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MARK J. MCBRIDE, ROBIN A. WEINBERG, AND DAVID R. ZUSMAN. Department of Microbiology and Immunology, University of California, Berkeley, CA 94720.
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 424-428, January 1989 Biochemistry

"Frizzy" aggregation genes of the gliding bacterium Myxococcus xanthus show sequence similarities to the chemotaxis genes of enteric bacteria (development/gliding motility/methyl-accepting chemotais proteins/ov factor)

MARK J. MCBRIDE, ROBIN A. WEINBERG, AND DAVID R. ZUSMAN Department of Microbiology and Immunology, University of California, Berkeley, CA 94720

Communicated by H. A. Barker, September 22, 1988

The fiz genes of Myxococcus xanthus are ABSTRACT necessary for proper aggregation of cells to form fruiting bodies. Mutations in the frz genes affect the frequency with which individual cells reverse their direction of movement. We have subdoned and determined the nucleotide sequence of three of theft genes. From the sequence we predict three open reading frames corresponding to frzA, fizB, and frzCD. The putative FrzA protein (17,094 Da) exhibits 28.1% amino acid identity with the CheW protein of Salmonella typhimurium. The putative FrzCD protein (43,571 Da) contains a region of about 250 amino acids which is similar to the C-terminal portions of the methyl-accepting chemotaxis receptor proteins of the enteric bacteria. FrzCD also contains a region with potentially significant similarity to the DNA-binding region of the Bacillus subtilis o43. The putative FrzB protein (12,066 Da) shares no significant identity with known chemotaxis proteins. The sequence similarities between the putative Frz proteins and the chemotaxis proteins of the enteric bacteria strongly support the hypothesis that the ftz genes define a system of signal transduction analogous to the enterobacterial chemotaxis systems.

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DNA Size (kbp)

FIG. 1. Physical organization of the M. xanthus frz genes. 9 denotes TnS insertion sites; kbp, kilobase pairs.

Myxococcus xanthus is a Gram-negative bacterium that moves by gliding on a solid surface (1). The mechanism responsible for gliding motility is unknown, but the cells do not have flagella. Cells aggregate to form fruiting bodies when the available food source is depleted. Within the fruiting bodies the cells develop into metabolically dormant myxospores. "Frizzy" (frz) mutants of M. xanthus aggregate into tangled filaments but fail to assemble into fruiting bodies (2). The frz mutations map to 5 complementation groups (frzA, -B, -C, -E, and -F) and are recessive to the wild-type alleles (3). Mutations in the closely linkedfrzD region are dominant and are not phenotypically frizzy. frzD mutants form nonspreading colonies although individual cells are motile. frzC and frzD are thought to define a single gene from the effect of mutations infrzD on thefrzC gene product in Escherichia coli maxicells (4). Mutations in the frz genes alter the gliding behavior of individual cells (5). Wild-type cells glide at a rate of about 2.0 gm per min and reverse their direction about every 7 min. Net movement occurs because cells spend more time gliding in one direction than in the other. Frizzy mutants reverse direction about every 2 hr. frzD mutants reverse directions about once every 2 min and do not show a bias in directional movement. These behavior patterns seem somewhat analogous to those of chemotaxis mutants of enteric bacteria, which swim smoothly and rarely tumble or tumble and rarely swim smoothly (6). The sequence analyses presented in this paper* provide strong evidence in favor of the hypothesis

that the frz genes define a system of signal transduction analogous to that involved in enterobacterial chemotaxis.

MATERIALS AND METHODS The frz genes were subcloned from pBB12 (3). Deletions were generated by exonuclease III digestion to allow sequencing of both strands (7). The nucleotide sequence was determined with Sequenase by the recommended procedures (United States Biochemical) except that 40%o (wt/vol) formamide was incorporated in all of the gels. To sequence the sites of insertion of the TnS mutations we used a synthetic oligonucleotide primer homologous to the TnS sequence and plasmid DNA in which one end of the TnS had been deleted. DNA and protein sequences were analyzed with the Intelligenetics programs.

