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Vira and Virg Activate the Ti Plasmid Repabc Operon, Elevating Plasmid Copy Number in Response to Wound-Released Chemical Signals

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Vira and Virg Activate the Ti Plasmid Repabc Operon, Elevating Plasmid Copy Number in Response to Wound-Released Chemical Signals VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals Hongbaek Cho and Stephen C. Winans* Department of Microbiology, Cornell University, Ithaca, NY 14853 Edited by Patricia C. Zambryski, University of California, Berkeley, CA, and approved August 10, 2005 (received for review April 27, 2005) The vir genes of Agrobacterium tumefaciens tumor-inducing (Ti) be described. It is also quite possible that VirG-regulated genes may plasmids direct the transfer of oncogenic portion of the Ti (tumor- exist on replicons other than the Ti plasmid (9). inducing) plasmid that is transferred to plant cells (T-DNA) into Ti plasmids are generally referred to by the opines they can plant cells and are coordinately induced by plant-released phenolic catabolize, and the two best studied are often referred to as the chemical signals. We have used DNA microarrays, representing all nopaline-type plasmid originally found in strain C58 and the genes of the octopine- and nopaline-type Ti plasmids, to identify octopine-type Ti plasmid, which is found in virtually identical forms all Ti-plasmid-encoded genes in the vir regulons of both plasmids. in strains such as A6, B6, 15955, Ach5, and R10. All known Ti Acetosyringone (AS) induced the expression of all known members plasmids contain (i) one or more T-DNAs, (ii)thevir genes, which of the vir regulons, as well as a small number of additional genes. direct the transfer of T-DNA into plant cells, (iii) a set of tra and Unexpectedly, AS also caused a modest induction of virtually every trb genes, which direct the conjugation of the Ti plasmid, (iv) a large Ti plasmid gene. This suggested that the copy number of the Ti number of genes that direct the uptake and catabolism of nutrients plasmid might increase in response to AS, a hypothesis confirmed called opines that are released from plant tumors, and (v)arepABC by DNA dot blotting. VirA and VirG were the only Vir proteins operon, which directs vegetative plasmid replication and partition- required for this copy number increase. Promoter resections and ing (8). Similar repABC operons are widespread among the plas- primer extension analysis of the repABC promoter region showed mids and some chromosomes of ␣-proteobacteria, but their regu- that expression of the promoter closest to repA (promoter P4) was lation is largely unexplored (10–12). induced by AS. We also identified a sequence resembling a con- In this study, we have used DNA microarrays to identify all sensus VirG-binding motif Ϸ70 nucleotides upstream from the P4 members of the vir regulons that lie on the two most widely studied transcription start site. Mutating this sequence blocked the AS- Ti plasmids. These microarrays contained all ORFs of the two Ti induced copy number increase of a RepABC-dependent miniplas- plasmids as well as Ϸ20 chromosomal genes thought to be consti- mid, indicating that phospho-VirG increases copy number solely by tutively expressed and several members of the putative SOS and enhancing repABC expression. heat-shock regulons. It seemed conceivable that either or both of these regulons might be AS-inducible, because phenolics are some- agrobacterium ͉ microarrays ͉ transcriptional profiling what toxic at the concentrations used (13), and the T-DNA is processed to a single-stranded form (14) that might possibly cause any microbes that associate with plant or animal hosts SOS induction (15). As described below, these transcriptional Mcoordinately regulate large sets of genes required for these profiling experiments confirmed the induction of virtually all associations and induce expression of these genes in response to known members of the vir regulons on the Ti plasmids and host-released chemical signals. For example, Sinorhizobium meliloti identified a small number of previously undescribed members. They induces expression of the nod regulon in response to flavonoids also led to the discovery that VirA and VirG directly activate released from host legumes (1), whereas Vibrio cholerae induces expression of the repABC operon, causing an increased copy expression of several toxins and adhesins in response to bile salts number of this plasmid in response to host-released chemical released into the gut of human or animal hosts (2). This phenom- signals. enon is also well illustrated by the plant pathogen Agrobacterium tumefaciens. The entire set of vir genes, whose products transfer Materials and Methods fragments of oncogenic portion of the tumor-inducing (Ti) plasmid Design of Oligonucleotides for PCR Amplification. Strains and plas- that is transferred to plant cells (T-DNA) from the large Ti plasmid mids used in this study are described in Table 2, which is published to the nuclei of host cells, is induced by chemical signals released as supporting information on the PNAS web site. Oligonucleotides from