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Applied and Environmental Microbiology, September 2008, p. 5841-5844, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01099-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

An Amino Acid Substitution at Position 740 in ⁷⁰ of Ralstonia solanacearum Strain OE1-1 Affects Its In Planta Growth

Ayami Kanda,^1, Kazuhiro Tsuneishi,¹ Ai Mori,¹ Kouhei Ohnishi,² Akinori Kiba,¹ and Yasufumi Hikichi^1*

Laboratory of Plant Pathology & Biotechnology, Kochi University, Nankoku, Kochi 783-8502, Japan,¹ Research Institute of Molecular Genetics, Kochi University, Nankoku, Kochi 783-8502, Japan²

Received 15 May 2008/ Accepted 11 July 2008

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in ⁷⁰ of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant ⁷⁰ over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.

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Ralstonia solanacearum strain OE1-1 shows virulence on tobacco, as well as tomato and eggplant, plants (17). OE1-1 possesses hrp genes, similar to R. solanacearum strain GMI1000, which is virulent on tomato plants and elicits a hypersensitivity response in infiltrated tobacco leaves (2, 5, 19, 34). After vigorous proliferation in intercellular spaces, OE1-1 invaded xylem vessels and then systemically infected the whole plant (17, 32, 33). The hrp mutants of OE1-1 lose their ability not only to proliferate in intercellular spaces but also to systemically infect the whole plant. Therefore, it is thought that proliferation of the bacteria in intercellular spaces after invasion of roots qualitatively control bacterial virulence.

The hrp genes encode proteins that construct the type III secretion system. In R. solanacearum, expression of hrp genes is regulated by the HrpB protein (2, 13, 34). Screening of genes controlled by HrpB has isolated many candidates for type III effector genes in R. solanacearum (9, 25). Moreover, by in vivo expression technology, genes expressed in R. solanacearum K60-infected tomato plants were isolated (6). However, the involvement of these candidates in the bacterium-host plant interactions remains to be elucidated. Furthermore, analysis by in vivo expression technology indicates that genes other than hrp and type III secretion system effector genes are involved in bacterial virulence (6, 7).

To elucidate the mechanism of vigorous growth of OE1-1 in roots in this study, we first selected mutants that lack systemic infectivity and do not provoke disease in tobacco plants. The suicide vector pUTSm/Sp (10) containing mini-Tn5, which includes the spectinomycin resistance gene, was transferred from Escherichia coli HB101 (Takara, Ohtsu, Japan) to OE1-1 by conjugation with E. coli HB101(pRK2013) (12). The roots of 8-week-old tobacco plants (Nicotiana tabacum cv. Bright Yellow) were soaked in a bacterial suspension (1.0 x 10⁸ CFU/ml) of 421 spectinomycin-resistant mutants derived from OE1-1 for 30 min, and then inoculated plants were grown in water culture pots (Yamato water culture pot no. 1; Yamato Plastic Co. Ltd., Tokyo, Japan) with one-fifth-strength Hoagland's solution in a growth room at 25°C under 10,000 lx for 16 h/day (root dipping) (17). Each assay was repeated in five successive trials, and within each trial we treated five plants with each strain. Only one mutant, OE1101, lacked the ability to cause wilt in tobacco plants. Seven days after inoculation by root dipping, the stems of five tobacco plants were cut into three pieces with razor blades (Fig. 1). The cut site of each piece was pressed onto Hara-Ono medium (15) for OE1-1 and onto Hara-Ono medium containing spectinomycin at 50 µg/ml for OE1101, and the media were incubated at 30°C for 3 days (plate-printing assay) (20). Though OE1-1 was detected at some sites, the mutant was never detected at sites (sites A and B) beyond the bacterium-inoculated area (site C). These results indicate that the mutant cannot systemically infect tobacco plants, leading to the loss of its ability to cause wilt in tobacco plants.

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FIG. 1. Cut sites on tobacco plants used in the plate-printing assay. Detection of R. solanacearum strains at cut sites (A, B, and C) on tobacco plants inoculated with bacteria by root dipping. The stems of the tobacco plants were cut into three pieces 7 days after inoculation. Dashed lines indicate cut sites.