RESULTS AND DISCUSSION Nucleotide Sequence of thefrzA, -B, and -CD Regions. The frz genes are contained on a 7.5-kilobase-pair (kbp) DNA fragment (Fig. 1). The direction of transcription of the genes that have been examined (frzA, -C, -E, and -F) is from left to right in this figure (4). We subcloned and sequenced the 2.5-kbp region between the Xho I site and the Pvu II site. This DNA encodes three complementation activities, frzA, frzB, and frzCD. From the nucleotide sequence (Fig. 2) we iden-

tified three likely candidates for translated reading frames based on the absence of stop codons and on the expected

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: MCP, methyl-accepting chemotaxis receptor protein. *The sequence reported in this paper is being deposited in the EMBL/GenBank data base (accession no. J04157). 424

425

Proc. Nati. Acad. Sci. USA 86 (1989)

Biochemistry: McBride et al.

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90

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Arg Ilie Gly Pro Arg Thr Arg Leu Phe Val Gly Val Thr Gly Ser Tyr Val Ala Gly Val Val Ala Asp Thr Val Leu Gly Leu Arg Arg 518 GGG ATG GGG CCG GGG ACG GGG GTG TTG GTG GGG GTG AGG GGG AGG TAG GTG GGC GGG GTG GTG GCG GAT ACG GTG CTG GGT CTG GGG CGG 130 120 110 Ilie Pro Val Ala Asp Ilie Leu Pro Pro Pro Leu Gly Gly Asp Ala Ala Ala Giu His Leu Leu Gly Val Val Gin Ala Ser Gly Asn

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Giu Asp Leu Thr Leu Ala Giu Leu Gly Leu Pro His Arg Gly Asn Arg Ala Ilie Val Phe Asp Thr Pro Giu Gly Giu Ala His Leu Lys 883 GAG GAG GTG AGG GTG GGG GAG GTG GGG GTG GGT GAG GGA GGG AAG GGG GGG ATG GTG TTG GAG AGG GGG GAG GGT GAA GGG GAG GTG AAG 60

70

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Val Asp Ala Val His Gly Val Arg Ser Ilie Pro Val Asn Ser Leu Arg Arg Met Pro Pro Thr Ala Gly Ala Ala Ala Tyr Ala Val Gly 973 GTG GAG GGG GTG GAG GGG GTG GGG TGG ATG GGG GTG AAT TGG TTG GGG CGGG ATG GGT GGG AGG GGG GGG GGG GGA GGG TAG GGA GTG GGT 90

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Gin Ala Leu Asp Ala Leu Ilie Gly Leu Val Arg Giu Gly Asp Leu Ser Arg Trp Asn Thr Thr Thr Glu Asp Pro Gin Leu Gly Pro Leu 1422 GAG GGG TTG GAT GGG GTG ATG GGG GTG GTG GGG GAG GGG GAG GTG TGG CGGG TGG AAG AGG AGG AGG GAA GAG GGG GAG GTG GGG GGG GTG 130

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160

170

180

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230

240

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250

260

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290

300

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380

390

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FIG. 2. Nucleotide sequence of frzA, frzB, and frzCD and deduced amino acid sequence of their gene products. Sites of insertion of TnS are indicated by 6 R (3' or right end of Tn5) and by 6 L (5' or left end of Tn5). OP and AM, opal and amber stop codons, respectively.

codon preferences. M. xanthus DNA contains approximately 68% G+C (1). Codon choices are expected to be heavily

biased toward use of G and C in silent positions, as in other bacteria containing DNA of high G+C content (8). The

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Proc. Natl. Acad. Sci. USA 86 (1989)

