Two DNA Invertases Contribute to Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium Strain LT2
http://www.100md.com
《细菌学杂志》
Graduate School of Natural Science and Technology and Department of Biology, Faculty of Science, Okayama University, Tsushima-Naka 3-1-1, Okayama 700-8530, Japan
ABSTRACT
Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.
INTRODUCTION
Diphasic Salmonella enterica strains possess two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments, and alternate expression between these two genes. This phenomenon is called flagellar phase variation, and cells expressing FliC are called phase 1 cells, whereas those expressing FljB are called phase 2 cells (14, 20). These two genes encode antigenically distinct flagellin molecules. For example, in S. enterica serovar Typhimurium, fliC and fljB encode flagellin proteins of antigenic types "i" and "1,2," respectively. The frequency of flagellar phase variation varies among different strains. Serovar Typhimurium strain C77 was reported to change from phase 1 to phase 2 and from phase 2 to phase 1 at frequencies of 1 x 10–5 and 3 x 10–4 per bacterial generation cycle, respectively (39). On the other hand, monophasic Salmonella serovars that express only either fliC or fljB are also known to exist (20). For example, serovar Abortusequi strains constantly express the fljB gene, which specifies flagellin of antigenic type "e,n,x" (16). Escherichia coli strains usually lack the fljB locus and express the fliC gene constitutively.
Flagellar phase variation is controlled by the recurrent inversion of a 996-bp DNA segment, called the H segment, which contains the promoter for the fljB gene (48, 49). The H segment is flanked by 26-bp inverted repetitious sequences, hixL and hixR, between which site-specific recombination occurs, leading to H inversion (18). This recombination is mediated by a DNA invertase, Hin, whose gene is located within the H segment (23, 38). The fljB gene constitutes an operon together with the fljA gene, which encodes a negative regulator for fliC expression (9). FljA has been shown to inhibit fliC expression through a posttranscriptional control mechanism (2, 47). When the H segment is in the "on" orientation, both fljB and fljA are transcribed, resulting in phase 2 flagellin being synthesized. On the other hand, when the H segment turns to the "off" orientation, neither fljB nor fljA is expressed, resulting in phase 1 flagellin being synthesized. Therefore, the frequency of phase variation directly reflects that of H inversion.
The molecular mechanism of Hin-mediated inversion of the H segment has been intensively studied both in vivo and in vitro (12, 18, 30, 35, 44). The inversion reaction requires a supercoiled DNA substrate containing the two hix sites and a 65-bp recombinational enhancer that locates within the hin gene. Hin assembles into a tripartite synaptic complex, called an invertasome, which contains these two hix sites and the Fis-bound enhancer. Fis activates Hin to catalyze DNA cleavage followed by strand exchange leading to recombination between the two hix sites. In this reaction, the DNA bending activity of the HU protein facilitates looping of the DNA between the enhancer and the hix sites.
Invertible DNA segments are not unique to flagellar phase variation. For example, bacteriophages Mu and P1 contain invertible G and C segments, respectively, whose inversion regulates the alternate expression between two sets of tail fiber genes and thus changes host specificity (4, 40). The cognate DNA invertase genes are called gin and cin, which locate just adjacent to the G and C segments, respectively (13, 19). These DNA invertases share sequence homology with each other and with Hin invertase. The DNA sequences of the recombination sites are also conserved among H, G, and C segments. Consistent with these homologies, the DNA invertase was shown to be interchangeable among these three inversion systems (23, 24). These facts collectively suggest that these inversion systems have evolved from a common ancestor (15). Some E. coli and Shigella strains also contain homologous inversion systems, which are located in cryptic Mu-like prophages (26, 33, 42, 43).
S. enterica serovar Abortusequi possesses a naturally occurring hin mutation (formally called vh2–) that results in this serovar being phase stable (16, 23). Derivatives of serovar Typhimurium whose hin-fljBA locus had been replaced with the counterpart from serovar Abortusequi by P22-mediated transduction became phase stable (16), suggesting that hin should be the sole DNA invertase gene responsible for H inversion in Salmonella. Surprisingly, however, a search of the whole genome sequence of LT2, a standard laboratory strain of serovar Typhimurium, revealed that it contains another gene (STM2702) that is highly homologous to the hin gene (28). This gene locates just adjacent to a 2.7-kb invertible DNA segment. Here, we refer to this gene and the invertible DNA as fin and F segment, respectively. The fin gene and F segment are located in a P2 phage-related resident prophage, Fels-2 (3, 41, 46), which exists 34.5 kb apart from the hin-fljBA locus. Strong sequence similarity between Hin and Fin (Fig. 1A) raised the possibility that the fin gene is also involved in flagellar phase variation. This work was carried out to test this possibility. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. We found that strains possessing either one of these mutations could manifest phase variation, whereas strains possessing both mutations could not. This result clearly indicates that these two different DNA invertases are both involved in flagellar phase variation in LT2. Furthermore, we showed that serovar Abortusequi lacks both of these two invertases, which makes this strain phase stable.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture media. Bacterial strains and plasmids used in this study are all listed in Table 1. Procedures for the construction of gene disruptants and recombinant plasmids are described below. Ordinary culture media, such as L broth, L agar plates, P22 broth, and motility agar plates, were prepared as described previously (22). If necessary, ampicillin, kanamycin, and chloramphenicol were added to the media at final concentrations of 50, 25, and 10 μg/ml, respectively.
Motility test. Single colonies were inoculated onto motility agar plates and incubated for 5 to 10 h at 30°C. Motility was detected as spreading growth on the plates. When phase variation ability was tested, each motility agar plate included 1 μl of the antiserum specific against either i- or 1,2-type flagellar filament per 25 ml of the medium. When a diphasic strain such as LT2 is used, phase 1 cells form spreading colonies on motility agar plates containing anti-1,2 serum and form compact colonies with halo-like spreading growth on motility agar plates containing anti-i serum. The halo-like spreading growth is due to the appearance of phase 2 cells during growth of phase 1 cells on the plate. Phase 2 cells of the diphasic strain give the opposite result; that is, they form spreading colonies on motility agar plates containing anti-i serum and form compact colonies with halo-like spreading growth on motility agar plates containing anti-1,2 serum. On the other hand, when a phase 1-fixed monophasic strain is used, the cells form spreading colonies on motility agar plates containing anti-1,2 serum and form compact colonies without a halo on motility agar plates containing anti-i serum. A phase 2-fixed monophasic strain gives the opposite result. Phase variation frequency was estimated according to the method described previously by Stocker (39).
Transduction and DNA manipulation. P22-mediated transduction was performed with P22HTint (36) as described previously (25). DNA manipulation and transformation were performed as described previously (25). Restriction enzymes and T4 DNA ligase were purchased from Toyobo (Osaka, Japan) or Takara (Kyoto, Japan). PCR amplification of DNA was carried out with an iCycler (Bio-Rad, CA) using KOD Dash DNA polymerase (Toyobo). Customized DNA primers were purchased from Hokkaido System Science (Sapporo, Japan). Their nucleotide sequences are listed in Table 2. Electroporation of DNA into Salmonella cells was carried out using a Gene Pulser system (Bio-Rad). DNA sequence was determined by the dideoxy chain termination method using a PRIZM 310NT genetic analyzer (Applied Biosystems, CA).
