Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yamada, K. Articles by Itoh, Y. Search for Related Content PubMed PubMed Citation Articles by Yamada, K. Articles by Itoh, Y. Agricola Articles by Yamada, K. Articles by Itoh, Y.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2008, p. 6017-6025, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.01297-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Binding Sequences for RdgB, a DNA Damage-Responsive Transcriptional Activator, and Temperature-Dependent Expression of Bacteriocin and Pectin Lyase Genes in Pectobacterium carotovorum subsp. carotovorum ^,

Kazuteru Yamada, Jun Kaneko, Yoshiyuki Kamio, and Yoshifumi Itoh^*

Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori Amamiya-machi 1-1, Aoba-ku, Sendai 981-8555, Japan

Received 10 June 2008/ Accepted 31 July 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23°C) differs from that for synthesis of Pnl (30°C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P₀, P₁, and P₂ promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K_d [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K_d = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23°C compared with that at 30°C. In contrast, the amount of pnl transcription tripled at 30°C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30°C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pectobacterium carotovorum subsp. carotovorum (formally Erwinia carotovora subsp. carotovora) is a causal agent of soft-rot disease in plants. This pathogen produces pectolytic enzymes, including pectate lyase, polygalacturonase, and pectin lyase (Pnl), which are crucial pathogenic determinants that can degrade pectin, a plant cell wall component (5). The regulatory mechanism underlying the synthesis of Pnl is exposure to DNA-damaging agents such as mitomycin C (MMC), nalidixic acid, and UV light (12, 14, 22, 23, 33). This differs from that underlying the synthesis of pectate lyase and polygalacturonase, the synthesis of which is stimulated by the substrate pectin (or pectic acid) through a quorum-sensing mechanism (5, 35). In P. carotovorum subsp. carotovorum strain Er and some other strains, DNA damage-inducible Pnl synthesis is accompanied by production of the bacteriocin carotovoricin (Ctv) (12, 15, 34, 38). The Ctv of strain Er has a phage tail-like morphology consisting of a sheath, core, base plate, and several tail fibers (11, 24). The Ctv genes (ctv) of strain Er cluster separately from the pnl locus on the chromosome, and they are transcribed in four units (36) (Fig. 1). The P₀ promoter precedes the lyt cassette, which contains the putative holin and murein hydrolase genes, responsible for cell lysis to liberate bacteriocin particles outside the cells (36). The P₁, P₂, and P₃ promoters precede the operons comprising the genes for bacteriocin components and proteins required for their assembly (36).

View larger version (5K): [in this window] [in a new window]

FIG. 1. Organization and transcription units of ctv genes. Open arrows indicate directions and sizes of putative ctv genes, orf1a though orf17. Putative functions of orf products have been assigned (36). Bent arrows denote transcription start sites and directions of promoters P0 to P3 of ctv operons; hairpins represent transcription terminators.

The recA, rdgA, and rdgB regulatory genes control DNA damage-inducible Pnl synthesis in P. carotovorum subsp. carotovorum strain 71 (17, 19, 23, 38). The rdgA and rdgB pair lies contiguously, but they are independently transcribed from their own promoters (17). RdgA is a repressor of rdgA and rdgB. Analogous to the RecA-LexA system of Escherichia coli (27), RecA in complexes with single-strand DNA molecules generated by DNA damage can assist the autocleavage (hence inactivation) of RdgA so that rdgB is expressed (19). RdgB is a transcriptional activator of the Mor/C family of proteins (16) and enhances pnl transcription through interaction with the pnl promoter (18).

Because Ctv production also requires RecA function, the RdgB downstream of RecA in the DNA damage-responsive regulatory circuit was also thought to direct Ctv induction (17). However, Nguyen et al. (25) noted that the production of Ctv and Pnl is maximal at 23°C and at 30°C, respectively, raising questions as to whether RdgB directly participates in Ctv induction and how the optimal temperatures for bacteriocin and enzyme synthesis are determined.

Here, we applied gel mobility shift assays to demonstrate the binding of RdgB to the promoters of the ctv operons and determined the binding sequences of RdgB on these promoters by DNase I footprinting. Quantitative reverse transcription-PCR (RT-PCR) revealed that more rdgB and ctv genes were transcribed at 23°C than at 30°C whereas the opposite was true for pnl. Knockout of rdgB caused an apparent reduction in pathogenicity to potato tubers. We considered the mechanism underlying the temperature-dependent production of Ctv and Pnl in conjunction with their biological roles based on these findings.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains, plasmids, media, and growth conditions.
Table 1 lists the bacterial strains and plasmids used in this study. Unless otherwise noted, Escherichia coli and P. carotovorum were cultured in Luria-Bertani (LB) medium (29) or on LB plates containing 1.5% (wt/vol) agar at 37°C and 30°C, respectively. When appropriate, antibiotics were added to medium at the following concentrations: ampicillin (Ap), 100 µg/ml; tetracycline (Tc), 10 µg/ml (for E. coli) or 1 µg/ml (for P. carotovorum subsp. carotovorum). The synthesis of bacteriocin and Pnl in response to DNA damage was induced by MMC (Wako Pure Chemicals, Osaka, Japan).

View this table: [in this window] [in a new window]

TABLE 1. Bacterial strains and plasmids used in this study

Cloning and sequencing of rdgAB.
A 1.5-kb rdgAB DNA fragment of P. carotovorum subsp. carotovorum Er (from the translation initiation codon of rdgA through the translation termination codon of rdgB) was amplified from chromosomal DNA of strain Er by PCR using primers RDGAS1 and RDGBS1 (Table 2), which were synthesized based on the published sequences of the P. carotovorum subsp. carotovorum 71 rdgAB (17). The flanking regions of rdgAB were amplified by inverse PCR using primers RDGAS2 and RDGBS2 (Table 2) and circularized EcoT22I fragments of Er chromosome DNA as templates. Nucleotides comprising rdgAB and their flanking regions were sequenced as described previously (36).

