Vol. 130, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, May 1977, p. 877-887 Copyright ©) 1977 American Society for Microbiology
Characterization of Lambda Escherichia coli Hybrids Carrying Chemotaxis Genes M. SILVERMAN, P. MATSUMURA, M. HILMEN, AND M. SIMON* Department of Biology (B-022), University of California, San Diego, La Jolla, California 92093 Received for publication 1 November 1976
Molecular cloning techniques were used to construct hybrid Escherichia coli lambda phage and isolate Col El factors that carried the cheB region of the E. coli genome. The products of these genes were examined by using a series of deletions in the phage to stimulate specific polypeptide synthesis in ultravioletirradiated cells and by using Col factor to program protein synthesis in minicells. Seven flagellar related polypeptides were synthesized. Three of these with apparent molecular weights of 38,000, 28,000, and 8,000 were associated with the cheB region; three polypeptides 63,000, 61,000, and 60,000 were associated with the region that maps between cheB and cheA. These bands were referred to as the triplet group. We suggest that these polypeptides are the same as the methyl-accepting chemotaxis protein described by Kort et al. (Proc. Natl. Acad. Sci. U.S.A. 72:3939-3943, 1975). Another polypeptide with a molecular weight of 12,000 is associated with the cheA region which also produces at least three gene products. We conclude that the cheA-cheB region in E. coli is complex. Further genetic and biochemical analyses are required to describe all of these products. More than 20 different genes in Escherichia coli are involved in flagellar structure and function. Examination of the phenotypes of flagellar mutants has revealed functional distinctions among these genes. Some of them (fla and hag) program the synthesis of the flagellar filament and basal structure and regulate the appearance of these structures (10, 16, 17). Other genes code for polypeptides that are distinct from the basal structure and are found in the inner membrane of the cell (15, 20). Some of these gene products appear to be specifically involved in flagellar rotation (19). A third group of genes (che) program the synthesis of products that are necessary for the integration of signals from chemoreceptors and the modulation of flagellar rotation to produce responsive swimming. In E. coli, four specific che genes have been defined (2, 3, 12) cheA, cheB, cheC, and cheD. cheC is identical to fZaA and may represent a protein that is a structural component of the flagellar apparatus (18). The cheD gene was found to map near the thr locus (12). The cheA gene is part of the "mocha" operon which is a group of genes that are co-transcribed and include motA, motB, and cheA. Figure 1 shows the localization on the E. coli map of the cheA and the cheB genes. The genetic system involved in controlling flagellar synthesis and function in Salmonella is analogous to that in E. coli (24). In Salmonella, a large number ofche mutants have been
analyzed and nine complementation groups were defined (22). At least five of these che genes were shown to map near the motB gene in Salmonella (5, 22). Very little is known about the products of the genes involved in chemotaxis. A specific membrane-bound polypeptide, which is reversibly methylated in E. coli (MCP) appears to be correlated with chemotaxis (9). However, the genes involved in the synthesis and modification of this protein have not been identified nor has the precise function of this polypeptide been demonstrated. To identify the products of the genes involved in flagellar formation and function, we have isolated these genes on a variety of specific vehicles and have studied their expression in ultraviolet (UV)-irradiated cells and in minicells. The flagellar genes were cloned in two ways. First, by isolating the specific deoxyribonucleotide acid (DNA) that carried many of these genes. This DNA was then treated with the EcoRI endonuclease restriction enzyme, and lambda hybrids carrying the restricted fragments were prepared (14). The second approach involved using the colony bank initially prepared by Clark and Carbon (4). They sheared the E. coli genome and isolated a large number of colonies which carried fragments of the genome annealed to the Col El factor. We selected from their collection clones that contained hybrid Col El factors carrying the E. coli flagellar genes. The Col El hybrids were 877
878
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FIG. 1. The construction of Xfla23. The top line shows the region of the E. coli genome that was treated with restriction enzymes. The vertical arrows and brackets indicate the relative positions of EcoRI endonuclease sites. The horizontal arrows indicate groups ofgenes that are co-transcribed. The dark line indicates the piece of E. coli DNA inserted into lambda, and the dotted line indicates the portion of the DNA that was removed by specific deletions.
transferred into a minicell-producing strain, ligase was similar to that of Thomas et al. (21). and the gene products synthesized in minicells Hybrid lambda was recovered by transfection into were characterized. Initially, three distinct E. E. coli C600 rK-, mK-. Transductional crosses were to characterize a large collection of hybrid coli lambda hybrid phages were isolated. used obtained by transfection. The hybrid Lambda flal was shown by complementation lambda lambda was cross-streaked on the surface of a motiland tests to carry the genes hag, flaN, flaB. ity agar plate on tester strains with defined flagellar Lambda fla2 carried the flal gene and part of mutations. The mutant tester strains were lysogenic the mocha group of genes including motA, for lambda to prevent lysis by the transfecting hymotB, and part of the cheA gene. In comple- brid lambda. Recombinants were recognized as mentation tests, the hybrid phage behaved as if swarms emanating from the region of infection, and the EcoRI endonuclease restriction site were in complementation was recognized by the presence of the middle of the cheA gene. Lambda fla3 car- trails in the motility agar (15). This paper describes the properties of a hybrid ried the other part of the cheA gene, as well as which complemented the genetic defects in the cheB gene, and the flaG and flaH genes. lambda CheB- strains el3pl and el4nl obtained from J. S. The capacity of a lambda hybrid or a lambda Parkinson. The hybrid lambda also gave recombihybrid deletion phage to synthesize specific nants with one CheA- mutant strain el4gl but did polypeptides was tested in UV-irradiated cells. not show complementation with this mutant. We The hybrid lambda carrying the hag gene was concluded, therefore, that this phage Xfla3M carried shown to program the synthesis of flagellin, the cheB gene and part of the cheA gene. The parent and more recently the technique was used to of Xfla3Al was Xfla3, which in addition to the cheB identify the gene products of the mocha operon gene and part of the cheA gene also carried the flaG flaH gene activities. Hybrid Xfla3 was un(motA, motB, and cheA) carried by the phage and the presumably because its genome was very lambda fla2. In this report, we will describe the stable, large and spontaneously gave rise to a population of initial characterization of the products of the deleted phages. This assumption was substantiated genes carried by the phage lambda fZa3Al, by testing a variety of single plaques derived from which included the cheB gene region. Xfla3. They were found to carry deletions resulting in MATERIALS AND METHODS
Construction of hybrid lambda. Hybrid lambda E. coli DNA molecules were constructed using AgtXc DNA as the vehicle to clone fragments obtained by EcoRI endonuclease digestion of an F factor carrying the region of the E. coli genome with most of the flagellar genes. The isolation of the F factor (MSF1338) has been described, and the procedure used in EcoRI restriction and ligation with T4 DNA
the loss of complementation activity for cheB or flaG and flaH. An extensive analysis of these deletions will be published elsewhere. To analyze the products of the genes on DNA inserted into lambda, we have developed a procedure that involves the following steps: (i) the preparation of lambdas that carry the hag gene fused to either the right arm or the left arm of lambda and have only a single EcoRI site; (ii) the insertion of the cloned piece of DNA onto this phage; (iii) the selection of a series of deletion mutants. With these mutants we could determine the
