Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A-tumefaciens rpoA gene, encoding the alpha subunit of RNA polymerase

被引:26
作者
Lohrke, SM
Nechaev, S
Yang, H
Severinov, K
Jin, SJ [1 ]
机构
[1] Univ Arkansas Med Sci, Dept Microbiol & Immunol, Little Rock, AR 72205 USA
[2] Rutgers State Univ, Waksman Inst, Dept Genet, Piscataway, NJ 08854 USA
关键词
D O I
10.1128/JB.181.15.4533-4539.1999
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However, virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the alpha subunit of RNA polymerase (RNAP), which confers significant expression of a virB promoter (virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulator virG gene. We conducted in vitro transcription assays using either reconstituted E. call RNAP or hybrid RNAP in which the alpha subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a sigma(70)- dependent E. coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBp in a virG-dependent manner. In addition, we provide evidence that the alpha subunit from A. tumefaciens, but not from E. coli, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the alpha subunit of A. tumefaciens and that the alpha subunit from E. coli is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examine vir gene expression as well as the T-DNA transfer process in E. coli.
引用
收藏
页码:4533 / 4539
页数:7
相关论文
共 66 条
[1]   T-DNA OF AGROBACTERIUM-TUMEFACIENS ENCODES AN ENZYME OF CYTOKININ BIOSYNTHESIS [J].
AKIYOSHI, DE ;
KLEE, H ;
AMASINO, RM ;
NESTER, EW ;
GORDON, MP .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (19) :5994-5998
[2]   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].
Altschul, SF ;
Madden, TL ;
Schaffer, AA ;
Zhang, JH ;
Zhang, Z ;
Miller, W ;
Lipman, DJ .
NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402
[3]   RECOMBINANT ESCHERICHIA-COLI RNA-POLYMERASE - PURIFICATION OF INDIVIDUALLY OVEREXPRESSED SUBUNITS AND IN-VITRO ASSEMBLY [J].
BORUKHOV, S ;
GOLDFARB, A .
PROTEIN EXPRESSION AND PURIFICATION, 1993, 4 (06) :503-511
[4]   Temperature-dependence of open-complex formation at two Escherichia coli promoters with extended - 10 sequences [J].
Burns, HD ;
Belyaeva, TA ;
Busby, SJW ;
Minchin, SD .
BIOCHEMICAL JOURNAL, 1996, 317 :305-311
[5]   SUGARS INDUCE THE AGROBACTERIUM VIRULENCE GENES THROUGH A PERIPLASMIC BINDING-PROTEIN AND A TRANSMEMBRANE SIGNAL PROTEIN [J].
CANGELOSI, GA ;
ANKENBAUER, RG ;
NESTER, EW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (17) :6708-6712
[6]   TRANSPOSITION AND FUSION OF LAC GENES TO SELECTED PROMOTERS IN ESCHERICHIA-COLI USING BACTERIOPHAGE-LAMBDA AND BACTERIOPHAGE-MU [J].
CASADABAN, MJ .
JOURNAL OF MOLECULAR BIOLOGY, 1976, 104 (03) :541-555
[7]   Mutations in the alpha and sigma-70 subunits of RNA polymerase affect expression of the mer operon [J].
Caslake, LF ;
Ashraf, SI ;
Summers, AO .
JOURNAL OF BACTERIOLOGY, 1997, 179 (05) :1787-1795
[8]   A CHROMOSOMALLY ENCODED 2-COMPONENT SENSORY TRANSDUCTION SYSTEM IS REQUIRED FOR VIRULENCE OF AGROBACTERIUM-TUMEFACIENS [J].
CHARLES, TC ;
NESTER, EW .
JOURNAL OF BACTERIOLOGY, 1993, 175 (20) :6614-6625
[9]   REPRESSION AND ACTIVATION OF TRANSCRIPTION BY GAL AND LAC REPRESSORS - INVOLVEMENT OF ALPHA-SUBUNIT OF RNA-POLYMERASE [J].
CHOY, HE ;
PARK, SW ;
AKI, T ;
PARRACK, P ;
FUJITA, N ;
ISHIHAMA, A ;
ADHYA, S .
EMBO JOURNAL, 1995, 14 (18) :4523-4529
[10]   DELINEATION OF THE REGULATORY REGION SEQUENCES OF AGROBACTERIUM-TUMEFACIENS VIRB OPERON [J].
DAS, A ;
PAZOUR, GJ .
NUCLEIC ACIDS RESEARCH, 1989, 17 (12) :4541-4550