View this table: [in this window] [in a new window]

TABLE 2. Oligonucleotide primers

Cloning ctv promoter regions.
Nucleotides (nt) –100 to +37 of P₀ (137 bp), nt –137 to –3 of P₁ (135 bp), nt –126 to +16 of P₂ (142 bp), nt –123 to +11 of P₃ (134 bp), and nt –73 to +83 of the pnl promoter (P_pnl; 156 bp) were separately amplified by PCR using Er chromosomal DNA as a template and the following pairs of oligonucleotide primers (Table 2): P0F and P0R, P₀ fragment; P1F and P1R, P₁ fragment; P2F and P2R, P₂ fragment; P3F and P3R, P₃ fragment; PNLP1 and PNLP2, P_pnl fragment. The amplified promoter fragments were cloned into the HincII site of plasmid pUC118 (37), resulting in plasmids pKY5, pKY6, pKY7, pKY8, and pYK9, respectively. Nucleotides comprising the inserts were verified by sequencing.

Construction of rdgB knockout mutant.
The 5'- and 3'-flanking regions of rdgB were separately amplified by PCR using P. carotovorum subsp. carotovorum Er chromosomal DNA as a template and oligonucleotide primers (Table 2). We used the RDGB1 and RDGB2 pair to amplify a 726-bp 5'-flanking region between nt –868 and –143 of the rdgB translation initiation codon and the RDGB3 and RDGB4 pair to amplify a 454-bp 3'-flanking region between nt 280 and 733 downstream of the rdgB coding sequence. The amplified 5'-flanking DNA fragment was cleaved with KpnI and inserted into plasmid pUC119 (37) at the corresponding site to produce plasmid pKY1. The 3'-flanking DNA fragment generated was digested with BamHI and cloned into the same site of pKY1 to yield plasmid pKY2. We isolated the Tc resistance gene of plasmid pBR322 (5) as a 1.4-kb AvaI and EcoRI fragment and inserted it, after converting the protruding ends to blunt ends using a blunting kination ligation kit (Takara Bio), into the SmaI site of pKY2, resulting in plasmid pKY3. The rdgB::Tc gene was then excised from pKY3 as a SacI-XbaI fragment, blunt ended, and inserted into the SmaI site on the suicide plasmid pYMD1 (sacB⁺) (Table 1). Because this plasmid requires the pir function for replication, its derivatives can be maintained in a pir⁺ host such as E. coli S17-1 but not in P. carotovorum subsp. carotovorum Er, which is devoid of pir. We introduced plasmid pKY11 into strain Er by electroporation and selected transformants harboring pKY11 integrated into the chromosome by single crossover on LB plates containing Ap and Tc. Finally, the rdgB knockout mutant strain KY1, which had lost the plasmid sequence on the chromosome by a second crossover, was selected on LB plates containing 5% (wt/vol) sucrose and Tc.

Construction of rdgB expression plasmids.
To prepare histidine-tagged RdgB (His₆-RdgB), a coding region (nt 2 to 351) of rdgB was PCR amplified using chromosomal DNA from strain Er and RDGB5 and RDGB6 primers (Table 2) and inserted into the blunted NdeI site of the expression vector pET-15b(+) (Novagen, Madison, WI) to yield plasmid pKY4. This construction created 16 additional amino acid residues (His₆-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His) at the N terminus of RdgB. To generate an isopropyl-β-D-thiogalactopyranoside (IPTG; Nacalai Tesque, Kyoto, Japan)-inducible rdgB fusion (Ptrc-rdgB), the nt 4 to 351 region of rdgB was PCR amplified from chromosomal DNA of strain Er using RDGB7 and RDGB8 primers (Table 2) and then inserted into the blunted NcoI site of plasmid pTrc99A (2) to produce plasmid pTrc-rdgB.

Expression and purification of His₆-RdgB.
Cultures of E. coli BL21(DE3) harboring pKY4 were incubated at 30°C in LB medium containing Ap until the optical density at 600 nm (OD₆₆₀) reached 0.7, when IPTG was added (final concentration, 1 mM). After incubation for an additional 2 h, the cells were harvested by centrifugation at 4°C and disrupted by passage through a French pressure cell at 1,000 lb/in². We loaded the cell extracts onto a HiTrap chelating HP column (Amersham Biosciences, Piscataway, NJ) and eluted His₆-RdgB using an imidazole gradient (150 to 300 mM; total, 60 ml). Homogeneous His₆-RdgB, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 14% (wt/vol) polyacrylamide gels, was eluted between 200 and 250 mM imidazole. After dialysis against binding buffer A (18) to remove imidazole, the purified protein was stored in the same buffer containing 20% glycerol at –20°C. Protein concentrations were determined using a Bio-Rad protein assay kit with bovine serum albumin (BSA) as the standard.

Western blotting.
Proteins were electrophoretically resolved on 12.5% acrylamide gels and electroblotted onto polyvinylidene difluoride membranes. Proteins on the membranes were cross-reacted with antiserum raised against the Ctv sheath protein or Pnl (25, 26). Sheath- or Pnl-antibody complexes were visualized using alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (Promega, Madison, WI), nitroblue tetrazolium (Wako Pure Chemicals), and 5-bromo-4-chloro-indolylphosphate (Wako Pure Chemicals).

