Pharmacia Biotech), 0·2 mM each dNTP, 5 µl Taq polymerase buffer (Pharmacia Biotech) and 10 or 30 ng B. cereus chromosomal DNA. The concentrations of both DNA template and primer were varied to rule out polymorphism in artefact bands. Four different PCR primers were used: HLWL74 (5'-ACGTATCTGC-3'), HLWL85 (5'-ACAACTGCTC-3'), M13 (5'-GAGGGTGGCGGCTCT-3') and OPE06 (5'-AAGACCCCTC-3'). Cycling was as follows: 30 cycles at 94 °C for 5 s, 36 °C for 1 min and 72 °C for 1·5 min, with a 10 min extension at 72 °C for the last cycle. Amplification reactions were performed in a Gene Amp PCR System 9600 (Perkin Elmer Cetus). An amplified fragment was ligated into the pGEM-T vector (Promega) to obtain pFEM that was propagated in E. coli JM109. Labelling of DNA by [32P]dCTP was achieved with the Multiprime DNA labelling kit (Amersham Life Science). Southern blotting experiments were performed as described by Sambrook et al. (1989) . Oligonucleotide primers were synthesized by Genset.
PCR amplification of fliY was performed with the primers YF1 (5'-CACACAAAAGGGGATACAAG-3') and YR1 (5'-ACATTTGGCGGCGTCATG-3'). Reaction mixtures (50 µl) contained 0·4 µM each primer, 2·5 U Pfu Turbo DNA Polymerase (Stratagene), 0·2 mM each dNTP, 5 µl Pfu Turbo buffer (Stratagene) and 10 ng B. cereus chromosomal DNA. Cycling was as follows: 30 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, with a 10 min extension at 72 °C for the last cycle.
Complementation of fliY.
fliY was amplified from strain NCIB 8122 using the primers 14FYU2 (5'-AGAAAAAGCTT-AATGAGGAGGCAG-3') and 14FYL2 (5'-CCAAAGGAT- CCCCAAATACTC-3'), which were designed based on sequences external to the coding region of B. cereus fliY (Celandroni et al., 2000 ). The primers carried synthetic HindIII and BamHI restriction sites (underlined), respectively. PCR conditions were as described above and cycling was as follows: one cycle at 94 °C for 2 min followed by 30 cycles at 95 °C for 30 s, 60 °C for 1 min and 72 °C for 1·5 min, with a 10 min extension at 72 °C for the last cycle. After amplification, the DNA fragment was digested with HindIII/BamHI, and inserted between the HindIII and BamHI sites of pHT304 (Arantes & Lereclus, 1991
), an E. coli/Bacillus thuringiensis shuttle and expression vector. The resulting vector, pHTY01, was propagated in E. coli TOP10 and introduced into strain MP01 by electroporation (Lereclus et al., 1989
) to produce strain MP04. Recombinant clones were screened on LB agar plates containing erythromycin.
DNA sequencing and nucleotide sequence analysis.
Nucleotide sequencing was performed by the dideoxy-chain termination method on double-stranded DNA, with the ALFexpress AutoRead Sequencing Kit (Pharmacia Biotech) and the ALFexpress automatic sequencer (Pharmacia Biotech). DNA sequences were processed using the ALF Manager version 3.02 (Pharmacia) and compared with available sequences using the BLAST programs implemented at the EMBL, GenBank and DDBJ nucleotide sequence databases. The nucleotide sequence determined in this study has been deposited in the EMBL database under the accession no. Y08031.
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RESULTS |
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Strain MP01 is a fliY null mutant
The RAPD-PCR technique is widely used for searching for sequence polymorphism exhibited by nearly identical bacterial genomes. It has been applied frequently to distinguish subgroups of closely related organisms including those showing a high degree of sequence conservation among isolates, as in the case of Bacillus anthracis (Henderson et al., 1994 ). In this study, RAPD-PCR was used to fingerprint the whole genome of B. cereus strains NCIB 8122 and MP01. Indistinguishable DNA banding patterns were obtained with three out of the four primers used (data not shown). Primer HLWL85 was the only one to generate a single polymorphic DNA fragment. A fragment of about 500 bp was amplified from strain NCIB 8122 (Fig. 5
); this fragment is absent in strain MP01. The polymorphic fragment was observed under all the reaction conditions tested, including primer concentrations of 1·0 and 3·0 µM, and DNA concentrations of 0·2 and 0·6 µg ml-1; additional polymorphic DNA bands were never detected even among the faint bands we obtained by adopting different reaction conditions. The polymorphic fragment was cloned into pGEM-T, generating pFEM. Analysis of the nucleotide sequence of the polymorphic fragment, determined in different E. coli transformants containing pFEM, revealed that the fragment (567 bp) was a portion of a unique putative ORF (accession no. Y08031). The predicted amino acid sequence was most homologous to a protein fragment encoded by fliY of B. cereus ATCC 10987 (accession no. AJ272332; 98% identity in 185 overlapping amino acids, 50% of FliY), B. cereus ATCC 14579 (accession no. AJ272330; 88% identity in 187 overlapping amino acids, 50% of FliY) and B. subtilis (accession no. P24073; 40% identity in 173 overlapping amino acids, 46% of FliY). The protein encoded by fliY (FliY) in B. subtilis is an essential component of the flagellar motor-switch complex (C-ring) and represents the Gram-positive counterpart of the Gram-negative FliN (Bischoff & Ordal, 1992
).
