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The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN1
Gene 189 (1997) 139–141
Short communication
The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN1 William J. Deakin, James L.C.I. Sanderson, Tapasree Goswami, Charles H. Shaw * Department of Biological Sciences, University of Durham, South Road, Durham, DH1 3LE, UK Received 30 July 1996; received in revised form 1 October 1996; accepted 4 October 1996; Received by A.M. Campbell
Abstract We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly. [ 1997 Elsevier Science B.V. All rights reserved. Keywords: Agrobacterium; Flagella; Switch protein; Motor; Motility
1. Introduction Crown Gall tumour formation by Agrobacterium tumefaciens involves the induction of the Ti plasmid virulence genes which mediate transfer of the T-DNA to the plant cell ( Winans, 1992; Zambryski, 1992). Plant wound phenolics attract the bacteria and mediate vir induction, both processes involving phosphate transfer in the two-component sensor-regulator system encoded by virA and virG (Palmer and Shaw, 1992; Shaw et al., 1988; Winans, 1992). To elucidate the molecular events underlying chemotaxis towards wounded plants, we have used transposon tagging to isolate genes concerned with motility and chemotaxis from A. tumefaciens. Eight of the transposon insertion sites map to a 25-kb region of the A. tumefaciens chromosome, contained in pDUB1900 (Shaw, 1996; Shaw et al., 1991). We have determined the nucleotide sequence of approx. 17 kb of * Corresponding author. Tel. +44 191 3742430; Fax +44 191 3742417; e-mail: [email protected] 1 On request, the authors will supply detailed experimental evidence for the conclusions reached in this Short Communication. Abbreviations: A., Agrobacterium; aa, amino acid(s); bp, base pair(s); C., Caulobacter; E., Escherichia; FliG, flagellar protein; fliG, gene encoding FliG; kb, kilobase(s) or 1000 bp; ORF, open reading frame; S., Salmonella; T-DNA, Ti plasmid transfer or tumour DNA; Ti plasmid, Agrobacterium tumour-inducing plasmid; vir, Agrobacterium virulence gene. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 53 - 0
pDUB1900, most of which appears to be concerned with flagellar structure and assembly. A 4-kb region towards the left end contains the A. tumefaciens homologues of the flagellar switch protein genes fliG, fliM and fliN Kihara et al., 1989; Kuo and Koshland, 1986; Malakooti et al., 1989), clustered into an apparent operon (see Fig. 1) commenced by the gene for the transmembrane proton channel motA (Blair and Berg, 1990).
2. Experimental and discussion The nucleotide sequence of 3978 bp of the motA operon ( Fig. 1) was completed (GenBank accession No. U63290). The operon is preceded by a class II flagellar consensus −10 sequence (GCCGATAT ) (Helmann and Chamberlin, 1988) followed 227 bp downstream by the start codon of motA. The A. tumefaciens motA is predicted to encode a protein of 290 aa, with a M of r 31 508, which shares 63% similarity with its Escherichia coli counterpart (Fig. 2). The predicted MotA has a hydropathic profile consistent with it being an integral membrane protein. The last four bases of motA overlap in an ATGA configuration with the start codon of fliM, whose protein product of 321 aa is predicted to have a M of 34 626, and shares 46% similarity with its r Caulobacter crescentus counterpart. An intergenic region
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W.J. Deakin et al. / Gene 189 (1997) 139–141
Fig. 1. Map of the 3978 bp presented here. The thin line represents the Agrobacterium chromosome, with restriction enzyme cleavage sites marked. Horizontal arrows represent the ORFs, and triangles signify the position of the Tn5 insertion sites. E, EcoRI; N, NruI; P, PstI; S, SacII; Sa, SalI; Sp, SpHI; X, XhoI.
Fig. 2. GAP alignment of A. tumefaciens MotA (top lines) and E. coli MotA (bottom lines). Vertical lines signify identity, two dots conservative substitutions, single dots semi-conservative substitutions.