Construction of gene disruptants. The -Red system described previously by Datsenko and Wanner (5) was used for the construction of gene disruption mutants. A hin disruption mutant was constructed as follows. The chloramphenicol resistance FRT (Flp recognition target) cassette was PCR amplified from pKD3 using primers hinf1P3 and hinr1P2. The amplified product was introduced into LT2 harboring pKD46 by electroporation. Recombinants were selected on L agar plates containing chloramphenicol. In the resulting hin disruptant, an 83-bp DNA in the middle of the hin gene was deleted and substituted with the chloramphenicol resistance FRT cassette, but the recombinational enhancer sequence was retained. This hin::cat allele was then moved to LT2 by transduction selecting for chloramphenicol resistance to obtain KK201. The chloramphenicol resistance cassette was removed through site-specific recombination between the flanking FRT sequences by transient exposure of KK201 to pCP20. In the resulting hin mutant, KK203, the H segment has the same size as that in LT2. The fin disruption mutant KK205 was constructed through the same procedure as described above, but in this case, the kanamycin resistance FRT cassette was amplified with primers 2702f4P1 and 2702r2P2 using pKD4 as a template. In KK205, the entire fin gene including the putative recombinational enhancer sequence was deleted and replaced with the kanamycin resistance FRT cassette. The hin fin double mutants were constructed by transduction of the fin::kan allele from KK205 into phase 1 and phase 2 cells of KK203. As described below, the resulting double mutants, KK211 and KK212, were fixed in phase 1 and phase 2, respectively.
Construction of plasmids carrying the hin gene. The genomic DNA of LT2 and pBR322 was digested with EcoRI, ligated, and introduced into EKK11 by transformation. Motile clones were selected on motility agar plates containing ampicillin. From a clone expressing the 1,2-type flagella, a plasmid was isolated and named pKK1001. This plasmid carries a 15-kb DNA fragment containing the hin and fljB genes from LT2. A 0.6-kb DNA fragment containing the hin gene was PCR amplified from pKK1001 with primers hinf3E and hinr3B. The amplified product was digested with EcoRI and BamHI and inserted into the corresponding site of pSTV29 to obtain pHN2.
Determination of H orientation. The orientation of the H segment was determined by PCR amplification of each fix site and its flanking sequences from genomic DNA using following primer pairs: hinf4 and HLr1 for hixL of the off-oriented H segment, HRf1 and hinr4 for hixR of the off-oriented H segment, hinr4 and HLr1 for hixL of the on-oriented H segment, and HRf1 and hinf4 for hixR of the on-oriented H segment. The amplified products were 543, 1,769, 1,125, and 1,188 bp long, respectively. Orientation of the H segment was further confirmed by DNA sequencing of the amplified products using primers hinf4 and hinr4.
Nucleotide sequence accession number. The DNA sequence data for the SL23 genome between the ssrA and fljA genes have been submitted to the DDBJ database under accession no. AB234611.
RESULTS
DNA invertase activity of the fin gene product. Figure 1A shows the similarity in amino acid sequence between the hin and fin gene products. Although Fin is five amino acids smaller than Hin, 62% of the amino acids are identical between the two. The similarity extends throughout the molecules, and most of the amino acids conserved in four authentic DNA invertases (Hin, Gin, Cin, and Pin) are also conserved in Fin. These facts suggest that Fin is also capable of mediating H inversion.
In order to test this, the fin gene was PCR amplified from the genomic DNA of LT2 with primers 2702f3E and 2702r1B, digested with EcoRI and BamHI, and inserted into the corresponding site of pBR322 to obtain pHN1. This plasmid was introduced into an E. coli strain, EKK15, by transformation. EKK15 possesses a defective fliC gene and an intact fljBA locus together with the H segment containing a defective hin gene originating from serovar Abortusequi. EKK15 is nonmotile, because it contains no DNA invertase activity and its H segment is fixed in the off orientation (26). However, EKK15 harboring pHN1 produced motile cells on motility agar plates (Fig. 2), indicating that the fin gene can mediate H inversion at least in the off-to-on direction.
Flagellar phase variation in the hin and fin mutants. According to the procedures described in Materials and Methods, hin and fin disruption mutants were constructed from LT2, and their phase variation abilities were examined on motility agar plates containing anti-i or anti-1,2 serum (Fig. 3). As expected, the fin disruption mutant KK205 showed motility on both plates, indicating that H inversion can occur in the absence of Fin. Interestingly, the hin disruption mutant KK203 could also show motility on both plates, indicating that flagellar phase variation can occur even in the absence of the Hin invertase. Next, the fin::kan mutation of KK205 was introduced into KK203 by P22-mediated transduction to obtain fin hin double mutants. We obtained two types of kanamycin-resistant transductants that differed in the behavior on motility agar plates containing anti-i or anti-1,2 serum. One mutant (KK211) formed spreading colonies on a motility agar plate containing anti-1,2 serum and compact colonies on that containing anti-i serum, indicating that this strain was fixed in phase 1. Conversely, the other mutant (KK212) formed spreading colonies on a motility agar plate containing anti-i serum and formed compact colonies on that containing anti-1,2 serum, indicating that this strain was fixed in phase 2. These results clearly indicate that both hin and fin gene products are involved in flagellar phase variation in LT2.
In order to correlate the flagellar phenotype to H inversion in these strains, DNA fragments containing each hix site and its flanking sequences were PCR amplified from the genomic DNAs, and the orientation of the H segment was determined by DNA sequencing of the amplified products (data not shown). From the genomic DNAs of LT2, KK203(hin), and KK205(fin), we obtained amplified products containing the H segment in each orientation, indicating that H inversion occurs in these three strains. In contrast, only DNA fragments containing the H segment in the off orientation were obtained from KK211(hin fin FljBoff), whereas only those containing the H segment in the on orientation were obtained from KK212(hin fin FljBon), indicating that H inversion does not occur in these two strains. Therefore, we conclude that H inversion is mediated by both Hin and Fin in LT2.
In order to know how much each of these two DNA invertases contributes to H inversion, we measured the frequencies of flagellar phase variation from phase 1 to phase 2 and from phase 2 to phase 1 in LT2, KK203(hin), and KK205(fin). The results obtained from representative experiments are summarized in Table 3. The frequency in KK203 was approximately 60 to 200 times lower than that of LT2, whereas KK205 underwent phase variation at a frequency almost equivalent to that of LT2. We also measured the phase variation frequency with LT2 and KK203 harboring pHN2, which carries the hin gene in a plasmid vector, pSTV29 (Table 3). Both strains were found to undergo phase variation at almost equal frequencies. This result excludes the possibility that the hin mutation in KK203 might have any cis-acting inhibitory effect on H inversion mediated by a DNA invertase supplied in trans. Therefore, we conclude that H inversion is mediated mainly by its cognate DNA invertase, Hin, although Fin invertase also contributes to H inversion.
Phase variation frequency was also examined with the hin fin double mutants (Table 3). As expected, KK211(hin fin FljBoff) did not produce cells of the opposite phase at a detectable frequency. On the other hand, KK212(hin fin FljBon) produced motile cells on motility agar plates containing anti-1,2 serum at a very low but detectable frequency. However, this phase change was found to be irreversible; that is, the phase 1 cells that emerged from KK212 never produced phase 2 cells, suggesting that the change from phase 2 to phase 1 in KK212 might have resulted from a spontaneous mutation that inactivated both fljB and fljA that was not from H inversion.
