Proc. Nati. Acad. Sci. USA Vol. 77, No. 7, pp. 4196-4200, July 1980 Genetics Analysis of the nucleotide sequence of an invertible controlling element (flagellar phase variation/recombinational switch/inverted repeat sequences/illegitimate recombination) JANINE ZIEG AND MELVIN SIMON Department of Biology, University of California, San Diego, La Jolla, California 92093 Communicated by Dan L. Lindsley, April 14,1980 ABSTRACr The nucleotide sequence of the inversion re- Mannheim. Hap II and Hae III were gifts from M. Hayashi and gion responsible for flagellar phase variation in Salmonela was R. Kolter, respectively. Pst I was prepared by the method of determined. Ihe inversion region is 995 base pairs (bp) in length P. Greene et al. (5). Enzymes used for labeling DNA fragments and is bounded by a 14-bp inverted repeat sequence. A homol- DNA large fragment (New England ogous recombination event between the 14-h inverted repeat included polymerase sequences would result in the inversion of the DNA segment BioLabs), bacterial alkaline phosphatase (Worthington), and between them. Sequence homologies with other systems suggest polynucleotide kinase (P-L Biochemicals). a-32P-Labeled de- that the 14-hp inverted repeat sequences may have some general oxynucleotide triphosphates were obtained from Amersham. significance as sites for specific recombinational events, The [,y-32P]ATP was either purchased from Amersham or prepared gene which specifies H2 ein synthesis begins 16 bp outside by modification of the Johnson and Walseth method (6). For the inversion region. Within the inversion region, an open the preparation of [ky-2P]ATP, 3sP-labeled phosphate was translational frame exists which could encode a low molecular obtained from ICN. weight polypeptide (190 amino acids). Plasmid Purification and Isolation of DNA Fragments. Hybrid phasmids were purified by the Sarkosyl lysis method of Phase transition in Salmonella is controlled by a novel kind of Bazaral and Helinski (7). Endonuclease digestion of plasmids regulatory unit in which a site-specific recombinational event was carried out in the buffer recommended by New England regulates gene expression (1, 2). An approximately 900-base- BioLabs for each enzyme. Slab gels containing 0.7-1.0% agarose pair (bp) DNA fragment adjacent to the H2 gene, which (SeaKem) or 9% acrylamide (BDH) were prepared in Tris/ specifies the synthesis of one of the flagellar antigens, can exist borate/EDTA buffer (TBE): 89 mM Tris, 89 mM boric acid, in either orientation with respect to the H2 structural gene. The and 2.5 mM EDTA. Gels were electrophoresed and treated as orientation of the inversion region controls expression of the H2 described by Bolivar et al. (8). gene-i.e., in one orientation the adjacent H2 gene is expressed DNA fragments were isolated from agarose or acrylamide and in the opposite orientation the H2 gene is not expressed. gels by the following procedures. Slices of agarose containing Genetic analysis of the invertible region has led to its subdi- DNA fragments were dissolved with 5 ml of 5 M Na perchlorate vision into five functional segments (3, 4). The deletion of two at 65°C for 40 min. DNA was bound to hydroxylapatite (Ha- small segments at opposite ends of the region completely patite C, Clarkson, Williamsport, PA), washed three times with eliminates inversion. Another segment which occupies about 5 mM NaPO4 (pH 7.4), and then eluted with 0.5 M NaPO4 (pH two-thirds of the inversion region controls the production of a 7.4). The eluent was dialyzed extensively against 10 mM Tris, factor which acts in trans to promote inversion. This has been pH 7.8/0.1 mM EDTA; then the DNA was precipitated with called the hin gene because its product is required for the in- ethanol. Alternatively, slices of acrylamide containing DNA version of the H2 controlling region. Finally, two other regions fragments were finely ground between layers of Parafilm. The map within the invertible segment. One is characterized by the pieces were then added to 1 ml of elution buffer (0.5 M NH4 observation that insertions and deletions generated by a 5-ki- acetate/0.01 M Mg acetate/0.1% NaDodSO4/0.1 mM EDTA) lobase transposable element within this region cause a 5-fold and incubated at 650C for at least 12 hr. The slurry was then decrease in the frequency of inversion. The second region filtered through glass wool and the DNA in the filtrate was corresponds to a promoter-like function; deletion of this region ethanol precipitated. Yields ranged from 75% to 95% for does not significantly affect inversion but eliminates formation fragments 700-10 bp long. of the H2 gene product. Labeling of DNA Fragments and Nucleotide Sequence In order to delineate precisely the genetic functions and to Determination. Labeling of 5' ends with polynucleotide kinase understand the molecular basis of the control of H2 gene ex- and [h-32P]ATP after bacterial alkaline phosphatase treatment pression and of the inversion process, it was necessary to de- was done as described by Maxam and Gilbert (9). 