Detection and Characterization of the Flagellar Master Operon in the Four Shigella Subgroups

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1 JOURNAL OF BACTERIOLOGY, July 1996, p Vol. 178, No /96/$ Copyright 1996, American Society for Microbiology Detection and Characterization of the Flagellar Master Operon in the Four Shigella Subgroups ABU AMAR M. AL MAMUN, AKIRA TOMINAGA, AND MASATOSHI ENOMOTO* Department of Biology, Faculty of Science, Okayama University, Okayama 700, Japan Received 22 January 1996/Accepted 18 April 1996 Strains in the genus Shigella are nonmotile, but they retain some cryptic flagellar operons whether functional or defective (A. Tominaga, M. A.-H. Mahmoud, T. Mukaihara, and M. Enomoto, Mol. Microbiol. 12: , 1994). To disclose the cause of motility loss in shigellae, the presence or defectiveness of the flhd and flhc genes, composing the master operon whose mutation causes inactivation of the entire flagellar regulon, was examined in the four Shigella subgroups. The flhd operon cloned from Shigella boydii and Shigella sonnei can activate, though insufficiently, the regulon in the Escherichia coli flhd or flhc mutant background. The clone from Shigella dysenteriae has a functional flhd gene and nonfunctional flhc gene, and its inactivation has been caused by the IS1 element inserted in its 5 end. The operon of Shigella flexneri is nonfunctional and has suffered an IS1-insertion mutation at the 5 end of the flhd gene. Comparison of restriction maps indicates that only the central 1.8-kb region, including part of the flhc gene and its adjacent mot operon, is conserved among the four Shigella subgroups as well as in E. coli, but in Salmonella typhimurium the whole map is quite different from the others. Motility loss in shigellae is not attributable to genetic damage in the master operon of a common ancestor, but it occurs separately in respective ancestors of the four subgroups, and in both S. dysenteriae and S. flexneri IS1 insertion in the master operon might be the primary cause of motility loss. The genus Shigella is divided into four subgroups: S. dysenteriae, S. flexneri, S. boydii and S. sonnei (31). One of the common characteristics of these subgroups is a lack of motility, i.e., no flagellation (8), although enteric bacteria in other genera are all motile. We have found, however, that some strains of S. flexneri and S. sonnei have the flic gene (39) encoding flagellin, the only component of a flagellar filament. The flic gene has been characterized after being cloned from these two subgroups (40) and from S. dysenteriae and S. boydii (unpublished data). Sequence comparison of the highly conserved regions of the flic genes suggests that the loss of motility in Shigella cells is a recent evolutionary event estimated to have occurred, at most, 10 5 years ago (40). Southern blot hybridization using as probes several flagellar genes from Escherichia coli and Salmonella typhimurium has revealed that each strain from the four subgroups retains most flagellar genes (6a). Now the question is how and why they lost motility. The flagellum-chemotaxis system of E. coli and S. typhimurium consists of more than 40 genes, which compose at least 13 operons to be integrated into one regulon (18, 25, 34). These operons are clustered in four regions, I, II, IIIa, and IIIb (14, 18, 20), on the chromosome and are organized in a complex transcriptional hierarchy of three classes (17, 20, 22). At the top (class 1) of this hierarchy is the flhd master operon in region II, which consists of the flhd and flhc genes and is adjacent to the mota operon containing motab and cheaw (22, 24). The flhd and flhc genes encode some positive transcription factor (13), and the complex of their products activates transcription of class 2 operons (2, 24), one of which contains the flia gene encoding F for class 3 transcription (21). Therefore, genetic damage of the flhd operon causes inactivation of all the flagellar operons. We focus our attention on the flhd operon to examine the cause of motility loss in the Shigella subgroups for two reasons. * Corresponding author. Phone: (81) (086) Fax: (81) (086) (i) Region II operons of the four subgroups are highly polymorphic in restriction fragment length when tested by Southern blotting with E. coli probes (unpublished data). (ii) Supposing