of 63 bp separates fliM from fliN, whose predicted product of M 18 791 (179 aa) shares 62% similarity r with its C. crescentus counterpart and is some 4-kDa larger than the enteric homologues. This is largely accounted for by a 41 aa N-terminal extension. 27 bp beyond fliN lies the start codon of fliG, predicted to encode a 334 aa protein of M 37 055, sharing 52% r similarity with FliG of C. crescentus. Two non-flagellate mutants map to fliG (Shaw et al., 1991) with Tn5 insertion sites in codons 29 ( fla-3) and 156 ( fla-10). This confirms that the operon is expressed and that fliG at least is required for flagellar assembly in A. tumefaciens. 118 bp downstream from the fliG stop codon lies a sequence with the potential to form a stem-loop structure, followed by a run of seven T residues out of nine. This is predicted by the TERMINATOR program (Brendel et al., 1986) to be a good transcriptional terminator. Analysis of the complete sequence by the program TESTCODE confirms that the four identified ORFs are in all probability coding regions. Moreover they all possess reasonable Shine-Dalgarno sequences. The A. tumefaciens switch protein genes, like their
enteric counterparts, fliG from Salmonella typhimurium ( Kihara et al., 1989) and fliM and fliN from E. coli and S. typhimurium ( Kihara et al., 1989; Kuo and Koshland, 1986; Malakooti et al., 1989), do not appear to be integral membrane proteins. In the enteric bacteria, chemotactic stimuli are channelled through a cytoplasmic phosphorylation pathway, the final phosphate acceptor being CheY (Bourret et al., 1989; Stock et al., 1990). CheY-P promotes CW rotation, and thus tumbling, its activity being antagonised by CheZ. Both CheY and CheZ interact with the flagellar switch proteins FliG, FliM and FliN (Parkinson et al., 1983; Yamaguchi et al., 1986a) which probably exist as a complex ( Yamaguchi et al., 1986a). This complex is almost certainly closely associated with the basal body (Aizawa, 1996), and interacts with the ‘motor’ proteins MotA and MotB ( Yamaguchi et al., 1986a,b). In E. coli, fliM and fliN are consecutive genes in the fliL operon, fliG is in the adjacent fliF operon, both mapping to the region IIIb flagellar genes at approx. 43∞ on the E. coli chromosome (Macnab, 1987, 1990). All three are class-2 genes, expressed early in the cascade of flagellar regulons, and their products incorporated into some of the earliest recognisable flagellar assembly intermediates (Macnab, 1990). Thus, mutations in these genes often produce a non-flagellated phenotype ( Yamaguchi et al., 1986a). In contrast, motA is the first gene in a class 3 operon mapping at 42∞ on the E. coli chromosome containing also motB, cheA and cheW (Macnab, 1990). Thus the organisation of flagellar operons in A. tumefaciens is significantly different from that of the enteric bacteria. Moreover, it would appear that in Agrobacterium MotA is required much earlier in the flagellar assembly pathway. Whether these differences are due to the altered motility and chemotactic behaviour of A. tumefaciens awaits subsequent analysis.
Acknowledgement The authors would like to thank David Humphreys and Nick Watkins for some early plasmid preparations, and Julia Bartley for assistance with DNA sequencing.
References Aizawa, S.-I. (1996) Flagellar assembly in Salmonella typhimurium. Mol. Microbiol. 19, 1–5. Blair, D.F. and Berg, H.C. (1990) The MotA protein of E. coli is a proton-conducting component of the flagellar motor. Cell 60, 439–449. Bourret, R.B., Hess, J.F., Borkovich, K.A., Pakula, A. and Simon, M.I. (1989) Protein phosphorylation in chemotaxis and two component regulatory systems of bacteria. J. Biol. Chem. 264, 7085–7088. Brendel, V., Hamm, G.H. and Trifonov, E.N. (1986) Terminators of
W.J. Deakin et al. / Gene 189 (1997) 139–141 transcription with RNA polymerase from Escherichia coli: what they look like and how to find them. J. Biomol. Struct. Dyn. 3, 705–723. Helmann, J.D. and Chamberlin, M.J. (1988) Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57, 839–872. Kihara, M., Homma, M., Kutsukake, K. and Macnab, R.M. (1989) Flagellar switch of Salmonella typhimurium: gene sequences and deduced protein sequences. J. Bacteriol. 171, 3247–3257. Kuo, S.C. and Koshland, D.E. (1986) Sequence of the flaA (cheC ) locus of Escherichia coli and discovery of a new gene. J. Bacteriol. 166, 1007–1012. Macnab, R.M. (1987) Flagella. In: Neidhardt, F.C. ( Ed.), E. coli and S. typhimurium. American Society for Microbiology, Washington, DC, pp. 70–83. Macnab, R.M. (1990) Genetics, structure and assembly of the bacterial flagellum. In: Armitage, J.P. and Lackie, J.M. ( Eds.), Biology of the Chemotactic Response. University of Cambridge Press, Cambridge, pp. 77–106. Malakooti, J., Komeda, Y. and Matsumura, P. (1989) DNA sequence analysis, gene product identification and localisation of flagellar motor components of Escherichia coli. J. Bacteriol. 171, 2728–2734. Palmer, A.C.V. and Shaw, C.H. (1992) The role of virA and G phosphorylation in acetosyringone chemotaxis. J. Gen. Microbiol. 138, 2509–2514. Parkinson, J.S., Parker, S.R., Talbert, P.B. and Houts, S.E. (1983) Interactions between chemotaxis genes and flagellar genes in Escherichia coli. J. Bacteriol. 155, 265–274.
