High-throughput Screening And Identification Of Bacteria Containing ACC Deaminase

When plants are exposed to stressful conditions, they often respond by producing ethylene. High level of ethylene inhibits plant growth. Plant growth-promoting bacteria that contain the enzyme 1-aminocyclopropane-l-carboxylate (ACC) deaminase can catalyze the cleavage of ACC, the immediate precursor of ethylene in plants, and thus lower the ethylene levels. Under current deteriorated field ecosystems in China, inoculation of bacteria containing ACC-deaminase often leads to a better plant growth than inoculation of some other plant growth-promoting bacteria, such as nitrogen-fixing bacteria. Hence, this study is focused on screening the bacteria containing ACC-deaminase from bacteria that are able to fix N2 or grow on nitrogen-free media and use them to promote crop production.Selective minimal media supplying ACC as the sole nitrogen source are widely used to isolate bacteria containing ACC-deaminase. However, this isolation method is not sensitive for bacteria that are able to grow on nitrogen-free media. And a high-throughput method is required to screen bacteria containing ACC deamianse from a large number of bacterial isolates that are able to grow on nitrogen-free media. This study developed a high-throughput colorimetric method based on ninhydrin reaction with ACC to measure bacterial consumption of ACC. In this method, ethylene glycol was used as the solvent to stabilize the ninhydrin regent and color development, ascorbic acid was used as the reducing regent to prevent the oxidation of the hydrindantin, and polypropylene chimney-top 96-well PCR plates were heated on boiling water bath, ACC concentration was determined by measurement of the absorbance at 570nm. By using this method six bacterial isolates consuming ACC were rapidly screened out from 43 nitrogen-fixing bacteria isolated from sugarcane and 68 bacterial isolates that were able to grow on nitrogen-free media and were isolated from maize. And the six isolates were demonstrated to possess ACC deaminase activity by using the colorimetric 2,4-dinitrophenyl-hydrazine assay. Therefore, the 96-well plate ninhydrin-ACC assay enables a high-throughput screening of bacteria containing ACC deaminase from large numbers of isolates. Furthermore, other 52 isolates containing ACC deaminase were screened out from 153 isolates that were able to grow on nitrogen-free medium and were isolated from sugarcane, and 268 isolates that were able to grow on minimal media containing ACC as the sole nitrogen source and were isolated from rice or Sedum alfredii.Molecular identification of bacteria containing ACC deaminase usually uses PCR amplification or DNA hybridization of the ACC deaminase structural gene (acdS). Previous studies designed degenerative primers based on a few available sequences. PCR amplification based on these primers was not as efficient as hybridization to detect acdS and yielded nonspecific amplicons whereas hybridization of different groups of acdS required different probes. This study designed broad-spectrum specific degenerative primers of CodehopACCf1,CodehopACCf2,CodehopACCf3 and CodehopACCr to amplify acdS based on the protein structures of ACC deaminases, differentiation of the conserved amino acids between ACC deaminase and D-cysteine desulfhydrase, and the CODEHOP strategy. PCR amplification based on the CODEHOP primers successfully amplified acdS from all the 58 bacterial isolates containing ACC deaminase and thus accomplished a high-throughput molecular method to identify bacteria containing ACC deaminase. Phylogenetic analysis of acdS showed that some acdS sequences obtained in this study were grouped into new acdS groups. And phylogenetic analysis of acdS and corresponding bacterial 16S rDNA revealed a broad horizontal gene transfer of acdS.This study further determined other plant growth-promoting traits including IAA secretion, nitrogen fixation, phosphate solubilization, siderophore secretion, and pathogen antagonism for 43 isolates isolated from maize and rice. They all showed more than one of these traits. Among them,16 isolates belonged to the genus Burkholderia were able to solubilize phosphate, secrete siderophore, and antagonize broad pathogenic fungi. Although no Burkholderia cepacia epidemic strain marker was detected from these isolates, they were close related to opportunistic pathogens Burkholderia cepacia complex and most of them were pathogenic to onion bulb. Pathogens may obtain ACC deaminase via horizontal transfer of acdS. Therefore, application of potential plant growth-promoting bacteria containing ACC deaminase requires strict pathogenic test.