JOURNAL OF BACTERIOLOGY, July 1990, p. 4106-4108 0021-9193/90/074106-03$02.00/0
Vol. 172, No. 7
Regulation of Escherichia coli OrF RNA Polymerase by flhD and flhC Flagellar Regulatory Genes DAVID N. ARNOSTIt Department of Chemistry, University of California, Berkeley, California 94720 Received 12 December 1989/Accepted 24 April 1990
The 7F RNA polymerase has been characterized biochemically and is known to transcribe several flagellar genes in Escherichwa coli. It was found that while the flagellar regulatory genesflhD andflhC are required for cr activity, the sizes of their corresponding gene products are inconsistent with their encoding (yF itself.
Biochemical characterization of the Escherichia coli crF factor indicated that this alternative sigma factor controls certain flageilar genes (1). Previous genetic studies showed that flagellar genes are organized in a complex transcriptional hierarchy (10-12, 15, 19). At the apex areflhD andflhC (9), whose sequences are homologous to portions of the flagellar-specific CD factor of Bacillus subtilis (2, 8, 18). A colleague and I suggested that FlhD and FlhC might constitute CrF (1); a similar two-component sigma factor controls late transcription in B. subtilis cells infected with phage SPOl (21). (F has an apparent molecular mass of 28 kilodaltons (kDa), larger than that expected for FlhC (22 kDa) (2). However, the mobility of sigma factors on sodium dodecyl sulfate (SDS)-polyacrylamide gels is often lower than expected (3, 6, 17). While no protein the size of FlhD (13 kDa) was required for reconstitution of uF activity (1), it is possible that this peptide was a minor contaminant in the core enzyme used. Alternatively, FlhD and FlhC may control UF activity or synthesis. To determine the relationship among crF, flhD, and flhC, I investigated the dependence of u on these genes and characterized their products. The initial characterization of CF indicated that the addition of a gel-purified 28-kDa peptide to core RNA polymerase reconstituted the activity in vitro (1). The core enzyme used was from a strain which may contain the FlhD and FlhC proteins, however. I determined that trace amounts of these proteins did not contribute to (rF activity in vitro by reconstituting the enzyme from core polymerase prepared from E. coli YK4519 (flhD::TnJO), which lacksfihD andflhC expression (data not shown). This result prompted me to investigate further the dependence of aF on flhD and flhC. Wild-type and mutant strains (Table 1) were grown in Luria broth (LB) (16) (30°C) with ampicillin (50 ,ug/ml) for strains carrying plasmids. One-liter cultures were harvested at the mid-logarithmic phase of growth (optical density at 600 nm, 0.8), extracts were prepared (5), and in vitro CF activity was assayed with a tar promoter-containing plasmid (1). Isogenic strains with flhD or flhC mutations lacked (rF activity (Fig. 1, lanes 3 to 6). (TheflhD mutation is not polar inflhC, as shown by complementation [Fig. 1, lanes 19 and 20]). The mixed FlhD-FlhC extract lacked activity (Fig. 1, lanes 7 and 8), suggesting that even when both products are combined in vitro, crF activity is not reconstituted. No inhibitory activity was detected when wild-type and mutant extracts were mixed (Fig. 1, lanes 9 to 12). Complementation of the mutations with appropriate plasmids (described be-
low) restored urF activity and motility, confirming that the lack of aF activity was indeed linked toflhD andflhC (Fig. 1, lanes 13 to 20). In this assay, one cannot distinguish between inactive crF and the absence of or'. The mixing experiments prove that no inhibitory substance could be detected, however; thus, the simplest interpretation is that a lesion inflhD or flhC results in a lack of (.F expression. Clearly, flhD andflhC are required for aF activity, yet the predicted sizes of their gene products differ from that of UF. flhD and flhC were cloned into an expression vector for characterization of the masses of the proteins (Fig. 2). This step was important for characterizing FlhD and for confirming that the protein identified by Bartlett et al. (2) was the physiological product of flhC. These investigators obtained no proteins when the entire flhD-flhC cistron was overexpressed, but a transcriptional fusion with only the latter portion led to the production of a 22-kDa protein. A 1.5kilobase DraI-HpaI fragment of pPM61 (2), which contains flhD andflhC and native promoter elements, was ligated into the HincII site of pUC18 (22). This construct, carryingflhD proximal to the EcoRI site, was digested with EcoRI and HindIII, and the fragment was ligated into the same sites of pT7-5, a plasmid containing a T7 promoter (20), to make pDNA40. A 510-base-pair SphI fragment containing most of flhC was removed to make pDNA402 (Fig. 2). To express flhD and flhC, E. coli MC4100 (flhD) was transformed with pGPl-2 (20), which contains the T7 RNA polymerase gene, and pDNA40, pDNA402, or pT7-5. Cells were grown in LB (30°C) with ampicillin and kanamycin (50 ,ug/ml) to an optical density at 600 nm of 0.5 and suspended in M9 medium (16) supplemented with thiamine (20 pLg/ml), amino acids (100 ,ug/ml) (except for cysteine and methioTABLE 1. E. coli strains and plasmids used in this study Strain or
Relevant genotype
Reference
plasmid
or comments
or source
Strains MC4100 YK0410 YK4136 YK4116 YK4519
Wild type
4 (flhD, 12) 10
flhC flhD flhD::TnlO
13 13 13
Plasmids pPM61 pDNA40 pDNA402
flhD-flhC T7 promoter-flhD-flhC T7 promoter-flhD
2
pT7-5
t Present address: Institute of Molecular Biology II, University of Zurich, Hoenggerberg, CH-8093 Zurich, Switzerland.
