Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB

Abstract

The activation of pectin lyase (Pnl) production in Enwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving r e d , d g A and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnlA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnlA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis w e have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnlA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the Em coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnlA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the d g B product is required for pnlA expression, establishes that d g B encodes a transcriptional factor which specifically interacts with the pnlA promoter/regulatory region.

Erwinia carotovoraSubspecies Produce Duplicate Variants of ExpR, LuxR Homologs That ActivatersmATranscription but Differ in Their Interactions withN-Acylhomoserine Lactone Signals

“…A 111-bp DNA probe was PCR amplified using 10 pmol of end-labeled primer 5Ј-GCTGAATTCCGTAAACTTAGACGATG GTAT-3Ј (corresponding to nucleotides Ϫ30 to Ϫ50 from transcriptional start site T2 of rsmA) and 10 pmol of unlabeled primer 5Ј-TGTAGATCTCAGTTC TGTTGTTGTGATAAC-3Ј (corresponding to nucleotides Ϫ140 to Ϫ119 from transcriptional start site T2). Labeling of the primer, the PCR, and the DNase I protection assays were carried out by using previously described procedures (27).…”

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

“…Another example is the pnlA gene of E. carotovora, whose expression is induced by DNA-damaging agents such as mitomycin C, nalidixic acid, or UV light but not by PGA (290). Transcription of pnlA is dependent on the recA, rdgA, and rdgB gene products (290,291,294). In the proposed model, in the presence of DNA-damaging agents, the activated proteolytic form of RecA processes RdgA to a form that activates rdgB expression, whose product in turn activates pnlA expression.…”

Temperature-dependent Production of Carotovoricin Er and Pectin Lyase in PhytopathogenicErwinia carotovorasubsp.carotovoraEr

“…The cells were grown overnight in a 500-ml ‰ask containing 100 ml LB medium (10 g of polypeptone, 5 g of NaCl, and 5 g of yeast extract in one liter, pH 7.2) at 309 C. The culture (50 ml) was transferred to a 3-liter ‰ask containing 1 liter of LB medium and was incubated at 309 C. After mitomycin C (0.4 mg W ml) was added at the logarithmic stage (OD＝0.6 at 660 nm), the culture was divided into 100-ml portions in nine 500-ml ‰asks. Each ‰ask was shaken at 15,17,20,23,26,30,32,34, or 379 C. After 2 h of incubation, during which cell lysis did not occur, cell densities in all samples were adjusted to 6×10 8 cell W ml (OD＝0.7 at 660 nm) with LB medium containing mitomycin C (0.4 mg W ml) and the incubation was continued for 4 h more at the diŠerent temperatures described above. Figure 1 shows the course of the decreased turbidity of cultures in the presence of mitomycin C at the temperatures indicated.…”

“…The turbidity of the cultures were found to be decreased in a similar way during 6 h of induction in the samples of 15, 23, 30 and 329 C, while at 34 and 379 C, the reduction of turbidity of the culture after 6 h of induction reached 50z and 10z of that at 309 C, respectively. After 6 h of induction at 15,17,20,23,26,30,32,34, and 379 C, the cultures were carotovora subsp. carotovora Er from 2 to 6 h After Induction by Mitomycin C at the Temperatures Indicated.…”

“…Previously, Liu et al showed the RdgBbinding sequence in the promoter region of the structural gene of pnl. 17) Taken together, the temperaturedependent expression of pnl may be controlled by RecA-Rdg system and global regulators such as Lrp, ppGpp, and cAMP-Crp.…”