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The sigma-70 subunit from Escherichia coli C differs from that of E. coli K-12
Gene, 162 (1995) 161-162 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
161
GENE 09051
The sigma-70 subunit from Escherichia coli C differs from that of E. coli K-12* (Nucleotide sequence; ~ factor; RNA polymerase)
Gail E. Christie and Sara B. Cale Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298-0678, USA Received by S.R. Kushner: 22 March 1995; Revised/Accepted: 13 April/23 April 1995; Received at publishers: 15 May 1995
SUMMARY
The nucleotide sequence of the rpoD gene (encoding the primary sigma-70 (~7o) subunit of RNA polymerase) from Escherichia coli C was determined. This gene differs from that of E. coli K-12 by a 30-bp deletion and five single-bp substitutions, resulting in a o 7° subunit with three amino acid (aa) changes and a deletion of ten acidic aa.
Strain C of E. coli lacks a type-I restriction/modification system, is a good host for a number of bacteriophages and recombines readily with E. coli K-12. Not surprisingly, the limited genetic mapping data available for E. coli C (Wiman et al., 1970) suggest that it is very similar to strain K-12. However, the transcriptional apparatus of E. coli C must differ from that of K-12, as evidenced by a difference in the phenotypes conferred by certain mutations (Sauer, 1979; G.E.C., unpublished observations) and a difference in the electrophoretic mobility of the primary ~ factors (Harris et al., 1977). Since cr7° of E. coli K-12 has recently been shown to be a target for some transcriptional regulatory proteins (Li et al., 1994; Kuldell and Hochschild, 1994), we were interested in comparing the primary cr factors from these two closely related bacterial strains. Sequence analysis of the E. coli C rpoD gene revealed several differences from E. coli K-12 (Fig. 1). The most Correspondence to: Dr. G.E. Christie, Department of Microbiology and Immunology, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA 23298-0678, USA. Tel. (1-804) 828-9093; Fax (1-804) 828-9946; e-mail: [email protected] *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); CAP, catabolite gene activator protein; nt, nucleotide(s); rpoD, gene encoding the primary sigma-70 (~7o) subunit of RNA polymerase. SSDI 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 3 3 3 - 9
striking variation was the deletion of a region encoding ten contiguous acidic aa. This deletion and one of the aa s u b s t i t u t i o n s , N 149 ~ D (K-12~C), lie between conserved
C K-12
268 268
Q CAA GAA E
90 270 270 90
C K-12
190 568 568 190
L G D D D S C T G G G C G A T G A C G A .............................. CAGC C TGGACGAT G A C G A A G A T G A A G A C G A A G A A G A T G G C G A T G A C G A C A G C L D D D E D E D E E D G D D D S
C K-12
H 1333 CAC 1363 CAT H
445 1335 1365 455
445 445
D GAT AAT N
H 1681 CAC 1711 TAC Y
i49 447 447 149
195 585 615 205
561 "-683 1713 571
Fig. 1. A comparison of the nt sequence of rpoD from E. coli C with that of E. coli K-12 (GenBank accession No. J01687) and the deduced aa sequence. The aa numbering is shown in italics. Only those regions containing differences are shown; nt changes are highlighted in bold. The single D TM ~ G change and the 10-aa deletion after D TM (in E. coli C) result in the net deletion of a stretch of ten consecutive acidic aa, as indicated by the underlined region. The GenBank accession No. for the complete nt sequence of the E. coli C rpoD gene is U23083; this gene encodes a ~ polypeptide of 69029 Da with a predicted pl of 4.70. Sequencing reactions were performed with fluorescent dideoxynucleotides using the ABI DyeDeoxy TM cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and analyzed on an ABI automated sequencer in the Nucleic Acids Core Facility, Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University. Sequencing primers were synthetic oligodeoxyribonucleotides corresponding to sequences of the E. coli K-12 rpoD gene. Sequence data were stored and analyzed using the Genetics Computer Group (1991) software package.