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orientation of the inserted piece of DNA with respect to the outside marker and also prepare a series of deletions, which removes successive portions of the inserted piece of DNA in one orientation or the other (18, 19). The steps involved in the construction of an appropriate hybrid lambda phage Xfla23 are outlined in Fig. 1. Hybrid Xfla3, Xfla2, and Aflal were constructed initially. The arrows indicate the position of the EcoRI restriction sites. Xfla3Al was isolated as a spontaneous deletion from Xfla3. XflalA4 was selected from Aflal by the pyrophosphate shock method of Parkinson and Huskey (13) and has the hag gene fused to the right arm of lambda. A mixture of EcoRI-digested Afla3Al and XflalA4 was annealed and ligated. A series of single plaques was picked and tested for cheB and hag activity. Subsequent examination of EcoRI and BamI endonuclease digests of deletions of one of these clones Xfla23 established the orientation of the gene shown in Fig. 1. Another independent isolate, Xfla42, had the same orientation as Xfla23. The two phages appear to be identical. The purification of the hybrid lambdas and the preparation of phage stocks has already been described (15). Protein synthesis and polyacrylamide gel electrophoresis. The infection and labeling of UV-irradiated cells with hybrid lambda have been described (14). [35S]methionine with specific activity of 320 Ci/ mmol (New England Nuclear) was used to label the protein. The host strain was E. coli K-12 strain 159 (X) obtained from H. Murialdo. The details of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography have been described elsewhere (14). Labeled proteins synthesized in minicells carrying Col E1-E. coli hybrids were compared to the proteins synthesized in UV-irradiated strain 159 carrying lambda. The hybrid Col El plasmid strains were selected from the colony bank prepared by Clark and Carbon (4). The hybrid plasmids were transferred by F-mediated conjugation to MinA MinB strain P678-54, with selection for Col El resistance. The minicell isolation was similar to that of Adler et al. (1). Labeling was done in RM medium (minimal medium with maltose) after a preincubation period of 20 min. Ten microcuries of [35S]methionine (350 ,Ci/mmol) was added to 0.5 ml of the minicell suspension with 3 x 109 minicells per ml, and the labeling was terminated after 30 min by the addition of unlabeled methionine. The cells were collected by centrifugation, and the pellet was washed and resuspended in SDS-polyacrylamide gel electrophoresis buffer (10). Preparation of MCP. Methyl-accepting chemotaxis protein (MCP) was used for comparison with the products of the flagellar genes carried on Xfla23. It was prepared by the method of Kort et al. (9) after labeling with [methyl-3H]methionine with a specific activity of 14 Ci/mmol (New England Nuclear) in the presence of chloramphenicol. The cells were collected and washed and either immediately processed, or the membrane fraction was isolated and the membranes were suspended in SDS-gel buffer, and samples were run on 22-cm acrylamide gels with gradients of 5 to 15% polyacrylamide. The gels were then dried and impregnated with scintillator,
879
and tritiated methyl counts were visualized by autoradiography. Endonuclease mapping or hybrid lambda. The enzyme EcoRI was prepared by the procedure of Green (6) and BamI endonuclease was prepared by the procedure of Wilson and Young (23). The enzyme digestions were done in 100 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane (pH 7.8), 10 mM MgCl2 at concentrations of 40 ,ug/ml of DNA. For sequential digestion, the DNA was incubated with EcoRI for 30 min at 37°C and then heated at 65°C for 5 min. Subsequently, BamI was added and the sample was digested for 1 h at 37°C, followed by heating at 65°C for 5 min. Sample buffer was added, and the samples were immediately loaded onto 6-mm 1.1% agarose gels.
RESULTS
Part of the genetic map of the lambda hybrids carrying flagellar genes is shown in Fig. 1. Xfla3 was shown to carry cheB, flaG, and flaH by complementation tests with a variety of mutant strains. This hybrid also gave recombinants, but no complementation with one cheA strain (el4ql, S. Parkinson). It was possible to restore the continuity of the cheA gene by constructing a hybrid lambda carrying motA and motB and part of the cheA gene from Afla2 and the other part of the cheA gene supplied by Xfla3. The properties of this phage, Xfla52 have been reported (20). Xfla3 behaved as if it were unstable. A larger number of single plaques were picked, and many of them were found to carry only part of the cloned fragment. We will describe the characterization of one deletion mutant, Xfla3Al (Fig. 1). It was shown by complementation activity to carry cheB and by recombination activity to carry part of cheA. The flaG and flaiI activities were deleted, but the EcoRI restriction sites were still present. To prepare and characterize a specific series of further deletions of Xfla3Al, we constructed a new phage which carried the hag gene as an outside marker. The construction of this phage is shown schematically in Fig. 1. Xfla23 and Xfla42 are two separate phages that have identical genetic characteristics, i.e., they carry cheB, cheA, and hag. Polypeptides programmed by Xfla23, Xfla42, and a series of deletions were detected after infecting and labeling UV-irradiated E. coli. The cells were lysogenic for lambda to prevent the expression of the lambda genes, and control experiments were performed with strains that carried mutations in the flaI gene. The flaI mutation prevents synthesis of flagellar specific products. Xfla23 programmed the synthesis of flagellin, the hag gene product (Fig. 2b), and a small polypeptide (Fig. 2e) whose presence depends upon the proximity of a functioning
880
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hag gene (this product will be discussed in detail elsewhere). There are a variety of other polypeptides including a group of bands which we will refer to as triplet. This includes bands corresponding to molecular weights of 63,000, 61,000, and 60,000 (Fig. 2a). They behave as a unit; deletions appear to remove all or none of them. Other flagellar-specific polypeptides include a band corresponding to 38,000 (Fig. 2c) and one corresponding to 28,000 (Fig. 2d) and a polypeptide with a molecular weight corresponding to 8,000 (Fig. 2f). To simplify the discussion, we will refer to these polypeptides with the prefix FP. Thus, the bands shown in Fig. 2 are: (a) FP63, FP61, FP60, or triplet; (b) FP54 or hag; (c) FP38; (d) FP28; (e) FP12, and (f) FP8. None of these proteins appears when the phage is used to infect UV-irradiated cells that carry mutations in the fYal gene. To correlate the specific polypeptides that appeared on the gels with the genes carried by the lambda phages, over 400 deletion mutations of Xfla23 and Xfla42 were isolated and characterized genetically. Forty of these deletions were then purified and tested for their capacity to program polypeptide synthesis. The deletion mutants Xfla23A1, A2, A5, A15, A21, and A23, as well as Xfla42A1, A4, and A9, all retained the capacity to complement both of the tester strains carrying cheB mutations. All of these
phages also have in common the presence of the bands FP38, FP28, and FP8, except for Xfla23A5 which has FP38 and FP8. Xfla23A5 appears to be missing FP28 and has a new band with an approximate molecular weight of 32,000. Two phages, X23A6 and X23A16, were able to complement the tester strain carrying the mutation el4nl but could not complement the strain carrying el3pl. These phages did not program the synthesis of FP38 or FP28, but they did synthesize FP8 (Fig. 2). To further examine the gene products of the cheB region, the hybrid Col El plasmids originally prepared by Clark and Carbon (4) were used. One of these plasmids (PFC21-2) was transferred to minicells. In genetic complementation tests this plasmid was shown to carry cheB gene activity. A control plasmid PLC13-12 which carries the flaD gene was also used. These plasmids were transferred to minicellproducing strains; minicells were collected and their pattern of polypeptide synthesis was examined. The plasmid PLC21-2 programmed the synthesis of FP38, FP28, and FP8 (Fig. 3). The control plasmid PLC13-12 did not stimulate the synthesis of these polypeptides. It is clear from an examination of the polypeptide patterns found with deletions of the phages Xfla23 and Xfla42, as well as Xfla52 (which does not have cheB gene activity), that
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SILVERMAN ET AL.