Southern blot analysis with Sm^r/Sp^r derived from pUTSm/Sp as the probe showed that mini-Tn5 was inserted at one site in the genomic DNA of OE1101 (data not shown). Nucleotide sequence analysis of pMINE, in which 3.4 kbp of genomic DNA of OE1101 including Sm^r/Sp^r was ligated into pUC118 (Takara), showed that mini-Tn5 was inserted into rpoD (which encodes ⁷⁰) (rpoD::mini-Tn5 Sm^r/Sp^r) at nucleotide position 2217 from the start codon (GTG). The bacterial RNA polymerase holoenzyme consists of a catalytic core enzyme with a sigma factor conferring on the holoenzyme the ability to initiate promoter-specific transcription (14). The primary sigma factor in E. coli, ⁷⁰, recognizes promoters that are needed for the transcription of general housekeeping genes and is responsible for most of the transcription during exponential growth (29). ⁷⁰ of OE1101 was predicted to be composed of 758 amino acid residues. The amino acid sequences of 19 residues at the C terminus were derived from mini-Tn5 (Fig. 2). ⁷⁰ shares four regions of similarity with primary sigma proteins of other prokaryotes (14, 23). When present in the RNA polymerase holoenzyme, ⁷⁰ sets the start site for transcription by recognizing various DNA elements, and residues within each of the four regions have been shown to interact with sequences in promoter DNA (4, 8, 11, 16, 26). The majority of E. coli promoters have –10 and –35 DNA elements, which are contacted by ⁷⁰ regions 2 and 4, respectively (8, 26). Specific base recognition of the –35 sequences arises through a DNA-binding helix-turn-helix (HTH) motif in region 4.2 (21, 27, 28). ⁷⁰ of OE1-1 also contained four conserved regions, and the amino acid sequences of region 4.2 of OE1-1 were identical to those of E. coli strain K-12 (Fig. 2). Arg740 was located adjacent to the C terminus of region 4.2 and the DNA binding HTH motif of OE1-1 ⁷⁰.

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FIG. 2. Comparison of the deduced amino acid sequences of the C terminus of region 4.2 in 70 of R. solanacearum strain OE1-1, an rpoD mutant version (OE1101) of OE1-1 derived by transposon mini-Tn5 insertion, a recombinant version (OE1102) of OE1-1 with Arg740Cys in 70, and E. coli strain K-12. The amino acids at positions 740 and 599 in 70 of R. solanacearum strains and E. coli, respectively, are underlined. H, T, and H represent the HTH motif in region 4.2.

To analyze the effect of the Arg740Cys substitution in ⁷⁰ on bacteria virulence, 3.0-kbp and 361-bp DNA fragments were PCR amplified from genomic DNA of OE1-1 with primers Xba-rpoD (5'-GCTCTAGACTTCGTCGAGCGCCTGGGCCAG-3') with an added XbaI site (underlined) and Mutant-FW (5'-TCCGAACGGCTCGGGTGACACAGTTTGCGC-3') and primers Mutant-RV (5'-AGGCGCTGCGCAAACTGTGTCACCCGAGCC-3') and Hind-rpoD (5'-CCCAAGCTTACCCTGCCGCCGGAGCCGCAT-3') with an added HindIII site (underlined), respectively. Each PCR product was mixed and PCR amplified with the Xba-rpoD and Hind-rpoD primer pairs. The resultant XbaI- and HindIII-digested 3.3-kbp DNA fragment was ligated into XbaI- and HindIII-digested pK18mobsacB (30) to create pK18mobsacBRS3. This plasmid was electroporated into OE1-1 cells (1, 33), and kanamycin-resistant and sucrose-sensitive recombinants were selected. The recombinant was incubated in a rich medium (PS medium) (17) for 6 h and a kanamycin-sensitive and sucrose-resistant recombinant, OE1102, was selected. DNA sequencing of PCR-amplified DNA fragments with Xba-rpoD and Hind-rpoD as primers was performed to verify the only Arg740Cys substitution in ⁷⁰ (data not shown). OE1102 grew in both PS medium and a minimal medium (Boucher medium) (5), similar to OE1-1, indicating that the Arg740Cys change in ⁷⁰ did not affect in vitro bacterial growth (data not shown).

Reduced sap flow caused by the presence of a large number of bacterial cells and exopolysaccharide (EPS) slime produced by the bacteria in some xylem vessels lead to extensive wilting in plants (31). EPS I content was quantified by measuring hexosamine with the Elson-Morgan reaction (18). The EPS I productivity of OE1102 was 448 µg of polymeric (>14-kDa) hexosamine/mg cell protein, similar to that of the wild type (432 µg/mg).