162 regions 1 a n d 2 in the stretch of aa that is lacking in the G r a m + p r i m a r y ~ factors ( H e l l m a n a n d Chamberlin, 1988). A second aa variation, E 9 ° ~ Q , is located within region 1, but at a residue that is not conserved. The third single aa change affects a residue in region 4 (H 561 in E.
coli C, equivalent to
y571 in
K-12)
immediately
N - t e r m i n a l to the putative recognition helix for the - 3 5 p r o m o t e r region. The same aa variation, H ~ Y , is also found at this position in the p r i m a r y ~ factors of a n u m b e r of other bacterial species, including Salmonella
typhimurium, Pseudomonas aeruginosa, Myxococcus xanthus, Stigmatella aurantiaca, Lactococcus lactis a n d Agrobacterium tumefaciens ( G e n B a n k accession Nos. M14427, D90118, M32347, M94370, X71493 and X69388, respectively). An E. coli K-12 rpoD m u t a t i o n isolated on the basis of increased C A P - i n d e p e n d e n t /ac expression affects the same aa, y571---~C, and results in increased levels of transcription from a n u m b e r of m u t a n t
lac promoters (Siegele et al., 1989). It remains to be determined whether the n a t u r a l variation at this position has consequences for the regulation of gene expression.
ACKNOWLEDGEMENT This work was supported by American Cancer Society grant N P - 8 6 9 A to G.E.C.
REFERENCES Harris, J.D., Martinez, 1.1. and Calendar, R.: A gene from Escherichia coli affecting the sigma subunit of RNA polymerase. Proc. Natl. Acad. Sci. USA 74 (1977) 1836-1840. Helmann, J.D. and Chamberlin, M.J.: Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57 (19881 839 872. Genetics Computer Group: Program Manual for the GCG Package, Version 7, April 1991. 575 Science Drive, Madison, WI 53711, USA, 1991. Kuldelk N. and Hochschild, A.: Amino acid substitutions in the 35 recognition motif of cyv° that result in defects in phage ~ repressorstimulated transcription. J. Bacteriol. 176 (1994) 2991-2998. Li, M, Moyle, H. and Susskind, M.M.: Target of the transcriptional activation function of phage lambda cI protein. Science 263 (1994) 75 77. Sauer, B.L.: Regulation of Late Gene Expression in the Temperate Coliphage P2. Ph.D. Dissertation, University of California. Berkeley, CA, 1979. Siegele, D.A., Hu, J.C., Walter, W.A. and Gross, C.A.: Altered promoter recognition by mutant forms of the ry7° subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 206 (1989) 591 603. Wiman, M., Bertani, G., Kelly, B. and Sasaki, I.: Genetic map of Escherichia coli strain C. Mol. Gen. Genet. 107 (1970} 1 31.
161
GENE 09051
The sigma-70 subunit from Escherichia coli C differs from that of E. coli K-12* (Nucleotide sequence; ~ factor; RNA polymerase)
Gail E. Christie and Sara B. Cale Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298-0678, USA Received by S.R. Kushner: 22 March 1995; Revised/Accepted: 13 April/23 April 1995; Received at publishers: 15 May 1995
SUMMARY
The nucleotide sequence of the rpoD gene (encoding the primary sigma-70 (~7o) subunit of RNA polymerase) from Escherichia coli C was determined. This gene differs from that of E. coli K-12 by a 30-bp deletion and five single-bp substitutions, resulting in a o 7° subunit with three amino acid (aa) changes and a deletion of ten acidic aa.
Strain C of E. coli lacks a type-I restriction/modification system, is a good host for a number of bacteriophages and recombines readily with E. coli K-12. Not surprisingly, the limited genetic mapping data available for E. coli C (Wiman et al., 1970) suggest that it is very similar to strain K-12. However, the transcriptional apparatus of E. coli C must differ from that of K-12, as evidenced by a difference in the phenotypes conferred by certain mutations (Sauer, 1979; G.E.C., unpublished observations) and a difference in the electrophoretic mobility of the primary ~ factors (Harris et al., 1977). Since cr7° of E. coli K-12 has recently been shown to be a target for some transcriptional regulatory proteins (Li et al., 1994; Kuldell and Hochschild, 1994), we were interested in comparing the primary cr factors from these two closely related bacterial strains. Sequence analysis of the E. coli C rpoD gene revealed several differences from E. coli K-12 (Fig. 1). The most Correspondence to: Dr. G.E. Christie, Department of Microbiology and Immunology, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA 23298-0678, USA. Tel. (1-804) 828-9093; Fax (1-804) 828-9946; e-mail: [email protected] *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); CAP, catabolite gene activator protein; nt, nucleotide(s); rpoD, gene encoding the primary sigma-70 (~7o) subunit of RNA polymerase. SSDI 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 3 3 3 - 9