Absence of Fels-2 in SL23. The H segment of phase-stable serovar Abortusequi strain SL23 was shown to undergo recurrent inversion, when a DNA invertase gene was supplied in trans (23). Because we showed as described above that another gene, fin, is also involved in phase variation, SL23 should lack not only a functional hin gene but also a functional fin gene. In order to test this, we examined the genomic DNA of SL23 by PCR amplification with primers 2702f3E and 2702r1B, specific for the fin gene. As expected, there was no specific DNA fragment amplified with these primers (data not shown). PCR amplification was also performed with primers 2739f1 and 2739r1, specific for another Fels-2 gene, STM2739. No specific DNA fragment was amplified either (data not shown), suggesting that SL23 lacks the entire Fels-2 element. Next, we performed PCR amplification with primers stssrAf1 and stssrAr1, specific for the tmRNA-encoding ssrA gene, because this gene locates just adjacent to Fels-2. This amplification yielded a specific DNA fragment of the expected length (0.7 kb) (data not shown), indicating that SL23 possesses the ssrA gene.
In order to know the genome structure of this region in serovar Abortusequi, we determined the entire DNA sequence between the ssrA and fljA genes in SL23. It was found that the DNA between these two genes is 7.0 kb long and shows no sequence homology with the corresponding region of serovar Typhimurium. In serovar Typhimurium, this region is 68.3 kb long and consists of a prophage, Fels-2, and a prophage-like element, Stm27X (3, 41, 46), both of which are absent from the SL23 genome (Fig. 4). Instead, this region of SL23 contains seven open reading frames (ORFs), four of which show sequence similarities with genes of another prophage-like elements found in the genomes of S. enterica serovars Paratyphi A (29) and Typhi Ty2 (6). Therefore, this region seems to consist of phage remnants in SL23.
DISCUSSION
Since its discovery in 1961 (16), Hin has long been believed to be the sole DNA invertase responsible for flagellar phase variation in LT2, a diphasic serovar Typhimurium strain. However, genome sequence information suggested that LT2 may encode another DNA invertase, Fin (Fig. 1A). Here, we prove experimentally that, in addition to Hin, Fin is involved in flagellar phase variation in LT2 (Fig. 3 and Table 3).
Although Fin can mediate H inversion, its contribution is much smaller than that of Hin (Table 3). In the Fin invertase, five amino acids that are conserved in all four authentic invertases (Hin, Gin, Cin, and Pin) are replaced with other amino acids (Fig. 1A). For example, Gln149, Pro161, Ala166, and Ile168 of Hin are replaced with Trp, Thr, Gly, and Leu in Fin, respectively. These four amino acids are known to be located within the region important for hix site recognition (7), suggesting that the difference in contribution to H inversion may be due to a difference in recombinase specificity or activity between the two DNA invertases. However, we cannot rule out a possibility that the difference may reflect a difference in expression level, because the fin gene seems to be expressed fully only after prophage induction (8).
Fin is encoded by a P2 phage-related prophage element, Fels-2, which is integrated at the ssrA gene and locates near the hin-fljBA locus (Fig. 4). SL23, a phase-stable serovar Abortusequi strain, was known to possess a naturally occurring hin mutation (16). In this study, we further show that SL23 lacks Fels-2 (Fig. 4). This indicates that SL23 possesses neither Hin nor Fin invertase, which ensures its phase stability.
SL23 shows no sequence similarity to LT2 in the intergenic region between the ssrA and fljA genes. However, the ORFs found in this region of SL23 show sequence similarities to genes of prophage-like elements from S. enterica serovars Paratyphi A and Typhi Ty2 (Fig. 4). These elements are related to the P4 phage and do not contain a gene for DNA invertase (6, 29). It is well known that the DNA sequence adjacent to the ssrA gene shows extensive heterogeneity among different serovars and even within the same serovars of S. enterica due to the integration of different prophages at the ssrA gene (10, 32, 34, 41, 46). Therefore, it is likely that some temperate phages other than Fels-2 might have been integrated into the ssrA gene, and extensive deletions of the prophage genes might have subsequently occurred during the genome diversification in serovar Abortusequi.
Iino (16) obtained phase-stable derivatives of serovar Typhimurium by replacing its hin-fljBA locus with its counterpart from serovar Abortusequi through P22-mediated transduction. Iino's result can be explained if we assume that the fin gene of serovar Typhimurium might have been removed simultaneously from the phase-stable recombinants. In this interserovar transduction, recombination was unlikely to occur in the region between the fljA and ssrA genes, because this region has no apparent sequence homology between these two serovars. Therefore, the entire DNA sequence between these two genes in serovar Typhimurium might have been replaced with that from serovar Abortusequi to yield phase-stable recombinants. This speculation is supported by the fact that this region of serovar Abortusequi consists of a 7.0-kb DNA (Fig. 4), which can be transferred by a single transductional event, because a single phage particle of P22 encapsidates a DNA fragment longer than 40 kb (21, 31).
The fin gene locates just adjacent to an invertible F segment in the Fels-2 prophage. Because almost all of the invertible DNA segments characterized so far are accompanied by genes for their cognate DNA invertases (11, 13, 23, 26, 33, 38), it is reasonable to postulate that the fin gene is involved in inversion of the F segment. This is further supported by the fact that the inverted repetitious sequences fixL and fixR flanking the F segment show sequence homology with those of the H segment (Fig. 1B). Our preliminary experiment showed that the F segment is actively inverting in LT2 and KK203, whereas it is fixed in one orientation in KK205 and KK211 (data not shown), suggesting that the fin gene is responsible for F inversion. The F segment contains two inversely oriented sets of two genes (STM2703 and STM2704, and STM2705 and STM2706) encoding tail fiber proteins (3, 28). Therefore, like G inversion in phage Mu (11), F inversion is likely to regulate the alternative expression of these genes.
There is an increasing amount of information indicating that the genomes of many S. enterica strains contain various prophage-like elements possessing genes homologous to hin and fin (3, 41, 46). Therefore, like in LT2, flagellar phase variation must be mediated by two or more different DNA invertases in at least some Salmonella strains. It is well known that many phages cause lysogenic conversion of O antigens (27). Our findings reported here indicate that prophages can also cause conversion of H antigens by mediating flagellar phase variation. This is an important aspect to be considered in the serotypic determination of clinical isolates. On the other hand, numerous virulence factors are known to be encoded by prophages (45). Because flagellar phase variation has been shown to have a significant effect on Salmonella virulence (17, 37), our findings also suggest that in addition to encoding virulence factors, prophages can play a role in virulence control through modulating flagellar phase variation frequency.
REFERENCES
Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113.
Bonifield, H. R., and K. T. Hughes. 2003. Flagellar phase variation in Salmonella enterica is mediated by a posttranscriptional control mechanism. J. Bacteriol. 185:3567-3574.
Canchaya, C., C. Proux, G. Fournous, A. Bruttin, and H. Brussow. 2003. Prophage genomics. Microbiol. Mol. Biol. Rev. 67:238-276.
Chow, L. T., and A. I. Bukhari. 1976. The invertible DNA segments of coliphages Mu and P1 are identical. Virology 74:242-248.
Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.
Deng, W., S.-R. Liou, G. Plunkett III, G. F. Mayhew, D. J. Rose, V. Burland, V. Kodoyianni, D. C. Schwartz, and F. R. Blattner. 2003. Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18. J. Bacteriol. 185:230-2337.
Feng, J.-A., R. C. Johnson, and R. E. Dickerson. 1994. Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions. Science 263:348-355.
Frye, J. G., S. Porwollik, F. Blackmer, P. Cheng, and M. McClelland. 2005. Host gene expression changes and DNA amplification during temperate phage induction. J. Bacteriol. 187:1485-1492.
Fujita, H., S. Yamaguchi, and T. Iino. 1973. Studies on H-O variants in Salmonella in relation to phase variation. J. Gen. Microbiol. 76:127-134.
