Polar localization of a bacterial chemoreceptor M.R.K. Alley, Janine R. Maddock, and Lucille Shapiro Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, California 94305-5427 USA

The bacterial chemotaxis signal transducer MCP is an integral membrane receptor protein. The chemoreceptor is localized at the flagellum-bearing pole of Caulobacter crescentus swarmer cells. Amino-terminal sequences of the MCP target the protein to the membrane while the carboxy-terminal portion of the protein is responsible for polar localization. The C. crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E. coli MCP is expressed in C. crescentus, it is targeted to the swarmer cell progeny. These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus. [Key Words: Caulobacter;

chemoreceptor; chemotaxis; polarity]

Received January 14, 1992; revised version accepted February 28, 1992.

The Caulobacter crescentus predivisional cell gives rise to distinctly different progeny: a stalked cell and a swarmer cell. The swarmer cell has a single polar flagellum, polar pili, and receptors for pole-specific phage. The flagellar components are synthesized in the predivisional cell and are specifically assembled at the nascent swarmer cell pole. Pilin, which is present throughout the predivisional cell (Smit 1987), is assembled at the pole of the swarmer cell upon ceil division (Sommer and Newton 1988). There are also several proteins that are synthesized in the predivisional cell that segregate to the nonmotile stalked cell. Among these are proteins involved in the heat shock response, including Lon, DnaK, and a putative RNA polymerase a-factor (Renter and Shapiro 1987). We have proposed that there are at least two mechanisms used to segregate newly synthesized proteins to specific progeny cells: transcriptional localization (Gober ct al. 1991a,b) and protein localization (Loewy et al. 1987). The transcription of several flagellar structural genes appears to be localized to the chromosome residing in the nascent swarmer cell portion of the predivisional cell (Milhausen and Agabian 1983; Gober et al. 1991b). However, transcriptional localization was not found for the flgf gene encoding the 29-kD flagellin (Loewy et al. 1987) nor for the mcpA gene encoding a chemoreceptor, yet both of these proteins segregate to the swarmer cell, suggesting that sequences within the protein are responsible for localization. The bacterial chemoreceptors are transmembrane methyl-accepting chemotaxis proteins (MCPs). The carboxy-terminal portion of these proteins is cytoplasmic and is differentially methylated and demethylated in response to ligand binding to the periplasmic portion of the

protein (Krikos et al. 1983). We have shown previously that the C. crescentus chemoreceptors are synthesized in the predivisional cell but are segregated to the progeny swarmer cell (Gomes and Shapiro 1984). In addition, indirect evidence using membrane vesicles prepared from the poles of predivisional cells suggests that newly synthesized MCPs are localized to the nascent swarmer cell portion of the predivisional cell (Nathan et al. 1986). In this study we demonstrate directly that the chemoreceptor, encoded by mcpA, is localized to the pole of the swarmer cell. We also show that the methylation involved in adaption of the chemotactic response is not needed for polar localization. A region in the carboxyl terminus of the protein is responsible for polar targeting and the consequent segregation to the swarmer cell progeny. Sequences within the carboxyl terminus are conserved in C. crescentus and Escherichia coli chemoreceptor proteins, and a chemoreceptor encoded by the E. coli tsr gene preferentially segregates to the swarmer cell progeny when expressed in C. crescentus.

Results Comparison of C. crescentus and chemoreceptors

enteric

The 2.3-kb EcoRl fragment shown in Figure 1 contains the promoter for a C. crescentus chemotaxis operon and encodes a protein that cross-reacts with antiserum raised against the Salmonella typhimurium chemoreceptor Tar (Alley et al. 1991). The D N A sequence of this £coRI fragment plus an adjoining 280-bp £coRI fragment is shown

GENES & DEVELOPMENT 6:825-836 © 1992 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/92 $3.00