the triplet polypeptides FP63, FP61, and FP60 are not correlated with cheB activity (Fig. 3). For example, Xfla23A22 and Xfla42A5 both show the triplet bands but do not show cheB activity in complementation tests. On the other hand, Xf7a42A9 and Xfla23A5 have cheB activity but do not show the triplet bands. However, since these polypeptides are subject to the regulation of the flaI gene, we presume that they have some role in flagellar function or flagellar structure. The three bands could be the result of a single gene or they may result from multiple genes. There is ample precedence for the product of a single gene appearing as a series of bands on acrylamide gels; flagellin, for example, can appear to have a molecular weight of 54,000, and under a variety of conditions of heating its apparent molecular weight changes to 56,000 and finally to 60,000. Intermediate mixtures of all three bands have been observed (8). Figure 4 shows the resolution of the triplet proteins and the flagellin protein on acrylamide gradient gels that were 23 cm long. The pattern of the protein in the triplet region can be resolved, and the multiple band pattern of the flagellin proteins can also be observed. The triplet protein generally appears as three bands. There is, however, some variation in the relative intensities of these bands. For example, in Xfla42A1, FP63 and FP61 are intense, whereas FP60 is faint. In the pattern obtained with X42A4 and A5, the 60,000 and 61,000 molecular weight bands appear to predominate, whereas the 63,000 appears to be very light. We have not as yet been able to correlate this change in intensity with either the method of preparation of the protein for acrylamide gel electrophoresis or with the genetic composition of specific deletions. To identify the triplet protein with one of the polypeptides involved in flagellar structure or function, it was compared to the polypeptide component of the flagellar basal structure which has an apparent molecular weight of 59,000 on acrylamide gels (7) and to the MCP described by Kort et al. (9) which has also been reported to have an apparent molecular weight of 60,000. These radioactive polypeptides were
compared by polyacrylamide gel electrophoresis under a variety of conditions. Figure 5 shows that MCP and the triplet protein have the same electrophoretic mobility. Both MCP and triplet show a distinctive three band pattern. The MCP pattern is seen when MCP is run alone or in a mixture with an extract from cells infected with Xfla7A5 which carried mot gene activity but not triplet. The triplet pattern is seen in extracts of cells infected with
J. BACTERIOL.
Xfla23A1 (Fig. 5 column 1 and 2 on left). When extracts containing both MCP and triplet are mixed and tested by electrophoresis, and when the film is exposed so that radioactivity corresponding to both triplet and MCP can be seen, the radioactive bands in the triplet region overlap completely with the MCP bands. In experiments involving two-dimensional acrylamide gels prepared by the technique of O'Farrell (11), both the MCP preparation and the triplet protein streaked in the isoelectric focusing direction. Even though there was heterogeneity, there was considerable overlap between MCP and the triplet bands, and both gave the same pattern. The flagellar basal component did not streak. Furthermore, in cell fractionation experiments both the 35S triplet protein and the methyl-3H-labeled MCP were found to be localized in the inner membrane of the cell. On the basis of these data, it is reasonable to propose the hypothesis that the triplet proteins are the same as MCP. The relative order of the cheB gene region, the region that codes for triplet and the distal portion of the cheA gene, can be deduced from the genetic properties of the deletion mutants. To test the hypothetical order, DNA was isolated from several hybrid lambda phages, and the endonuclease digestion pattern was compared to predictions made from genetic and complementation tests and protein synthesis experiments. In Fig. 6a, the EcoRI and BamI endonuclease fragments of DNA from the deletions of Xfla23 and Xfla42 are shown. The positions of the EcoRI and BamI digestion sites are known and it is therefore possible to assign fractional lengths to the fragments shown on the gels. The two EcoRI sites result from the insertion of the cheB cheA fragment into XflalA4. Two of the BamI sites are in the lambda portion of the DNA. The third site that generates a 2.8 fragment with one end derived from an EcoRI cut and the other end from BamI occurs close to or directly within the hag gene. The inserted piece of DNA corresponds to the 16.8 fragment. Deletions may occur within this fragment, thus changing its mobility on the gel. For example, in Xfla23A6, a region corresponding to approximately 7% of the total lambda genome was removed from the 16.8 fragment. Deletions that included the endonuclease cleavage site resulted in the fusion of two fragments; for example, in Xfla23A5, the 16.8 fragment and the 2.8 fragment are fused, generating a new band representing about 9% lambda. A similar but smaller deletion in Xfla23A1 gives a band corresponding to about 13% lambda. An approximate estimate of the
CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
VOL. 130, 1977
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CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
VOL. 130, 1977
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42A13 FIG. 6. Endonuclease mapping of DNA deletions in Xfla23 and Xf!a42. (a) The results of agarose gel electrophoresis after digestion with both EcoRI and BamI endonuclease. The numbers are the relative percentage of the length of wild-type lambda represented by each fragment. (b) The map derived from the endonuclease digestion, genetic complementation, and polypeptide synthesis experiments. The black bars represent the activities that are apparently deleted from each phage. Their length is not an accurate map length.
size of the deletion can be made from the loca- lated genes. The data derived from the analysis tion of the modified band on the agarose gel. of this phage taken together with previous reThe map shown in Fig. 6b is consistent with the sults allows us to develop an hypothesis about pattern in 6a. The length of the black line only the gene products involved in chemotaxis in E. roughly corresponds to actual distances on the coli. The evidence suggests that cheB is a complex locus made up of a number of genes, at map, but it indicates the functions that are missing in either genetic tests (cheB comple- least three. The following findings support this mentation and cheA recombination) or in pro- notion. (i) Most of the phages that carry cheB activity also synthesize, FP38, FP28, and FP8. tein synthesis. (ii) The phages Xf7a23A6 and Xfla23Al6 have DISCUSSION genetic complementation activity when tested The initial characterization of Xfla3Al indi- against MS5031, a cheB strain that carries the cates that it carried a number of flagellar-re- mutation el4nl, but they do not complement
886
SILVERMAN ET AL.