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Based on the CI values recorded, mannitol (CIMann, 60·8) was the most powerful attractant for B. cereus. Taking into consideration the observation that high concentrations (in the millimolar range) of an attractant may saturate its own receptor and inhibit chemotaxis (Burkart et al., 1998 ), we wondered whether sensing of a mannitol gradient could play a critical role in swarming motility other than in swimming by B. cereus. Fig. 7(a)
shows that inhibition of the chemotactic response in strain NCIB 8122 was produced by increasing mannitol concentrations (from 2 to 20 mM), which was deduced by measuring the reduction of bacterial migration occurring in swim plates. The swarming response of this strain, on the contrary, did not exhibit any change when the concentration of mannitol in swarm TrA (1% agar) plates was varied from 0·2 to 20 mM (Fig. 7b
). These results suggest that chemotaxis itself, at least toward mannitol, is unlikely to play a role in B. cereus swarming motility.
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DISCUSSION |
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The present study demonstrates that B. cereus is able to exhibit two alternate forms of flagellum-driven motility, swimming and swarming, depending on whether the bacterium is grown in liquid or solid media. While swimming motility is brought about by individual swimmer cells that are short oligoflagellated rods, swarming is an organized and collective movement of differentiated swarm cells, which, in B. cereus, are 34 times longer and 40 times more flagellated than the swimmer cells. The swarming response of B. cereus was induced by surface sensing but different from other swarming bacteria (Eberl et al., 1996 ; Harshey & Matsuyama, 1994
; Young et al., 1999
), and occurred at a wide range of medium viscosity (from 0·4 to 2% agar) and temperatures (2538 °C). Swarm-cell differentiation was also observed in minimal media, although colonies appeared small and rhizoid (data not shown). B. cereus swarm colonies never exhibited macroscopically layered consolidation phases due to regularly spaced cycles of swarming migration and consolidation. As also reported for Y. enterocolitica, S. liquefaciens and Clostridium septicum (Eberl et al., 1999
; Macfarlane et al., 2001
; Young et al., 1999
), B. cereus appeared to swarm continuously at the colony rim, while the colony centre apparently consisted of a unique consolidation phase.
An important finding emerging from this study was that a spontaneous mutant of B. cereus carrying a deletion in fliY was non-swarming and non-chemotactic, and such phenotypic defects could be complemented by a plasmid harbouring the fliY gene. This gene has been identified previously in B. subtilis (Bishoff & Ordal, 1992 ), where it encodes a protein that is a component of the flagellar motor-switch complex, which consists of FliG (Albertini et al., 1991
), FliM (Zuberi et al., 1991
) and FliY (Bishoff & Ordal, 1992
). B. subtilis FliY was shown to be partly homologous (Bishoff & Ordal, 1992
) to the E. coli and S. typhimurium switch proteins FliM and FliN. However, FliY differs from FliN in Gram-negative bacteria in that it appeared to act as a bifunctional protein, being able to co-operate with FliG and FliM in constituting the switch complex and also interact with other proteins in modulating intracellular chemotactic signals (Bishoff & Ordal, 1992
). The fliY null mutant of B. subtilis was non-chemotactic, as was the fliY mutant of B. cereus. However, in contrast to B. subtilis, the fliY mutant of B. cereus was flagellated and motile. The inability of the B. cereus mutant to respond to chemoattractants suggests that FliY is a flagellar motor-switch protein also in this species. Such a role for FliY is supported by the observation that the fliY mutant of B. cereus was impaired in swimming motility, resulting in more tumbling than the wild-type strain.
The lack of swarming differentiation in the fliY mutant of B. cereus is consistent with the fact that all the mutants characterized by defects in any one of the flagellar genes, including those encoding the motor switch proteins (Belas et al., 1995 ; Burkart et al., 1998
), are impaired in swarming migration. However, as already described for E. coli (Burkart et al., 1998
), a direct involvement of chemotaxis in swarming motility was not demonstrated, since swarming of the wild-type strain was unaffected by the addition of compounds that acted as attractants in liquid media.
Another observation pertains to the role of fliY in the assembly of the flagellar filament. In Gram-negative bacteria as well as in B. subtilis, the switch complex is required for the completion of the flagellar assembly (Bishoff & Ordal, 1992 ; Macnab, 1996
). Therefore, the finding that the fliY mutant of B. cereus is flagellated and motile raises the question whether a peculiar regulatory pathway governing the assembly of flagellar proteins or the expression of flagellar genes acts in this species.
Finally, the demonstration that B. cereus strain NCIB 8122 produces the L2 component of haemolysin BL only in the swarm-cell state opens new perspectives in considering swarming motility potentially coupled to the virulence of this organism. An association between swarming motility and virulence has been shown for several Gram-negative and -positive bacteria. In P. mirabilis, swarming has been associated with the ability to invade urothelial cells and to express higher levels of virulence factors, such as intracellular urease, extracellular haemolysin and metalloprotease (Allison et al., 1992a , b
, 1994
). Swarming by S. liquefaciens was found to be accompanied by an increase in the expression of the phospholipase gene (Eberl et al., 1996
), and C. septicum was capable of producing DNase, hyaluronidase and neuraminidase only during the swarm-cell state (Macfarlane et al., 2001
). The fact that the B. cereus mutant harbouring an expression plasmid containing fliY regained the ability to swarm and to produce the L2 component of HBL strongly suggests that a relationship between expression of virulence factors and differentiation of swarm cells also exists in this organism.
Because bacterial motility may be crucial to seek nutrients, to rapidly colonize surfaces in the natural environment, to establish infection inside a host, and, presumably, to produce toxins and other virulence factors, the dependence of swarming, chemotaxis and L2 secretion on the activity of fliY reveals a major role for this gene in different adaptive responses elicited by B. cereus to its environment.
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ACKNOWLEDGEMENTS |
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Received 5 November 2001;
revised 25 January 2002;
accepted 31 January 2002.