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in Figure 2. The 2.3-kb EcoRl fragment contains an open reading frame (ORF) that encodes a predicted 70-kD protein composed of 657 ammo acid residues. This ORF originates just upstream of the BaniHl site (Figs. 1 and 2) and ends exactly at the EcoRI site (Fig. 21 Figure 3 is a comparison of the deduced amino acid sequences of the C. crescentus mcpA gene and four E. coli chemoreceptors, Tap (ecotap), Trg (ecotrg), Tsr (ecotsr), and Tar (ccotar), and two chemoreceptors from Enterobacter aerogenes, Tse (earotsl and Tas (earota). Amino acid sequence similarities at the amino terminus are rather sparse, with the exception of the first eight ammo acids. These residues are part of the first transmembrane portion (TMl), which is thought to act as a signal sequence (Gebert et al. 1988). The high sequence conserv^ation might indicate an additional function for this region. Differences among the various chemotaxis transducers in the amino-terminal domain are expected, as this region contains the periplasmic receptor for different chemoattractants as well as the transmembrane-spanning portions of the protein. Using the Wisconsin GCG package (Devereux et al. 1984) we identified two hydrophobic regions in McpA at positions analogous to the predicted transmembrane domains of the E. coli chemoreceptors. A fusion protein that contains the ammo terminus of McpA fused at the Sail site (between T M l and TM21 to p-lactamase is membrane localized. As expected for fusions that place ^-lactamase in the periplasm (BroomeSmith and Spratt 1986), strains containing this McpAp-lactamase fusion exhibit high levels of ampicillin resistance. However, a p-lactamase protein fusion that contains both predicted transmembrane domains of McpA (MCL2, Fig. 4) and is therefore predicted to localize p-lactamase cytoplasmically has a much lower level of ampicillin resistance (data not shown). The predicted amino acid sequence of the C. crescentus McpA protein has an unusually large linker region between TM2 and the first methylation region Kl. In addition, this region contains a string of 7 alanines at residues 312-318, followed by a glutamine and a glutamic acid residue, reminiscent of the sequence for the Rl methylation region of several chemoreceptors. Thus, it may be that the C. crescentus chemoreceptor contains

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GENES & DEVELOPMENT

an additional methylation region. The McpA Kl and Rl methylation regions shown in Figure 3 are very similar to the enteric chemoreceptors. The Kl region is more highly conserved, perhaps reflecting the larger number of potential methylation sites. A striking feature in this amino acid comparison is the large stretch of identical amino acid residues from residue 474 to 524 (Fig. 3), referred to as the highly conserved domain (HCD). The conservation of this domain among a diverse group of bacteria suggests a functional role for the region. The HCD within the E. coli transducer Tsr chemoreceptor has been demonstrated genetically to interact with the CheW protein (Liu and Parkinson 1991). Segregation of McpA-jS-lactamase progeny swarmer cell

fusions to the

We have shown that C, crescentus MCPs are synthesized m predivisional cells and are then segregated to the swarmer cell progeny upon cell division (Gomes and Shapiro 1984). Analysis of membrane vesicles derived from predivisional cells showed that the C. crescentus MCPs preferentially accumulate at the flagellated pole of the predivisional cell (Nathan et al. 1986). To determine whether McpA segregates to swarmer cells, and if so, which portion of the protein is responsible for this segregation pattern, we constructed two chromosomal xMcpA-p-lactamase protein fusions, with different lengths of McpA fused to (B-lactamase (Fig. 4A). The (3-lactamase gene used in these fusions lacks its own signal sequence and therefore relies on the McpA protein for membrane insertion. The longest McpA-p-lactamase fusion has the entire mcpA gene fused to (3-lactamase (MCL722), whereas the other fusion (MCL2) lacks the carboxy-terminal coding region, including the HCD and the Rl methylation region (Fig. 4A). The segregation of these two fusions to the swarmer cell upon cell division was assayed by pulselabeling predivisional cells with [^^S]methionine and collecting the separated progeny after cell division (shown diagrammatically in Fig. 9, below). The protein fusions from each cell type were immunoprecipitated with anti-p-lactamase antibody. Parallel immunoprecipitations with anti-flagellin antiserum were used as a control and showed that the flagellins segregated to the swarmer cell, as reported previously (Loewy et al. 1987; Renter and Shapiro 1987). The full-length fusion (MCL722) segregates specifically to the swarmer cells, whereas the deletion fusion (MCL2) is distributed equally to both cell types (Fig. 4B). These results suggest that sequences within the carboxyl terminus are required for the asymmetric segregation of McpA to the progeny swarmer cell. We determined that both fusions are membrane localized by separating membrane and cytosolic fractions from cells harboring chromosomal copies of either MCL722 or MCL2. Immunoblots with anti-|3-lactamase antibody showed that both protein fusions were membrane localized (Fig. 4C), as might be expected because both fusions retain the two transmembrane domains. We

Chemoreceptor polar localization

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GENES & DEVELOPMENT

827

Alley et al.