Garaizar, J., S. Porwollik, A. Echeita, A. Rementeria, S. Herrera, R. M. Wong, J. Frye, M. A. Usera, and M. McClelland. 2002. DNA microarray-based typing of an atypical monophasic Salmonella enterica serovar. J. Clin. Microbiol. 40:2074-2078.
Giphart-Gassler, M., R. H. A. Plasterk, and P. van de Putte. 1982. G inversion in bacteriophage Mu: a novel way of gene splicing. Nature 297:339-342.
Heichman, K. A., and R. C. Johnson. 1990. The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer. Science 249:511-517.
Iida, S., J. Meyer, K. E. Kennedy, and W. Arber. 1982. A site-specific, conservative recombination system carried by bacteriophage P1. Mapping the recombinase gene cin and the cross-over sites cix for the inversion of the C segment. EMBO J. 1:1445-1453.
Iino, T. 1969. Genetics and chemistry of bacterial flagella. Bacteriol. Rev. 33:454-475.
Iino, T., and K. Kutsukake. 1983. Flagellar phase variation in Salmonella: a model system regulated by flip-flop DNA inversions, p. 395-406. In K. Mizobuchi, I. Watanabe, and J. D. Watson (ed.), Nucleic acid research, future development. Academic Press, New York, N.Y.
Iino, T. A. 1961. Stabilizer of antigenic phases in Salmonella abortus-equi. Genetics 46:1465-1469.
Ikeda, J. S., C. K. Schmitt, S. C. Darnell, P. R. Watson, J. Bispham, T. S. Wallis, D. L. Weinstein, E. S. Metcalf, P. Adams, C. D. O'Connor, and A. D. O'Brien. 2001. Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis. Infect. Immun. 69:3021-3030.
Johnson, R. C., and M. I. Simon. 1985. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombination enhancer. Cell 41:781-791.
Kahmann, R., F. Rudt, C. Koch, and G. Mertens. 1985. G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor. Cell 41:771-780.
Kauffmann, F. 1964. Das Kaufmann-White schema, p. 21-66. In E. van Oye (ed.), The world problem of salmonellosis. Dr. W. Junk Publishers, The Hague, The Netherlands.
Kemper, J. 1974. Gene order and co-transduction in the leu-ara-fol-pyrA region of the Salmonella typhimurium linkage map. J. Bacteriol. 117:94-99.
Kutsukake, K. 1997. Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis. J. Bacteriol. 179:1268-1273.
Kutsukake, K., and T. Iino. 1980. A trans-acting factor mediates inversion of a specific DNA segment in flagellar phase variation of Salmonella. Nature 284:479-481.
Kutsukake, K., and T. Iino. 1980. Inversions of specific DNA segments in flagellar phase variation of Salmonella and inversion systems of bacteriophages P1 and Mu. Proc. Natl. Acad. Sci. USA 77:7338-7341.
Kutsukake, K., S. Iyoda, K. Ohnishi, and T. Iino. 1994. Genetic and molecular analyses of the interaction between the flagellum-specific sigma and anti-sigma factors in Salmonella typhimurium. EMBO J. 13:4568-4576.
Kutsukake, K., T. Nakao, and T. Iino. 1985. A gene for DNA invertase and an invertible DNA in Escherichia coli K-12. Gene 34:343-350.
Lerouge, I., and J. Vanderleyden. 2001. O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. FEMS Microbiol. Rev. 26:17-47.
McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-856.
McClelland, M., K. E. Sanderson, S. W. Clifton, P. Latreille, S. Porwollik, A. Sabo, R. Meyer, T. Bieri, P. Ozersky, M. McLellan, C. R. Harkins, C. Wang, C. Nguyen, A. Berghoff, G. Elliott, S. Kohlberg, C. Strong, F. Du, J. Carter, C. Kremizki, D. Layman, S. Leonard, H. Sun, L. Fulton, W. Nash, T. Miner, P. Minx, K. Delehaunty, C. Fronick, V. Magrini, M. Nhan, W. Warren, L. Florea, J. Spieth, and R. K. Wilson. 2004. Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid. Nat. Genet. 36:1268-1274.
Merickel, S. K., and R. C. Johnson. 2004. Topological analysis of Hin-catalyzed DNA recombination in vivo and in vitro. Mol. Microbiol. 51:1143-1154.
Pedulla, M. L., M. E. Ford, T. Karthikeyan, J. M. Houtz, R. W. Hendrix, G. F. Hatfull, A. R. Poteete, E. B. Gilcrease, D. A. Winn-Stapley, and S. R. Casjens. 2003. Corrected sequence of the bacteriophage P22 genome. J. Bacteriol. 185:1475-1477.
Pelludat, C., S. Mirold, and W.-D. Hardt. 2003. The SopE phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage. J. Bacteriol. 185:5182-5191.
Plasterk, R. H. A., A. Brinkman, and P. van de Putte. 1983. DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems. Proc. Natl. Acad. Sci. USA 80:5355-5358.
Reen, F. J., E. F. Boyd, S. Porwollik, B. P. Murphy, D. Gilroy, S. Fanning, and M. McClelland. 2005. Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray. Appl. Environ. Microbiol. 71:1616-1625.
Sanders, E. R., and R. C. Johnson. 2004. Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution. J. Mol. Biol. 340:753-766.
Schmieger, H. 1972. Phage P22 mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119:75-88.
Schmitt, C. K., S. C. Darnell, and A. D. O'Brien. 1996. The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin. J. Bacteriol. 178:2911-2915.
Silverman, M., and M. Simon. 1980. Phase variation: genetic analysis of switching mutants. Cell 19:845-854.
Stocker, B. A. D. 1949. Measurements of rate of mutation of flagellar antigenic phase in Salmonella typhimurium. J. Hyg. 47:398-413.
Symonds, N., and A. Coelho. 1978. Role of the G segment in the growth of phage Mu. Nature 271:573-574.
Thomson, N., S. Baker, D. Pickard, M. Fookes, M. Anjum, N. Hamlin, J. Wain, D. House, Z. Bhutta, K. Chan, S. Falkow, J. Parkhill, M. Woodward, A. Ivens, and G. Dougan. 2004. The role of prophage-like elements in the diversity of Salmonella enterica serovars. J. Mol. Biol. 339:279-300.
Tominaga, A. 1997. The site-specific recombinase encoded by pinD in Shigella dysenteriae is due to the presence of a defective Mu prophage. Microbiology 143:2057-2063.
Tominaga, A., S. Ikemizu, and M. Enomoto. 1991. Site-specific recombinase genes in three Shigella subgroups and nucleotide sequences of a pinB gene and an invertible B segment from Shigella boydii. J. Bacteriol. 173:4079-4087.
Wada, M., K. Kutsukake, T. Komano, F. Imamoto, and Y. Kano. 1989. Participation of the hup gene product in site-specific DNA inversion in Escherichia coli. Gene 76:345-352.
Wagner, P. L., and M. K. Waldor. 2002. Bacteriophage control of bacterial virulence. Infect. Immun. 70:3985-3993.
Williams, K. P. 2003. Traffic at the tmRNA gene. J. Bacteriol. 185:1059-1070.
Yamamoto, S., and K. Kutsukake. 2006. FljA-mediated posttranscriptional control of phase 1 flagellin expression in flagellar phase variation of Salmonella enterica serovar Typhimurium. J. Bacteriol. 188:958-967.
Zieg, J., M. Silverman, M. Hilmen, and M. Simon. 1977. Recombinational switch for gene expression. Science 196:170-172.
