Influence Of PH And Ionic Conditions On LysR-type Regulatory Gene SalR In Acinetobacter Baylyi ADP1

Acinetobacter spp. (family Moraxellaceae) is widespread in the natural environment. The non-pathogenic soil bacterium Acinetobacter baylyi ADP1 closely relates to Pseudomonas aeruginosa and Pseudomonas putida. But unlike these bacteria, Acinetobacter baylyi ADP1 is highly competent for natural transformation that facilitates genetic engineering. In Acinetobacter baylyi ADP1, salicylate is hydroxylated into catechol which entersβ-ketoadipate pathway, then converted to acetyl CoA and succinyl CoA which enter tricarboxylic acid cycle. The sal operon is composed of four open reading frames: salA, salR, salE, and salD. The product of salR gene,which is a member of LysR-type transcription regulator, regulates the expression of salA but not salE. A salicylate biosensor strain Acinetobacter ADPWH_lux had been constructed. This biosensor strain contains a chromosomally located fusion of the salA gene at the Acinetobacter baylyi ADP1 sal operon with the promoterless bioluminescence gene cassette luxCDABE from Photorhabdus luminescens. The bacterium produces bioluminescence specifically induced by salicylate, which does not require exogenous substrate, and is proportional to the concentration of the salicylate within a wide range.In this paper, the conditions where the biosensor can be applied were optimized. The bacteria were cultured under various environment with different pH values or ions to simulate the environmental stress which bacteria were often encountered in natural environment. The expression of salR gene in Acinetobacter baylyi ADP1 was quantitatively analysised by bioluminescence combined with RT-PCR to confirm transcription of the salA gene. This biosensor was used to detect the salicylate concentrations of insect-resistant transgenic rice"MF86"containing both cry1Ac and CpTI genes and the control"MingHui86"when they were bitted by insects. The ecological community structures of phyllosphere microbe in both transgenic and non-transgenic rice were also anlysised by plate counts and DGGE in this process.As a result, the expression of salR gene was very stable in pH 5.54-9.05 in exponential phase and pH 5.79-9.79 in stationary phase. Bioluminescence maintained at >80% (exponential) and >60% (stationary) levels of the highest value within these two ranges, respectively. More extreme pH environment strongly inhibit the bioluminescence of the bacteria. In 27 kinds of different ions conditions, most heavy metal ions in high concentrations strongly inhibited the growth and bioluminescence of the bacteria. There is only Ba2+ could enhance the bioluminescence of the bacteria in exponential phase. So far, there is no sufficient information about the influence of different pH and ionic conditions on the LTTR activity so this paper revealed some unknown features of a LTTR regulated gene expression and demonstrated that the biosensor is a sensitive tool for understanding salR regulation. It maybe difficult to use the salicylate biosensor in the rice, and the results of plate counts and DGGE demonstrated the kinds of phyllosphere bacteria were nearly the same in different test groups, but more experiments are needed to verify the results.The application of the biosensor has been optimized, convenient for detecting the changes of salicylic acid in transgenic rice. And the ecological community structure of phyllosphere microbe in transgenic rice was studied to finally provide theoretical reference and experimental basis for the biosafety evaluation of transgenic rice.