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828

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ccmcpA ecotap ecotrg ecotsr earots earota ecotar

ccmcpA ecotap ecotrg ecotsr earots earota ecotar

290

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on facing page.)

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Chemoreceptor polai localization Figure 4. Analysis of progeny segregation, membrane localization, and methylation of the McpA protein and its truncated derivative fused to a p-lactamase reen z I, I J I I, I. I I, porter protein. (A) A restriction map of the mcpA gene with the specific regions of the protein denoted below. (TMl) Transmembrane domain 1; (TM2) transmembrane domain 2; (Kl and Rl) methylation re+ - gions; (HCD) highly conserved domain. (Below) Diagram of the McpA-^-lactamase protein fusions showing the extent nCL722 of the McpA protein fused to p-lactamase. [B] Segregation patterns of the McpA-plactamase fusions. Predivisional cells were ST SW Cyt, Memb. pulse labeled at 110 min into the cell cycle with ['^^Slmethionine and allowed to dinCL722 nCL722 vide, and the swarmer (SW) and stalked (ST) cell progeny were isolated by centrifMCL2 MCL2 ugation through a Ludox density gradient, as described in Materials and methods. The McpA-p-lactamase protein fusions synthesized in the predivisional cell were immunoprecipitated from each progeny cell type with anti-p-lactamase antibody. (C) Membrane localization of the McpA-^-lactamase fusions. Membrane (Memb) and cytosolic (Cyt) fractions of strains MCL722 and MCL2 were prepared as described previously (Shaw et al. 1983) and immunoblotted with anti-^lactamase antibody. (D) In vivo methylation of the McpA-(3-lactamase fusions was carried out as described by Gomes and Shapiro (1984). Labeled cultures were lysed and immunoprecipitated with anti-p-lactamase antibody as described in Materials and methods. The arrow at 100 kD denotes the size of the MCL722 McpA-p-lactamase fusion.

conclude, therefore, that membrane localization alone is not sufficient for segregation to tlie swarmer cell. To address whether methylation might be involved in the segregation process we examined the methylation of these protein fusions (Fig. 4D). T h e full-length fusion was methylated^ but the shorter fusion was not, even though MCL2 retains the Kl methylation region. Although the loss of McpA segregation correlates with the loss of protein carboxy-methylation, methylation is probably not causal, because a full-length McpA-^-galactosidase fusion protein is localized to the swarmer pole even in a methylation-minus mutant, SCI 130N (see

Fig. 8B, below). This strain has a T n 5 insertion in t h e first gene in the general chemotaxis operon [mcpA] and, owing to the polarity of this insertion, is deficient in methyesterase and methyltransferase activity (Alley et al. 1991; Fig. 1). Intracellular

ccmcpA ecotap ecocrg ecotsr earots earota ecotar

M

[A|

R

T E A V D V T E A V IX A D A V [A T Q A V S A

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of McpA

The intracellular localization of McpA was determined by immunoelectron microscopy. Antisera were raised against the glO-McpA fusion protein purified from an overproducing E. coli strain, as described in Materials

630 ccmcpA ecotap ecocrg ecotsr earcLs earcta ecocar

localization

635

G[i]srslYARPAV

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P P|A]Q P[R]LR

I [A][E][£) D P N W E [T][?]

