Analysis of the Pleiotropic Regulation of Flagellar and Chemotaxis Gene

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1341-1345, March 1984 Biochemistry Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in Caulobacter crescentus by using plasmid complementation (TnS insertion mutations/chemotaxis methylation/flageilins) RUTH BRYAN, MARY PURUCKER, SUELY LOPES GOMES*, WILLIAM ALEXANDER, AND LUCILLE SHAPIROt Department of Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, NY 10461 Communicated by Harry Eagle, October 31, 1983 ABSTRACT The biosynthesis of the single polar flagellum genetic evidence that certain che gene products directly in- and the proteins that comprise the chemotaxis methylation teract with at least two components of the flagellar machin- machinery are both temporally and spacially regulated during ery (5). This kind of direct interaction may be part of the the Caulobacter crescentus cell-division cycle. The genes in- mechanism by which che gene expression is governed within volved in these processes are widely separated on the chromo- the flagellar gene regulatory cascade (3). some. The region of the chromosome defined by flaE muta- The biogenesis of the Caulobacter crescentus single polar tions contains at least one flagellin structural gene and appears flagellum requires at least 26 genes (refs. 6 and 7; B. Ely, to regulate flagellin synthesis and flagellar assembly. The pro- personal communication). The fla, mot, and che genes are tein product of the adjacentflaY gene was found to be required located both singly and in several clusters widely scattered to regulate the expression of several flagellin proteins and the on the chromosome (see Fig. LA). The flagellum has been assembly of a functional flagellum. We demonstrate here that shown to be synthesized and assembled at a given time in the each of these genes is also required for the expression of che- cell-division cycle and to be released from the cell surface at motaxis methylation genes known to map elsewhere on the a specific time later in the cell cycle (8-11). Some of us re- chromosome. In order to study the regulation of these genes, cently have dembnstrated that the biochemical machinery plasmids were constructed that contain either an intactflaYE that mediates chemotaxis exists only when the cell is able to region or deletions in the region ofJfY. These plasmids were respond to a chemotactic signal (7). This coincident turn-on mated into a wild-type strain and into strains containing vari- of the fla and the che genes in C. crescentus may be analo- ous TnS insertion and deletion mutations and a temperature- gous to the highly integrated control ofthese genes seen in E. sensitive mutation in theflaYE region. The presence of a plas- coli and Salmonella. However, the loss of the che functions mid containing theflaYE region allowed the mutant strains to in C. crescentus coincident with the physical release of the swim and to exhibit chemotaxis, to synthesize increased flagellum from the cell surface indicates additional levels of amounts of the flagellins, to methylate their "methyl-accepting control probably involving structural constraints. chemotaxis proteins" (MCPs), and to regain wild-type levels of C. crescentus contains several genes for flagellin proteins methyltransferase activity. Chromosomal deletions that ex- (12-14). The flagellar filament is composed of two major fla- tend beyond the cloned region were not complemented by this gellins [25 and 27.5 kilodaltons (kDa)], and a 29-kDa flagellin plasmid. Plasmids containing small deletions in theflaY region appears to be a minor component required for filament as- failed to restore to anyflaY orflaE mutants the ability to swim sembly (12, 14, 15). The flagellin genes appear to be located or to assemble a flagellar filament. When mated into a wild- at two distinct regions on the chromosome (14). The flaYE type strain, plasmids bearing deletions in theflaY region were region of the chromosome includes at least one gene that found to be recessive. The pleiotropic regulation of flagellin encodes a flagellin protein (14, 16, 17). The sequence of the synthesis, assembly, and chemotaxis methylation functions ex- flaE gene has been determined recently and shown to en- hibited by both theflaY andflaE genes suggest that their gene code the 29-kDa flagellin (ref. 14; see Fig. 1C). Other flagel- products function in a regulatory hierarchy that controls both lin genes do not occur in the region directly 3' to this gene flagellar and chemotaxis gene expression. (14). Several Tn5 insertions 3' to this structural gene and deletions that are missing sequences in the flaYE region (see Bacterial flagella are composed of approximately 15 struc- Fig. 1 B and C) have been shown to result in a general de- tural proteins and require on the order of 30 genes for