Zieg, J., and M. Simon. 1980. Analysis of the nucleotide sequence of an invertible controlling element. Proc. Natl. Acad. Sci. USA 77:4196-4200.(Kazuhiro Kutsukake, Hisas)
ABSTRACT
Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.
INTRODUCTION
Diphasic Salmonella enterica strains possess two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments, and alternate expression between these two genes. This phenomenon is called flagellar phase variation, and cells expressing FliC are called phase 1 cells, whereas those expressing FljB are called phase 2 cells (14, 20). These two genes encode antigenically distinct flagellin molecules. For example, in S. enterica serovar Typhimurium, fliC and fljB encode flagellin proteins of antigenic types "i" and "1,2," respectively. The frequency of flagellar phase variation varies among different strains. Serovar Typhimurium strain C77 was reported to change from phase 1 to phase 2 and from phase 2 to phase 1 at frequencies of 1 x 10–5 and 3 x 10–4 per bacterial generation cycle, respectively (39). On the other hand, monophasic Salmonella serovars that express only either fliC or fljB are also known to exist (20). For example, serovar Abortusequi strains constantly express the fljB gene, which specifies flagellin of antigenic type "e,n,x" (16). Escherichia coli strains usually lack the fljB locus and express the fliC gene constitutively.
Flagellar phase variation is controlled by the recurrent inversion of a 996-bp DNA segment, called the H segment, which contains the promoter for the fljB gene (48, 49). The H segment is flanked by 26-bp inverted repetitious sequences, hixL and hixR, between which site-specific recombination occurs, leading to H inversion (18). This recombination is mediated by a DNA invertase, Hin, whose gene is located within the H segment (23, 38). The fljB gene constitutes an operon together with the fljA gene, which encodes a negative regulator for fliC expression (9). FljA has been shown to inhibit fliC expression through a posttranscriptional control mechanism (2, 47). When the H segment is in the "on" orientation, both fljB and fljA are transcribed, resulting in phase 2 flagellin being synthesized. On the other hand, when the H segment turns to the "off" orientation, neither fljB nor fljA is expressed, resulting in phase 1 flagellin being synthesized. Therefore, the frequency of phase variation directly reflects that of H inversion.
The molecular mechanism of Hin-mediated inversion of the H segment has been intensively studied both in vivo and in vitro (12, 18, 30, 35, 44). The inversion reaction requires a supercoiled DNA substrate containing the two hix sites and a 65-bp recombinational enhancer that locates within the hin gene. Hin assembles into a tripartite synaptic complex, called an invertasome, which contains these two hix sites and the Fis-bound enhancer. Fis activates Hin to catalyze DNA cleavage followed by strand exchange leading to recombination between the two hix sites. In this reaction, the DNA bending activity of the HU protein facilitates looping of the DNA between the enhancer and the hix sites.
Invertible DNA segments are not unique to flagellar phase variation. For example, bacteriophages Mu and P1 contain invertible G and C segments, respectively, whose inversion regulates the alternate expression between two sets of tail fiber genes and thus changes host specificity (4, 40). The cognate DNA invertase genes are called gin and cin, which locate just adjacent to the G and C segments, respectively (13, 19). These DNA invertases share sequence homology with each other and with Hin invertase. The DNA sequences of the recombination sites are also conserved among H, G, and C segments. Consistent with these homologies, the DNA invertase was shown to be interchangeable among these three inversion systems (23, 24). These facts collectively suggest that these inversion systems have evolved from a common ancestor (15). Some E. coli and Shigella strains also contain homologous inversion systems, which are located in cryptic Mu-like prophages (26, 33, 42, 43).
S. enterica serovar Abortusequi possesses a naturally occurring hin mutation (formally called vh2–) that results in this serovar being phase stable (16, 23). Derivatives of serovar Typhimurium whose hin-fljBA locus had been replaced with the counterpart from serovar Abortusequi by P22-mediated transduction became phase stable (16), suggesting that hin should be the sole DNA invertase gene responsible for H inversion in Salmonella. Surprisingly, however, a search of the whole genome sequence of LT2, a standard laboratory strain of serovar Typhimurium, revealed that it contains another gene (STM2702) that is highly homologous to the hin gene (28). This gene locates just adjacent to a 2.7-kb invertible DNA segment. Here, we refer to this gene and the invertible DNA as fin and F segment, respectively. The fin gene and F segment are located in a P2 phage-related resident prophage, Fels-2 (3, 41, 46), which exists 34.5 kb apart from the hin-fljBA locus. Strong sequence similarity between Hin and Fin (Fig. 1A) raised the possibility that the fin gene is also involved in flagellar phase variation. This work was carried out to test this possibility. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. We found that strains possessing either one of these mutations could manifest phase variation, whereas strains possessing both mutations could not. This result clearly indicates that these two different DNA invertases are both involved in flagellar phase variation in LT2. Furthermore, we showed that serovar Abortusequi lacks both of these two invertases, which makes this strain phase stable.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture media. Bacterial strains and plasmids used in this study are all listed in Table 1. Procedures for the construction of gene disruptants and recombinant plasmids are described below. Ordinary culture media, such as L broth, L agar plates, P22 broth, and motility agar plates, were prepared as described previously (22). If necessary, ampicillin, kanamycin, and chloramphenicol were added to the media at final concentrations of 50, 25, and 10 μg/ml, respectively.
Motility test. Single colonies were inoculated onto motility agar plates and incubated for 5 to 10 h at 30°C. Motility was detected as spreading growth on the plates. When phase variation ability was tested, each motility agar plate included 1 μl of the antiserum specific against either i- or 1,2-type flagellar filament per 25 ml of the medium. When a diphasic strain such as LT2 is used, phase 1 cells form spreading colonies on motility agar plates containing anti-1,2 serum and form compact colonies with halo-like spreading growth on motility agar plates containing anti-i serum. The halo-like spreading growth is due to the appearance of phase 2 cells during growth of phase 1 cells on the plate. Phase 2 cells of the diphasic strain give the opposite result; that is, they form spreading colonies on motility agar plates containing anti-i serum and form compact colonies with halo-like spreading growth on motility agar plates containing anti-1,2 serum. On the other hand, when a phase 1-fixed monophasic strain is used, the cells form spreading colonies on motility agar plates containing anti-1,2 serum and form compact colonies without a halo on motility agar plates containing anti-i serum. A phase 2-fixed monophasic strain gives the opposite result. Phase variation frequency was estimated according to the method described previously by Stocker (39).
Transduction and DNA manipulation. P22-mediated transduction was performed with P22HTint (36) as described previously (25). DNA manipulation and transformation were performed as described previously (25). Restriction enzymes and T4 DNA ligase were purchased from Toyobo (Osaka, Japan) or Takara (Kyoto, Japan). PCR amplification of DNA was carried out with an iCycler (Bio-Rad, CA) using KOD Dash DNA polymerase (Toyobo). Customized DNA primers were purchased from Hokkaido System Science (Sapporo, Japan). Their nucleotide sequences are listed in Table 2. Electroporation of DNA into Salmonella cells was carried out using a Gene Pulser system (Bio-Rad). DNA sequence was determined by the dideoxy chain termination method using a PRIZM 310NT genetic analyzer (Applied Biosystems, CA).