striking variation was the deletion of a region encoding ten contiguous acidic aa. This deletion and one of the aa s u b s t i t u t i o n s , N 149 ~ D (K-12~C), lie between conserved
C K-12
268 268
Q CAA GAA E
90 270 270 90
C K-12
190 568 568 190
L G D D D S C T G G G C G A T G A C G A .............................. CAGC C TGGACGAT G A C G A A G A T G A A G A C G A A G A A G A T G G C G A T G A C G A C A G C L D D D E D E D E E D G D D D S
C K-12
H 1333 CAC 1363 CAT H
445 1335 1365 455
445 445
D GAT AAT N
H 1681 CAC 1711 TAC Y
i49 447 447 149
195 585 615 205
561 "-683 1713 571
Fig. 1. A comparison of the nt sequence of rpoD from E. coli C with that of E. coli K-12 (GenBank accession No. J01687) and the deduced aa sequence. The aa numbering is shown in italics. Only those regions containing differences are shown; nt changes are highlighted in bold. The single D TM ~ G change and the 10-aa deletion after D TM (in E. coli C) result in the net deletion of a stretch of ten consecutive acidic aa, as indicated by the underlined region. The GenBank accession No. for the complete nt sequence of the E. coli C rpoD gene is U23083; this gene encodes a ~ polypeptide of 69029 Da with a predicted pl of 4.70. Sequencing reactions were performed with fluorescent dideoxynucleotides using the ABI DyeDeoxy TM cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and analyzed on an ABI automated sequencer in the Nucleic Acids Core Facility, Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University. Sequencing primers were synthetic oligodeoxyribonucleotides corresponding to sequences of the E. coli K-12 rpoD gene. Sequence data were stored and analyzed using the Genetics Computer Group (1991) software package.
162 regions 1 a n d 2 in the stretch of aa that is lacking in the G r a m + p r i m a r y ~ factors ( H e l l m a n a n d Chamberlin, 1988). A second aa variation, E 9 ° ~ Q , is located within region 1, but at a residue that is not conserved. The third single aa change affects a residue in region 4 (H 561 in E.
coli C, equivalent to
y571 in
K-12)
immediately
N - t e r m i n a l to the putative recognition helix for the - 3 5 p r o m o t e r region. The same aa variation, H ~ Y , is also found at this position in the p r i m a r y ~ factors of a n u m b e r of other bacterial species, including Salmonella
typhimurium, Pseudomonas aeruginosa, Myxococcus xanthus, Stigmatella aurantiaca, Lactococcus lactis a n d Agrobacterium tumefaciens ( G e n B a n k accession Nos. M14427, D90118, M32347, M94370, X71493 and X69388, respectively). An E. coli K-12 rpoD m u t a t i o n isolated on the basis of increased C A P - i n d e p e n d e n t /ac expression affects the same aa, y571---~C, and results in increased levels of transcription from a n u m b e r of m u t a n t
lac promoters (Siegele et al., 1989). It remains to be determined whether the n a t u r a l variation at this position has consequences for the regulation of gene expression.
ACKNOWLEDGEMENT This work was supported by American Cancer Society grant N P - 8 6 9 A to G.E.C.
REFERENCES Harris, J.D., Martinez, 1.1. and Calendar, R.: A gene from Escherichia coli affecting the sigma subunit of RNA polymerase. Proc. Natl. Acad. Sci. USA 74 (1977) 1836-1840. Helmann, J.D. and Chamberlin, M.J.: Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57 (19881 839 872. Genetics Computer Group: Program Manual for the GCG Package, Version 7, April 1991. 575 Science Drive, Madison, WI 53711, USA, 1991. Kuldelk N. and Hochschild, A.: Amino acid substitutions in the 35 recognition motif of cyv° that result in defects in phage ~ repressorstimulated transcription. J. Bacteriol. 176 (1994) 2991-2998. Li, M, Moyle, H. and Susskind, M.M.: Target of the transcriptional activation function of phage lambda cI protein. Science 263 (1994) 75 77. Sauer, B.L.: Regulation of Late Gene Expression in the Temperate Coliphage P2. Ph.D. Dissertation, University of California. Berkeley, CA, 1979. Siegele, D.A., Hu, J.C., Walter, W.A. and Gross, C.A.: Altered promoter recognition by mutant forms of the ry7° subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 206 (1989) 591 603. Wiman, M., Bertani, G., Kelly, B. and Sasaki, I.: Genetic map of Escherichia coli strain C. Mol. Gen. Genet. 107 (1970} 1 31.