R S S G S A A L A Q A P A S D G W E E F

Figure 3. Amino acid sequence comparison of the predicted McpA protein from C. ciescentus, four E. coli chemotaxis transducers, and two £. aerogenes transducers. The C. ciescentus McpA sequence is indicated as ccmcpA; the E. coli sequences, as ecotap (Krikos et al. 1983), ecotrg (Bollinger et al. 1984) with recent corrections (G. Hazelbauer, pers. comm.), ecotsr (Boyd et al. 1983), and ecotar (Krikos et al. 1983); the E. aeiogenes sequences, as earots and earota (Dahl et al. 1989). Comparative analysis was performed by the TULLA program (Subbiah and Harrison 1989). The boxed residues denote amino acid identity with the C, ciescentus McpA protein. (TMl) transmembrane domain 1; (TM2) transmembrane domain 2; (Kl and Rl) methylation regions proposed for the £. coli Tsr chemoreceptor (Kehry et al. 1983). The bold asterisks helow the amino acid sequence denote potential methylation sites, on the basis of the reported methylation sites for the £. coli Tsr chemoreceptor (Kehry et al. 1983). The C. crescentus MCP is methylated at glutamate/glutamine residues (Gomes and Shapiro 1984).

GENES & DEVELOPMENT

829

Alley et al. and methods. This antiserum was shown to react with the 70-kD McpA protein in CB15N cell extracts, whereas no 70-kD band was observed in SC1130N cell extracts (Fig. 5A). The strain SC1130N carries a Tn5 insertion in the mcpA gene. However, in control experiments, the 70-kD protein band was still observed in SC1062 protein extracts; the strain SC1062 carries a Tn5 insertion in flaE. The antisera also detected a 70-kD protein in E. coli (KO607) contammg a derivative of the overproducing plasmid pMCP2 (see Table II. Swarmer cells were isolated from CB15N wild-type cultures, and the location of McpA was determined by immunoelectron microscopy of fixed and sectioned cells using anti-McpA antibody (Fig. 5B), McpA is localized at the swarmer cell pole of wild-type CB15N, but virtually no signal was present m strain SC1130N (data not shown). In addition to the intense labeling at the flagellated pole, we occasionally observed a small amount of labeling (1-4 gold particles) at the other pole. One interpretation of this result is that both poles of the new swarmer cell are competent for McpA retention. However, the pole bearing the flagellum is the only swarmer pole present when the nicpA gene is expressed m the predivisional cell (Gomes and Shapiro 1984); thus, the flagellated pole would retain the majority of the newly synthesized McpA. Very little chemoreceptor is synthesized following cell division (Gomes and Shapiro 1984), but perhaps a small amount of McpA not localized before cell division can be positioned at either pole after cell division. To determine the intracellular distribution of the McpA-(3-lactamase fusion (MCL2), which segregates to

both daughter progeny, we first determined the positioning of two full-length McpA fusion constructs: M c p A 3-galactosidase (CEE281) and McpA-p-lactamase (MCL722), shown diagrammatically in Figure 6. Using immunoelectron microscopy we found that both the McpA-p-galactosidase fusion (Fig. 7A,B) and the M c p A (3-lactamase fusion protein (Fig. 7C,D) appear predominantly at the flagellated pole of the swarmer cell. Thus, like the native McpA protein, both (i-galactosidase and (B-lactamase fusions to the full-length McpA protein were localized to the cell pole. The intracellular positioning of the shorter McpA-p-lactamase fusion (MCL2) was then examined (Fig. 6). This protein fusion lacks the carboxyl one-third of the McpA protein and is still membrane localized. However, unlike the full-length fusion protein, it is distributed evenly around the swarmer cell (Fig. 7E,F), implicating the carboxy-terminal portion of the protein in segregation to the swarmer cell and in polar localization of the protein. To determine whether carboxy-methylation of McpA is required for polar localization, we examined the positioning of a full-length McpA-p-galactosidase fusion protein encoded by mcpA-lacZ carried on a plasmid (pRCHZlO) m the carboxy-methylation-minus mutant SC1130N. Because this plasmid-borne fusion is present at two to three copies per cell, we first ascertained whether the plasmid-borne McpA-p-galactosidase fusion protein is localized polarly in the C. crescentus wild-type strain CB15N. As expected, the McpA-p-galactosidase fusion protein was positioned predominantly at the flagellated pole of CB15N swarmer cells (Fig. 8A). Thus, both chromosomal and plasmid-encoded McpA

,y.^M

^9

70 kD

Figure 5. Polar localization of the C. crescentus McpA chemoreceptor by immunoelectron microscopy. [A] Immunoblots (Towbin et al. 1979) of-C. crescentus cell extracts of wild-type C, crescentus CB15N, mutant strains SC1130N and SC1062, and an E. coli strain KO607, carrying the plasmid-borne McpA gene jpMCP2) using antisera raised against T7 glO-McpA, as described in Materials and methods. [B] Electron micrographs of fixed and sectioned wild-type swarmer cells treated with a 1/800 dilution of anti-McpA antibody, and visualized by using goat anti-rabbit 10 nM colloidal gold conjugates as the secondary antibody. Bar, 0.5 |JLM.