that a common ancestor of the four subgroups has undergone some stable mutation in the master operon, its descendants will have more chance to survive in adverse circumstances than will those of other flagellar mutants with futile expression of some flagellar operons. We report here the results of analysis showing that the flhd operon of S. dysenteriae and S. flexneri undergoes insertion (IS) mutations in different sites and that in the two other subgroups the operon still retains weak function to activate subordinate operons. MATERIALS AND METHODS Strains and plasmids. The strains and plasmids used are shown in Table 1. Strains EJ2902 and EJ2903 were made by transducing (reca-srl)306 srl-301:: Tn10 of CSH126 (28) into strains YK4136 and YK4116 (18), respectively. Plasmid ptsd610 carries the 7.6-kb S. dysenteriae fragment with part of IS600 and the pind gene (37a). Media. TLY broth, nutrient agar, and nutrient semisolid agar for the motility test have been described elsewhere (7). Tetracycline (Sigma Chemical Co.) and ampicillin (Sigma) were used at final concentrations of 25 and 50 mg/liter, respectively. DNA manipulation. Isolation and purification of genomic DNA followed the methods previously described (4, 39). Isolation of plasmid DNA, the cloning procedure, and restriction analysis of cloned fragments were performed by the standard methods (32). Restriction endonucleases, calf intestine alkaline phosphatase, T4 DNA ligase, and Klenow fragment were purchased from Takara Shuzo Co. DNA probes and hybridization. To examine flhd operons in Shigella subgroups, E. coli probes were used. The E. coli flhd operon and its adjacent genes have previously been cloned and analyzed in this laboratory. In short, genomic DNA from strain W3110 (23) and pbr322 (5) were spliced after digestion with EcoRI, transformed into nonmotile mutant EJ2903 (flhd), and spread on nutrient semisolid medium with ampicillin. Plasmid pedc1, isolated from one of the motile transformants, had a 5.5-kb EcoRI fragment, which was analyzed by restriction mapping and subcloning (Fig. 1A). The 1.8-kb BglII fragment was found to carry the flhdc genes by complementation tests with the flhd and flhc mutants. The exact locations of flhd, flhc, and other genes were determined by comparing the restriction map of pedc1 with the published sequence data of these genes (2, 6, 15, 37). Fragments (E1 to E4) with part of each gene were used as probes (Fig. 1A). Restriction fragments from seven IS elements were also used as probes (Fig. 2) to survey cloned Shigella fragments for the presence of IS elements. Probes were prepared by the random-primed method (9) with a non- 3722

2 VOL. 178, 1996 FLAGELLAR MASTER OPERON IN THE SHIGELLA SUBGROUPS 3723 TABLE 1. Strains and plasmids used Strain or plasmid Relevant genotype or description Reference or source E. coli RP3098 (flhc-flha) 35 EJ2902 flhc4136 (reca-srl)306 srl-301::tn10 This study EJ2903 flhd4116 (reca-srl)306 srl-301::tn10 This study Shigella spp. S. dysenteriae S. flexneri IID S. boydii NCTC 9733 National Institute of Health, Japan S. sonnei IID Plasmids pmw119 Cloning vector 3, 42 pkk1211 pbr322 with the flhd and mota operons from S. typhimurium 21 psam3 puc18 with IS1 from S. sonnei 27 psam42 puc18 with iso-is2 from S. sonnei 27 pnc155 phsg398 with IS3 from E. coli 19 ptsd610 pbr322 with IS600 from S. dysenteriae This laboratory psam629 puc18 with IS629 from S. sonnei 27 psam31 puc18 with IS630 from S. sonnei 27 psam41 puc18 with IS640 from S. sonnei 27 radioactive DNA labelling kit (Boehringer Mannheim). Hybridization was performed by standard methods (32, 36), and hybrid bands were detected by the digoxigenin enzyme-linked immunosorbent assay method (9) with a kit. Colony blot hybridization to detect Shigella flhdc clones followed the method described previously (9, 12, 32). RESULTS AND DISCUSSION Cloning of the flhd operon from strains of the four Shigella subgroups. Genomic DNAs from four strains, representing each subgroup (Table 1), were first tested for the presence of the flhd operon by Southern blotting with probe E1, which includes most of the E. coli flhd gene (Fig. 1A). The S. dysenteriae DNA digested with BamHI, S. flexneri and S. boydii DNAs digested with EcoRI, and S. sonnei DNA digested with KpnI were examined as described above, and each digest