Gel mobility shift assay.
Probe DNA segments of P₀, P₁, P₂, P₃, and P_pnl were amplified by PCR using plasmids pKY5, pKY6, pKY7, pKY8, and pYK9 (Table 1), respectively, as templates and M13 primers M4 and Rv (Takara Bio, Otsu, Japan). The amplified DNA segments were labeled at the 3' ends with digoxigenin (DIG) using a DIG gel shift kit (second generation; Roche Diagnostics, Basel, Switzerland). DIG-labeled DNA fragments (0.1 nM) were incubated at 20°C for 20 min with various amounts of His₆-RdgB in 20 µl of binding buffer A (18) containing salmon sperm DNA (1 µg) and BSA (2 µg). Free and His₆-RdgB-bound DNA fragments were electrophoretically separated on 5% (wt/vol) polyacrylamide gels and electroblotted onto Hybond N+ membranes. Thereafter, DIG-labeled DNA fragments were detected on X-ray films using DIG-AP antibody and chemiluminescent reagents according to the supplier's protocol.

DNase I footprinting.
DNase I footprinting proceeded as described by Machida et al. (20) with a minor modification. We labeled P₀, P₁, and P₂ DNA segments with infrared dye IRD800 at the 5' ends of either the coding or the noncoding strand by PCR using a pair of M13 sequencing primers, one of which had IRD800 at the 5' end, and plasmids pKY5, pKY6, and pKY7 as templates. After unreacted nucleotides were removed by ethanol precipitation, end-labeled DNA probes (30 ng) were incubated at 20°C for 20 min with various amounts of His₆-RdgB in binding buffer A (total, 50 µl) containing salmon sperm DNA (2.5 µg) and BSA (5 µg) to allow the protein to bind to the DNA probes. After addition of 50 µl of a mixture of Ca²⁺-Mg²⁺ solution (5 mM CaCl₂ and 10 mM MgCl₂), the reaction mixtures were incubated at 20°C for 1 min, and then the DNA fragments were digested with DNase I (3.0 x 10^–2 U) at 20°C for 1 min. Reactions were terminated by adding 100 µl of stop solution (200 mM NaCl, 20 mM EDTA, and 1.0% sodium dodecyl sulfate). After extraction with phenol-chloroform, precipitation with ethanol, and washing with 80% (vol/vol) ethanol, the digested DNA fragments were dissolved in 20 µl of 95% (vol/vol) formamide containing 20 mM EDTA, 0.1% (wt/vol) bromophenol blue, and 0.05% (wt/vol) xylene cyanol. After denaturation at 95°C for 2 min, DNase I-digested fragments were resolved along with sequence ladders on denatured 6.0% Long Ranger gels and detected using a model 4000 sequencer equipped with BaseImagIR, version 2.30 (Li-Cor, Lincoln, NE). We created DNA sequence ladders by dideoxy chain termination using relevant plasmid DNA as a template, 5'-IRD800-labeled M13 primers, and a Thermo Sequenase cycle sequencing kit (USB Corporation, Cleveland, OH).

Real-time RT-PCR.
Real-time PCR proceeded using LightCycler and LightCycler FastStart DNA Master Sybr green I kits (Roche Diagnostic), according to the manufacturer's instructions. Total RNA (250 ng) was prepared from Er cells by the hot-phenol method described by Aiba et al. (1) and used as a template to synthesize cDNA using avian myeloblastosis virus reverse transcriptase XL (Takara Bio) and the oligonucleotide primers (Table 2) CTV1 (complementary between nt +520 and +540 of orf1a), CTV2 (complementary between nt +462 and +481 of orf4), PNLR1 (complementary between nt +694 and +717 of pnl), RDGB9 (complementary between nt +401 and +431 of rdgB), and 16S-1 (complementary between nt +479 and +498 of 16S rRNA) (numbers are relative to transcriptional start sites [+1] of the relevant genes and denote nucleotides of the P. carotovorum subsp. carotovorum 16S rRNA gene [accession no. AB437348]). Regions of interest were amplified by PCR in 20-µl mixtures (containing 2 µl of cDNA, 0.1 µM of the appropriate primers [see below], and 3 mM MgCl₂) as follows: denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s. The primers and amplified DNA regions were as follows: CTV3 and CTV4, +48 to +267 of orf1a (220 bp); CTV5 and CTV6, nt +129 to +419 of orf4 (291 bp); PNLR2 and PNLR3, nt +205 to +508 of pnl (304 bp); RDGB10 and RDGB11, nt +16 to +335 of rdgB (319 bp); 16S-1 and 16S-2, nt +82 to +381 of 16S rRNA (300 bp). Fluorescence emitted from Sybr green I incorporated into PCR products was measured at the end of each cycle to determine the amplification kinetics of each cDNA. Reactions proceeded in triplicate with RNA samples prepared from three independent cultures. All data were normalized to those of 16S rRNA, an internal control, and analyzed by Student's t test.

Ctv and virulence assays.
Ctv particles precipitated from 1 ml of culture supernatants by centrifugation at 4°C at 160,000 x g for 30 min were suspended in the same volume of phosphate-buffered saline buffer (25). The activity of Ctv was determined by the double-layer method using strain 645Ar as an indicator, and reciprocals of maximum dilutions that completely inhibited indicator growth were defined as killing units (24). To assess virulence, 10 µl of Er (wild-type) and KY1 (rdgB::Tc) cell suspensions (10⁸ cells/ml) were spotted onto sections of potato tubers cut at the center into two pieces. After incubation in moist chambers at 23°C or 30°C for 24 h, the diameters of at least three lesions that developed on the tuber sections were measured. Values are averages of three independent tests, and differences were statistically analyzed using Student's t test.