830

GENES & DEVELOPMENT

f •f

fM:

A*#^'"

Chemoreceptor polar localization Table 1. Bacterial strains and plasmids Bacterial strains

Genotype or description

Reference

E. coli

K0607 S17-1 MG1655

Ato-7021, A(tar-tap] 5201, Atrg-100, recA56 M294::RP4,2-Tc::Mu, Km::Tn7 F X'

Oosawaet al. (1988) Simon etal. (1983) Guyeret al. (1980)

C. crescentus

CB15N SC1130N

synchronizable wild type synchronizable derivative of SCI 130 mcpA::Tn5; methylesterase and methyltransferase deficient flaE178::Tn5 Pro A Str-104 CB15N::pMCL722 CB15N::pMCL2 CB15N::pCEE281

Evinger and Agabian (1977) Alley etal. (1991)

pUC 19 derivative pUC19 derivative T7 expression plasmid 5.6-kb BamHl fragment cloned into pET3C contains the T7 RNA polymerase gene under the control of the PL promoter pIC20R polylinker upstream of the ^-lactamase gene without its signal sequence (Broome-Smith and Spratt 1986) contains a 2.3-kb £coRI fragment in pJY43A contains a 1.7-kb EcoRl-Sacl fragment in pJY43A p-galactosidase translational fusion vector contains a 2.3-kb £coRl fragment in pJBZ281 IncPl plasmid a derivative of pCEE281 inserted into pRK290 a helper plasmid for conjugal transfer of pJY43A and PJBZ281 derivative 5.1-kb f/iridlll-EcoRI fragment from pPA63 containing the E. coll tsr gene was inserted into pRK290KS2 5.1-kb HmdIII-£coRl fragment contains the E. coli tsi gene inpUC19 pBSKS(II) polylinker inserted into the £coRI site of pRK290

Stratagene Marsh etal. (1984) Studier et al. (1990) this study Tabor and Richardson 1985)

SCI 062 SU203 (MCL722) SU204(MCL2) SU205 (CEE281) Plasmids

pBS(II)KS( + ) pIC20H pET3C pMCPl pGl-2 pJY43A pMCL722 pMCL2 pJBZ281 pCEE281 pRK290 pRCHZlO pLVC9 pRTSRl pPA63 pRK290KS2

protein fusions are localized to the cell pole, suggesting that polar sites are not saturated in the CB15N/ pRCHZlO strain. As shown in Figure 8B, the fusion protein is localized to the cell pole in strain SC1130N. Strain SCI 130N lacks the enzymes to carry out carboxymethylation (Alley et al. 1991), and thus localization does not depend upon the carboxy-methylation of the McpA moiety.

Purucker et al. (1982) this study this study Alley etal. (1991)

J. Yu (unpubl.) this study this study Alley etal. (1991) Alley etal. (1991) Ditta et al. (1980) this study G. Warren (unpubl.) this study J. Parkinson (unpubl. Alley etal. (1991)

a plasmid-borne E. coli tsr gene (pRTSRI) and asked whether the Tsr protein specifically segregates to the progeny swarmer cell. The newly synthesized Tsr protein, labeled in the predivisional cell, segregates predominantly to the swarmer cell (Fig. 9). Therefore, E. coli Tsr contains all of the information necessary for swarmer cell-specific segregation. Discussion

Asymmetric segregation of the E.coli Tsr expressed in C. crescentus

chemoreceptor

The amino acid sequence of the carboxy-terminal portions of the C. crescentus McpA and the E. coli Tsr chemotaxis transducer is highly conserved. Because we have shown that this region of McpA is required for swarmer cell segregation and for polar localization, we determined the fate of the E. coli tsr gene product when it is expressed in C. crescentus. We chose the Tsr chemoreceptor because we had demonstrated previously that tsr is transcribed in C. crescentus from its native start site in a cell cycle pattern similar to the endogenous C. crescentus chemoreceptor (Frederikse and Shapiro 1989). We generated a strain of C. crescentus that carries