M85032, which carries the cheB mutation el3pl. They make FP8 but do not synthesize FP38 or FP28, suggesting that FP8 is sufficient to complement one of the cheB mutations but not the other. (iii) The phage Xfla23A5 shows genetic complementation with both tester strains. However, FP28 is missing and is replaced by a new band. One possible interpretation is that FP28 represents the product of another independent gene previously grouped as part of the cheB complex. (iv) These products are not restricted to the cheB genes carried on lambda. When the cheB gene is carried on the Col El vehicle, the same three polypeptides are synthesized. Thus, the cheB region specifies three polypeptides that are presumably encoded by three distinct genes. Futthermore, on the basis of the endonuclease maps, we can suggest that the genes are contiguous and that the gene coding for FP28 is adjacent to the triplet gene, the gene coding for FP38 would be next, and, finally, the gene coding for FP8 would be close to the restriction site (Fig. 6b). It is, of course, possible that there are even more genes included in this cheB complex, since the deletion that removed flaG and flaH may have also removed cheB genes. The relative complexity of this region suggests that it may be analogous to the che genes described in Salmonella by Warrick et al. (22). The triplet does not correspond to any known phenotype, and we have not as yet found mutants that map in this region. It is possible that the "triplet" region includes a number of genes; on the other hand, the multiple bands observed on acrylamide gels may be the result of modification of a single polypeptide. The bands appear to be the same as the MCP described by Kort et al. (9). In our experiments, the MCP shows a number of bands on acrylamide gels, and the triplet protein runs together with MCP both on SDS-gels, which separate on the basis of size, and on isoelectric focusing gels, which separate on the basis of charge. Furthermore, both the triplet and MCP are found exclusively in the inner membrane fraction of the cell. Finally, the synthesis of both proteins is regulated by the flagellar regulatory system. The identification of the position of the gene may allow specific mutants to be prepared. The phenotypes of such mutants could help in deducing the function of the polypeptides. The cheA gene group may also be complex. We have shown that when the integrity of the cheA region that had been interrupted by endonuclease restriction is restored, three polypeptides are produced. Thus, the cheA region may also contain more than one gene. In fact, FP12, which was observed as a product of polypeptide
J. BACTERIOL.
synthesis programmed by many of the phages described in this report, appears to be the same as FP12 previously demonstrated to be a product of the cheA region. Deletions that remove hag activity and part of the cheA region also result in the loss of FP12 synthesis (e.g., Xfla23A1, A2, A5, A15, A22; Fig. 2). FP12 may be associated directly with the hag gene or it may be the product of another che gene in the cheA region whose expression is stimulated by the adjacent hag promoter. All of the data argue that this region of the E. coli genome carries the genetic information to code for many flagellar-related polypeptides. Previous work has shown that there is a group of co-transcribed genes called mocha which program the synthesis of a motA, motB, and three che gene products. Adjacent to this cheA region is a triplet region that codes for three polypeptide bands. This may be the result of a single gene or as many as three genes. This is followed by the cheB region which appears to include at least three distinct genes. There may be more flagellar-related genes in the cheB region, since the experiments described in this paper used a phage which carried a deletion removing the region between "cheB" and flaG. It is apparent that a careful comparison of the genetic properties of the cloned lambda fragments and their deletions with the chemotaxis mutants will be necessary to completely analyze this region. There may be as many as 12 and no fewer than 8 gene products involved in flagellar function that are specified by the region of the genome included between the map positions of the flal gene and the flaG gene. This region in E. coli appears to be as complex as the analogous region described in Salmonella. ACKNOWLEDGMENTS This work was supported by grant BMS 13-01606 from the National Science Foundation. P. Matsumura is a NRSA Postdoctoral Research Fellow of National Institute of General Medical Sciences (GM 05249-01). We thank L. Clarke and J. Carbon for providing their hybrid plasmidcontaining strains and J. S. Parkinson for the chemotaxis strains.
LITERATURE CITED 1. Adler, H. I., W. D. Fisher, A. Cohen, and A. A. Ardigree. 1967. Miniature Escherichia coli cells deficient in DNA. Proc. Natl. Acad. Sci. U.S.A. 57:321-326. 2. Armstrong, J. B., and J. Adler. 1967. Genetics of motility in Escherichia coli: complementation of paralyzed
mutants. Genetics 56:363-373. 3. Armstrong, J. B., and J. Adler. 1969. Complementation of nonchemotactic mutants of Escherichia coli. Genetics 61:61-66. 4. Clark, L., and J. Carbon. 1976. A colony bank contain-
ing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell 9:91-99. 5. Collins, A., L. Tsui, and B. A. D. Stocker. 1975. Salmo-
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6.
7.
8.
9.
10. 11.
CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
nella typhimurium mutants generally defective in chemotaxis. J. Bacteriol. 128:754-765. Green, P. J., M. Betlach, H. M. Goodman, and H. Boyer. 1974. The EcoRI restriction nuclease, p. 87111. In R. B. Wickner (ed.), Molecular biology series: DNA replication and biosynthesis, vol. 7, Marcel Dekker, New York. Hilmen, M., M. Silverman, and M. Simon. 1974. The regulation of flagellar formation. J. Supramol. Struct. 2:360-371. Kondoh, H., and H. Hotani. 1974. Flagellin fromEscherichia coli K-12: polymerization and molecular weight in comparison with Salmonella flagellins. Biochim. Biophys. Acta 336:117-139. Kort, E. N., M. F. Goy, S. H. Larsen, and J. Adler. 1975. Methylation of a membrane protein involved in bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 72:3939-3943. Matsumura, P., M. Silverman, and M. Simon. 1977. Expression of the flagellar hook gene on hybrid-plasmids in minicells. Nature (London) 265:758-760. O'Farrell, F. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-
4016. 12. Parkinson, J. S. 1976. cheA, cheB, and cheC genes of Escherichia coli and their role in chemotaxis. J. Bacteriol. 126:758-770. 13. Parkinson, J. S., and R. J. Huskey. 1971. Deletion mutants of bacteriophage lambda. J. Mol. Biol. 56:369-384. 14. Silverman, M., P. Matsumura, R. Draper, S. Edwards, and M. Simon. 1976. Expression of flagellar genes carried by bacteriophage lambda. Nature (London) 261:248-250.