showed one hybrid band of 7 to 11 kb (data not shown). Then the bands corresponding to the hybrid bands were eluted from the gels, inserted into the respective sites of puc118 (41), and transformed into strain RP3098 lacking the flhc gene (35). Transformants with the cloned flhd operon were identified by colony blot hybridization with probe E2, carrying part of the E. coli flhc gene (Fig. 1A), to eliminate the influence of hybridization with the chromosomal flhd operon. Plasmids were isolated from one of the positive clones in each transformation and verified by Southern blotting with probe E1 to carry the flhd gene. Plasmids with inserts from S. dysenteriae, S. flexneri, S. boydii, and S. sonnei were named pddc1071, pfdc35, pbdc7, and psdc16, respectively (each beginning with the first letter of the appropriate species name). Mapping of the cloned fragments and map comparison. The four cloned fragments were mapped first for various restriction sites and then for loci of cloned genes. In addition to the flhdc genes, the genes in the mota operon were located. A number of restriction fragments that comprise the entire length of the cloned fragment were made by digesting a recombinant plasmid with a different kind of restriction enzyme or two enzymes combined, electrophoresed, and blotted with each of the five probes, E1 to E4 (Fig. 1A) and S1 (Fig. 1F). Probe S1 (motb), of S. typhimurium origin (21), was used in place of an E. coli probe because suitable restriction sites to excise this gene were not detected in the E. coli fragment. The hybrid bands were integrated with other restriction fragments onto the restriction map, and finally the five genes were aligned in the order of flhd-flhc-mota-motb-chea in three subgroups (all except S. dysenteriae) as in E. coli and S. typhimurium (Fig. 1). In the fragment from S. dysenteriae the chea gene was missing. The hybridization tests also showed that the E. coli probes can hybridize with the Shigella genes as strongly as they do with the E. coli genes, but probe S1 hybridizes rather weakly with the Shigella motb genes. Comparison of the restriction maps revealed that the central 1.8-kb region from the SphI toclai sites, including the 3 end of flhc, mota, and the 5 end of motb, is conserved among the four Shigella subgroups as well as in E. coli, but its flanking regions are more or less different from one another (Fig. 1A to E). The S. typhimurium fragment does not have PstI and ClaI sites, and its restriction map was quite different from the others (Fig. 1F). This finding, together with low intensity of the hybrid bands with probe S1, indicates that the genus Shigella is more closely related to the genus Escherichia than to the genus Salmonella, as has been suggested elsewhere (29). Motility restoration to E. coli flhd and flhc mutants by cloned fragments. To test whether the flhdc genes cloned from the four subgroups have undergone some genetic defect or still retain their function, motility restoration by these clones to E. coli flhd and flhc mutants was examined. The fragments were recloned to the low-copy-number plasmid pmw119 to eliminate the effect of gene dosage and transformed into the two mutants (Table 2). The plasmid with the S. dysenteriae operon restored motility to the flhd mutant but not the flhc mutant, and the plasmid with the S. flexneri operon failed to restore motility to the two mutants, but those with the operon from S. boydii or S. sonnei conferred motility on both the mutants. However, swarms produced by the flhdc genes from S. boydii and S. sonnei were very small and composed of many dots (small nonmotile colonies). Whatever phenotype they show, the presence of the functional flhdc genes in the two subgroups does not support the idea that some stable mutation in the master operon of a shigella common ancestor might be the primary cause of motility loss. Detection of IS elements in the nonfunctional flhd operon. To disclose what kinds of mutations caused defects in the S. dysenteriae flhc and S. flexneri flhdc genes, Southern blotting with IS probes (Fig. 2) was carried out. It has been reported that the four Shigella subgroups all harbor a large number of copies of several IS elements (26, 27), and these elements