We have determined that the chemoreceptor McpA is spatially constrained to the pole of the C. crescentus swarmer cell. The cell pole that bears the flagellum, and to which the chemotaxis MCPs are localized, is the division site of the previous cell division. It is possible that epigenetic information is passed on at the cell poles by the deposition of specific proteins at the division plane. Huguenel and Newton (1982) have reported that cell division events in the previous cell cycle are required for the assembly of structures at the cell pole. Both membrane flagellin pools (Huguenel and Newton 1984) and MCPs (Nathan et al. 1986) are preferentially associated with membrane vesicles prepared from the cell poles, suggesting that C. crescentus has unique polar domains.

GENES & DEVELOPMENT

831

Alley et al.

CDCO

(/iCDCQD-

m u

ro

TMl

TM2

Kl

MCL722 h-

HCD

:S C/)

Rl

(O

(C

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[^J"

Figure 6. A restriction map of the mcpA gene with the specific regions of the protein denoted below, as described in the legend to Fig. 4. [Below] Diagram of McpA protein fusions to p-lactamase or to p-galactosidase, accompanied by a summary of the segregation data presented m Fig. 4B and the immunoelectron microscopy locaUzation data shown in Fig. 7. (NT) Not tested.

There are at least two mechanisms by which polar localization of McpA could occur. First, McpA could be inserted randomly in the inner membrane and could reach the cell pole by diffusion. Once it has reached the cell pole, it could be constrained. Retention of McpA at the pole could be the result of interactions with one or more polar proteins or by a physical barrier that allows McpA to enter, but not exit, the pole. Our second model predicts that McpA is inserted only at the pole of the cell via a specific localized secretory pathway. This model also requires McpA retention at the pole, either by interactions with polar proteins or by a physical barrier. Evidence for such a barrier, the polar periseptal annulus, has been reported for E. coh (Cook et al. 1986). A chemoreceptor lacking the native carboxy-termmal region is inserted m the membrane but is not localized to the cell pole. How can these models account for the random cellular distribution of the McpA with a deleted carboxyl terminus? Both models require that McpA is sequestered at the pole. Therefore, the deleted region is responsible, in part, for specific protein interactions and/ or retention by a physical barrier. The second model demands that the choice of secretory pathway is determined by domains in the carboxyl terminus. The random membrane insertion of the deleted protein would then, in effect, occur by a default pathway, perhaps by using the secA-dependent membrane insertion pathway of the E. coli Tsr protein (Gebert et al. 19881, which requires an amino-termmal signal sequence. A carboxyterminal signal has been shown to be sufficient for membrane targeting and translocation of the £. coh hemolysin protein (HylA) (Koronakis et al. 1989). Because our experiments suggest that sequences within the carboxyl terminus of McpA are responsible for specific polar localization, we compared McpA sequences with those of the other chemoreceptors (Fig. 3). Within this region are the methylation domains as well as the HCD. Both methylation regions Rl and Kl are conserved but not to the same extent as the HCD. Two

832

GENES & DEVELOPMENT

proteins interact with the Rl and Kl regions, the methylesterase and the methyltransf erase, involved in removing and adding methyl groups to glutamic acid residues at certain sites in the Rl and Kl regions. The truncated McpA-p-lactamase fusion is not methylated, although it retains an intact Kl methylation region. A similar finding was reported using a truncated Tar protein that had the Rl region deleted (Krikos et al. 1985). Although there is a correlation between the absence of methylation and the inability of the truncated McpA-^-lactamase fusion to be polarly localized in swarmer cells, we found using a methylation-deficient mutant, SC1130N, that methylation of McpA is not required for polar localization. The transposon insertion in this mutant strain is polar, and therefore other chemotaxis proteins present in this operon (Alley et al. 1991), such as cheA, cheW, methylesterase, and methyltransferase, are not required for polar localization of McpA. The most striking feature of the chemoreceptor amino acid sequence comparison is the near identity of the cytoplasmic HCD in enterics and the phylogenetically distant C. crescentus. This suggests a strong selective pressure to maintain this region, perhaps owing to a number of protein interactions with this domain. It has been inferred from genetic data that cheA (J.S. Parkinson, pers. comm.) and cheW {Liu and Parkinson 1991) interact with the chemoreceptor at the highly conserved domain. The sequence similarity between E. coli and C. crescentus chemotaxis proteins may be sufficient for these proteins to be functionally equivalent. We have found that when the E. coli chemoreceptor Tsr is expressed in C. crescentus, it segregates to the swarmer cell. Sequences in the HCD might interact with a complex of proteins at the cell pole, as well as with the cytoplasmic CheW and CheA proteins involved in chemotaxis signal transduction. C. crescentus utilizes a number of mechanisms to position cell type-specific proteins to the correct cell progeny, as well as to the correct region of the cell. Some