Nucleotide sequence accession numbers.
The nucleotide sequences of rdgAB and their flanking regions and of pYMD1 have been deposited in the DDJ/GenBank/EMBL databases under accession no. AB298803 and AB304880, respectively.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synthesis of Ctv requires rdgB.
We initially examined whether or not the RecA-RdgAB pathway controls the production of DNA damage-inducible Ctv like that of Pnl. We accordingly derived an rdgB knockout mutant (strain KY1) from P. carotovorum subsp. carotovorum Er (see Materials and Methods) and compared the amounts of DNA damage-inducible Ctv and Pnl synthesis between this mutant and the wild-type strains at 23°C. The OD₆₆₀s measured indicated that wild-type cultures started to lyse after 4 h in the presence of MMC (Fig. 2A) and concomitantly produced active Ctv particles in the medium. Bacteriocin levels became maximal (150 killing units/ml) (data not shown) in parallel with cell lysis (Fig. 2A) during an additional incubation for 4 h. In contrast, the rdgB mutant continued to proliferate in culture without detectable lysis in the presence of MMC (Fig. 2A) and produced no measurable amounts of bacteriocin for up to 8 h. In the presence of MMC, Western blotting using antisera raised against the Ctv sheath protein or Pnl detected the respective proteins in wild-type but not mutant cultures (Fig. 2B). These results verified that RdgB mediates the MMC-inducible synthesis of Pnl in strain Er, as in strain 71 (17, 18), and supported the notion that the RecA-RdgAB regulatory pathway also controls the MMC-inducible synthesis of Ctv.

View larger version (19K): [in this window] [in a new window]

FIG. 2. MMC-induced cell lysis (A) and Ctv synthesis (B) require rdgB. (A) Cultures of strains Er (wild type) and KY1 (rdgB) grown at 30°C to an OD660 of 0.3 were incubated at 23°C (optimal temperature for Ctv production) with or without of MMC (0.5 µg/ml). OD660s were measured after various incubation periods. (B) Proteins in Er and KY1 cultures (each 10 µl) incubated with MMC for 8 h were resolved on 12.5% polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes. Membrane Ctv sheath protein and Pnl were detected by Western blotting using appropriate antisera.

Binding of RdgB to the ctv promoter regions.
The ctv genes are included in four operons that are preceded by promoters P₀, P₁, P₂, and P₃ (36) (Fig. 1). P₀, P₁, and P₂ are MMC inducible and have sequences homologous to the –35 and –10 regions, but P₃ is a constitutive and weak promoter lacking canonical –35 and –10 sequences (36). We examined whether RdgB can directly interact with these MMC-inducible ctv promoters by gel mobility shift assays using recombinant His₆-RdgB protein and the P₀, P₁, P₂, and P₃ DNA fragments as probes. His₆-RdgB retarded the migration of the P₀, P₁, and P₂ fragments (Fig. 3A) but not the P₃ fragment and a 135-bp fragment upstream of the P₀ fragment (see Fig. S1 in the supplemental material), proving that His₆-RdgB specifically binds to the P₀, P₁, and P₂ fragments. The P₀ and P₂ probes shifted as one DNA band, whereas the P₁ probe was retarded as four distinct bands depending on the His₆-RdgB concentration (Fig. 3A), suggesting that the first two have single RdgB-binding sites whereas the last has four sites with different levels of binding affinity for the regulatory protein.

View larger version (31K): [in this window] [in a new window]

FIG. 3. Gel mobility shift assays (A) and determination of Kd values (B) for His6-RdgB binding to ctv and pnl promoters. (A) P0, P1, P2, and Ppnl DNA probes labeled with DIG at their 3' ends (0.1 nM) were incubated with various amounts of His6-RdgB (lanes 1 to 14, 0, 10, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 800, and 1,000 nM of His6-RdgB, respectively). Free and His6-RdgB-bound probes were separated by electrophoresis using 5% (wt/vol) polyacrylamide gels, blotted onto Hybond N+ membranes, and detected using DIG detection kits. (B) Amounts of free and bound forms of P0, P1, P2, and Ppnl probes on mobility shift assay gels from panel A were quantified using NIH Image, and relative amounts of free to total probes were calculated. Values are averages of three experiments.

Affinity of RdgB for binding to the ctv and pnl promoters.
His₆-RdgB shifted a P_pnl DNA fragment at 50 nM, but higher concentrations (200 nM) were required to retard the P₀, P₁, and P₂ DNA fragments (Fig. 3A), suggesting that the P_pnl promoter has higher affinity for RdgB than the ctv promoters have. We accordingly determined the K_d (dissociation constant) values for the binding of these promoter DNA fragments to His₆-RdgB (Fig. 3B). An excess (10 nM) of His₆-RdgB was included in the assay mixtures relative to DNA probes (0.1 nM). As relative amounts of His₆-RdgB to those of the DNA fragments were negligible, the His₆-RdgB concentrations required for half-maximal binding approximately corresponded to the K_d (7). The determined K_d values for the binding of the P₀, P₁, P₂, and P_pnl fragments to His₆-RdgB were 350, 250, 325, and 150 nM, respectively.

RdgB-binding sequences of the P₀, P₁, and P₂ promoters.
We then applied DNase I footprinting to determine RdgB-binding sequences of the P₀, P₁, and P₂ promoters. His₆-RdgB (1 µM) protected single DNA regions in these promoters: a –53 to –27 region of the P₀ coding and noncoding strands; –47 to –19 and –38 to –20 regions of the coding and noncoding strands of P₁, respectively; and a –57 to –29 region in the coding strand and a –57 to –30 region of the noncoding strand of P₂ (Fig. 4). This transcriptional factor also started to protect the P₁ fragment from DNase I digestion with increasing His₆-RdgB concentrations at regions –120 to –101, –85 to –70, and –65 to –48 of the coding strand and at regions –111 to –106, –87 to –75, and –64 to –49 of the noncoding strand (Fig. 4).