their crease in the synthesis of all flagellins and to yield a Fla- biogenesis and function (1, 2). A group of genes are also in- phenotype (16, 18). A point mutation in flaE was found to volved in chemotaxis functions. The genes devoted to the result in the loss of the 29-kDa flagellin and simultaneously biogenesis of flagella likely mediate the complex regulation to lower the levels of the other flagellins. We show here that of their assembly, the coordination of their biogenesis with the mutant strains containing these insertions and deletions the cell-division cycle, and their positioning on the cell sur- also curtail methylation functions associated with chemotax- face. The expression of groups of flagellar and chemotaxis is. The genes involved in chemotaxis methylation have been genes has been shown in Escherichia coli to be controlled, in identified and mapped to other regions of the chromosome part, by a regulatory cascade (3). It has been demonstrated (7). Mutations in flaY and in flaYE resulted in a decrease in that the transcription of one group offla genes is required for both methyltransferase activity and methyl-acceptor activity the expression of another group, and so on, in an hierarchi- of the "methyl-accepting chemotaxis proteins" (MCPs). cal manner. The order of afla gene in the cascade appears to As a first step in the analysis of these regulatory interac- correspond to the position of its protein product in the flagel- tions, we report here plasmid complementation of this de- lar morphogenetic pathway (3, 4). Analysis in E. coli of sec- fined set of deletion and insertion mutants in the flaYE gene ond-site suppressors of specific che mutants has provided Abbreviations: MCPs, methyl-accepting chemotaxis proteins; kDa, kilodalton(s). The publication costs of this article were defrayed in part by page charge *On leave from the Instituto de Quimica, Universidade de Sao Pau- payment. This article must therefore be hereby marked "advertisement" lo, Sao Paulo, Brasil. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1341 Downloaded by guest on September 30, 2021 1342 Biochemistry: Bryan et aL Proc. NatL. Acad. Sci USA 81 (1984) cluster (see Fig. 1A). A temperature-sensitive mutation in cheA cheF che G,C flaY was also complemented. Mutant strains altered in the chIEmotBISe motA mot C cheB flaYE region but carrying a plasmid with an intactflaYE gene A. cluster were found to swim and undergo chemotaxis, to qfI |flaI|JH floA flaZ flaR floP | make increased amounts of flagellins, to methylate their flaoH MCPs, and to have wild-type levels of methyltransferase activity. The flaY gene product, which is required for normal levels of flagellin synthesis and flagellar assembly, also is flaWOM J L Q N K floBCD required for chemotaxis methylation. These regulatory events are effected either directly (in trans) or indirectly by a cascade mechanism to control distalfla and che gene expres- fla Y E F-G sion, demonstrating the coordinate control of these genes in C. crescentus. I~ ~ ~ MATERIALS AND METHODS CB1_n N a B. 1p1.1 p.1 15 7 1-5 2. 15 C 1 Materials. Calf alkaline phosphatase was obtained from Hpol XhI HpIXhX T-, I Hpol Boehringer Mannheim. DNA polymerase I, T4 DNA ligase, I:C11SCu: 31133 and all restriction enzymes were obtained from New En- RI Bam Bar Bam R1 gland BioLabs. 5'[a-32P]dCTP (3000 Ci/mmol; 1 Ci = 37 1.55~SKbr ASC520 GBq) and S-adenosyl-[methyl-3H]methionine (15 Ci/mmol) 2.\\&i9Ksb\\\ ASC512 were purchased from Amersham. [methyl-3H]methionine (80 ~29Kflogellin < '' £ - Ci/mmol) was obtained from New England Nuclear, and C. flaY floE floF manu- I ~ 14C-reconstituted protein hydrolysate (21 Ci/mmol, ~~~~~ CB1 D. 2.4 1.3 0.7'61 1.6 2.6 1 2.6 CB13 facturer's mixture) was from Schwarz/Mann. Kanamycin Xh Hpo RN Hpl BaI Barn R sulfate and trimethoprim were purchased from Sigma, and XholI tetracycline hydrochloride was from Calbiochem. pS7 psi pS6 -.A-IC Bacterial Strains. C. crescentus CB15 spontaneous or TnS- induced fla mutants were obtained from B. Ely (6, 19). SC512, SC520, SC1062, SC1133, and SC1121 all contain mutations (Fig. 1) that cause a flagella-minus phenotype. Mu- tant strain SC274 is a temperature-sensitive mutant with a /1 partial flagellar structure (6, 12, 18). E. coli strain HB101 RI~~~' I~ // was obtained from Cold Spring Harbor Laboratories. pRBI Plasmids. pRK29O and pRK2013 were obtained from G. Ditta (20). Plasmid pMP7 was constructed by ligating pBR325 and DNA from the flaYE gene cluster from C. cres- I /J centus SC1062 as described (15). Mutant SC1062 contains a Tn5 insertion mutation in theflaE region of strain CB15 (Fig. 1B). DNA homologous to the CB15 flaYE region was isolat- ed from a C. crescentus CB13 XWES clone bank (15) and pRK 290 then recloned into pBR325 to give the plasmid pA8 (Fig.

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