Construction of gene disruptants. The -Red system described previously by Datsenko and Wanner (5) was used for the construction of gene disruption mutants. A hin disruption mutant was constructed as follows. The chloramphenicol resistance FRT (Flp recognition target) cassette was PCR amplified from pKD3 using primers hinf1P3 and hinr1P2. The amplified product was introduced into LT2 harboring pKD46 by electroporation. Recombinants were selected on L agar plates containing chloramphenicol. In the resulting hin disruptant, an 83-bp DNA in the middle of the hin gene was deleted and substituted with the chloramphenicol resistance FRT cassette, but the recombinational enhancer sequence was retained. This hin::cat allele was then moved to LT2 by transduction selecting for chloramphenicol resistance to obtain KK201. The chloramphenicol resistance cassette was removed through site-specific recombination between the flanking FRT sequences by transient exposure of KK201 to pCP20. In the resulting hin mutant, KK203, the H segment has the same size as that in LT2. The fin disruption mutant KK205 was constructed through the same procedure as described above, but in this case, the kanamycin resistance FRT cassette was amplified with primers 2702f4P1 and 2702r2P2 using pKD4 as a template. In KK205, the entire fin gene including the putative recombinational enhancer sequence was deleted and replaced with the kanamycin resistance FRT cassette. The hin fin double mutants were constructed by transduction of the fin::kan allele from KK205 into phase 1 and phase 2 cells of KK203. As described below, the resulting double mutants, KK211 and KK212, were fixed in phase 1 and phase 2, respectively.
Construction of plasmids carrying the hin gene. The genomic DNA of LT2 and pBR322 was digested with EcoRI, ligated, and introduced into EKK11 by transformation. Motile clones were selected on motility agar plates containing ampicillin. From a clone expressing the 1,2-type flagella, a plasmid was isolated and named pKK1001. This plasmid carries a 15-kb DNA fragment containing the hin and fljB genes from LT2. A 0.6-kb DNA fragment containing the hin gene was PCR amplified from pKK1001 with primers hinf3E and hinr3B. The amplified product was digested with EcoRI and BamHI and inserted into the corresponding site of pSTV29 to obtain pHN2.
Determination of H orientation. The orientation of the H segment was determined by PCR amplification of each fix site and its flanking sequences from genomic DNA using following primer pairs: hinf4 and HLr1 for hixL of the off-oriented H segment, HRf1 and hinr4 for hixR of the off-oriented H segment, hinr4 and HLr1 for hixL of the on-oriented H segment, and HRf1 and hinf4 for hixR of the on-oriented H segment. The amplified products were 543, 1,769, 1,125, and 1,188 bp long, respectively. Orientation of the H segment was further confirmed by DNA sequencing of the amplified products using primers hinf4 and hinr4.
Nucleotide sequence accession number. The DNA sequence data for the SL23 genome between the ssrA and fljA genes have been submitted to the DDBJ database under accession no. AB234611.
RESULTS
DNA invertase activity of the fin gene product. Figure 1A shows the similarity in amino acid sequence between the hin and fin gene products. Although Fin is five amino acids smaller than Hin, 62% of the amino acids are identical between the two. The similarity extends throughout the molecules, and most of the amino acids conserved in four authentic DNA invertases (Hin, Gin, Cin, and Pin) are also conserved in Fin. These facts suggest that Fin is also capable of mediating H inversion.
In order to test this, the fin gene was PCR amplified from the genomic DNA of LT2 with primers 2702f3E and 2702r1B, digested with EcoRI and BamHI, and inserted into the corresponding site of pBR322 to obtain pHN1. This plasmid was introduced into an E. coli strain, EKK15, by transformation. EKK15 possesses a defective fliC gene and an intact fljBA locus together with the H segment containing a defective hin gene originating from serovar Abortusequi. EKK15 is nonmotile, because it contains no DNA invertase activity and its H segment is fixed in the off orientation (26). However, EKK15 harboring pHN1 produced motile cells on motility agar plates (Fig. 2), indicating that the fin gene can mediate H inversion at least in the off-to-on direction.
Flagellar phase variation in the hin and fin mutants. According to the procedures described in Materials and Methods, hin and fin disruption mutants were constructed from LT2, and their phase variation abilities were examined on motility agar plates containing anti-i or anti-1,2 serum (Fig. 3). As expected, the fin disruption mutant KK205 showed motility on both plates, indicating that H inversion can occur in the absence of Fin. Interestingly, the hin disruption mutant KK203 could also show motility on both plates, indicating that flagellar phase variation can occur even in the absence of the Hin invertase. Next, the fin::kan mutation of KK205 was introduced into KK203 by P22-mediated transduction to obtain fin hin double mutants. We obtained two types of kanamycin-resistant transductants that differed in the behavior on motility agar plates containing anti-i or anti-1,2 serum. One mutant (KK211) formed spreading colonies on a motility agar plate containing anti-1,2 serum and compact colonies on that containing anti-i serum, indicating that this strain was fixed in phase 1. Conversely, the other mutant (KK212) formed spreading colonies on a motility agar plate containing anti-i serum and formed compact colonies on that containing anti-1,2 serum, indicating that this strain was fixed in phase 2. These results clearly indicate that both hin and fin gene products are involved in flagellar phase variation in LT2.
In order to correlate the flagellar phenotype to H inversion in these strains, DNA fragments containing each hix site and its flanking sequences were PCR amplified from the genomic DNAs, and the orientation of the H segment was determined by DNA sequencing of the amplified products (data not shown). From the genomic DNAs of LT2, KK203(hin), and KK205(fin), we obtained amplified products containing the H segment in each orientation, indicating that H inversion occurs in these three strains. In contrast, only DNA fragments containing the H segment in the off orientation were obtained from KK211(hin fin FljBoff), whereas only those containing the H segment in the on orientation were obtained from KK212(hin fin FljBon), indicating that H inversion does not occur in these two strains. Therefore, we conclude that H inversion is mediated by both Hin and Fin in LT2.
In order to know how much each of these two DNA invertases contributes to H inversion, we measured the frequencies of flagellar phase variation from phase 1 to phase 2 and from phase 2 to phase 1 in LT2, KK203(hin), and KK205(fin). The results obtained from representative experiments are summarized in Table 3. The frequency in KK203 was approximately 60 to 200 times lower than that of LT2, whereas KK205 underwent phase variation at a frequency almost equivalent to that of LT2. We also measured the phase variation frequency with LT2 and KK203 harboring pHN2, which carries the hin gene in a plasmid vector, pSTV29 (Table 3). Both strains were found to undergo phase variation at almost equal frequencies. This result excludes the possibility that the hin mutation in KK203 might have any cis-acting inhibitory effect on H inversion mediated by a DNA invertase supplied in trans. Therefore, we conclude that H inversion is mediated mainly by its cognate DNA invertase, Hin, although Fin invertase also contributes to H inversion.
Phase variation frequency was also examined with the hin fin double mutants (Table 3). As expected, KK211(hin fin FljBoff) did not produce cells of the opposite phase at a detectable frequency. On the other hand, KK212(hin fin FljBon) produced motile cells on motility agar plates containing anti-1,2 serum at a very low but detectable frequency. However, this phase change was found to be irreversible; that is, the phase 1 cells that emerged from KK212 never produced phase 2 cells, suggesting that the change from phase 2 to phase 1 in KK212 might have resulted from a spontaneous mutation that inactivated both fljB and fljA that was not from H inversion.
Absence of Fels-2 in SL23. The H segment of phase-stable serovar Abortusequi strain SL23 was shown to undergo recurrent inversion, when a DNA invertase gene was supplied in trans (23). Because we showed as described above that another gene, fin, is also involved in phase variation, SL23 should lack not only a functional hin gene but also a functional fin gene. In order to test this, we examined the genomic DNA of SL23 by PCR amplification with primers 2702f3E and 2702r1B, specific for the fin gene. As expected, there was no specific DNA fragment amplified with these primers (data not shown). PCR amplification was also performed with primers 2739f1 and 2739r1, specific for another Fels-2 gene, STM2739. No specific DNA fragment was amplified either (data not shown), suggesting that SL23 lacks the entire Fels-2 element. Next, we performed PCR amplification with primers stssrAf1 and stssrAr1, specific for the tmRNA-encoding ssrA gene, because this gene locates just adjacent to Fels-2. This amplification yielded a specific DNA fragment of the expected length (0.7 kb) (data not shown), indicating that SL23 possesses the ssrA gene.