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Shaw, C.H. (1996) The early molecular events in the Agrobacteriumplant interaction. In: Anderson, H. ( Ed.), Plant Roots – Cells to Systems. Kluwer, Dordrecht. Shaw, C.H., Ashby, A.M., Brown, A.P., Royal, C., Loake, G.J. and Shaw, C.H. (1988) virA and G are the Ti-plasmid functions required for chemotaxis of Agrobacterium tumefaciens toward acetosyringone. Mol. Microbiol. 2, 413–418. Shaw, C.H., Loake, G.J., Brown, A.P., Garrett, C.S., Deakin, W., Alton, G., Hall, M., Jones, S.A., O’Leary, M. and Primavesi, L. (1991) Isolation and characterisation of behavioural mutants and genes from Agrobacterium tumefaciens. J. Gen. Microbiol. 137, 1939–1953. Stock, J.B., Stock, A.M. and Mottonen, J.M. (1990) Signal transduction in bacteria. Nature 344, 395–400. Winans, S.C. (1992) Two-way chemical signalling in Agrobacteriumplant interactions. Microbiol. Rev. 56, 12–31. Yamaguchi, S., Aizawa, S.-I., Kihara, M., Isomura, M., Jones, C.J. and Macnab, R.M. (1986a) Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium. J. Bacteriol. 168, 1172–1179. Yamaguchi, S., Fujita, H., Ishihara, A., Aizawa, S.-I. and Macnab, R.M. (1986b) Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation and switching. J. Bacteriol. 166, 187–193. Zambryski, P. (1992) Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu. Rev. Plant Physiol. 43, 465–490.
Short communication
The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN1 William J. Deakin, James L.C.I. Sanderson, Tapasree Goswami, Charles H. Shaw * Department of Biological Sciences, University of Durham, South Road, Durham, DH1 3LE, UK Received 30 July 1996; received in revised form 1 October 1996; accepted 4 October 1996; Received by A.M. Campbell
Abstract We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly. [ 1997 Elsevier Science B.V. All rights reserved. Keywords: Agrobacterium; Flagella; Switch protein; Motor; Motility
1. Introduction Crown Gall tumour formation by Agrobacterium tumefaciens involves the induction of the Ti plasmid virulence genes which mediate transfer of the T-DNA to the plant cell ( Winans, 1992; Zambryski, 1992). Plant wound phenolics attract the bacteria and mediate vir induction, both processes involving phosphate transfer in the two-component sensor-regulator system encoded by virA and virG (Palmer and Shaw, 1992; Shaw et al., 1988; Winans, 1992). To elucidate the molecular events underlying chemotaxis towards wounded plants, we have used transposon tagging to isolate genes concerned with motility and chemotaxis from A. tumefaciens. Eight of the transposon insertion sites map to a 25-kb region of the A. tumefaciens chromosome, contained in pDUB1900 (Shaw, 1996; Shaw et al., 1991). We have determined the nucleotide sequence of approx. 17 kb of * Corresponding author. Tel. +44 191 3742430; Fax +44 191 3742417; e-mail: [email protected] 1 On request, the authors will supply detailed experimental evidence for the conclusions reached in this Short Communication. Abbreviations: A., Agrobacterium; aa, amino acid(s); bp, base pair(s); C., Caulobacter; E., Escherichia; FliG, flagellar protein; fliG, gene encoding FliG; kb, kilobase(s) or 1000 bp; ORF, open reading frame; S., Salmonella; T-DNA, Ti plasmid transfer or tumour DNA; Ti plasmid, Agrobacterium tumour-inducing plasmid; vir, Agrobacterium virulence gene. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 53 - 0
pDUB1900, most of which appears to be concerned with flagellar structure and assembly. A 4-kb region towards the left end contains the A. tumefaciens homologues of the flagellar switch protein genes fliG, fliM and fliN Kihara et al., 1989; Kuo and Koshland, 1986; Malakooti et al., 1989), clustered into an apparent operon (see Fig. 1) commenced by the gene for the transmembrane proton channel motA (Blair and Berg, 1990).