pGPl-2
4106
flhD
T7 promoter T7 RNA polymerase
This study This study 20 20
NOTES
VOL. 172, 1990
motility
-
+
+
+
flhC flhC flh D flhC + F wild+ + type flh C flh D flh D w.t. w.t. D/C D
4107
+ flh D
D/C
D A
"ar ..........
1
2
3 4
5 6
.'s
W-P
7 8 9 10 1112 13 14 15 16 17 18 19 20
FIG. 1. Primer extension reactions showing crF activity in extracts of wild-type E. coli and flagellar mutants. Extract preparation and in vitro transcription were performed as described in the text. Extract samples contained 51 ,ug (odd lanes) or 153 ,ug (even lanes) of protein. Lanes: 1 and 2, E. coli YK0410 (wild type); 3 and 4, YK4136 (flhC); 5 and 6, YK4116 (flhD); 7 and 8, 1:1 mixture of YK4136 and YK4116; 9 and 10, 1:1 mixture of YK4136 and YK0410; 11 and 12, 1:1 mixture of YK4116 and YK0410; 13 and 14, YK4136 transformed with pDNA40 (flhD and flhC); 15 and 16, YK4136 transformed with pDNA402 (flhD); 17 and 18, YK4116 transformed with pDNA40; 19 and 20, YK4116 transformed with pDNA402. The motility of the bacteria was determined by microscopy and is indicated by + or -. w.t., Wild type.
nine), and antibiotics. Growth was allowed to continue (30°C, 30 min), cells were heat shocked (42°C, 15 min) to induce T7 RNA polymerase synthesis, rifampin (200 ,ug/ml) was added, and cells were incubated at 42°C (10 min) and then shifted to 30°C (60 min). Samples (1 ml) were labeled by the addition of [35S]methionine (10 ,uCi, 1,129 Ci/mmol; Dupont, NEN Research Products, Boston, Mass.) for 5 min. Pulse-chase labeling was performed by the addition of [35S]methionine (10 ,uCi), incubation (30°C, 2 min), and the addition of L-methionine (to 0.2 mg/ml) for 0, 5, 15, or 45 min. Samples were centrifuged, and pellets were boiled in Laemmli buffer (14) prior to electrophoresis. Two prominent bands of 13 and 22 kDa were present in lysates of cells transformed with pDNA40 (flhD and flhC) (Fig. 3, lane 2). No proteins were synthesized in cells containing only pT7-5 (control, Fig. 3, lane 1). Only the 13-kDa band was present in cells carrying pDNA402 (flhD) (Fig. 3, lane 3); thus, the 22-kDa band corresponds to FlhC. These masses are exactly those predicted by the DNA sequences (2), and the mass of FlhC is identical to that obtained by Bartlett et al. (2). A less abundant 20-kDa
protein was associated with the intactflhC gene. Pulse-chase analysis demonstrated that the 22- and 13-kDa species did not exhibit a precursor-product relationship (Fig. 3, lanes 4 to 7). The aF factor migrated in this gel system (with identical molecular weight standards) at 28 kDa, a mass distinctly different from those of FlhD and FlhC. This result suggests thatflhD andflhC are not the structural genes for (rF but that they are regulators of the expression of the activity of CF. This study indicates that rF is integrated into the regulatory hierarchy of flagellar genes, subordinate to flhD and flhC. The aF structural gene, still unidentified, probably corresponds to a gene at an intermediate level of the transcriptional hierarchy, since CF iS thought to control genes, such asfliC (flagellin), at the lowest levels (1, 7). I found that extracts prepared from a fliC mutant contained cF activity, while a fliA mutant (fliA is an intermediate-level regulatory locus) lacked CrF activity (data not shown). Identification of the (rF gene will be an important step in determining how this factor functions in the flagellar gene hierarchy.
pPM61
mEco
33
1 kb.