Table 1. Percentage G + C at codon positions in frz open reading frames % G+C at codon

selected the one with the best Shine-Dalgarno sequence upstream. We do not have extensive information concerning translational regulation of M. xanthus genes, but the sequences of M. xanthus and E. coli 16S rRNAs are identical in the region containing the Shine-Dalgarno complementary sequence (9). We examined the regions upstream of the frzA, frzB, and frzCD open reading frames and the region downstream of frzCD (upstream of frzE) for possible promoter sites. The regions upstream of frzA, frzCD, and frzE were relatively A+T rich (40-50% A+T over 40 to 50 nucleotides). We did not detect any sites that were obviously similar to the E. coli o.70 consensus promoter (10), the Bacillus subtilis fy28_ dependent promoters (11), or the NtrA-dependent promoters (12). Sequence Similarities Between thefrz Genes and Chemotaxis Genes from Enteric Bacteria. The proposed frzA reading frame codes for a 160 amino acid protein with a calculated molecular mass of 17,094 Da (Fig. 2). The putative FrzA protein shares considerable sequence similarity with CheW of Salmonella typhimurium (13) (Fig. 3). The FrzA sequence contains 45 identical residues (28.1% amino acid identity over the entire FrzA sequence). The striking similarity between FrzA and CheW suggests that they are of common evolutionary origin and perform similar functions in directing cell movement. The proposedfrzCD open reading frame (Fig. 2) codes for a 417 amino acid protein of 43,571 Da. This size agrees reasonably well with the previous estimate of 47,000 Da obtained from sodium dodecyl sulfate gel electrophoresis of labeled proteins produced in maxicells (4). The FrzCD protein contains a large region of similarity to the methylaccepting chemotaxis receptor proteins (MCPs) of the enteric bacteria (14-16) (Fig. 4). The regions of strongest similarity are those that are also highly conserved among the enteric MCPs. FrzCD and S. typhimurium Tar exhibit 40% amino acid identity over 141 residues between positions 157 and 306 of FrzCD. In addition to the identical amino acids there are a large number of conservative replacements throughout the sequences. Three gaps were introduced into Tar to allow for optimal alignment. These gaps span integral numbers of helical turns of FrzCD as predicted by the Chou and Fasman algorithm (17). This may indicate that the conserved regions between S. typhimurium Tar and M. xanthus FrzCD are similarly juxtaposed in the two proteins and that this orientation is necessary for proper functioning. MCPs are involved in chemotactic signal transduction in a wide variety of flagellated eubacteria (18). Enteric MCPs are transmembrane proteins of approximately 60,000 Da that contain an N-terminal periplasmic chemoeffector-binding domain, two membrane-spanning regions, and a C-terminal cytoplasmic domain. According to one model for MCP function, when the periplasmic portion of a receptor binds its

position

Open reading frame

2 48 46 48

1 76 73 68

frzA frzB frzCD

3

91 83 %

reading frames that are identified in Fig. 2 demonstrate the expected bias (Table 1). The third and to a lesser extent the first codon positions are strongly biased toward G and C, while the second positions contain approximately 50% G+C. In the frzA and frzCD reading frames the expected bias extends from the proposed start codon to the stop codon. The codon bias is less apparent in the last 40 nucleotides offrzB. This region is relatively A+T rich (45%), which suggests that it may also function as a regulatory region of thefrzCD gene. As discussed below, A+T-rich regions lie 5' to several of the frz genes and may be important for gene expression. The open reading frames predicted above correspond in size and position to the three complementation groups previously defined by analysis of TnS insertion mutations in the frz genes (3). The exact sites of the TnS insertions were determined by sequence analysis to more precisely define the boundaries of the three complementation groups (Fig. 2). Insertion f1212 does not yield a frizzy phenotype and thus provides a 5' boundary to thefrz region. Insertion mutations f1213 and 11216, which define the frzA complementation group, lie at opposite ends of the frzA open reading frame. Insertion f1216 lies within the termination codon of thefrzA open reading frame and should result in the formation of a protein identical to that deduced for frzA except for the addition of cysteine at the C terminus. Apparently this altered product is unstable or nonfunctional. The frzB open reading frame falls between insertions f1216 and f1217 as previously predicted by complementation analysis. The frzCD open reading frame spans the region containing insertions Q1217 to Q1225. Insertions Q1217 to f1223 define the frzC complementation group. Complementation analysis with the frzD insertions, f1224 and Q1225, could not be performed because these insertions were dominant to the wild-type allele. Previous work indicated thatfrzC and frzD might define a single gene (4). The present analysis supports this suggestion, since insertions f1224 and Q1225 were located in the C-terminal end of the frzCD open reading frame. Insertion f1226 withinfrzE lies 98 bp 3' to the final nucleotide, position 2507, of Fig. 2. Start codons were assigned to the three reading frames on the basis of the TnS insertion data and the extent of the reading frame predicted by the codon bias. Several GTG start codons are possible for thefrzB open reading frame. We have FrzA

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FIG. 3. Comparison of the deduced amino acid sequences (in standard one-letter symbols) of M. xanthus FrzA and S. typhimurium CheW. Regions of amino acid identity are boxed.