2. Experimental and discussion The nucleotide sequence of 3978 bp of the motA operon ( Fig. 1) was completed (GenBank accession No. U63290). The operon is preceded by a class II flagellar consensus −10 sequence (GCCGATAT ) (Helmann and Chamberlin, 1988) followed 227 bp downstream by the start codon of motA. The A. tumefaciens motA is predicted to encode a protein of 290 aa, with a M of r 31 508, which shares 63% similarity with its Escherichia coli counterpart (Fig. 2). The predicted MotA has a hydropathic profile consistent with it being an integral membrane protein. The last four bases of motA overlap in an ATGA configuration with the start codon of fliM, whose protein product of 321 aa is predicted to have a M of 34 626, and shares 46% similarity with its r Caulobacter crescentus counterpart. An intergenic region
140
W.J. Deakin et al. / Gene 189 (1997) 139–141
Fig. 1. Map of the 3978 bp presented here. The thin line represents the Agrobacterium chromosome, with restriction enzyme cleavage sites marked. Horizontal arrows represent the ORFs, and triangles signify the position of the Tn5 insertion sites. E, EcoRI; N, NruI; P, PstI; S, SacII; Sa, SalI; Sp, SpHI; X, XhoI.
Fig. 2. GAP alignment of A. tumefaciens MotA (top lines) and E. coli MotA (bottom lines). Vertical lines signify identity, two dots conservative substitutions, single dots semi-conservative substitutions.
of 63 bp separates fliM from fliN, whose predicted product of M 18 791 (179 aa) shares 62% similarity r with its C. crescentus counterpart and is some 4-kDa larger than the enteric homologues. This is largely accounted for by a 41 aa N-terminal extension. 27 bp beyond fliN lies the start codon of fliG, predicted to encode a 334 aa protein of M 37 055, sharing 52% r similarity with FliG of C. crescentus. Two non-flagellate mutants map to fliG (Shaw et al., 1991) with Tn5 insertion sites in codons 29 ( fla-3) and 156 ( fla-10). This confirms that the operon is expressed and that fliG at least is required for flagellar assembly in A. tumefaciens. 118 bp downstream from the fliG stop codon lies a sequence with the potential to form a stem-loop structure, followed by a run of seven T residues out of nine. This is predicted by the TERMINATOR program (Brendel et al., 1986) to be a good transcriptional terminator. Analysis of the complete sequence by the program TESTCODE confirms that the four identified ORFs are in all probability coding regions. Moreover they all possess reasonable Shine-Dalgarno sequences. The A. tumefaciens switch protein genes, like their
enteric counterparts, fliG from Salmonella typhimurium ( Kihara et al., 1989) and fliM and fliN from E. coli and S. typhimurium ( Kihara et al., 1989; Kuo and Koshland, 1986; Malakooti et al., 1989), do not appear to be integral membrane proteins. In the enteric bacteria, chemotactic stimuli are channelled through a cytoplasmic phosphorylation pathway, the final phosphate acceptor being CheY (Bourret et al., 1989; Stock et al., 1990). CheY-P promotes CW rotation, and thus tumbling, its activity being antagonised by CheZ. Both CheY and CheZ interact with the flagellar switch proteins FliG, FliM and FliN (Parkinson et al., 1983; Yamaguchi et al., 1986a) which probably exist as a complex ( Yamaguchi et al., 1986a). This complex is almost certainly closely associated with the basal body (Aizawa, 1996), and interacts with the ‘motor’ proteins MotA and MotB ( Yamaguchi et al., 1986a,b). In E. coli, fliM and fliN are consecutive genes in the fliL operon, fliG is in the adjacent fliF operon, both mapping to the region IIIb flagellar genes at approx. 43∞ on the E. coli chromosome (Macnab, 1987, 1990). All three are class-2 genes, expressed early in the cascade of flagellar regulons, and their products incorporated into some of the earliest recognisable flagellar assembly intermediates (Macnab, 1990). Thus, mutations in these genes often produce a non-flagellated phenotype ( Yamaguchi et al., 1986a). In contrast, motA is the first gene in a class 3 operon mapping at 42∞ on the E. coli chromosome containing also motB, cheA and cheW (Macnab, 1990). Thus the organisation of flagellar operons in A. tumefaciens is significantly different from that of the enteric bacteria. Moreover, it would appear that in Agrobacterium MotA is required much earlier in the flagellar assembly pathway. Whether these differences are due to the altered motility and chemotactic behaviour of A. tumefaciens awaits subsequent analysis.