887
15. Silverman, M., P. Matsumura, and M. Simon. 1976. The identification of the mot gene product with Escherichia coli-lambda hybrids. Proc. Natl. Acad. Sci. U.S.A. 73:3126-3130. 16. Silverman, M., and M. Simon. 1972. Flagellar assembly mutants in Escherichia coli. J. Bacteriol. 112:986993. 17. Silverman, M., and M. Simon. 1973. Genetic analysis of flagellar mutants in Escherichia coli. J. Bacteriol. 113:105-113. 18. Silverman, M., and M. Simon. 1973. Genetic analysis of bacteriophage Mu-induced flagellar mutations in Escherichia coli. J. Bacteriol. 116:114-122. 19. Silverman, M., and M. Simon. 1974. Flagellar rotation and the mechanism of bacterial motility. Nature (London) 249:73-74. 20. Silverman, M., and M. Simon. 1976. Genes controlling motility and chemotaxis in E. coli. Nature (London) 264:577-580. 21. Thomas, M., J. R. Cameron, and R. W. Davis. 1974. Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. U.S.A. 71:4579-4583. 22. Warrick, H. M., B. L. Taylor, and D. E. Koshland, Jr. 1977. Chemotactic mechanisms of Salmonella typhimurium: mapping and characterization of mutants. J. Bacteriol. 130:223-231. 23. Wilson, G., and F. E. Young. 1975. Isolation of a sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens II. J. Mol. Biol. 97:123-125. 24. Yamaguchi, S., T. Iino, T. Horiguchi, and K. Ohta. 1972. Genetic analysis of fla and mot cistrons closely linked to Hi in Salmonella abortusegui and its derivatives. J. Gen. Microbiol. 70:59-75.
JOURNAL OF BACTERIOLOGY, May 1977, p. 877-887 Copyright ©) 1977 American Society for Microbiology
Characterization of Lambda Escherichia coli Hybrids Carrying Chemotaxis Genes M. SILVERMAN, P. MATSUMURA, M. HILMEN, AND M. SIMON* Department of Biology (B-022), University of California, San Diego, La Jolla, California 92093 Received for publication 1 November 1976
Molecular cloning techniques were used to construct hybrid Escherichia coli lambda phage and isolate Col El factors that carried the cheB region of the E. coli genome. The products of these genes were examined by using a series of deletions in the phage to stimulate specific polypeptide synthesis in ultravioletirradiated cells and by using Col factor to program protein synthesis in minicells. Seven flagellar related polypeptides were synthesized. Three of these with apparent molecular weights of 38,000, 28,000, and 8,000 were associated with the cheB region; three polypeptides 63,000, 61,000, and 60,000 were associated with the region that maps between cheB and cheA. These bands were referred to as the triplet group. We suggest that these polypeptides are the same as the methyl-accepting chemotaxis protein described by Kort et al. (Proc. Natl. Acad. Sci. U.S.A. 72:3939-3943, 1975). Another polypeptide with a molecular weight of 12,000 is associated with the cheA region which also produces at least three gene products. We conclude that the cheA-cheB region in E. coli is complex. Further genetic and biochemical analyses are required to describe all of these products. More than 20 different genes in Escherichia coli are involved in flagellar structure and function. Examination of the phenotypes of flagellar mutants has revealed functional distinctions among these genes. Some of them (fla and hag) program the synthesis of the flagellar filament and basal structure and regulate the appearance of these structures (10, 16, 17). Other genes code for polypeptides that are distinct from the basal structure and are found in the inner membrane of the cell (15, 20). Some of these gene products appear to be specifically involved in flagellar rotation (19). A third group of genes (che) program the synthesis of products that are necessary for the integration of signals from chemoreceptors and the modulation of flagellar rotation to produce responsive swimming. In E. coli, four specific che genes have been defined (2, 3, 12) cheA, cheB, cheC, and cheD. cheC is identical to fZaA and may represent a protein that is a structural component of the flagellar apparatus (18). The cheD gene was found to map near the thr locus (12). The cheA gene is part of the "mocha" operon which is a group of genes that are co-transcribed and include motA, motB, and cheA. Figure 1 shows the localization on the E. coli map of the cheA and the cheB genes. The genetic system involved in controlling flagellar synthesis and function in Salmonella is analogous to that in E. coli (24). In Salmonella, a large number ofche mutants have been
analyzed and nine complementation groups were defined (22). At least five of these che genes were shown to map near the motB gene in Salmonella (5, 22). Very little is known about the products of the genes involved in chemotaxis. A specific membrane-bound polypeptide, which is reversibly methylated in E. coli (MCP) appears to be correlated with chemotaxis (9). However, the genes involved in the synthesis and modification of this protein have not been identified nor has the precise function of this polypeptide been demonstrated. To identify the products of the genes involved in flagellar formation and function, we have isolated these genes on a variety of specific vehicles and have studied their expression in ultraviolet (UV)-irradiated cells and in minicells. The flagellar genes were cloned in two ways. First, by isolating the specific deoxyribonucleotide acid (DNA) that carried many of these genes. This DNA was then treated with the EcoRI endonuclease restriction enzyme, and lambda hybrids carrying the restricted fragments were prepared (14). The second approach involved using the colony bank initially prepared by Clark and Carbon (4). They sheared the E. coli genome and isolated a large number of colonies which carried fragments of the genome annealed to the Col El factor. We selected from their collection clones that contained hybrid Col El factors carrying the E. coli flagellar genes. The Col El hybrids were 877
878
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SILVERMAN ET AL.
flaH flaG
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FIG. 1. The construction of Xfla23. The top line shows the region of the E. coli genome that was treated with restriction enzymes. The vertical arrows and brackets indicate the relative positions of EcoRI endonuclease sites. The horizontal arrows indicate groups ofgenes that are co-transcribed. The dark line indicates the piece of E. coli DNA inserted into lambda, and the dotted line indicates the portion of the DNA that was removed by specific deletions.