3 3724 AL MAMUN ET AL. J. BACTERIOL. FIG. 1. Physical and genetic maps of the cloned fragments with the flhd and mota operons. (A) pedc1, with the 5.5-kb fragment from E. coli; (B) pddc1071, with the 7.4-kb fragment from S. dysenteriae; (C) pfdc35, with the 10.6-kb fragment from S. flexneri; (D) pbdc7, with the 7.9-kb fragment from S. boydii; (E) psdc16, with the 9.3-kb fragment from S. sonnei; (F), pkk1211, with the 9-kb fragment from S. typhimurium. The maps are arranged to be comparable with one another for the segment including the flhc, mota, and motb genes. The transcription direction of each operon is shown by an arrow. The fragments of pedc1, pfdc35, and pbdc7 are lacking the 3 end of the chea gene. Elements IS1 and IS600 are depicted by open and hatched boxes, respectively, and the arrow under the box shows the 5 -to-3 direction (27, 30) (Fig. 2). Solid bars above the maps of pedc1 and pkk1211 are restriction fragments used for probes. A partial digestion site for HincII and sites for HpaI were examined only in pedc1. Likewise, sites for VspI and NruI have been examined only in pkk1211 (21). Abbreviations for restriction sites: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII; Hc, HincII; Hp, HpaI; K, KpnI; M, MluI; N, NruI; P, PstI; RV, EcoRV; S, SalI; Sp, SphI; and V, VspI. might cause insertion mutations or mediate the restriction map difference shown in Fig. 1. Plasmid pddc1071 was found to hybridize with probes IS1 and IS600, and pfdc35 showed hybridization with probe IS1, but the two other plasmids do not hybridize with any of the probes tested (data not shown). pddc1071 was digested with various restriction enzymes, electrophoresed, and blotted with probes IS1 and IS600. The 1.2-kb HindIII-SphI fragment, containing the functional flhd gene and part of the flhc gene, hybridized with probe IS1. The flhc gene is nonfunctional, and so the IS1 seems to reside in the 5 coding region of the flhc gene. However, the PstI and MluI sites, each of which is the unique site of IS1 (30), were not detected in this region, suggesting that this element is a mutant form of IS1. To test this possibility, the 1.2-kb fragment was further examined with probes IS1-5 and IS1-3 (Fig. 2A) and found to hybridize only with IS1-5. This result suggests that in this fragment the 3 moiety of IS1 has been deleted, probably along with the 5 end of the flhc gene, by an unknown mechanism. Probe IS600 hybridized with the 2.5-kb ClaI-BamHI fragment of pddc1071. In this fragment there are HindIII and MluI sites, each representing the unique site of IS600 (27), and this positioned IS600 downstream of the motb gene (Fig. 1B). As described above, pddc1071 does not carry the chea gene, although the fragment is long enough. This experiment showed that the chea gene is replaced with IS600. In addition, the restriction pattern downstream of the element is much different from the corresponding regions of the three other Shigella fragments. It is assumed that at first IS600 had transposed downstream of the motb gene and then IS600-mediated gene rearrangement, including deletion and/or inversion, occurred in this region. Analysis of the pfdc35 fragment showed that probe IS1 hybridizes with the 1.4-kb BamHI-SphI fragment which carries the flhd gene and the 5 region of the flhc gene (Fig. 1C). The MluI site, the unique site of IS1, was detected in the 5 end of the flhd gene, but another unique site, PstI, was not detected. Probes IS1-3 and IS1-5 formed hybrid bands with the 0.4-kb BamHI-MluI and 1.0-kb MluI-SphI fragments, respectively, showing that a full length of IS1 lies in this region. This result, together with the finding that both the flhd and FIG. 2. IS probes used. Each box numbered at both ends shows an IS element or part of it. Numbers indicate the nucleotide coordinates in the 5 -to-3 direction (see references in Table 1). Stippled regions were used as probes after cleavage at the restriction sites shown. (A) Probe IS1 from plasmid psam3. This includes short sequences other than IS1 at both ends. Probes IS1-5 and IS1-3, representing the 5 and 3 fragments, are shown above the IS1 probe. (B) Probe iso-is2 from psam42. This also includes a short sequence other than the element at the 5 end. (C) Probe IS3 (916 bp) from pnc155; (D) probe IS600 (679 bp) from ptsd610; (E) probe IS629 (1,288 bp) from psam629; (F) probe IS630 (372 bp) from psam31; (G) probe IS640 (571 bp) from psam41.