In order to know the genome structure of this region in serovar Abortusequi, we determined the entire DNA sequence between the ssrA and fljA genes in SL23. It was found that the DNA between these two genes is 7.0 kb long and shows no sequence homology with the corresponding region of serovar Typhimurium. In serovar Typhimurium, this region is 68.3 kb long and consists of a prophage, Fels-2, and a prophage-like element, Stm27X (3, 41, 46), both of which are absent from the SL23 genome (Fig. 4). Instead, this region of SL23 contains seven open reading frames (ORFs), four of which show sequence similarities with genes of another prophage-like elements found in the genomes of S. enterica serovars Paratyphi A (29) and Typhi Ty2 (6). Therefore, this region seems to consist of phage remnants in SL23.
DISCUSSION
Since its discovery in 1961 (16), Hin has long been believed to be the sole DNA invertase responsible for flagellar phase variation in LT2, a diphasic serovar Typhimurium strain. However, genome sequence information suggested that LT2 may encode another DNA invertase, Fin (Fig. 1A). Here, we prove experimentally that, in addition to Hin, Fin is involved in flagellar phase variation in LT2 (Fig. 3 and Table 3).
Although Fin can mediate H inversion, its contribution is much smaller than that of Hin (Table 3). In the Fin invertase, five amino acids that are conserved in all four authentic invertases (Hin, Gin, Cin, and Pin) are replaced with other amino acids (Fig. 1A). For example, Gln149, Pro161, Ala166, and Ile168 of Hin are replaced with Trp, Thr, Gly, and Leu in Fin, respectively. These four amino acids are known to be located within the region important for hix site recognition (7), suggesting that the difference in contribution to H inversion may be due to a difference in recombinase specificity or activity between the two DNA invertases. However, we cannot rule out a possibility that the difference may reflect a difference in expression level, because the fin gene seems to be expressed fully only after prophage induction (8).
Fin is encoded by a P2 phage-related prophage element, Fels-2, which is integrated at the ssrA gene and locates near the hin-fljBA locus (Fig. 4). SL23, a phase-stable serovar Abortusequi strain, was known to possess a naturally occurring hin mutation (16). In this study, we further show that SL23 lacks Fels-2 (Fig. 4). This indicates that SL23 possesses neither Hin nor Fin invertase, which ensures its phase stability.
SL23 shows no sequence similarity to LT2 in the intergenic region between the ssrA and fljA genes. However, the ORFs found in this region of SL23 show sequence similarities to genes of prophage-like elements from S. enterica serovars Paratyphi A and Typhi Ty2 (Fig. 4). These elements are related to the P4 phage and do not contain a gene for DNA invertase (6, 29). It is well known that the DNA sequence adjacent to the ssrA gene shows extensive heterogeneity among different serovars and even within the same serovars of S. enterica due to the integration of different prophages at the ssrA gene (10, 32, 34, 41, 46). Therefore, it is likely that some temperate phages other than Fels-2 might have been integrated into the ssrA gene, and extensive deletions of the prophage genes might have subsequently occurred during the genome diversification in serovar Abortusequi.
Iino (16) obtained phase-stable derivatives of serovar Typhimurium by replacing its hin-fljBA locus with its counterpart from serovar Abortusequi through P22-mediated transduction. Iino's result can be explained if we assume that the fin gene of serovar Typhimurium might have been removed simultaneously from the phase-stable recombinants. In this interserovar transduction, recombination was unlikely to occur in the region between the fljA and ssrA genes, because this region has no apparent sequence homology between these two serovars. Therefore, the entire DNA sequence between these two genes in serovar Typhimurium might have been replaced with that from serovar Abortusequi to yield phase-stable recombinants. This speculation is supported by the fact that this region of serovar Abortusequi consists of a 7.0-kb DNA (Fig. 4), which can be transferred by a single transductional event, because a single phage particle of P22 encapsidates a DNA fragment longer than 40 kb (21, 31).
The fin gene locates just adjacent to an invertible F segment in the Fels-2 prophage. Because almost all of the invertible DNA segments characterized so far are accompanied by genes for their cognate DNA invertases (11, 13, 23, 26, 33, 38), it is reasonable to postulate that the fin gene is involved in inversion of the F segment. This is further supported by the fact that the inverted repetitious sequences fixL and fixR flanking the F segment show sequence homology with those of the H segment (Fig. 1B). Our preliminary experiment showed that the F segment is actively inverting in LT2 and KK203, whereas it is fixed in one orientation in KK205 and KK211 (data not shown), suggesting that the fin gene is responsible for F inversion. The F segment contains two inversely oriented sets of two genes (STM2703 and STM2704, and STM2705 and STM2706) encoding tail fiber proteins (3, 28). Therefore, like G inversion in phage Mu (11), F inversion is likely to regulate the alternative expression of these genes.
There is an increasing amount of information indicating that the genomes of many S. enterica strains contain various prophage-like elements possessing genes homologous to hin and fin (3, 41, 46). Therefore, like in LT2, flagellar phase variation must be mediated by two or more different DNA invertases in at least some Salmonella strains. It is well known that many phages cause lysogenic conversion of O antigens (27). Our findings reported here indicate that prophages can also cause conversion of H antigens by mediating flagellar phase variation. This is an important aspect to be considered in the serotypic determination of clinical isolates. On the other hand, numerous virulence factors are known to be encoded by prophages (45). Because flagellar phase variation has been shown to have a significant effect on Salmonella virulence (17, 37), our findings also suggest that in addition to encoding virulence factors, prophages can play a role in virulence control through modulating flagellar phase variation frequency.
REFERENCES
Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113.
Bonifield, H. R., and K. T. Hughes. 2003. Flagellar phase variation in Salmonella enterica is mediated by a posttranscriptional control mechanism. J. Bacteriol. 185:3567-3574.
Canchaya, C., C. Proux, G. Fournous, A. Bruttin, and H. Brussow. 2003. Prophage genomics. Microbiol. Mol. Biol. Rev. 67:238-276.
Chow, L. T., and A. I. Bukhari. 1976. The invertible DNA segments of coliphages Mu and P1 are identical. Virology 74:242-248.
Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.
Deng, W., S.-R. Liou, G. Plunkett III, G. F. Mayhew, D. J. Rose, V. Burland, V. Kodoyianni, D. C. Schwartz, and F. R. Blattner. 2003. Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18. J. Bacteriol. 185:230-2337.
Feng, J.-A., R. C. Johnson, and R. E. Dickerson. 1994. Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions. Science 263:348-355.
Frye, J. G., S. Porwollik, F. Blackmer, P. Cheng, and M. McClelland. 2005. Host gene expression changes and DNA amplification during temperate phage induction. J. Bacteriol. 187:1485-1492.
Fujita, H., S. Yamaguchi, and T. Iino. 1973. Studies on H-O variants in Salmonella in relation to phase variation. J. Gen. Microbiol. 76:127-134.
Garaizar, J., S. Porwollik, A. Echeita, A. Rementeria, S. Herrera, R. M. Wong, J. Frye, M. A. Usera, and M. McClelland. 2002. DNA microarray-based typing of an atypical monophasic Salmonella enterica serovar. J. Clin. Microbiol. 40:2074-2078.