_
X~'~ 3 -
(1
)0.
=
CL1 2:
-
Q
cn)
a.
I la I
I
pDNA40 pDNA402
FIG. 2. Portions of pPM61 used for construction of pDNA40 and pDNA402. The flhD and flhC genes were subcloned from pPM61 into pT7-5 behind a T7 RNA polymerase promoter. The resulting plasmids, pDNA40 (flhD and flhC) and pDNA402 (flhD), allowed low-level expression of these gene products from the native transcriptional signals located in the 0.5 kilobase (kb.) of DNA 5' to flhD or high-level expression from the T7 promoter.
4108
NOTES
J. BACTERIOL.
pulse-chase -
D/C D
0 0
5
1 15
45
kDa
1509266 51-
36-
26-
14-
._.._
KIMAlo
6 7 4 5 1 3 2 FIG. 3. Autoradiogram of 12% sodium dodecyl sultate-polyacrylamide gel containing [35S]methionine-labeled proteins. Lanes: 1, extracts of E. coli MC4100 (flhD) cells transformed with pGP1-2 (T7 RNA polymerase) and pT7-5 (vector); 2, pGP1-2-pDNA40 (flhD and flhC) transformants; 3, pGP1-2-pDNA402 (flhD) transformants; 4 to 7, pulse-chase analysis of [355]methionine-labeled proteins; chase periods of 0 (lane 4), 5 (lane 5), 15 (lane 6), or 45 (lane 7) min were used. Details of the procedure are described in Materials and Methods. Molecular mass standards were 150 kDa (,B and 1' subunits of E. coli RNA polymerase); 92 kDa (rabbit phosphorylase a); 66 kDa (bovine serum albumin); 51 kDa (Aspergillus a-amylase); 36 kDa (a subunit of E. coli RNA polymerase); 26 kDa (rabbit triosephosphate isomerase); and 14 kDa (horse cytochrome c).
Strains and plasmids used in this work were kindly provided by Sydney Kustu, Phil Matsumura, and Robert Macnab. Members of the Kustu laboratory provided helpful advice on the construction of expression plasmids. This research was supported by Public Health Service research grant GM 12010 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Arnosti, D. N., and M. J. Chamberlin. 1989. Secondary sigma factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. Proc. Natl. Acad. Sci. USA 86:830-834. 2. Bartlett, D. H., B. B. Frantz, and P. Matsumura. 1988. Flagellar transcriptional activators flbB and flaL: gene sequences and 5' consensus sequences of operons underflbB and flaI control. J. Bacteriol. 170:1575-1581. 3. Burton, Z., B. B. Burgess, J. Lin, D. Moore, S. Holder, and C. Gross. 1981. The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit for E. coli K-12. Nucleic Acids Res. 9:2889-2903.