OE1-1 infiltration of tobacco leaves induced necrotic lesions, which is dependent on hrp genes, at the sites of infection 60 h after inoculation (19). OE1102 induced necrotic lesions in the infiltrated area of tobacco leaves at 60 h after inoculation, as well as the parent strain.

The hrpY and popA genes encode a protein constructing hrp pili and the type III effector protein PopA, respectively. To analyze the influence of an Arg740Cys substitution in ⁷⁰ on the expression of rpoD, hrpY, and popA by reverse transcription-PCR, total RNA was isolated from five of each set of tobacco leaves at 0, 1, 3, and 6 h after infiltration with 50 µl bacterial suspension (1.0 x 10⁸ CFU/ml) of R. solanacearum strains, and DNase I (Applied Biosystems, Tokyo, Japan) treatment was used to remove the genomic DNA from the RNA preparation (20). rpoD, hrpY, and popA cDNA fragments were synthesized from total RNA (6 µg) with primers rpoD-FW (5'-CGCCTTCGCTTCGATCTGG-3'), hrpY-Bam (5'-CGGGATCCTTAGCTGATCAGGTCCTTGGC-3'), and popA-SQ- (5'-GTTGGCACCGTTGACATCGC-3'), respectively. PCR was performed with primers rpoD-FW and rpoD-RV (5'-TGCAGGAAACCGGCAACG-3') for the amplification of a 370-bp DNA fragment specific to rpoD, hrpY-Bam and Nde-hrpY (5'-GGAATTCATATGGCAGGCGTTC CGAAAC-3') for the amplification of a 250-bp DNA fragment specific to hrpY, and PopA-SQ– and popA-SQ+ (5'-CTGGTGAAGCTGCTGAAGGC-3') for the amplification of a 320-bp DNA fragment specific to popA. When the RNA treated with DNase I was used as the template in PCRs, no product was observed. The expression of mutated rpoD in OE1102-infiltrated tobacco leaves was constitutively detected, as well as that of rpoD in OE1-1-infiltrated leaves (Fig. 3). Furthermore, expression of hrpY and popA in OE1102-infiltrated tobacco leaves was detected immediately after inoculation and 3 h after infiltration, respectively, similar to that in OE1-1-infiltrated leaves (Fig. 3). These results indicated that OE1102 retained its activity to express hrp genes, similar to OE1-1.

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FIG. 3. Reverse transcription-PCR analysis of rpoD, hrpY, and popA of R. solanacearum strain OE1-1 and a recombinant version (OE1102) of OE1-1 with Arg740Cys in 70 infecting tobacco leaves. Total RNA was isolated from tobacco leaves at 1, 3, and 6 h after infiltration with R. solanacearum strains.

Tobacco plants inoculated with OE1-1 wilted 7 days after inoculation. In contrast, OE1102 lacked virulence on tobacco plants, as did OE1101. To analyze the virulence of R. solanacearum strains on other solanaceous plants, 8-week-old tomato (Lycopersicon esculentum cv. Ohgata-Fukuju) and eggplant (Solanum melongenae cv. Senryo-nigou) plants were inoculated with a bacterial suspension at 1.0 x 10⁸ CFU/ml by root dipping. Though OE1-1 showed virulence on eggplant and tomato plants 7 days after inoculation, OE1101 and OE1102 lacked virulence on both solanaceous plants. These results indicate that an Arg740Cys substitution in ⁷⁰ leads to a loss of bacterial virulence.

To analyze the bacterial population in roots, roots were excised daily from five of each set of tobacco plants, from 0 to 5 days after inoculation with bacteria, and ground with a mortar and pestle. The original solution and 10-fold serial dilutions of it were spread onto three plates of Hara-Ono medium. The colonies were counted after 2 days of incubation at 30°C. The population of OE1-1 in roots increased to 7.0 x 10⁷ CFU/g 5 days after inoculation (Fig. 4). In contrast, the population of OE1102 in roots drastically decreased to 3.0 x 10² CFU/g 5 days after inoculation. The plate-printing assay showed that OE1102 was detected in the only bacterium-inoculated area, as well as OE1101. These findings indicate that an Arg740Cys substitution in ⁷⁰ results in loss of the bacterial ability to grow in roots, leading to loss of systemic infectivity.