Giphart-Gassler, M., R. H. A. Plasterk, and P. van de Putte. 1982. G inversion in bacteriophage Mu: a novel way of gene splicing. Nature 297:339-342.
Heichman, K. A., and R. C. Johnson. 1990. The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer. Science 249:511-517.
Iida, S., J. Meyer, K. E. Kennedy, and W. Arber. 1982. A site-specific, conservative recombination system carried by bacteriophage P1. Mapping the recombinase gene cin and the cross-over sites cix for the inversion of the C segment. EMBO J. 1:1445-1453.
Iino, T. 1969. Genetics and chemistry of bacterial flagella. Bacteriol. Rev. 33:454-475.
Iino, T., and K. Kutsukake. 1983. Flagellar phase variation in Salmonella: a model system regulated by flip-flop DNA inversions, p. 395-406. In K. Mizobuchi, I. Watanabe, and J. D. Watson (ed.), Nucleic acid research, future development. Academic Press, New York, N.Y.
Iino, T. A. 1961. Stabilizer of antigenic phases in Salmonella abortus-equi. Genetics 46:1465-1469.
Ikeda, J. S., C. K. Schmitt, S. C. Darnell, P. R. Watson, J. Bispham, T. S. Wallis, D. L. Weinstein, E. S. Metcalf, P. Adams, C. D. O'Connor, and A. D. O'Brien. 2001. Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis. Infect. Immun. 69:3021-3030.
Johnson, R. C., and M. I. Simon. 1985. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombination enhancer. Cell 41:781-791.
Kahmann, R., F. Rudt, C. Koch, and G. Mertens. 1985. G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor. Cell 41:771-780.
Kauffmann, F. 1964. Das Kaufmann-White schema, p. 21-66. In E. van Oye (ed.), The world problem of salmonellosis. Dr. W. Junk Publishers, The Hague, The Netherlands.
Kemper, J. 1974. Gene order and co-transduction in the leu-ara-fol-pyrA region of the Salmonella typhimurium linkage map. J. Bacteriol. 117:94-99.
Kutsukake, K. 1997. Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis. J. Bacteriol. 179:1268-1273.
Kutsukake, K., and T. Iino. 1980. A trans-acting factor mediates inversion of a specific DNA segment in flagellar phase variation of Salmonella. Nature 284:479-481.
Kutsukake, K., and T. Iino. 1980. Inversions of specific DNA segments in flagellar phase variation of Salmonella and inversion systems of bacteriophages P1 and Mu. Proc. Natl. Acad. Sci. USA 77:7338-7341.
Kutsukake, K., S. Iyoda, K. Ohnishi, and T. Iino. 1994. Genetic and molecular analyses of the interaction between the flagellum-specific sigma and anti-sigma factors in Salmonella typhimurium. EMBO J. 13:4568-4576.
Kutsukake, K., T. Nakao, and T. Iino. 1985. A gene for DNA invertase and an invertible DNA in Escherichia coli K-12. Gene 34:343-350.
Lerouge, I., and J. Vanderleyden. 2001. O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. FEMS Microbiol. Rev. 26:17-47.
McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-856.
McClelland, M., K. E. Sanderson, S. W. Clifton, P. Latreille, S. Porwollik, A. Sabo, R. Meyer, T. Bieri, P. Ozersky, M. McLellan, C. R. Harkins, C. Wang, C. Nguyen, A. Berghoff, G. Elliott, S. Kohlberg, C. Strong, F. Du, J. Carter, C. Kremizki, D. Layman, S. Leonard, H. Sun, L. Fulton, W. Nash, T. Miner, P. Minx, K. Delehaunty, C. Fronick, V. Magrini, M. Nhan, W. Warren, L. Florea, J. Spieth, and R. K. Wilson. 2004. Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid. Nat. Genet. 36:1268-1274.
Merickel, S. K., and R. C. Johnson. 2004. Topological analysis of Hin-catalyzed DNA recombination in vivo and in vitro. Mol. Microbiol. 51:1143-1154.
Pedulla, M. L., M. E. Ford, T. Karthikeyan, J. M. Houtz, R. W. Hendrix, G. F. Hatfull, A. R. Poteete, E. B. Gilcrease, D. A. Winn-Stapley, and S. R. Casjens. 2003. Corrected sequence of the bacteriophage P22 genome. J. Bacteriol. 185:1475-1477.
Pelludat, C., S. Mirold, and W.-D. Hardt. 2003. The SopE phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage. J. Bacteriol. 185:5182-5191.
Plasterk, R. H. A., A. Brinkman, and P. van de Putte. 1983. DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems. Proc. Natl. Acad. Sci. USA 80:5355-5358.
Reen, F. J., E. F. Boyd, S. Porwollik, B. P. Murphy, D. Gilroy, S. Fanning, and M. McClelland. 2005. Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray. Appl. Environ. Microbiol. 71:1616-1625.
Sanders, E. R., and R. C. Johnson. 2004. Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution. J. Mol. Biol. 340:753-766.
Schmieger, H. 1972. Phage P22 mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119:75-88.
Schmitt, C. K., S. C. Darnell, and A. D. O'Brien. 1996. The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin. J. Bacteriol. 178:2911-2915.
Silverman, M., and M. Simon. 1980. Phase variation: genetic analysis of switching mutants. Cell 19:845-854.
Stocker, B. A. D. 1949. Measurements of rate of mutation of flagellar antigenic phase in Salmonella typhimurium. J. Hyg. 47:398-413.
Symonds, N., and A. Coelho. 1978. Role of the G segment in the growth of phage Mu. Nature 271:573-574.
Thomson, N., S. Baker, D. Pickard, M. Fookes, M. Anjum, N. Hamlin, J. Wain, D. House, Z. Bhutta, K. Chan, S. Falkow, J. Parkhill, M. Woodward, A. Ivens, and G. Dougan. 2004. The role of prophage-like elements in the diversity of Salmonella enterica serovars. J. Mol. Biol. 339:279-300.
Tominaga, A. 1997. The site-specific recombinase encoded by pinD in Shigella dysenteriae is due to the presence of a defective Mu prophage. Microbiology 143:2057-2063.
Tominaga, A., S. Ikemizu, and M. Enomoto. 1991. Site-specific recombinase genes in three Shigella subgroups and nucleotide sequences of a pinB gene and an invertible B segment from Shigella boydii. J. Bacteriol. 173:4079-4087.
Wada, M., K. Kutsukake, T. Komano, F. Imamoto, and Y. Kano. 1989. Participation of the hup gene product in site-specific DNA inversion in Escherichia coli. Gene 76:345-352.
Wagner, P. L., and M. K. Waldor. 2002. Bacteriophage control of bacterial virulence. Infect. Immun. 70:3985-3993.
Williams, K. P. 2003. Traffic at the tmRNA gene. J. Bacteriol. 185:1059-1070.
Yamamoto, S., and K. Kutsukake. 2006. FljA-mediated posttranscriptional control of phase 1 flagellin expression in flagellar phase variation of Salmonella enterica serovar Typhimurium. J. Bacteriol. 188:958-967.
Zieg, J., M. Silverman, M. Hilmen, and M. Simon. 1977. Recombinational switch for gene expression. Science 196:170-172.
Zieg, J., and M. Simon. 1980. Analysis of the nucleotide sequence of an invertible controlling element. Proc. Natl. Acad. Sci. USA 77:4196-4200.(Kazuhiro Kutsukake, Hisas)