4. Casadaban, M. J. 1976. Transposition and fusion of the lac
genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104:541-555. 5. Farr, S. F., D. N. Arnosti, M. J. Chamberlin, and B. N. Ames. 1989. An apaH mutation causes AppppA to accumulate and affects motility and catabolite repression in Escherichia coli. Proc. Natl. Acad. Sci. USA 86:4061-4065. 6. Gift, M. A., L.-F. Wang, and R. H. Doi. 1985. A strong homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli. J. Biol. Chem. 260:7178-7185. 7. Hemann, J. D., and M. J. Chamberlin. 1987. DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc. Natl. Acad. Sci. USA 84:6422-6424. 8. Helmann, J. D., L. M. Marquez, and M. J. Chamberlin. 1988. Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene. J. Bacteriol. 170:1568-1574. 9. Iino, T., Y. Komeda, K. Kutsukake, R. M. Macnab, P. Matusumura, J. S. Parkinson, M. I. Simon, and S. Yamaguchi. 1988. New unified nomenclature for the flagellar genes of Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 52:533-535. 10. Komeda, Y. 1982. Fusions of flagellar operons to lactose genes on a Mu lac bacteriophage. J. Bacteriol. 150:16-26. 11. Komeda, Y. 1986. Transcriptional control of flagellar genes in Escherichia coli. J. Bacteriol. 168:1315-1318. 12. Komeda, Y., and T. Iino. 1979. Regulation of expression of the flagellin gene (hag) in Escherichia coli K-12: analysis of hag-lac gene fusions. J. Bacteriol. 139:7221-7229. 13. Komeda, Y., K. Kutsukake, and T. Iino. 1980. Definition of additional flagellar genes in Escherichia coli K12. Genetics 94:277-290. 14. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. J. Mol. Biol. 80:575-579. 15. Macnab, R. M. 1987. Flagella, p. 70-83. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, and M. Schaechter (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Merrich, M. J., and J. R. Gibbins. 1985. The nucleotide sequence of the nitrogen-regulated gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res. 13:76077620. 18. Mirel, D. B., and M. J. Chamberlin. 1989. The Bacillus subtilis flagellin gene (hag) is transcribed by the sigma 28 form of RNA polymerase. J. Bacteriol. 171:3095-3101. 19. Silvermann, M., and M. Simon. 1977. Bacterial flagella. Annu. Rev. Microbiol. 31:397-419. 20. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled expression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074-1078. 21. Tjian, R., and J. Pero. 1976. Bacteriophage SPOl regulatory proteins directing late gene transcription in vitro. Nature (London) 262:753-757. 22. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.
Vol. 172, No. 7
Regulation of Escherichia coli OrF RNA Polymerase by flhD and flhC Flagellar Regulatory Genes DAVID N. ARNOSTIt Department of Chemistry, University of California, Berkeley, California 94720 Received 12 December 1989/Accepted 24 April 1990
The 7F RNA polymerase has been characterized biochemically and is known to transcribe several flagellar genes in Escherichwa coli. It was found that while the flagellar regulatory genesflhD andflhC are required for cr activity, the sizes of their corresponding gene products are inconsistent with their encoding (yF itself.
Biochemical characterization of the Escherichia coli crF factor indicated that this alternative sigma factor controls certain flageilar genes (1). Previous genetic studies showed that flagellar genes are organized in a complex transcriptional hierarchy (10-12, 15, 19). At the apex areflhD andflhC (9), whose sequences are homologous to portions of the flagellar-specific CD factor of Bacillus subtilis (2, 8, 18). A colleague and I suggested that FlhD and FlhC might constitute CrF (1); a similar two-component sigma factor controls late transcription in B. subtilis cells infected with phage SPOl (21). (F has an apparent molecular mass of 28 kilodaltons (kDa), larger than that expected for FlhC (22 kDa) (2). However, the mobility of sigma factors on sodium dodecyl sulfate (SDS)-polyacrylamide gels is often lower than expected (3, 6, 17). While no protein the size of FlhD (13 kDa) was required for reconstitution of uF activity (1), it is possible that this peptide was a minor contaminant in the core enzyme used. Alternatively, FlhD and FlhC may control UF activity or synthesis. To determine the relationship among crF, flhD, and flhC, I investigated the dependence of u on these genes and characterized their products. The initial characterization of CF indicated that the addition of a gel-purified 28-kDa peptide to core RNA polymerase reconstituted the activity in vitro (1). The core enzyme used was from a strain which may contain