JOURNAL OF BACTERIOLOGY, Jan. 1996, p. 542–545 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 178, No. 2
Salmonella typhimurium Fimbrial Phase Variation and FimA Expression STEVEN CLEGG,* LISA S. HANCOX,
AND
KUANG-SHENG YEH
Department of Microbiology, University of Iowa, College of Medicine, Iowa City, Iowa 52242 Received 24 July 1995/Accepted 3 November 1995
Bacteria in a nonfimbriate phase because of continuous aeration of liquid cultures produce FimA in amounts similar to those produced by fimbriate bacteria. However, relatively low FimA production was observed in nonfimbriate-phase cultures obtained by growth on solid media or by anaerobic incubation. Regardless of the fimbrial phase of Salmonella typhimurium, the fimA promoter region was always oriented in the direction that might allow fimA transcription. the culture express levels of FimA equivalent to those found in cultures without aeration. Therefore, it is likely that the lack of fimbrial expression in cultures aerated by shaking is not due to limiting amounts of the fimA gene product. The absence of detectable fimbriae on the surfaces of these cultures may be due to effects on the regulation of additional fim or non-fim genes. Alternatively, the physical agitation of aerated cultures could produce conditions that prevent the optimal assembly of intact fimbrial appendages on the bacterial surface. Since nonfimbriate-phase S. typhimurium can also be produced by growth on solid medium incubated aerobically, as well as in static liquid cultures incubated anaerobically, the expression of fimA under these growth conditions was also studied. Unlike the nonfimbriate-phase bacteria grown in aerated liquid cultures, the bacteria resulting from anaerobic incubation of broth cultures or from serial subculture on agar express significantly less FimA polypeptide than fimbriatephase bacteria (Table 2). Agar-grown and anaerobic broth cultures express approximately 5- to 10-fold less FimA than aerobically grown broth cultures. Throughout our studies, the presence of surface-associated fimbriae appeared to be associated with detectable levels equal to or greater than approximately 70 Miller units (17) of b-galactosidase activity. Therefore, the relatively low levels of enzyme activity observed in the agar-grown and anaerobic cultures are consistent with the nonfimbriate phenotype of these strains. The b-galactosidase activity of nonfimbriate-phase bacteria is relatively high in aerated broth cultures but low in cultures produced by other methods of cultivation. Therefore, it is likely that the absence of fimbriation in bacteria grown under these different conditions is due to two distinct mechanisms that decrease fimbrial expression. Since FimA expression is low in bacteria incubated on agar or anaerobically, the lack of detectable fimbrial appendages in these strains may be due to low levels of the FimA subunit. However, our results do not eliminate the possibility that effects on additional genes may also play a role in the lack of fimbrial expression by agar-grown bacteria. The relatively high level of FimA expression in shaken broth cultures suggests that limitation of FimA under these conditions is not responsible for the absence of fimbriae on the surfaces of these bacteria. Consequently, the nonfimbriate phase of S. typhimurium LB5010 cannot always be associated with low levels of fimA expression. Orientation of the fimA promoter in fimbriate- and nonfimbriate-phase S. typhimurium. Since the fimA promoter region of S. typhimurium has been shown to be flanked by a 10-bp inverted repeat sequence (22), inversion of this region could
Phenotypic variation of the expression of type 1 fimbriae in Salmonella typhimurium was initially observed and characterized by Old and coworkers (18, 19). These investigators described a biphasic growth pattern associated with the outgrowth of fimbriate bacteria incubated aerobically as a static, liquid broth culture. Fimbrial phase variation has been observed in a large number of enteric bacteria that express type 1 fimbriae (5, 7, 8). In general, strongly fimbriate-phase bacteria are isolated following serial subculture as described for S. typhimurium. However, some strains of enterobacteria are constitutively fimbriate and do not exhibit fimbrial phase variation (6, 12). The phenotypic expression of fimbriae is the result of the interaction and cooperativity of a large number of gene products (4, 14, 21). It is unknown whether the expression of specific fim gene products in Salmonella species is altered during fimbrial phase variation. The molecular events associated with fimbrial phase variation in Escherichia coli have been investigated (1, 9, 10). It has been demonstrated that fimA expression is in part dependent upon the orientation of a DNA region possessing the fimA promoter (2, 13, 16). In addition, E. coli fimbrial phase variation has been shown to occur independently of the orientation of the invertible DNA fragment (15). In S. typhimurium, initial evidence indicated that the fimA promoter region was not invertible (4, 23). Therefore, in order to determine whether the phenotypic fimbrial phase variation observed in S. typhimurium is reflective of the amount of FimA subunit produced by the bacteria, we examined phase variation using a fimA-lacZ fusion. Also, PCR analysis was used to determine whether inversion of the S. typhimurium fimA promoter region occurred at a low frequency. Expression of fimA during phase variation. In order to determine whether changes in fimA production are observed during fimbrial phase variation, we used a previously described S. typhimurium lfimA-lacZ lysogen (Table 1) in which expression of b-galactosidase acts as a reporter of fimA expression (23). Strongly fimbriate bacterial cultures derived by growth in static liquid media demonstrated enzyme activity similar to that of phenotypically nonfimbriate cultures which were incubated with shaking (Fig. 1). An increase in FimA production was not associated with the phase of secondary growth observed for fimbriate cultures, indicating that bacteria which are in a nonfimbriate phase produced by continuous aeration of
* Corresponding author. Phone: (319) 335-7778. Fax: (319) 3359006. Electronic mail address:
[email protected]. 542
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TABLE 1. Bacterial strains used in this study Strain
S. typhimurium LB5010 IS145 LBZ100 S7471 S2231 S. enteritidis DMSO34 S. abony DMSO001 S. dublin DMSO003 S. senftenberg DMSO073 S. ibadan DMSO041 S. napoli DMSO053 a
Relevant fimbrial phenotypea
Fimbriate and phase variable LB5010 lfimA-lacZ lysogen; fimbriate and phase variable fimZ mutant of LB5010; nonfimbriate FIRN; nonfimbriate Non-FIRN; nonfimbriate Fimbriate and phase variable Constitutively fimbriate Constitutively fimbriate Constitutively fimbriate Nonfimbriate Fimbriate and phase variable
Source or reference
3 24 15 D. C. Old (20) D. C. Old (20) J. P. Duguid J. P. Duguid J. P. Duguid J. P. Duguid J. P. Duguid J. P. Duguid
‘‘Phase variable’’ indicates that fimbrial expression occurs following growth in static liquid media and that no fimbriae are detected on agar-grown cultures.
facilitate regulation of fimA expression. In E. coli, two genes, fimB and fimE, mediate inversion of the promoter-containing region, and cultures of nonfimbriate-phase bacteria contain a large number of organisms possessing this DNA region in the ‘‘off’’ position (1, 9, 10). The fim gene cluster of S. typhimurium possesses no genes related to fimB or fimE of E. coli (13). Therefore, if inversion of the DNA fragment containing the S. typhimurium fimA promoter does occur, it is mediated by gene products unrelated to FimB or FimE. Southern hybridization results indicated that in both fimbriate- and nonfimbriate-phase cultures of S. typhimurium LB5010, the promoter region of fimA is oriented in the ‘‘on’’ position (Fig. 2). Therefore, following restriction of genomic DNA with enzymes selected to detect a possible inversion of the DNA region containing the fimA promoter (11), the digests were probed with a DNA fragment encompassing the fimA promoter and the N terminus of FimA (23). Two representative Southern blots are shown in Fig. 2. Regardless of the fimbrial phenotype from which the DNA was prepared, the Southern hybridization profiles were identical. For example, the probe hybridized to two TaqI DNA fragments (738 and 171
bp) regardless of fimbrial phase. The sizes of these fragments are consistent with the orientation of the fimA promoter region being such that transcription of fimA could occur. If this region was inverted in nonfimbriate-phase bacteria, two DNA fragments of 650 and 259 bp should have been detected. Similar results were obtained for HpaI digests in which the sizes of DNA fragments (609 and 102 bp) hybridizing to the probe indicate that the fimA promoter is oriented in the ‘‘on’’ position (Fig. 2). Further analysis of this region with different DNA probes and restriction digests that were designed to detect inversion demonstrated that all genomic DNA preparations were composed of sequences arranged in the ‘‘on’’ orientation. In addition to S. typhimurium, a phase-variable strain of Salmonella enteritidis was also used in these studies. As for S. typhimurium, DNA preparations from fimbriate- and nonfimbriate-phase cultures demonstrated identical Southern hybridization profiles. In all cases, the results were consistent with the fimA promoter being able to mediate transcription. Therefore, for both strains of Salmonella, even though most of the bacteria in the nonfimbriate-phase cultures do not express fimbriae
FIG. 1. Growth curves of S. typhimurium lIS145 incubated as a shaken (f——f) or static (j——j) broth culture. The secondary phase of growth in static broth cultures can be observed at approximately 24 h for this strain. b-Galactosidase activities of shaken (f----f) and static (j----j) cultures in Miller units are also shown.