transferred into a minicell-producing strain, ligase was similar to that of Thomas et al. (21). and the gene products synthesized in minicells Hybrid lambda was recovered by transfection into were characterized. Initially, three distinct E. E. coli C600 rK-, mK-. Transductional crosses were to characterize a large collection of hybrid coli lambda hybrid phages were isolated. used obtained by transfection. The hybrid Lambda flal was shown by complementation lambda lambda was cross-streaked on the surface of a motiland tests to carry the genes hag, flaN, flaB. ity agar plate on tester strains with defined flagellar Lambda fla2 carried the flal gene and part of mutations. The mutant tester strains were lysogenic the mocha group of genes including motA, for lambda to prevent lysis by the transfecting hymotB, and part of the cheA gene. In comple- brid lambda. Recombinants were recognized as mentation tests, the hybrid phage behaved as if swarms emanating from the region of infection, and the EcoRI endonuclease restriction site were in complementation was recognized by the presence of the middle of the cheA gene. Lambda fla3 car- trails in the motility agar (15). This paper describes the properties of a hybrid ried the other part of the cheA gene, as well as which complemented the genetic defects in the cheB gene, and the flaG and flaH genes. lambda CheB- strains el3pl and el4nl obtained from J. S. The capacity of a lambda hybrid or a lambda Parkinson. The hybrid lambda also gave recombihybrid deletion phage to synthesize specific nants with one CheA- mutant strain el4gl but did polypeptides was tested in UV-irradiated cells. not show complementation with this mutant. We The hybrid lambda carrying the hag gene was concluded, therefore, that this phage Xfla3M carried shown to program the synthesis of flagellin, the cheB gene and part of the cheA gene. The parent and more recently the technique was used to of Xfla3Al was Xfla3, which in addition to the cheB identify the gene products of the mocha operon gene and part of the cheA gene also carried the flaG flaH gene activities. Hybrid Xfla3 was un(motA, motB, and cheA) carried by the phage and the presumably because its genome was very lambda fla2. In this report, we will describe the stable, large and spontaneously gave rise to a population of initial characterization of the products of the deleted phages. This assumption was substantiated genes carried by the phage lambda fZa3Al, by testing a variety of single plaques derived from which included the cheB gene region. Xfla3. They were found to carry deletions resulting in MATERIALS AND METHODS
Construction of hybrid lambda. Hybrid lambda E. coli DNA molecules were constructed using AgtXc DNA as the vehicle to clone fragments obtained by EcoRI endonuclease digestion of an F factor carrying the region of the E. coli genome with most of the flagellar genes. The isolation of the F factor (MSF1338) has been described, and the procedure used in EcoRI restriction and ligation with T4 DNA
the loss of complementation activity for cheB or flaG and flaH. An extensive analysis of these deletions will be published elsewhere. To analyze the products of the genes on DNA inserted into lambda, we have developed a procedure that involves the following steps: (i) the preparation of lambdas that carry the hag gene fused to either the right arm or the left arm of lambda and have only a single EcoRI site; (ii) the insertion of the cloned piece of DNA onto this phage; (iii) the selection of a series of deletion mutants. With these mutants we could determine the
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CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
orientation of the inserted piece of DNA with respect to the outside marker and also prepare a series of deletions, which removes successive portions of the inserted piece of DNA in one orientation or the other (18, 19). The steps involved in the construction of an appropriate hybrid lambda phage Xfla23 are outlined in Fig. 1. Hybrid Xfla3, Xfla2, and Aflal were constructed initially. The arrows indicate the position of the EcoRI restriction sites. Xfla3Al was isolated as a spontaneous deletion from Xfla3. XflalA4 was selected from Aflal by the pyrophosphate shock method of Parkinson and Huskey (13) and has the hag gene fused to the right arm of lambda. A mixture of EcoRI-digested Afla3Al and XflalA4 was annealed and ligated. A series of single plaques was picked and tested for cheB and hag activity. Subsequent examination of EcoRI and BamI endonuclease digests of deletions of one of these clones Xfla23 established the orientation of the gene shown in Fig. 1. Another independent isolate, Xfla42, had the same orientation as Xfla23. The two phages appear to be identical. The purification of the hybrid lambdas and the preparation of phage stocks has already been described (15). Protein synthesis and polyacrylamide gel electrophoresis. The infection and labeling of UV-irradiated cells with hybrid lambda have been described (14). [35S]methionine with specific activity of 320 Ci/ mmol (New England Nuclear) was used to label the protein. The host strain was E. coli K-12 strain 159 (X) obtained from H. Murialdo. The details of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography have been described elsewhere (14). Labeled proteins synthesized in minicells carrying Col E1-E. coli hybrids were compared to the proteins synthesized in UV-irradiated strain 159 carrying lambda. The hybrid Col El plasmid strains were selected from the colony bank prepared by Clark and Carbon (4). The hybrid plasmids were transferred by F-mediated conjugation to MinA MinB strain P678-54, with selection for Col El resistance. The minicell isolation was similar to that of Adler et al. (1). Labeling was done in RM medium (minimal medium with maltose) after a preincubation period of 20 min. Ten microcuries of [35S]methionine (350 ,Ci/mmol) was added to 0.5 ml of the minicell suspension with 3 x 109 minicells per ml, and the labeling was terminated after 30 min by the addition of unlabeled methionine. The cells were collected by centrifugation, and the pellet was washed and resuspended in SDS-polyacrylamide gel electrophoresis buffer (10). Preparation of MCP. Methyl-accepting chemotaxis protein (MCP) was used for comparison with the products of the flagellar genes carried on Xfla23. It was prepared by the method of Kort et al. (9) after labeling with [methyl-3H]methionine with a specific activity of 14 Ci/mmol (New England Nuclear) in the presence of chloramphenicol. The cells were collected and washed and either immediately processed, or the membrane fraction was isolated and the membranes were suspended in SDS-gel buffer, and samples were run on 22-cm acrylamide gels with gradients of 5 to 15% polyacrylamide. The gels were then dried and impregnated with scintillator,
879
and tritiated methyl counts were visualized by autoradiography. Endonuclease mapping or hybrid lambda. The enzyme EcoRI was prepared by the procedure of Green (6) and BamI endonuclease was prepared by the procedure of Wilson and Young (23). The enzyme digestions were done in 100 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane (pH 7.8), 10 mM MgCl2 at concentrations of 40 ,ug/ml of DNA. For sequential digestion, the DNA was incubated with EcoRI for 30 min at 37°C and then heated at 65°C for 5 min. Subsequently, BamI was added and the sample was digested for 1 h at 37°C, followed by heating at 65°C for 5 min. Sample buffer was added, and the samples were immediately loaded onto 6-mm 1.1% agarose gels.