the FlhD and FlhC proteins, however. I determined that trace amounts of these proteins did not contribute to (rF activity in vitro by reconstituting the enzyme from core polymerase prepared from E. coli YK4519 (flhD::TnJO), which lacksfihD andflhC expression (data not shown). This result prompted me to investigate further the dependence of aF on flhD and flhC. Wild-type and mutant strains (Table 1) were grown in Luria broth (LB) (16) (30°C) with ampicillin (50 ,ug/ml) for strains carrying plasmids. One-liter cultures were harvested at the mid-logarithmic phase of growth (optical density at 600 nm, 0.8), extracts were prepared (5), and in vitro CF activity was assayed with a tar promoter-containing plasmid (1). Isogenic strains with flhD or flhC mutations lacked (rF activity (Fig. 1, lanes 3 to 6). (TheflhD mutation is not polar inflhC, as shown by complementation [Fig. 1, lanes 19 and 20]). The mixed FlhD-FlhC extract lacked activity (Fig. 1, lanes 7 and 8), suggesting that even when both products are combined in vitro, crF activity is not reconstituted. No inhibitory activity was detected when wild-type and mutant extracts were mixed (Fig. 1, lanes 9 to 12). Complementation of the mutations with appropriate plasmids (described be-
low) restored urF activity and motility, confirming that the lack of aF activity was indeed linked toflhD andflhC (Fig. 1, lanes 13 to 20). In this assay, one cannot distinguish between inactive crF and the absence of or'. The mixing experiments prove that no inhibitory substance could be detected, however; thus, the simplest interpretation is that a lesion inflhD or flhC results in a lack of (.F expression. Clearly, flhD andflhC are required for aF activity, yet the predicted sizes of their gene products differ from that of UF. flhD and flhC were cloned into an expression vector for characterization of the masses of the proteins (Fig. 2). This step was important for characterizing FlhD and for confirming that the protein identified by Bartlett et al. (2) was the physiological product of flhC. These investigators obtained no proteins when the entire flhD-flhC cistron was overexpressed, but a transcriptional fusion with only the latter portion led to the production of a 22-kDa protein. A 1.5kilobase DraI-HpaI fragment of pPM61 (2), which contains flhD andflhC and native promoter elements, was ligated into the HincII site of pUC18 (22). This construct, carryingflhD proximal to the EcoRI site, was digested with EcoRI and HindIII, and the fragment was ligated into the same sites of pT7-5, a plasmid containing a T7 promoter (20), to make pDNA40. A 510-base-pair SphI fragment containing most of flhC was removed to make pDNA402 (Fig. 2). To express flhD and flhC, E. coli MC4100 (flhD) was transformed with pGPl-2 (20), which contains the T7 RNA polymerase gene, and pDNA40, pDNA402, or pT7-5. Cells were grown in LB (30°C) with ampicillin and kanamycin (50 ,ug/ml) to an optical density at 600 nm of 0.5 and suspended in M9 medium (16) supplemented with thiamine (20 pLg/ml), amino acids (100 ,ug/ml) (except for cysteine and methioTABLE 1. E. coli strains and plasmids used in this study Strain or
Relevant genotype
Reference
plasmid
or comments
or source
Strains MC4100 YK0410 YK4136 YK4116 YK4519
Wild type
4 (flhD, 12) 10
flhC flhD flhD::TnlO
13 13 13
Plasmids pPM61 pDNA40 pDNA402
flhD-flhC T7 promoter-flhD-flhC T7 promoter-flhD
2
pT7-5
t Present address: Institute of Molecular Biology II, University of Zurich, Hoenggerberg, CH-8093 Zurich, Switzerland.
pGPl-2
4106
flhD
T7 promoter T7 RNA polymerase
This study This study 20 20
NOTES
VOL. 172, 1990
motility
-
+
+
+
flhC flhC flh D flhC + F wild+ + type flh C flh D flh D w.t. w.t. D/C D
4107
+ flh D
D/C
D A
"ar ..........
1
2
3 4
5 6
.'s
W-P
7 8 9 10 1112 13 14 15 16 17 18 19 20
FIG. 1. Primer extension reactions showing crF activity in extracts of wild-type E. coli and flagellar mutants. Extract preparation and in vitro transcription were performed as described in the text. Extract samples contained 51 ,ug (odd lanes) or 153 ,ug (even lanes) of protein. Lanes: 1 and 2, E. coli YK0410 (wild type); 3 and 4, YK4136 (flhC); 5 and 6, YK4116 (flhD); 7 and 8, 1:1 mixture of YK4136 and YK4116; 9 and 10, 1:1 mixture of YK4136 and YK0410; 11 and 12, 1:1 mixture of YK4116 and YK0410; 13 and 14, YK4136 transformed with pDNA40 (flhD and flhC); 15 and 16, YK4136 transformed with pDNA402 (flhD); 17 and 18, YK4116 transformed with pDNA40; 19 and 20, YK4116 transformed with pDNA402. The motility of the bacteria was determined by microscopy and is indicated by + or -. w.t., Wild type.