Chemoreceptor polar localization

D •JM-':

Figure 7. Localization of McpA fusion proteins within C. crescentus swarmer cells by immunoelectron microscopy. {A,B] Wild-type CB15N swarmer cells containing a chromosomal full-length McpA-p-galactosidase fusion (CEE281); the sectioned cells are treated with antip-galactosidase antibody, as the primary antibody. {C,D] A full-length McpA-plactamase fusion (MCL722). [E,F] A truncated McpA-p-lactamase fusion (MCL2) treated with anti-p-lactamase antibody as the primary antibody. The positions of these fusion proteins are visualized by 10 nM colloidal gold spots conjugated to goat anti-rabbit antibody. The arrow in C identifies a rare cross section through a flagellum. Bar, 0.5 fxM.

proteins are positioned owing to cell type-specific transcription (Gobcr et al. 1991b). Other proteins, such as pilins, are dispersed throughout the predivisional cell and are assembled only at the swarmer pole (Smit 1987). In this report we have demonstrated polar positioning of the newly synthesized chemoreceptor McpA, which is dependent on sequences in the carboxy-terminal domain of the protein. McpA is still localized polarly in mutants that fail to assemble polar structures such as pili and flagella, and in mutants that have a paralyzed flagellum (J. Maddock, unpubl.). We do not know whether McpA is positioned at the pole of the swarmer cell because of a functional relationship with the flagellar motor or whether the polar positioning reflects a general mechanism used by prokaryotes to localize specific categories of proteins. Experiments by Engstrom and Hazclbauer (1982) suggest that the MCPs are not tightly associated with the basal bodies of the peritrichous flagella in E. coli, thus arguing against a spatial requirement for func-

tional interaction. The high degree of sequence similarity and functional equivalency between the C. crescentus and £. coli chemotaxis receptor proteins leads us to speculate that the E. coli MCPs may be associated with the poles of the cell as they are in C. crescentus.

Materials and methods BacLerial strains, plasmids, and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. C. crescentus was grown at 32°C in either PYE (Poindexter 1964) or minimal M2 medium (lohnson and Ely 1977). Plasmids were transferred from E. coli to C. crescentus by conjugal transfer as described by Alley et al. (1991). Immunoprecipitation assays of synchronized cells Swarmer cells were collected from Ludox density gradients, as described by Evinger and Agabian (1977). The swarmer cells

GENES & DEVELOPMENT

833

Alley et al.

•^}^ •s'*^*^

•J^

•S

B • •!«S^^^S3!3av*Sf'

•^.; Figure 8. Subcellular localization of McpA in wild-type and chromotaxis methylation mutants. The distribution of the fusion protein encoded by the fulllength McpA gene fused to lacZ on plasmid pRCHZlO was monitored in swarmer cells from wild-type C, crescentus CB15N [A] and in the die strain SC1130N [B). The McpA-fi-galactosidase fusion was stained with anti-^-galactosidase antibody and visualized by colloidal gold conjugated to goat anti-rabbit antibody. Bar, 0.5 fxM.