View larger version (49K): [in this window] [in a new window]

FIG. 4. DNase I footprint analysis of His6-RdgB binding sites on ctv promoters. Coding (C) and noncoding (N) strands of P0, P1, and P2 fragments were labeled with IRD800 at 5' ends. DNA probes (6 nM) were incubated at 20°C with 0, 1, 4, or 10 µM of His6-RdgB (lanes 1 to 4, respectively) in 50-µl binding assay mixtures for 20 min and then with DNase I (3.0 x 10–2 U) for 1 min. DNA sequences protected by RdgB are indicated on the right. Numbers are relative to the transcriptional start sites (+1) of the relevant operons. Arrows show common 3-bp inverted repeats in the regions protected by RdgB. Dots on the right of the P1 promoter footprints indicate regions protected by higher concentrations of His6-RdgB. Boxes indicate –35 sequences of each promoter.

Alignment of the DNA sequences protected with 1 µM His₆-RdgB revealed imperfect 7-bp palindromes with a loop of six bases, 5'-TA/TNPyTAA-[N₆]-TTAAA/TAT/G/A-3' (underlined) (Fig. 5). Three DNA sequences in the P₁ promoter protected by higher concentrations of His₆-RdgB had no palindromic structure. These sequences were TA rich like the palindrome sequence and contained 5'-TAA-3' or 5'-TTA-3' trinucleotides (data not shown).

View larger version (15K): [in this window] [in a new window]

FIG. 5. Comparison of RdgB-binding sequences. RdgB-binding sequences of P0, P1, and P2 were compared with that of Ppnl and the Mor protein-binding sequence of the bacteriophage Mu Pm promoter (3, 28). Arrows show inverted repeats. Perfectly conserved trinucleotides TAA and TTA in repeats are underlined.

Temperature-dependent transcription of pnl, ctv, and rdgB.
Although Ctv and Pnl are simultaneously synthesized in response to DNA-damaging agents, they are optimally synthesized at 23°C and at 30°C, respectively (25). We examined whether the temperature-dependent synthesis of Ctv and Pnl is controlled at the transcription level and whether rdgB, which is also inducible by DNA-damaging agents (17), is actively transcribed at either temperature. We thus used RT-PCR to measure transcripts of orf1a and orf4 of ctv (Fig. 1), pnl, and rdgB in Er cells that had been incubated with MMC at 23°C or 30°C. After adding MMC (final concentration, 0.5 µg/ml), we separated log-phase Er cultures (OD₆₆₀ = 0.3) into one portion that was incubated at 30°C and another that was incubated at 23°C. After an additional 3-h incubation, cells were harvested from each culture to isolate total RNA. RT-PCR of the total RNA samples showed that the amounts of orf1a and orf4 transcripts at 23°C were 1.9- and 1.5-fold increased compared with those at 30°C, respectively (Fig. 6). The amounts of rdgB transcripts at 23°C were double those at 30°C. On the other hand, the amounts of pnl transcripts produced at these temperatures were inverse, that is, about triple at 30°C compared with 23°C (Fig. 6). These relative amounts of the transcripts closely correlated with the relative amounts of Ctv and Pnl synthesized in MMC-induced cultures at the relevant temperatures (25).

View larger version (11K): [in this window] [in a new window]

FIG. 6. Relative transcription levels of orf1a, orf4, rdgB, and pnl genes induced by MMC at 23 and 30°C. Log-phase cultures (OD660 = 0.3) of strain Er were incubated at 23°C or at 30°C with MMC (0.5 µg/ml), and cells were harvested every hour from each culture to isolate total RNA. Amounts of orf1a, orf4, rdgB, and pnl transcripts at 0 h (immediately before adding MMC) and after a 3-h incubation were determined by real-time RT-PCR using total RNA samples as templates and normalized to those of 16S rRNA as an internal reference. Values are amounts of transcripts after 3 h of incubation relative to those at 0 h, calculated as averages of three RT-PCRs using total RNA samples from three independent cultures. Error bars represent standard deviations. Differences in relative amounts of all tested transcripts at 23°C and at 30°C are significant (P < 0.01; Student's t test).

Amounts of rdgB transcripts control temperature response of Ctv production.
The above results indicated that temperature-sensitive Ctv synthesis is controlled at the transcriptional level and suggested that the temperature-sensitive transcription of rdgB correlates with reduced transcription of the ctv operons at 30°C. We then examined whether the amounts of Ctv production and accompanying cell lysis are determined by transcription levels of rdgB rather than by temperature as follows. We generated plasmid pTrc-rdgB (Ptrc::rdgB) by inserting rdgB downstream of the IPTG-inducible trc promoter on the expression vector pTrc99A (2) and measured cell lysis and Ctv sheath protein synthesis directed by the plasmid-borne fusion. After adding IPTG (final concentration, 0.3 mM), a log-phase culture (OD₆₆₀ = 0.3) of P. carotovorum subsp. carotovorum Er/pTrc-rdgB was split into two portions for incubation at either 23°C or 30°C. Cells induced by IPTG at 30°C began to lyse after 1 h (Fig. 7A), while cells in the MMC-induced culture began to lyse after 4 h (Fig. 2A). Cell lysis started after 3 h at 23°C. Western blotting showed that IPTG induced 2.1-fold more Ctv sheath protein synthesis at 30°C than at 23°C (Fig. 7B). The extensive cell lysis and increased Ctv protein synthesis elicited by IPTG-induced RdgB at the higher temperature supports the notion that ctv transcription is not temperature sensitive but rather depends on RdgB levels.

View larger version (9K): [in this window] [in a new window]

FIG. 7. Ptrc::rdgB promotes cell lysis and Ctv synthesis at 30°C. (A) Log-phase cultures (OD660 = 0.3) of P. carotovorum subsp. carotovorum Er/pTrc-rdgB were incubated with 0.3 mM IPTG at 23°C or at 30°C, and their OD660s were measured. (B) Ctv and Pnl in 5-µl portions of the cultures incubated at 23°C or 30°C for 5 h were detected by Western blotting using antisera against Ctv sheath or Pnl protein and quantified using NIH Image software. Average amounts of each protein at 30°C relative to those at 23°C in three measurements with standard deviations (vertical bars) are presented. Differences in Ctv and Pnl synthesized at 23°C and at 30°C are significant (P < 0.01 according to Student's t test).