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TABLE 2. Expression of b-galactosidase and type 1 fimbriae by S. typhimurium IS145 Method of cultivation
b-Galactosidase activitya
Production of type 1 fimbriaeb
Static liquid medium with O2 Static liquid medium without O2 Solid medium with O2
120 10 20
1 2 2
a
Miller units. Fimbriae detected by hemagglutination and reactivity with specific antiserum. b
(6), the isolated genomic DNA possesses no detectable fragments in the ‘‘off’’ position. Since inversion may occur in a small proportion of bacteria in nonfimbriate-phase cultures, the sensitivity of detecting this event was increased with PCR technology. The primers used in these experiments were designed to differentiate between the ‘‘on’’ and ‘‘off’’ orientations. As indicated in Fig. 3, the primers used in these studies were constructed so that in the ‘‘on’’ orientation, with genomic DNA as the template, PCR products obtained with primer 1 plus primer 2 would be 399 bp, whereas the use of primers 2 and 3 would result in a 153-bp product. Under these conditions, primers 1 and 3 would result in no PCR product. If the putative invertible DNA region was in the ‘‘off’’ orientation, the use of primers 1 and 2 would not generate a DNA fragment from the chromosomal DNA template. The results of the PCR analysis with template DNA from either fimbriate- or nonfimbriate-phase S. typhimurium LB5010 are shown in Fig. 4. In both cases, the promoter region of fimA is oriented in the ‘‘on’’ position. The PCR products of primers 1 and 2 and of primers 2 and 3 are approximately 399 and 153 bp, respectively, whereas no products were observed with primers 1 and 3. The constitutively nonfimbriate FIRN and non-FIRN strains of S. typhimurium (20) were also shown to possess fimA promoters in the ‘‘on’’ orientation, as was the nonfimbriate fimZ mutant (24) of S. typhimurium LB5010 (Table 1). However, the mutation resulting in the nonfimbrial phenotype has not been characterized for either the FIRN or the non-FIRN strains, and these strains may be unable to produce fimbriae because of a mutation in an unidentified fim or non-fim gene. We have recently characterized the fimZ mutant of S. typhimurium LB5010 and demonstrated that FimZ is a transcriptional activator of fimA (24). Our results indicate that the lack of FimZ production does not result in a phase-locked ‘‘off’’ orientation of the fimA promoter. In addition to S. typhimurium, a num-
FIG. 2. Southern hybridization analysis of fimbriate-phase (lanes A and C) and nonfimbriate-phase (lanes B and D) cultures of S. typhimurium. Genomic DNA was digested with TaqI (lanes A and B) or HpaI (lanes C and D) and probed with fimA-specific DNA sequences (see text). The sizes (in base pairs) of the restriction fragments hybridizing to the probe are indicated, and they are consistent with the promoter region of fimA being in the ‘‘on’’ position regardless of the fimbrial phenotype.