RESULTS
Part of the genetic map of the lambda hybrids carrying flagellar genes is shown in Fig. 1. Xfla3 was shown to carry cheB, flaG, and flaH by complementation tests with a variety of mutant strains. This hybrid also gave recombinants, but no complementation with one cheA strain (el4ql, S. Parkinson). It was possible to restore the continuity of the cheA gene by constructing a hybrid lambda carrying motA and motB and part of the cheA gene from Afla2 and the other part of the cheA gene supplied by Xfla3. The properties of this phage, Xfla52 have been reported (20). Xfla3 behaved as if it were unstable. A larger number of single plaques were picked, and many of them were found to carry only part of the cloned fragment. We will describe the characterization of one deletion mutant, Xfla3Al (Fig. 1). It was shown by complementation activity to carry cheB and by recombination activity to carry part of cheA. The flaG and flaiI activities were deleted, but the EcoRI restriction sites were still present. To prepare and characterize a specific series of further deletions of Xfla3Al, we constructed a new phage which carried the hag gene as an outside marker. The construction of this phage is shown schematically in Fig. 1. Xfla23 and Xfla42 are two separate phages that have identical genetic characteristics, i.e., they carry cheB, cheA, and hag. Polypeptides programmed by Xfla23, Xfla42, and a series of deletions were detected after infecting and labeling UV-irradiated E. coli. The cells were lysogenic for lambda to prevent the expression of the lambda genes, and control experiments were performed with strains that carried mutations in the flaI gene. The flaI mutation prevents synthesis of flagellar specific products. Xfla23 programmed the synthesis of flagellin, the hag gene product (Fig. 2b), and a small polypeptide (Fig. 2e) whose presence depends upon the proximity of a functioning
880
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hag gene (this product will be discussed in detail elsewhere). There are a variety of other polypeptides including a group of bands which we will refer to as triplet. This includes bands corresponding to molecular weights of 63,000, 61,000, and 60,000 (Fig. 2a). They behave as a unit; deletions appear to remove all or none of them. Other flagellar-specific polypeptides include a band corresponding to 38,000 (Fig. 2c) and one corresponding to 28,000 (Fig. 2d) and a polypeptide with a molecular weight corresponding to 8,000 (Fig. 2f). To simplify the discussion, we will refer to these polypeptides with the prefix FP. Thus, the bands shown in Fig. 2 are: (a) FP63, FP61, FP60, or triplet; (b) FP54 or hag; (c) FP38; (d) FP28; (e) FP12, and (f) FP8. None of these proteins appears when the phage is used to infect UV-irradiated cells that carry mutations in the fYal gene. To correlate the specific polypeptides that appeared on the gels with the genes carried by the lambda phages, over 400 deletion mutations of Xfla23 and Xfla42 were isolated and characterized genetically. Forty of these deletions were then purified and tested for their capacity to program polypeptide synthesis. The deletion mutants Xfla23A1, A2, A5, A15, A21, and A23, as well as Xfla42A1, A4, and A9, all retained the capacity to complement both of the tester strains carrying cheB mutations. All of these
phages also have in common the presence of the bands FP38, FP28, and FP8, except for Xfla23A5 which has FP38 and FP8. Xfla23A5 appears to be missing FP28 and has a new band with an approximate molecular weight of 32,000. Two phages, X23A6 and X23A16, were able to complement the tester strain carrying the mutation el4nl but could not complement the strain carrying el3pl. These phages did not program the synthesis of FP38 or FP28, but they did synthesize FP8 (Fig. 2). To further examine the gene products of the cheB region, the hybrid Col El plasmids originally prepared by Clark and Carbon (4) were used. One of these plasmids (PFC21-2) was transferred to minicells. In genetic complementation tests this plasmid was shown to carry cheB gene activity. A control plasmid PLC13-12 which carries the flaD gene was also used. These plasmids were transferred to minicellproducing strains; minicells were collected and their pattern of polypeptide synthesis was examined. The plasmid PLC21-2 programmed the synthesis of FP38, FP28, and FP8 (Fig. 3). The control plasmid PLC13-12 did not stimulate the synthesis of these polypeptides. It is clear from an examination of the polypeptide patterns found with deletions of the phages Xfla23 and Xfla42, as well as Xfla52 (which does not have cheB gene activity), that
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CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
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882
SILVERMAN ET AL.
the triplet polypeptides FP63, FP61, and FP60 are not correlated with cheB activity (Fig. 3). For example, Xfla23A22 and Xfla42A5 both show the triplet bands but do not show cheB activity in complementation tests. On the other hand, Xf7a42A9 and Xfla23A5 have cheB activity but do not show the triplet bands. However, since these polypeptides are subject to the regulation of the flaI gene, we presume that they have some role in flagellar function or flagellar structure. The three bands could be the result of a single gene or they may result from multiple genes. There is ample precedence for the product of a single gene appearing as a series of bands on acrylamide gels; flagellin, for example, can appear to have a molecular weight of 54,000, and under a variety of conditions of heating its apparent molecular weight changes to 56,000 and finally to 60,000. Intermediate mixtures of all three bands have been observed (8). Figure 4 shows the resolution of the triplet proteins and the flagellin protein on acrylamide gradient gels that were 23 cm long. The pattern of the protein in the triplet region can be resolved, and the multiple band pattern of the flagellin proteins can also be observed. The triplet protein generally appears as three bands. There is, however, some variation in the relative intensities of these bands. For example, in Xfla42A1, FP63 and FP61 are intense, whereas FP60 is faint. In the pattern obtained with X42A4 and A5, the 60,000 and 61,000 molecular weight bands appear to predominate, whereas the 63,000 appears to be very light. We have not as yet been able to correlate this change in intensity with either the method of preparation of the protein for acrylamide gel electrophoresis or with the genetic composition of specific deletions. To identify the triplet protein with one of the polypeptides involved in flagellar structure or function, it was compared to the polypeptide component of the flagellar basal structure which has an apparent molecular weight of 59,000 on acrylamide gels (7) and to the MCP described by Kort et al. (9) which has also been reported to have an apparent molecular weight of 60,000. These radioactive polypeptides were
compared by polyacrylamide gel electrophoresis under a variety of conditions. Figure 5 shows that MCP and the triplet protein have the same electrophoretic mobility. Both MCP and triplet show a distinctive three band pattern. The MCP pattern is seen when MCP is run alone or in a mixture with an extract from cells infected with Xfla7A5 which carried mot gene activity but not triplet. The triplet pattern is seen in extracts of cells infected with