4 VOL. 178, 1996 FLAGELLAR MASTER OPERON IN THE SHIGELLA SUBGROUPS 3725 TABLE 2. Restoration of motility of the E. coli mutants by the cloned Shigella flhd operon a Source of flhd plasmid flhd mutant Motility restoration b of: flhc mutant S. dysenteriae S. flexneri S. boydii S. sonnei E. coli pmw119 only a Plasmid pmw119 carrying each of the cloned fragments (Fig. 1) was transformed into strains EJ2902 (flhc4136) and EJ2903 (flhd4116) and selected for ampicillin resistance. Five transformants from each experiment were streaked onto the same selective medium, and single colonies were stabbed into nutrient semisolid medium containing ampicillin and incubated at 30 C for 12 h. b Motility:, large confluent swarms around the inoculation site;, small dotted swarms;, no swarm. flhc genes are nonfunctional (Table 2), suggests that the IS1 element is inserted in the regulatory region or the 5 end of the flhd gene and inactivates the operon by promoter disruption or by exerting its polar effect. However, we cannot exclude the possibility that some gene rearrangement has occurred in the upstream region of the flhd operon by mediation of this IS1. Motility loss in the ancestors of the subgroups. This experiment did not support the idea that motility loss in shigellae might have been caused by some stable mutation in the flhd master operon of a common ancestor of the four Shigella subgroups. However, the IS1 elements were detected in the different sites of the master operon of S. dysenteriae and S. flexneri. IS1is one of the most prevalent insertion elements in Shigella spp. and has more than 20 copies per genome (26). Supposing that 20 copies of IS1 are randomly distributed in the S. flexneri genome of the same size as E. coli (4,700 kb) (16), a fragment 235 kb long will contain one copy of IS1 on average. This size is more than 20 times larger than the fragment (10.6 kb, pfdc35) with flhd::is1 from S. flexneri. This also is the case for the fragment (7.7 kb, pddc1071) from S. dysenteriae. This bias in distribution may be interpreted by random transposition of IS1 followed by selective preservation of inserts in some evolutionary age. Flagellar genes are nonessential to life, so that bacteria with IS1 inserted in these genes can survive while those with inserts in essential genes are eliminated from population. In addition, bacteria with stable mutations in the flhd operon seem to have a greater advantage for survival in adverse circumstances than do other flagellar mutants for the reason described above. Such consideration leads us to the possibility that S. dysenteriae and S. flexneri have independently lost their motility by IS1 insertion in the flhd operons of their respective ancestors. However, to make this possibility more definite, other strains in these two subgroups should be examined for the presence of IS1 in this operon, since only one strain was used for each subgroup in this study and variation might be present among different strains. For the two other subgroups, S. boydii and S. sonnei, the cloned flhd operon was functional, but it caused insufficient expression of subordinate operons (24) to result in small dotted swarms in the E. coli mutant backgrounds. It is interesting that the same type of swarms is produced by the two cloned operons. When observed under a microscope, almost all bacteria from swarms are nonmotile and a few bacteria are poorly motile. It seems that these operons have some mutations in the regulatory region to reduce their expression or they have mutations in the coding regions to produce the modified gene products. For complete loss of motility in these two subgroups, other mutations, possibly caused by IS elements, must have occurred in other flagellar operons. After we finished writing this paper, we noticed a recently published paper (10) reporting that two fresh clinical isolates of S. sonnei and some prototypic strains of Shigella spp. are pauci-flagellate to show poor motility. This finding demonstrates that our idea, motility loss in a common shigella ancestor, is incorrect, but the conclusion shown here that some ancestors of shigellae have independently lost motility by stable mutations is not contradictory to the above finding. However, the existence of motile shigellae raises some new questions of whether the generally accepted concept that Shigella spp. are nonmotile is applicable only to some laboratory strains preserved for a long time by successive inoculation and all fresh clinical isolates are motile without exception. If so, motility must confer a significant selective advantage on shigellae in searching for infection sites on the intestinal mucosa (10) while in adverse circumstances like stock culture flagellation and motility will be burdens for survival owing to their energetically high costs, and thus, a culture might be replaced by a nonmotile population after repeated passages of a motile clinical sample. In such a case, IS sites will be different among strains of the same subgroup even if mutations occur in the flhd master operon. When some fresh clinical isolates are motile and others are nonflagellate, motility cannot be a significant survival factor in natural circumstances in the host. 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