were allowed to proceed synchronously through the cell cycle m fresh media, and at 110 min jof a 180 min generation time) predivisional cells were incubated with ["^^Slmethiomne \3 jxCi/ ml) for 10 min, chased with 2 mM methionine and 0.2% iwt/vol) Bacto-peptone, and allowed to divide. The progeny swarmer and stalked cells were separated by Ludox density centrifugation. Cell extracts were prepared and immunoprecipitated with antip-lactamase antibody, as described previously (Gomes and Shapiro 1984). Proteins were separated by electrophoresis through 10% fwt/vol) (SDS-PAGE). Labeled proteins were detected by autoradiography. DNA sequencing

strategy

The 2.3-kb £coRI fragment was cloned into pBS(II)KSl + 1 m both directions, and deletions were created by exonuclease 111 and SI nuclease digestion (Pharmacia nested deletion kit) to create nested deletions in both directions. These deletions were sequenced using double-stranded templates in Sanger dideoxynu-

834

GENES & DEVELOPMENT

-\

cleotide sequencing reactions (Sanger et al. 1977) with Sequenase (U.S. Biochemical) and 7-deaza-dGTP. D N A sequences that had ambiguous GC compressions were subcloned into M13 m p l 8 / m p l 9 (Norrander et al. 1983) and sequenced using dITP. The adjoining 280-bp £coRI fragment was cloned into M13, m p l 8 , and m p l 9 and sequenced in both directions. Purification of the C. crescentus McpA protein generation of anti-McpA antibody

and

McpA was overexpressed in E. coli (MG1655) using a T7 expression system (Tabor and Richardson 1985). The 5.6-kb BamHl fragment shown in Figure 1 was inserted into pET3C to create pMCPl, a translational fusion between glO (T7 capsid protein) and mcpA under the control of a T7 promoter. The plasmid pMCPl was then introduced into the E. coli strain MG1655 bearing the T7 RNA polymerase gene, pGl-2. Overexpression of the fusion protein was performed as described by Tabor and Richardson (1985). Isolation of membranes from the disrupted

Chemoreceptor polar localization

pRTSRl

I

J.R.M. is an NIH postdoctoral fellow. We thank Dan Koshland for the anti-Tar antisera and Bob Bourret for his generous gifts of E. coli strains, plasmids, and antibodies. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

r

TMI

TM2

Kl

HCD

Rl

References

ST

T

SW

T

S-METHIONINE

Figure 9. Progeny cell segregation of the E.coli Tsr chemoreceptor expressed in C. crescentus. Predivisional cells of C. ciescentus bearing a plasmid containing the £. coli tsr gene (pRTSRI) were pulse labeled with p'^Sjmethionine. The label was chased with cold methionine, and swarmer and stalked cell progeny were separated following cell division, as described in Materials and methods. Labeled proteins were immunoprecipitated from stalked (ST) and swarmer (SW) cells using anti-Tar antiserum. As a control, immunoprecipitation was also performed using flagellin antiserum (data not shown).

cells was performed as described by Burrows et al. (1989). The isolated membranes were subjected to SDS-PAGE and the fusion protein was then visualized using (0.05% wt/vol) Coomassie brilliant blue m water. The corresponding protein band was excised and used to immunize a rabbit, performed by Josman Laboratories (Napa, CA). ImmunoelectTon

microscopy

Swarmer cells were collected and fixed in cold 3 % formaldehyde, 0.1% glutaraldehyde, in 20 mw phosphate buffer for 1-2 hr on ice. Dehydration, infiltration, polymerization of LR White resin, preparation of "sticky" grids, and antibody reactions are described in Wright and Rine 1989. A 1 : 500 dilution, in PEST (140 mM NaCl, 30 mw KCl, 8 mM NA2HPO4, 1.5 mM KH^VO^, and 0.05% Tween-20) plus 2% BSA of primary antibody was typically used. The secondary antibody was a 1 : 30 dilution of goat anti-rabbit 10 UM colloidal gold conjugates (Jackson ImmunoResearch, West Grove, PA) in PBST plus 2% BSA. The background signal that we observed under these conditions using either anti-p-lactamase or anti-p-galactosidase antibodies was 0-3 gold particles/cell. The anti-McpA antibody had virtually no cross-reactivity with SC1130N (0-2 gold particles/cell). Secondary antibody only resulted in 0-1 gold particles/cell. Sections were poststained with saturated uranyl acetate (in 50% ETOH) and examined at 60 kV on a Phillips 300 electron microscope.

Acknowledgments This investigation was supported by U.S. Public Health Service grant GM32506 from the National Institutes of Health (NIH).

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