Acknowledgement The authors would like to thank David Humphreys and Nick Watkins for some early plasmid preparations, and Julia Bartley for assistance with DNA sequencing.
References Aizawa, S.-I. (1996) Flagellar assembly in Salmonella typhimurium. Mol. Microbiol. 19, 1–5. Blair, D.F. and Berg, H.C. (1990) The MotA protein of E. coli is a proton-conducting component of the flagellar motor. Cell 60, 439–449. Bourret, R.B., Hess, J.F., Borkovich, K.A., Pakula, A. and Simon, M.I. (1989) Protein phosphorylation in chemotaxis and two component regulatory systems of bacteria. J. Biol. Chem. 264, 7085–7088. Brendel, V., Hamm, G.H. and Trifonov, E.N. (1986) Terminators of
W.J. Deakin et al. / Gene 189 (1997) 139–141 transcription with RNA polymerase from Escherichia coli: what they look like and how to find them. J. Biomol. Struct. Dyn. 3, 705–723. Helmann, J.D. and Chamberlin, M.J. (1988) Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57, 839–872. Kihara, M., Homma, M., Kutsukake, K. and Macnab, R.M. (1989) Flagellar switch of Salmonella typhimurium: gene sequences and deduced protein sequences. J. Bacteriol. 171, 3247–3257. Kuo, S.C. and Koshland, D.E. (1986) Sequence of the flaA (cheC ) locus of Escherichia coli and discovery of a new gene. J. Bacteriol. 166, 1007–1012. Macnab, R.M. (1987) Flagella. In: Neidhardt, F.C. ( Ed.), E. coli and S. typhimurium. American Society for Microbiology, Washington, DC, pp. 70–83. Macnab, R.M. (1990) Genetics, structure and assembly of the bacterial flagellum. In: Armitage, J.P. and Lackie, J.M. ( Eds.), Biology of the Chemotactic Response. University of Cambridge Press, Cambridge, pp. 77–106. Malakooti, J., Komeda, Y. and Matsumura, P. (1989) DNA sequence analysis, gene product identification and localisation of flagellar motor components of Escherichia coli. J. Bacteriol. 171, 2728–2734. Palmer, A.C.V. and Shaw, C.H. (1992) The role of virA and G phosphorylation in acetosyringone chemotaxis. J. Gen. Microbiol. 138, 2509–2514. Parkinson, J.S., Parker, S.R., Talbert, P.B. and Houts, S.E. (1983) Interactions between chemotaxis genes and flagellar genes in Escherichia coli. J. Bacteriol. 155, 265–274.
141
Shaw, C.H. (1996) The early molecular events in the Agrobacteriumplant interaction. In: Anderson, H. ( Ed.), Plant Roots – Cells to Systems. Kluwer, Dordrecht. Shaw, C.H., Ashby, A.M., Brown, A.P., Royal, C., Loake, G.J. and Shaw, C.H. (1988) virA and G are the Ti-plasmid functions required for chemotaxis of Agrobacterium tumefaciens toward acetosyringone. Mol. Microbiol. 2, 413–418. Shaw, C.H., Loake, G.J., Brown, A.P., Garrett, C.S., Deakin, W., Alton, G., Hall, M., Jones, S.A., O’Leary, M. and Primavesi, L. (1991) Isolation and characterisation of behavioural mutants and genes from Agrobacterium tumefaciens. J. Gen. Microbiol. 137, 1939–1953. Stock, J.B., Stock, A.M. and Mottonen, J.M. (1990) Signal transduction in bacteria. Nature 344, 395–400. Winans, S.C. (1992) Two-way chemical signalling in Agrobacteriumplant interactions. Microbiol. Rev. 56, 12–31. Yamaguchi, S., Aizawa, S.-I., Kihara, M., Isomura, M., Jones, C.J. and Macnab, R.M. (1986a) Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium. J. Bacteriol. 168, 1172–1179. Yamaguchi, S., Fujita, H., Ishihara, A., Aizawa, S.-I. and Macnab, R.M. (1986b) Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation and switching. J. Bacteriol. 166, 187–193. Zambryski, P. (1992) Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu. Rev. Plant Physiol. 43, 465–490.