nine), and antibiotics. Growth was allowed to continue (30°C, 30 min), cells were heat shocked (42°C, 15 min) to induce T7 RNA polymerase synthesis, rifampin (200 ,ug/ml) was added, and cells were incubated at 42°C (10 min) and then shifted to 30°C (60 min). Samples (1 ml) were labeled by the addition of [35S]methionine (10 ,uCi, 1,129 Ci/mmol; Dupont, NEN Research Products, Boston, Mass.) for 5 min. Pulse-chase labeling was performed by the addition of [35S]methionine (10 ,uCi), incubation (30°C, 2 min), and the addition of L-methionine (to 0.2 mg/ml) for 0, 5, 15, or 45 min. Samples were centrifuged, and pellets were boiled in Laemmli buffer (14) prior to electrophoresis. Two prominent bands of 13 and 22 kDa were present in lysates of cells transformed with pDNA40 (flhD and flhC) (Fig. 3, lane 2). No proteins were synthesized in cells containing only pT7-5 (control, Fig. 3, lane 1). Only the 13-kDa band was present in cells carrying pDNA402 (flhD) (Fig. 3, lane 3); thus, the 22-kDa band corresponds to FlhC. These masses are exactly those predicted by the DNA sequences (2), and the mass of FlhC is identical to that obtained by Bartlett et al. (2). A less abundant 20-kDa
protein was associated with the intactflhC gene. Pulse-chase analysis demonstrated that the 22- and 13-kDa species did not exhibit a precursor-product relationship (Fig. 3, lanes 4 to 7). The aF factor migrated in this gel system (with identical molecular weight standards) at 28 kDa, a mass distinctly different from those of FlhD and FlhC. This result suggests thatflhD andflhC are not the structural genes for (rF but that they are regulators of the expression of the activity of CF. This study indicates that rF is integrated into the regulatory hierarchy of flagellar genes, subordinate to flhD and flhC. The aF structural gene, still unidentified, probably corresponds to a gene at an intermediate level of the transcriptional hierarchy, since CF iS thought to control genes, such asfliC (flagellin), at the lowest levels (1, 7). I found that extracts prepared from a fliC mutant contained cF activity, while a fliA mutant (fliA is an intermediate-level regulatory locus) lacked CrF activity (data not shown). Identification of the (rF gene will be an important step in determining how this factor functions in the flagellar gene hierarchy.
pPM61
mEco
33
1 kb.
_
X~'~ 3 -
(1
)0.
=
CL1 2:
-
Q
cn)
a.
I la I
I
pDNA40 pDNA402
FIG. 2. Portions of pPM61 used for construction of pDNA40 and pDNA402. The flhD and flhC genes were subcloned from pPM61 into pT7-5 behind a T7 RNA polymerase promoter. The resulting plasmids, pDNA40 (flhD and flhC) and pDNA402 (flhD), allowed low-level expression of these gene products from the native transcriptional signals located in the 0.5 kilobase (kb.) of DNA 5' to flhD or high-level expression from the T7 promoter.
4108
NOTES
J. BACTERIOL.
pulse-chase -
D/C D
0 0
5
1 15
45
kDa
1509266 51-
36-
26-
14-
._.._
KIMAlo
6 7 4 5 1 3 2 FIG. 3. Autoradiogram of 12% sodium dodecyl sultate-polyacrylamide gel containing [35S]methionine-labeled proteins. Lanes: 1, extracts of E. coli MC4100 (flhD) cells transformed with pGP1-2 (T7 RNA polymerase) and pT7-5 (vector); 2, pGP1-2-pDNA40 (flhD and flhC) transformants; 3, pGP1-2-pDNA402 (flhD) transformants; 4 to 7, pulse-chase analysis of [355]methionine-labeled proteins; chase periods of 0 (lane 4), 5 (lane 5), 15 (lane 6), or 45 (lane 7) min were used. Details of the procedure are described in Materials and Methods. Molecular mass standards were 150 kDa (,B and 1' subunits of E. coli RNA polymerase); 92 kDa (rabbit phosphorylase a); 66 kDa (bovine serum albumin); 51 kDa (Aspergillus a-amylase); 36 kDa (a subunit of E. coli RNA polymerase); 26 kDa (rabbit triosephosphate isomerase); and 14 kDa (horse cytochrome c).
Strains and plasmids used in this work were kindly provided by Sydney Kustu, Phil Matsumura, and Robert Macnab. Members of the Kustu laboratory provided helpful advice on the construction of expression plasmids. This research was supported by Public Health Service research grant GM 12010 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Arnosti, D. N., and M. J. Chamberlin. 1989. Secondary sigma factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. Proc. Natl. Acad. Sci. USA 86:830-834. 2. Bartlett, D. H., B. B. Frantz, and P. Matsumura. 1988. Flagellar transcriptional activators flbB and flaL: gene sequences and 5' consensus sequences of operons underflbB and flaI control. J. Bacteriol. 170:1575-1581. 3. Burton, Z., B. B. Burgess, J. Lin, D. Moore, S. Holder, and C. Gross. 1981. The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit for E. coli K-12. Nucleic Acids Res. 9:2889-2903.
4. Casadaban, M. J. 1976. Transposition and fusion of the lac
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