Cloning And Functional Confirmation Of The TetR Related To Antagonistic Activity In Delftia Tsuruhatensis MTQ3

Take the Ralstonia solanacearum as target bacteria,23isolates which have notableantagonistic activity were got in plate confrontation test. By morphologic observation,physiological, phenotypic characterization and16S rDNA sequencing, MTQ3was identifiedas Delftia tsuruhatensis and FGQ5was identified as Delftia acidovorans. The Genbanknumber of MTQ3and FGQ5are HQ327477and HQ327476, respectively.Tested by the nutrition agar plate with muliple gradient of antibiotics, the result showsthat MTQ3and FGQ5were sensitive to kanamycin, but could tolerate10μg/mL rifampicin.We construct useful Delftia sp. Tn5-1063insertion library by triparental mating, MTQ3andFGQ5were used as recipient stains while E.coli DH5α (pRL1063a)used as donor stain andE.coli DH5α (pRK2013) as helperee stain,8896mutants were obtained. The mutated coloniescarrying the transposon on their chromosome were tested for antibacterial activity onsemi-solid PDA plates containing R. solanacearum. After tested all the mutants of MTQ3,23mutants had a bigger inhibition zone than the wild type,4mutants had a smaller inhibitionzone than the wild type, and only one mutant, MTQ3-ΔT, completely lost its antibacterialactivity. Of all the mutants of FGQ5, we obtained12mutants had a bigger inhibition zonethan the wild type, and5mutants had a smaller inhibition zone than the wild type. Themutants with Tn5-1063were confirmed by amplification of the sequence of the luxA with theluxA1and luxA2primers. The amplification results indicated that all of the mutants had theinsertion of Tn5-1063. In addition, the16S rRNA gene of MTQ3-ΔT was amplified andsequenced, confirming it was the mutant from parental MTQ3.According to the upstream sequence of Tn5-1063, three specific primers were designed,seven arbitrary degenerate primers were also designed. Using TAIL-PCR, the upstreamflanking sequence of Tn5-1063were obtained from the mutant MTQ3-ΔT, and was identifiedas the downstream of tetR gene. Then, according to the obtained sequence, another threespecific primers were designed for the upstream of tetR gene. Finally, by assembling theupstream and the downstream sequences, a sequence of1492bp was obtained containing thewhole open reading frame of tetR gene. This sequence was deposited in the GeneBank(accession number: JQ425693). The gene product is TetR family of transcriptional regulators, which consists a223-amino-acid protein TetR. The reported genomes of Delftia sp. Cs1-4andDelftia acidovorans SPH-1were all contained the gene of tetR, identical at93%level, andtheir protein sequence have identities of96%and95%.To examine whether the observed phenotype was due to tetR gene disruption in mutantMTQ3-ΔT, pBR322with the tetR gene containing its promoter region was constructed andintroduced into the MTQ3-ΔT mutant by triparental mating. The antagonistic activity of theresult strain MTQ3-T which complement MTQ3-ΔT with pBR322-T was tested in plateconfrontation test, with the parent strain MTQ3served as a positive control, MTQ3-ΔT-Pcomplement MTQ3-ΔT with vector pBR322, which served as a negative control. Theantagonistic activity of the mutant could be restored by complementation of a single copy oftetR gene, demonstrating that TetR is an important regulator of the antagonistic activity ofgenus Delftia. Although the entire genome sequences of Delftia acidovorans SPH-1andDelftia sp. Cs1-4have recently been available, their mechanisms of action against plantpathogens are poorly understood. Therefore, this may be a novel regulatory mechanism of theTetR family in Delftia sp. and a new antibiotic biosynthetic gene or a new disease resistancemechanism.