J. BACTERIOL.
Xfla23A1 (Fig. 5 column 1 and 2 on left). When extracts containing both MCP and triplet are mixed and tested by electrophoresis, and when the film is exposed so that radioactivity corresponding to both triplet and MCP can be seen, the radioactive bands in the triplet region overlap completely with the MCP bands. In experiments involving two-dimensional acrylamide gels prepared by the technique of O'Farrell (11), both the MCP preparation and the triplet protein streaked in the isoelectric focusing direction. Even though there was heterogeneity, there was considerable overlap between MCP and the triplet bands, and both gave the same pattern. The flagellar basal component did not streak. Furthermore, in cell fractionation experiments both the 35S triplet protein and the methyl-3H-labeled MCP were found to be localized in the inner membrane of the cell. On the basis of these data, it is reasonable to propose the hypothesis that the triplet proteins are the same as MCP. The relative order of the cheB gene region, the region that codes for triplet and the distal portion of the cheA gene, can be deduced from the genetic properties of the deletion mutants. To test the hypothetical order, DNA was isolated from several hybrid lambda phages, and the endonuclease digestion pattern was compared to predictions made from genetic and complementation tests and protein synthesis experiments. In Fig. 6a, the EcoRI and BamI endonuclease fragments of DNA from the deletions of Xfla23 and Xfla42 are shown. The positions of the EcoRI and BamI digestion sites are known and it is therefore possible to assign fractional lengths to the fragments shown on the gels. The two EcoRI sites result from the insertion of the cheB cheA fragment into XflalA4. Two of the BamI sites are in the lambda portion of the DNA. The third site that generates a 2.8 fragment with one end derived from an EcoRI cut and the other end from BamI occurs close to or directly within the hag gene. The inserted piece of DNA corresponds to the 16.8 fragment. Deletions may occur within this fragment, thus changing its mobility on the gel. For example, in Xfla23A6, a region corresponding to approximately 7% of the total lambda genome was removed from the 16.8 fragment. Deletions that included the endonuclease cleavage site resulted in the fusion of two fragments; for example, in Xfla23A5, the 16.8 fragment and the 2.8 fragment are fused, generating a new band representing about 9% lambda. A similar but smaller deletion in Xfla23A1 gives a band corresponding to about 13% lambda. An approximate estimate of the
CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
VOL. 130, 1977
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CHARACTERIZATION OF LAMBDA E. COLI HYBRIDS
VOL. 130, 1977
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42A13 FIG. 6. Endonuclease mapping of DNA deletions in Xfla23 and Xf!a42. (a) The results of agarose gel electrophoresis after digestion with both EcoRI and BamI endonuclease. The numbers are the relative percentage of the length of wild-type lambda represented by each fragment. (b) The map derived from the endonuclease digestion, genetic complementation, and polypeptide synthesis experiments. The black bars represent the activities that are apparently deleted from each phage. Their length is not an accurate map length.
size of the deletion can be made from the loca- lated genes. The data derived from the analysis tion of the modified band on the agarose gel. of this phage taken together with previous reThe map shown in Fig. 6b is consistent with the sults allows us to develop an hypothesis about pattern in 6a. The length of the black line only the gene products involved in chemotaxis in E. roughly corresponds to actual distances on the coli. The evidence suggests that cheB is a complex locus made up of a number of genes, at map, but it indicates the functions that are missing in either genetic tests (cheB comple- least three. The following findings support this mentation and cheA recombination) or in pro- notion. (i) Most of the phages that carry cheB activity also synthesize, FP38, FP28, and FP8. tein synthesis. (ii) The phages Xf7a23A6 and Xfla23Al6 have DISCUSSION genetic complementation activity when tested The initial characterization of Xfla3Al indi- against MS5031, a cheB strain that carries the cates that it carried a number of flagellar-re- mutation el4nl, but they do not complement
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M85032, which carries the cheB mutation el3pl. They make FP8 but do not synthesize FP38 or FP28, suggesting that FP8 is sufficient to complement one of the cheB mutations but not the other. (iii) The phage Xfla23A5 shows genetic complementation with both tester strains. However, FP28 is missing and is replaced by a new band. One possible interpretation is that FP28 represents the product of another independent gene previously grouped as part of the cheB complex. (iv) These products are not restricted to the cheB genes carried on lambda. When the cheB gene is carried on the Col El vehicle, the same three polypeptides are synthesized. Thus, the cheB region specifies three polypeptides that are presumably encoded by three distinct genes. Futthermore, on the basis of the endonuclease maps, we can suggest that the genes are contiguous and that the gene coding for FP28 is adjacent to the triplet gene, the gene coding for FP38 would be next, and, finally, the gene coding for FP8 would be close to the restriction site (Fig. 6b). It is, of course, possible that there are even more genes included in this cheB complex, since the deletion that removed flaG and flaH may have also removed cheB genes. The relative complexity of this region suggests that it may be analogous to the che genes described in Salmonella by Warrick et al. (22). The triplet does not correspond to any known phenotype, and we have not as yet found mutants that map in this region. It is possible that the "triplet" region includes a number of genes; on the other hand, the multiple bands observed on acrylamide gels may be the result of modification of a single polypeptide. The bands appear to be the same as the MCP described by Kort et al. (9). In our experiments, the MCP shows a number of bands on acrylamide gels, and the triplet protein runs together with MCP both on SDS-gels, which separate on the basis of size, and on isoelectric focusing gels, which separate on the basis of charge. Furthermore, both the triplet and MCP are found exclusively in the inner membrane fraction of the cell. Finally, the synthesis of both proteins is regulated by the flagellar regulatory system. The identification of the position of the gene may allow specific mutants to be prepared. The phenotypes of such mutants could help in deducing the function of the polypeptides. The cheA gene group may also be complex. We have shown that when the integrity of the cheA region that had been interrupted by endonuclease restriction is restored, three polypeptides are produced. Thus, the cheA region may also contain more than one gene. In fact, FP12, which was observed as a product of polypeptide
J. BACTERIOL.
synthesis programmed by many of the phages described in this report, appears to be the same as FP12 previously demonstrated to be a product of the cheA region. Deletions that remove hag activity and part of the cheA region also result in the loss of FP12 synthesis (e.g., Xfla23A1, A2, A5, A15, A22; Fig. 2). FP12 may be associated directly with the hag gene or it may be the product of another che gene in the cheA region whose expression is stimulated by the adjacent hag promoter. All of the data argue that this region of the E. coli genome carries the genetic information to code for many flagellar-related polypeptides. Previous work has shown that there is a group of co-transcribed genes called mocha which program the synthesis of a motA, motB, and three che gene products. Adjacent to this cheA region is a triplet region that codes for three polypeptide bands. This may be the result of a single gene or as many as three genes. This is followed by the cheB region which appears to include at least three distinct genes. There may be more flagellar-related genes in the cheB region, since the experiments described in this paper used a phage which carried a deletion removing the region between "cheB" and flaG. It is apparent that a careful comparison of the genetic properties of the cloned lambda fragments and their deletions with the chemotaxis mutants will be necessary to completely analyze this region. There may be as many as 12 and no fewer than 8 gene products involved in flagellar function that are specified by the region of the genome included between the map positions of the flal gene and the flaG gene. This region in E. coli appears to be as complex as the analogous region described in Salmonella. ACKNOWLEDGMENTS This work was supported by grant BMS 13-01606 from the National Science Foundation. P. Matsumura is a NRSA Postdoctoral Research Fellow of National Institute of General Medical Sciences (GM 05249-01). We thank L. Clarke and J. Carbon for providing their hybrid plasmidcontaining strains and J. S. Parkinson for the chemotaxis strains.
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