host plants (3). The primary signal molecules that stimulate were designed by using the PRIMER3 program (16). We used default expression of the vir regulon are phenolic compounds [including settings except for the following parameters: product size range, acetosyringone (AS), which is widely used in many laboratories as 501–900 (most preferred) or 300–500 (acceptable) and %GC, a vir gene inducer], particular monosaccharides, and extracellular 40–60; maximum polyX, 3; and CG clamp, 1. The primers were acidity (4). synthesized by IDT Technologies (Coralville, IA) and are listed in The expression of vir genes is controlled by the periplasmic Table 3, which is published as supporting information on the PNAS sugar-binding protein ChvE, the transmembrane histidine protein web site. PCR reactions were carried out by using genomic DNA kinase VirA, and the cytoplasmic response regulator VirG (4). from derivatives of strains R10 or C58 as templates and Platinum MICROBIOLOGY Phospho-VirG activates expression of at least 14 vir operons, TaqDNA polymerase (Invitrogen). PCR products were purified by containing a total of 30 genes (3, 5, 6) on the octopine-type Ti using 96-well multiscreen filter plates (Millipore). plasmid, whereas far fewer VirG-inducible genes have been iden- tified on the nopaline-type Ti plasmid (7). The products of some vir genes play direct roles in T-DNA transfer, whereas the products of This paper was submitted directly (Track II) to the PNAS office. other vir genes are not required for tumorigenesis and probably play Abbreviations: Ti, tumor-inducing; AS, acetosyringone; T-DNA, portion of the Ti plasmid other roles during the initial stages of infection (8). To date, all that is transferred to plant cells. genes known to be in the vir regulon are located on Ti plasmids. It *To whom correspondence should be addressed. E-mail: [email protected]. is quite plausible that additional members of this regulon remain to © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503458102 PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14843–14848 Downloaded by guest on September 28, 2021 Spotting PCR Products onto Glass Slides. Purified PCR products were (16 bit) were analyzed by using the SCANALYZE 2.50 program transferred to 384-well plates, and water was removed by evapo- (http:͞͞rana.lbl.gov͞EisenSoftware.htm). A normalization coeffi- ration. DNA fragments were resuspended in 3ϫSSC buffer sup- cient was computed by averaging the signal intensity ratio of 21 plemented with 1.5 M betaine (17) and spotted on glass slides chromosomally encoded genes on each array. Four replicate ex- coated with aminosilane (CMT-GAPSII, Corning). Each DNA periments (with independent bacterial culturing, RNA preparation, sample was spotted three times per slide by using a MicroGrid II cDNA probe synthesis, dye coupling, and hybridizations) were DNA Arrayer (Matrix Science, Boston). DNA was crosslinked to performed with dyes swapped in two trials for each strain. the slides using heat and UV irradiation (18). DNA Dot Blotting. Strains were cultured for 24 h in the presence or Bacterial Culturing and RNA Extraction. Strains were cultured for 24 h absence of 100 ␮M AS and harvested at midexponential phase in acidified AB minimal medium (19) in the presence or absence of (OD600 ϭ 0.4). Cells were resuspended in TE buffer (10 mM 100 ␮M AS. The initial OD600 of each culture was 0.25, and cultures Tris⅐HCl͞1 mM EDTA, pH 8.0) and lysed by incubation at 55°C were diluted as needed to keep the OD600 at or below 0.5. Two with 1.25% SDS͞0.6 M NaCl͞0.6 mg/ml proteinase K for 30 min. milliliters of the resulting cultures were mixed with 4 ml of RNA The lysate was extracted with phenol-chloroform, and the nucleic Protect Bacteria Reagent (Qiagen, Valencia, CA) and processed as acids were ethanol-precipitated and resuspended in TE buffer recommended by the manufacturer. RNA was purified by using supplemented with RNase (20 ␮g͞ml). DNA was purified, and RNeasy mini columns (Qiagen). Before elution, RNA was treated 3-fold serial dilutions were spotted onto Zeta-Probe Blotting mem- with DNase I to remove contaminating DNA. Integrity of RNA was branes (Bio-Rad), using a Minifold I Dot Blot System (Schleicher monitored by agarose gel electrophoresis. & Schuell). Each filter was probed with radiolabeled PCR products containing the virD5, bapA, kdpD,orrpoD genes to detect the Ti cDNA Synthesis and Hybridization. Fluorescent cDNA probes were plasmid, pAtC58, the linear chromosome, and the circular chro- synthesized, purified, and used to probe microarrays following a mosome, respectively. Radioactivity was quantified by using a published procedure (18). Briefly, RNA samples were treated with Storm B840 Phosphorimager and IMAGEQUANT software (Molec- Superscript II reverse transcriptase (Invitrogen) in the presence of ular Dynamics). dNTPs (Invitrogen), aminoallyl-dUTP (Sigma), and random hex- amers (Invitrogen). The resulting cDNA was purified with PCR Primer Extension Analysis, Plasmid Copy Number Estimation, and S1 purification columns (Qiagen), coupled to either Cy3 or Cy5 dyes Nuclease Protection Assays.
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