FIG. 3. S. typhimurium fimA promoter region. The sites and directions of PCR extensions of primers 1, 2, and 3 are indicated by the large arrows. The inverted repeat (IR) DNA sequences flanking the fimA promoter are shown by the open boxes. The transcription and translation initiation sites are shown by the small arrows, and the transcription initiation site is designated as 11.
ber of Salmonella serovars (Table 1) were examined by PCR analysis. Salmonella abony DMSO001, Salmonella dublin DMSO0030, and Salmonella senftenberg DMSO073 are fimbriate strains that do not exhibit phase variation. Therefore, fimbriate bacteria were detected following growth on solid media as well as growth in broth. Salmonella ibadan DMSO041 was never observed to produce detectable amounts of type 1 fimbriae, even after serial subculture in broth. Salmonella napoli DMSO053 exhibited fimbrial phase variation, with strongly fimbriate cultures being detected after subculture in broth but nonfimbriate-phase bacteria being produced by serial subculture on agar. When DNA isolated from each of the fimbrial phenotypes was used as the template in PCR analysis, the results indicated that the DNA flanked by the inverted repeats was always in a single orientation. This orientation was consistent with the direction of fimA transcription, and in none of the strains was the promoter region of fimA directed away from the FimA-coding sequences. The data presented herein suggest that, unlike type 1 fimbrial expression in E. coli, Salmonella fimbrial phase variation is not mediated by an invertible DNA fragment. However, it is possible that in vivo recombination at the sites of the 10-bp inverted repeats may occur, but this event was not detected under the conditions of our experimentation. Although E. coli fimbrial phase variation is associated with inversion of a DNA
FIG. 4. PCR analysis of fimA promoter region. The location and direction of extension of PCR products are as indicated in Fig. 1. PCR products were detected following agarose gel electrophoresis and ethidium bromide staining. The template of mixtures of primers (as shown for lanes A, B, and C) was genomic DNA prepared from fimbriate-phase (fim1) or nonfimbriate-phase (fim2) S. typhimurium. In all cases, PCR products were detected with primers 1 and 2 (399-bp fragment in lanes A) or 2 and 3 (153-bp product in lanes C) but not with primers 1 and 3 (lanes B).
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element, it has also been shown that this phenomenon can occur independently of this inversion event (15). Consequently, E. coli fimA expression exhibits at least two mechanisms of regulation, one dependent upon the orientation of the invertible region and one independent of this process. In S. typhimurium, the primary method of fimA regulation may be independent of the orientation of the fimA promoter. The low levels of b-galactosidase activity in nonfimbriate-phase cultures produced by serial subculture on agar or anaerobic incubation is consistent with the lack of inversion to the ‘‘off’’ position. If the fimA promoter was oriented in the ‘‘off’’ position, no enzyme activity should be detected. We have recently demonstrated that the fim gene products that affect fimA expression are not related to FimB or FimE of E. coli (23, 24). At least one of these gene products acts as a transcriptional activator of fimA in S. typhimurium and does not demonstrate recombinase activity on the 10-bp inverted repeat region. Currently, we are determining whether the 10-bp inverted region of S. typhimurium plays any role in fimA expression by using site-directed mutagenesis of this region. However, it is clear that DNA isolated from cultures of predominantly nonfimbriate S. typhimurium, in which 60 to 70% of the bacteria are phenotypically nonfimbriate (6), does not possess the promoter-containing region as an invertible fragment. This work was supported by grant 94-37204-0854 from the USDA NRICGP. REFERENCES 1. Abraham, J. A. M., C. S. Freitag, J. R. Clements, and B. I. Eisenstein. 1985. An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli. Proc. Natl. Acad. Sci. USA 82:5724–5727. 2. Blomfield, I. C., M. S. McClain, J. A. Princ, P. J. Calie, and B. I. Eisenstein. 1991. Type 1 fimbriation and fimE mutants of Escherichia coli K-12. J. Bacteriol. 173:5298–5307. 3. Bullas, L. R., and J.-I. Ryu. 1983. Salmonella typhimurium LT2 strains which are r2 m1 for all three chromosomally located systems of DNA restriction and modification. J. Bacteriol. 156:471–474. 4. Clegg, S., and D. L. Swenson. 1994. Salmonella fimbriae, p. 105–113. In P. Clemm (ed.), Fimbriae: adhesion, genetics, biogenesis, and vaccines. CRC Press, Boca Raton, Fla. 5. Duguid, J. P. 1959. Fimbriae and adhesive properties in Klebsiella strains. J. Gen. Microbiol. 21:271–286. 6. Duguid, J. P., E. S. Anderson, and I. Campbell. 1966. Fimbriae and adhesive
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