Proc. Natl. Acad. Sci. USA 86 (1989)

Biochemistry: McBride et al. FrzCDInG Tar

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FIG. 4. Comparison of the deduced amino acid sequences of M. xanthus FrzCD and S. typhimurium Tar. Identical amino acid residues are boxed. Arrows indicate the methylatable residues of Tar. The regions boxed with broken lines denote similarity between the fourth methylatable site of Tar and a site in FrzCD. Sites of insertion of Tn5 in the FrzCD sequence are indicated by 9).

chemoeffector the conformation of the cytoplasmic portion of the MCP is altered, allowing it to send a signal via the other Che proteins that influences the direction of rotation of the flagella, resulting in the cell's either moving forward or tumbling (6). The MCPs adapt to the new concentration of effector over a period lasting from seconds to minutes. Adaptation involves covalent modification of the cytoplasmic portion of the protein. Specific glutamate residues are modified by methylation and demethylation during the adaptive response. The second glutamate of the consensus sequence, Glu-Glu-Xaa-Xaa-Ala-Thr/Ser is the site of methylation (ref. 19; Fig. 4). This position can also originate as a glutamine residue which is subsequently deamidated to glutamate by CheB. FrzCD contains sites which are distinctly similar to and are aligned with the first three methylatable sites of the enteric MCPs (between positions 167 and 187 of the FrzCD sequence). A fourth site in FrzCD at position 348 (Val-Gln-Glu-Thr-Ser-Asn-Ala-Ala) (boxed with broken lines in Fig. 4) is very similar to the fourth methylatable site of Tar at position 489 (Val-Gln-Glu-Ser-Ala-Ala-Ala-Ala). We do not know if the sites in FrzCD are methylated in vivo. FrzCD does not contain any regions similar to the periplasmic domains of the MCPs. Analyses of the amino acid sequences of the MCPs predict two extended hydrophobic regions that anchor the proteins in the cytoplasmic membrane (14). There are no similar predicted hydrophobic regions of FrzCD. We do not know where the FrzCD protein is localized in the cell. FrzCD might interact with membrane proteins to form a connection between the periplasm and the cell interior. Alternatively, FrzCD might be a soluble protein that responds to some change within the cell. It has recently been reported that a cytoplasmic fragment of Tar retains the ability to respond to internal stimuli such as a change in the internal pH (20). Mutants of enteric bacteria containing MCPs truncated near the C terminus often result in a dominant phenotype in

which the cells are biased towards tumbling or smooth swimming (21). TnS insertions in frzD also result in a dominant phenotype. These insertions may result in the formation of stable truncated proteins which do not have normal biological activity but can interfere with the functioning of the wild-type product. Truncated enteric MCPs are poor substrates for methylation even though they contain all of the methylatable sites (14, 21). Cells containing truncated Tar are able to respond to a stimulus, but cannot adapt, presumably because of sluggish methylation. Inability to adapt could explain the frzD phenotype, in which cells reverse direction very frequently and make very little net movement (5). FrzD mutants might continually transmit a signal which results in a change in direction of movement. Sequence Similarity Between FrzCD and B. subtilis RpoD. The N-terminal region of the FrzCD protein shows little similarity to known chemotaxis proteins but does show a striking similarity to a region of B. subtilis RpoD (0,43). This region of RpoD has been implicated in the binding of the or factor to DNA (22). FrzCD and B. subtilis RpoD show 31.6% amino acid identity over 79 residues (Fig. 5). Interaction of FrzCD with a specific DNA sequence might regulate gene expression or might be important for signal transduction. Analysis of the frzB Sequence. The proposed frzB reading frame codes for a 112 amino acid protein of 12,066 Da (Fig. 2). We could not detect any significant similarities between FrzB and any of the sequences in the GenBank, National Biomedical Research Foundation (NBRF), or European Molecular Biology Laboratory (EMBL) databases as of Oct. 15, 1988. We suspect that this region does code for a protein, since it is genetically distinct from frzA and frzCD (3) and shows the expected codon bias. This protein may represent a novel component in the signal transduction system of M. xanthus. Such components should be expected, considering the many differences between the enteric and myxobacterial systems of motility.