3'-End la- termine the nucleotide sequences that correspond to each of beling of DNA fragments with DNA polymerase large frag- these functions. In this paper, we present the nucleotide se- ment and [a-32P]dNTPs was also performed by the procedure quence of the inversion region and correlate parts of the se- of Maxam and Gilbert (9). Single end-labeled fragments were quence with these genetically defined functions. obtained by either secondary cleavage with a restriction enzyme or by strand separation. The strand separation procedure in- MATERIALS AND METHODS volved resuspending the double-strand DNA pellet in dimethyl Enzymes and Radioactive Isotopes. Alu I, Hae II, Hha I, sulfoxide/half-strength TBE, 60:40 (vol/vol). The solution was HinfI, Hpa II, and Taq I were purchased from New England boiled for 5 min and then added immediately to a 5% nonde- from naturing acrylamide gel. The gel and running buffer were BioLabs; Hpa II and Alu I were obtained Boehringer prepared in half-strength TBE. All sequence determination was done by the method of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Maxam and Gilbert (9, 10). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s). 4196 Genetics: Zieg and Simon Proc. Nati. Acad. Sci. USA 77 (1980) 4197 Amino Acid Analysis of H2 Flagellin. H2-enx flagellin was Fig. 3 illustrates a possible mechanism for the inversion isolated from cells harboring pJZ1OO (11) as described (12). The process involving these repeated sequences. A homologous re- purified protein was subjected to solid-phase amino acid combination event between the inverted repeat sequences could analysis by the thioacetylation stepwise degradation procedure result in an inversion of the DNA segment. By using restriction (13) by R. Doolittle (Department of Chemistry, University of endonuclease analysis, we have found that pJZ143 and pJZ121 California at San Diego, La Jolla, CA). retain the ability to invert (unpublished observations). Thus, sequences on the arm segments are not required for the inver- RESULTS AND DISCUSSION sion event. It is possible that the 14-bp inverted repeats alone Strategy for Sequence Determination. The restriction en- are required as sites for the inversion event. donuclease cleavage maps of plasmids pJZ121 and pJZ143 that Analysis of the 14-bp inverted repeat sequence revealed that were used to determine the nucleotide sequence are shown in it shares homology with DNA regions in other systems that are Fig. 1. These plasmids are in vdvo recombinants derived from involved in recombination and transposition. For example, the populations of the plasmid pJZllO, and their construction has A att core sequence resembles this sequence, as do regions on been described (2, 11). pJZll0 carries a fragment derived from the A P and A P' arms (14, 15). Two regions within the TnlO the Salmonella genome which includes the inversion region inverted repeat are also strikingly similar to the 14-bp repeat and flanking sequences. It is capable of inverting, and it is (16). Also, we find homology between the 14-bp sequence and shown in Fig. 1 in the orientation that leads to the expression a DNA region just outside the inverted repeat within the Tn3 of H2 gene function (i.e., the H2 "on" configuration). Plasmid transposon (17). Table 1 shows the extent of these sequence pJZ121 contains the entire inversion region with the H2 en- homologies. It is possible that the phase-variation inverted re- coding arm on both sides. Plasmid pJZ143 also contains the peat sequences may have evolved from some general site which entire inversion region flanked by the opposite or non-H2 en- takes part in various specific recombinational events. A simple coding arm. Fig. 1 also illustrates the strategy for determining recombination event within the inverted repeats can account the nucleotide sequence from the endonuclease cleavage map. for inversion in the phase variation system, as illustrated in Fig. The nucleotide sequence of the inversion region and proximal 3. arm regions is shown in Fig. 2. The G-loop region of bacteriophage Mu and the C loop of Determination of the Ends of the Inversion Region. It was bacteriophage P1 are genetic elements capable of inversion (18). possible to determine the extent of the inversion region by In addition, it is known that the orientation of the G loop reg- comparing sequences of fragments that contained the ends of ulates specific gene expression in Mu. It is possible that the in- the inversion region from both pJZ121 and pJZ143. For ex- version events in Mu and P1 are similar to the inversion event ample, by comparing the sequences of fragments a and b shown in phase variation.