Pathogenicity of the rdgB mutant.
We examined whether Pnl plays a role in the pathogenicity of P. carotovorum subsp. carotovorum Er by comparing soft-rot symptoms on potato tubers induced by wild-type Er and the KY1 mutant (rdgB::Tc), which is defective in Pnl synthesis (Fig. 2B). The wild-type strain caused diseased spots with a 2.2-fold-larger diameter at 30°C than at 23°C (Fig. 8A and B). Likewise, the mutant strain also created 1.9-fold-larger lesions at 30°C than at 23°C. However, the lesions caused by the mutant were 2.4- and 2.8-fold smaller than those cause by the wild-type strain at both 23°C and 30°C, respectively (Fig. 8A and B). The differences in virulence in terms of temperature and of rdgB function were significant (P < 0.01).

View larger version (41K): [in this window] [in a new window]

FIG. 8. Pathogenicity of a rdgB knockout mutant. (A) Sections of potato tubers were incubated with 10 µl of Er (wild-type) and KY1 (rdgB::Tc) cell suspensions (108 cells/ml) in moist chambers for 24 h at 23°C or 30°C. (B) Diameters of diseased areas were measured. Values are average diameters of three or more diseased areas elicited by each strain at the relevant temperature with standard deviations (vertical bars). Student's t test showed that differences in tissue lesions caused by two strains are significant at both 23°C (P < 0.001) and 30°C (P < 0.01).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The RecA-RgA-RdgB regulatory pathway appears to control Ctv and Pnl synthesis in response to DNA damage. RdgB belongs to the Mor/C family of proteins (16). The Mor protein of bacteriophage Mu is a homodimer, which binds to a 16-bp DNA region (5'-AgTAA-[N₆]-TTAaT-3'; lowercase letters indicate mismatch bases) located between nt –51 and –36 relative to the transcription start site (+1) of the middle promoter (P_m) of the Mu phage (3) (Fig. 5). Mor binding to the operator facilitates recruitment of an RNA polymerase holoenzyme to the promoter through interaction with the C-terminal domains (CTD) of the (-CTD) and (-CTD) subunits of the holoenzyme (4). RdgB binds to the palindromic RdgB box of 5'-TTATTAA-[N₆]-TTAATAA-3' between nt –56 and –36 of P_pnl of P. carotovorum subsp. carotovorum 71 (18). This sequence is perfectly conserved in the same (–56 to –36) region of the P. carotovorum subsp. carotovorum Er P_pnl (28) (accession no. M65057) (Fig. 5). DNase I footprinting identified nucleotide sequences that were similar to those of the P_pnl RdgB boxes around the –35 regions of the P₀, P₁, and P₂ promoters of ctv. The RdgB box of P₁ has two mismatches, and those of P₀ and P₂ have three (Fig. 5). Mutational analysis has confirmed that dyad symmetry is crucial for Mor protein binding and hence for transcriptional activation (3). The RdgB boxes of P₀, P₁, and P₂ had lower affinity for binding to RdgB than that of P_pnl (Fig. 3B), supporting the notion that RdgB recognizes and binds to the palindrome with 7-bp dyad symmetry and that base changes in the dyad symmetry decrease RdgB-binding affinity. Since the three-base internal symmetry sequence 5'-TAA-[N₆]-TTA-3' is conserved in all RdgB boxes of the ctv promoters, this structure should be critical for RdgB binding (Fig. 5).

The transcription rates of pnl increased at high temperature, 30°C (Fig. 6), which is optimal for the growth of P. carotovorum subsp. carotovorum Er (25). In contrast, transcription of the ctv and rdgB genes decreases as the temperature rises. The parallel expression of rdgB and the ctv operons suggests that cellular concentrations of RdgB determine the transcription frequency of the ctv operons. In E. coli, the SOS-responsive expression of sfiA, which is under the control of the LexA repressor, is enhanced at low temperatures (8) and the synthesis of RecA, which is also repressed by LexA, is stimulated after a temperature downshift (13). RecA-dependent LexA cleavage would be enhanced at low temperatures, thus stimulating transcription of the LexA-repressible genes. RdgA structurally resembles LexA, and these proteins both function as transcriptional repressors (17). The expression of rdgB at 23°C is over double that at 30°C, allowing efficient transcription of the ctv operons (Fig. 6) and the consequently higher production of Ctv at the lower temperature (25). In contrast, Pnl production is stimulated more at 30°C than at 23°C (25). In fact, pnl transcription is tripled at the higher temperature (Fig. 6).

We considered that the high binding affinity of the RdgB box of P_pnl for RdgB can explain why pnl is transcribed more abundantly than the ctv genes, although less RdgB transcriptional activator is formed at 30°C. Even if the cellular RdgB concentration is halved at 30°C owing to the high binding affinity of the P_pnl RdgB box for RdgB, P_pnl can efficiently drive transcription at the higher temperature. Metabolism should be more active at 30°C (optimal growth temperature) than at 23°C (10); hence, the amounts of the pnl gene transcribed and translated would increase (Fig. 6). A decrease in rdgB transcript levels at 30°C must be critical for ctv promoters with RdgB boxes that have low binding affinity. Therefore, the transcription frequency of the ctv promoters would be reduced, limiting Ctv production at high temperatures. Indeed, when RdgB is supplied from the IPTG-inducible Ptrc::rdgB gene on a plasmid, strain Er can produce more Ctv at 30°C than at 23°C (Fig. 7B), in accordance with the notion that the amount of RdgB in the MMC-induced Er cells limits transcription of the ctv genes at 30°C. On the other hand, the relative amounts of Pnl protein (or pnl transcripts) synthesized at 23°C and 30°C by MMC- and IPTG-induced cells are similar (Fig. 6 and 7B), indicating that the amount of RdgB in MMC-induced cells cultured at 30°C is sufficient for fully expressing the pnl gene.