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Biochemistry: McBride et al. FrzCD RpoD

Proc. Natl. Acad. Sci. USA 86 (1989)

V A A Q E IDD2AL D~AMlI GM~V FR-E1G D rW LL DN FQ V L D F AAPE H RK HAYEL[LKELJL VDT[T D[ JE N LG IDEJQJEEJT(PSDHA

FrzCD L V RpoD

107 325

N T T T EP Q L G P L7G S14W FrGXV|I E T L TFVRRE N E AIA L RI 147 R F G L DDJGR V|G K V|F G V TIE R ISQmE A K|A L R 364 3R6T4LEE

LML

Potential DNA Binding Region

FIG. 5. Comparison of the deduced amino acid sequences of M. xanthus FrzCD and B. subtilis RpoD (a43).

Are the frz Gene Products Involved in Chemotaxis? The results presented in this paper show that FrzA and FrzCD of M. xanthus share significant sequence similarities with CheW and the MCPs, respectively, of the enteric bacteria. Recently we have found that the putative frzE gene product shares sequence similarities with CheA and CheY ofS. typhimurium and E. coli (W. McCleary and D.R.Z., unpublished data). The extensive similarities between the amino acid sequences of these proteins suggest that there are also similarities in their functions within their respective signal transduction networks. In the enteric bacteria CheY and CheZ control the direction of flagellar rotation (23). Chemoeffector concentration is monitored by the MCPs which transmit a signal, probably via CheW and CheA, to CheY and CheZ. Recent evidence indicates that this transmission may involve a protein phosphorylation cascade in which CheA plays a central role (23, 24). It seems likely that FrzCD responds to some stimulus and transmits a signal via the other Frz proteins which alters the behavior of the cell. Complementation analysis of frz mutants suggested an interaction between FrzCD and FrzE (3, 5). The sequence similarities between the MCPs and FrzCD and between CheA and FrzE make such an interaction seem likely. The function of CheW, the putative homologue of FrzA, in chemotactic signal transduction is incompletely understood. CheW may interact with the MCPs and with CheA. By analogy, we predict that FrzA may interact with FrzCD and with FrzE. We do not know whether thefrz gene products are involved in a chemotactic system. These genes are involved in the directional control of the cells, but the nature of the signal is unknown. Cells may respond to a gradient of a chemical released by other cells to coordinate aggregation. Chemotaxis of myxobacterial cells towards diffusible signals has been challenged by Dworkin and Eide (25). They argue that the slow movement of individual cells is inconsistent with a sensory system for responding to a concentration gradient of a diffusible molecule. However, it is possible that myxobacterial cells respond to slowly diffusible compounds or to signals which remain bound to cell surfaces or to the substratum. Alternatively, cells might respond to physiological changes brought about by cell aggregation which are independent of extracellular chemical signals. The mechanisms of the cell-cell interactions and of the gliding motility of myxobacteria are incompletely understood. It is likely that the firz gene products are associated with both of these processes. Further exploration of the function of the frz genes of M. xanthus may provide insight into cell-Cell communication and signal transduction during developmental aggregation and into the nature of the motility apparatus of this unique group of bacteria.

Note Added in Proof. In reviewing the amino acid sequence of FrzCD, we noted a fifth potential methylation site (from position 370-375; Fig. 4). This sequence, which was not aligned with the Tar methylation sites, conforms well with the enterobacterial consensus sequence for methylatable sites.

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