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FIG. 4. Populations of R. solanacearum strain OE1-1 (black columns) and a recombinant (OE1102; white columns) of OE1-1 with Arg740Cys in 70 in roots of tobacco plants inoculated by soaking the roots in a bacterial suspension (1.0 x 108 CFU/ml) for 20 min. Values represent the mean ± the standard deviation of five separate experiments. Asterisks denote values significantly different from the population of OE1-1 (*; P < 0.05) by the Student t test.

To test if the rpoD mutation leading to an Arg740Cys substitution is dominant or recessive, the dominant negative phenotype was analyzed with the rpoD mutation located on the plasmid or on the recipient chromosome. DNA fragments of 3.4 kbp were PCR amplified with OE1102 genomic DNA and OE1-1 genomic DNA as templates and Xba-rpoD and Hind-rpoD as primers, and the resultant XbaI- and HindIII-digested fragments were ligated into the XbaI and HindIII sites of pUCD3101 (17) to create pUCD3101rpoDRS3 and pUCD3101rpoD, respectively. OE1-1 and OE1102 were transformed with pUCD3101rpoDRS3 and pUCD3101rpoD by electroporation to create kanamycin-resistant transformants OE1103 and OE1104, respectively. Both OE1103 and OE1104 lacked virulence on tobacco plants, similar to OE1102. Furthermore, the plate-printing assay with Hara-Ono medium containing kanamycin at 50 µg/ml showed that both transformants were never detected at sites beyond the bacterium-inoculated area, suggesting that both OE1103 and OE1104 lacked systemic infectivity. Transformants of OE1-1 and OE1102 with pUCD3101 showed the same virulence phenotype as OE1-1 and OE1102, respectively. These results indicate the negative dominance of mutant ⁷⁰ over the wild-type protein.

The presence of a good match to both the –10 and –35 canonical sequences of promoter DNA is usually sufficient for E. coli ⁷⁰ to recognize a promoter without the aid of additional factors. However, activation factors can be needed when either sequence element deviates significantly from the consensus. Many activators in E. coli work through class I or class II activation mechanisms (3, 22). Class I activators interact with residues in the C-terminal domain of the subunit of polymerase. Class II activators interact with residues in ⁷⁰ region 4, on the basis of mutations in ⁷⁰ that selectively eliminated the function of an activator without altering basal transcription. These activator-specific mutations cluster in two regions of ⁷⁰: a segment at the beginning of the first helix (amino acids 573 to 580) and a segment downstream from the second helix (amino acids 584 to 598) of the HTH motif in E. coli ⁷⁰ region 4.2. The five basic residues, including Arg599, in a narrow region of E. coli ⁷⁰ (residues 590 to 603) are implicated in activation, and the residues are either in the C terminus of a long recognition helix that includes residues recognizing the –35 hexamer region of the promoter or in the subsequent loop (24). The Arg599 residue in ⁷⁰ of E. coli, which corresponds to Arg740 in ⁷⁰ of OE1-1, is reportedly the contact point with the cyclic AMP receptor protein, which regulates catabolic genes under aerobic conditions. Therefore, an Arg740Cys substitution may lead to changes in interactions between the class II activators and ⁷⁰, resulting in differences in the expression of genes that are required for vigorous in planta growth of the bacteria. The effects of interactions between the class II activators and ⁷⁰ on the expression of genes involved in bacterial in planta growth remains to be elucidated.

Taken together, our results indicate that the potential of ⁷⁰ with an Arg740Cys substitution to suppress bacterial growth in planta may lead to elucidation of the mechanism of bacterial growth in planta, which plays a key role in bacterial virulence.

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rpoD nucleotide sequences of R. solanacearum strains OE1-1 and OE1101, which was derived from OE1-1 with synthetic transposon mini-Tn5 Sm/Sp, were assigned to the DDGJ database under accession numbers AB358977 and AB445124, respectively.

 
This work was supported by Grants-in-Aid for Scientific Research awarded to Y.H. (16380037, 20380029), A.K. (16780031, 18780029), and K.O. (17380031, 20017020) from the Ministry of Education, Science, Sports and Culture, Japan, and a grant from the Asahi Glass Foundation to A.K.

 
* Corresponding author. Mailing address: Faculty of Agriculture, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502, Japan. Phone: 81-88-864-5218. Fax: 81-88-864-5200. E-mail: yhikichi{at}cc.kochi-u.ac.jp

Published ahead of print on 18 July 2008.

Present address. National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan.

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Applied and Environmental Microbiology, September 2008, p. 5841-5844, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01099-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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