Strains of Pectobacterium spp. are frequently isolated from soils at between 17 and 25°C, depending on species (32), implying that they prevail in natural environments under lower temperatures than their optimal growth temperatures in vitro (10, 32). Although most other P. carotovorum subsp. carotovorum strains, including strain Er, optimally grow between 28 and 31°C (10, 32), they may share natural niches with other Pectobacterium spp. Bacteriocins kill competitive bacteria of the same or closely related species (15, 26). Bacteriocinogenic strains would have an advantage over their competitors if they produce bactericides at temperatures favorable for their competitors (i.e., 23°C). Pnl appears to accelerate plant tissue degradation (Fig. 8A and B), and infective pathogens would utilize nutrients in macerating plant tissues. The increased production of Pnl at 30°C would promote tissue maceration, thereby supplying actively growing Er cells with nutrients. Thus, the temperature-dependent regulation of Ctv and Pnl synthesis probably fulfills the biological functions of these proteins in nature.

The pnl gene is distributed among many, if not all, strains of soft-rotting Pectobacterium spp. (22), and Pnl synthesis is induced in plant tissues, including potato tubers, presumably by innate detrimental compounds that potentially damage DNA (38). These findings suggest that this pectolytic enzyme plays a role in the virulence of this group of bacteria. P. carotovorum subsp. carotovorum Er caused more-pronounced soft-rot symptoms on potato tubers at 30°C than at 23°C (Fig. 8A and B). The virulence of an rdgB mutant defective in Pnl production was significantly reduced compared with that of the wild-type strain at both 23°C and 30°C, but the effect of rdgB knockout on virulence was more apparent at 30°C than 23°C (Fig. 8), in accordance with the fact that more Pnl is produced at 30°C than at 23°C (Fig. 6 and 7B). Our preliminary experiments revealed that the pnl knockout mutant exhibits virulence that is intermediate between that of the wild type and that of the rdgB mutant, proving that Pnl is a virulence factor of strain Er and that RdgB controls another unknown virulence gene(s). Understanding the activation mechanism of rdgB in host plants and identification of the genes under the control of this regulatory gene should provide further insights into the pathogenicity of P. carotovorum spp.

 
We thank the National BioResource Project (NIG, Japan) for E. coli S17-1(pir) and the plasmid pLOI2223.

 
* Corresponding author. Mailing address: Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori Amamiya-machi 1-1, Aoba-ku, Sendai 981-8555, Japan. Phone: 81-22-717-8779. Fax: 81-22-717-8780. E-mail: yosifumi{at}biochem.tohoku.ac.jp

Published ahead of print on 8 August 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Department of Human Health and Nutrition, Graduate School of Comprehensive Human Sciences, Shokei Gakuin University, Yurigaoka 4-10-1, Natori 981-1295, Japan.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Aiba, H., S. Sdhya, and B. de Crombrugghe. 1981. Evidence for two functional gal promoters in intact Escherichia coli cells. J. Biol. Chem. 256:11905-11910.[Abstract/Free Full Text]
2
2. Amann, E., B. Ochs, and K. J. Abel. 1988. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301-315.[CrossRef][Medline]
3
3. Artsimovitch, I., and M. M. Howe. 1996. Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in P_m identifies a new region required for promoter function. Nucleic Acids Res. 24:450-457.[Abstract/Free Full Text]
4
4. Artsimovitch, I., K. Murakami, A. Ishihama, and M. M. Howe. 1996. Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both and ⁷⁰ subunits of Escherichia coli RNA polymerase. J. Biol. Chem. 271:32343-32348.[Abstract/Free Full Text]
5
5. Barras, F., F. Vangijsegem, and A. K. Chatterjee. 1994. Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu. Rev. Phytopathol. 32:201-234.
6
6. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113.[Medline]
7
7. Carey, J. 1991. Gel retardation. Methods Enzymol. 208:103-117.[Medline]
8
8. Dri, A. M., and P. L. Moreau. 1993. Phosphate starvation and low temperature as well as ultraviolet irradiation transcriptionally induce the Escherichia coli LexA-controlled gene sfiA. Mol. Microbiol. 8:697-706.[CrossRef][Medline]
9
9. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.[Medline]
10
10. Hasegawa, H., A. Chatterjee, Y. Cui, and A. K. Chatterjee. 2005. Elevated temperature enhances virulence of Erwinia carotovora subsp. carotovora strain EC153 to plants and stimulates production of the quorum sensing signal, N-acyl homoserine lactone, and extracellular proteins. Appl. Environ. Microbiol. 71:4655-4663.[Abstract/Free Full Text]
11
11. Itoh, Y., K. Izaki, and H. Takahashi. 1978. Purification and characterization of a bacteriocin from Erwinia carotovora. J. Gen. Appl. Microbiol. 24:27-39.[CrossRef]
12
12. Itoh, Y., K. Izaki, and H. Takahashi. 1980. Simultaneous synthesis of pectin lyase and carotovoricin induced by mitomycin C, nalidixic acid or ultraviolet light irradiation in Erwinia carotovora. Agric. Biol. Chem. 44:1135-1140.
13
13. Jones, P. G., R. A. VanBogelen, and F. C. Neidhardt. 1987. Induction of proteins in response to low temperature in Escherichia coli. J. Bacteriol. 169:2092-2095.[Abstract/Free Full Text]
14
14. Kamimiya, S., K. Izaki, and H. Takahashi. 1972. A new pectolytic enzyme in Erwinia aroideae formed in the presence of nalidixic acid. Agric. Biol. Chem. 36:2367-2372.
15
15. Kamimiya, S., K. Izaki, and H. Takahashi. 1977. Bacteriocin in Erwinia aroideae with tail like structure of bacteriophages. Agric. Biol. Chem. 41:911-912.
16
16. Kumaraswami, M., M. M. Howe, and H. W. Park. 2004. Crystal structure of the Mor protein of bacteriophage Mu, a member of the Mor/C family of transcription activators. J. Biol. Chem. 279:16581-16590.[Abstract/Free Full Text]
17
17. Liu, Y., A. Chatterjee, and A. K. Chatterjee. 1994. Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. carotovora in response to DNA-damaging agents. Mol. Microbiol. 14:999-1010.[CrossRef][Medline]
18
18. Liu, Y., Y. Cui, A. Mukherjee, and A. K. Chatterjee. 1997. Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnlA: a role for RdgB. Microbiology 143:705-712.[Abstract/Free Full Text]
19
19. Liu, Y., X. Wang, A. Mukherjee, and A. K. Chatterjee. 1996. RecA relieves negative autoregulation of rdgA, which specifies a component of the RecA-Rdg regulatory circuit controlling pectin lyase production in Erwinia carotovora ssp. carotovora. Mol. Microbiol. 22:909-918.[CrossRef][Medline]
20
20. Machida, M., H. Kamio, and D. Sorensen. 1997. Long-range and highly sensitive DNase I footprinting by an automated infrared DNA sequencer. BioTechniques. 23:300-303.[Medline]
21
21. Martinez-Morales, F., A. C. Borges, A. Martinez, K. T. Shanmugam, and L. O. Ingram. 1999. Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction. J. Bacteriol. 181:7143-7148.[Abstract/Free Full Text]
22
22. McEvoy, J. L., H. Murata, and A. K. Chatterjee. 1990. Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents. J. Bacteriol. 172:3284-3289.[Abstract/Free Full Text]
23
23. McEvoy, J. L., H. Murata, and A. K. Chatterjee. 1992. Genetic evidence for an activator required for induction of pectin lyase in Erwinia carotovora subsp. carotovora by DNA-damaging agents. J. Bacteriol. 174:5471-5474.[Abstract/Free Full Text]
24
24. Nguyen, A. H., T. Tomita, M. Hirota, T. Sato, and Y. Kamio. 1999. A simple purification method and morphology and component analyses for carotovoricin Er, a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er. Biosci. Biotechnol. Biochem. 63:1360-1369.[CrossRef][Medline]
25
25. Nguyen, H. A., J. Kaneko, and Y. Kamio. 2002. Temperature-dependent production of carotovoricin Er and pectin lyase in phytopathogenic Erwinia carotovora subsp. carotovora Er. Biosci. Biotechnol. Biochem. 66:444-447.[CrossRef][Medline]
26
26. Nguyen, H. A., T. Tomita, M. Hirota, J. Kaneko, T. Hayashi, and Y. Kamio. 2001. DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er. J. Bacteriol. 183:6274-6281.[CrossRef]
27
27. Ogawa, H., and T. Ogawa. 1990. Regulation in repressor inactivation by RecA protein. Adv. Biophys. 26:33-49.[CrossRef][Medline]
28
28. Ohnishi, H., T. Nishida, A. Yoshida, Y. Kamio, and K. Izaki. 1991. Nucleotide sequence of pnl gene from Erwinia carotovora Er. Biochem. Biophys. Res. Commun. 176:321-327.[CrossRef][Medline]
29
29. Sambrook, J., and D. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
30
30. Schweizer, H. P. 1992. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol. Microbiol. 6:1195-1204.[Medline]
31
31. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784-791.[CrossRef]
32
32. Smadja, B., X. Latour, S. Trigui, J. F. Burini, S. Chevalier, and N. Orange. 2004. Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.). Can. J. Microbiol. 50:19-27.[CrossRef][Medline]
33
33. Tomizawa, H., and H. Takahashi. 1971. Stimulation of pectolytic enzyme formation of Erwinia aroidea by nalidixic acid, mitomycin C and bleomycin. Agric. Biol. Chem. 35:191-200.
34
34. Tsuyumu, S., and A. K. Chatterjee. 1984. Pectin lyase production in Erwinia chrysanthemi and other soft-rot Erwinia species. Physiol. Plant Pathol. 24:291-302.[CrossRef]
35
35. Von Bodman, S. B., W. D. Bauer, and D. L. Coplin. 2003. Quorum sensing in plant-pathogenic bacteria. Annu. Rev. Phytopathol. 41:455-482.[CrossRef][Medline]
36
36. Yamada, K., M. Hirota, Y. Niimi, H. A. Nguyen, Y. Takahara, Y. Kamio, and J. Kaneko. 2006. Nucleotide sequence and organization of the genes for carotovoricin (Ctv) from Erwinia carotovora indicate that Ctv evolved from the same ancestor as Salmonella tyhpi prophage. Biosci. Biotechnol. Biochem. 70:2236-2247.[CrossRef][Medline]
37
37. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.[CrossRef][Medline]
38
38. Zink, R. T., J. K. Engwall, J. L. McEvoy, and A. K. Chatterjee. 1985. recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp. carotovora. J. Bacteriol. 164:390-396.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, October 2008, p. 6017-6025, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.01297-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yamada, K. Articles by Itoh, Y. Search for Related Content PubMed PubMed Citation Articles by Yamada, K. Articles by Itoh, Y. Agricola Articles by Yamada, K. Articles by Itoh, Y.