Cloning And Functional Identification HdtS Gene Encoding AHL Synthase From Oxalobacter Formigenes

Kidney stone disease is one of the common urological diseases, and calcium oxalate （CaOx） stonesare the most common form of five kinds of kidney stones, accounting for more than80%of kidneystones. The probability of occurrence of calcium oxalate stones is positively correlated with theconcentration of oxalic acid in human urine. Oxalic acid can be used as the only carbon and energysources by Oxalobacter formigenes （OxF）, which is Gram-negative and strictly anaerobic. Study showsthat one of the effective means of prevention and treatment of kidney stone disease is taking the OxFagents and improving their proportion of colonization in intestines. However, the facters that influencetheir colonization rate are the restricting the development of them in the clinical application. Studieshave shown that colonization rate of a lot of intestinal bacterial is controlled by quorum sensing （QS）system. QS is a process by which bacteria communicate via the secretion of chemical signalingmolecules termed autoinducers.QS in Gram-negative bacteria is commonly composed of a AHL synthase which could produceautoinducer and the corresponding regulator. So far, based on the difference of the synthase and theregulatory protein, QS can be divided into three types: the LuxI-LuxR system, LuxM-LuxN/AinS-AinRsystem and HdtS system. For the first two types, they have been deeply identified. However, the thirdone, HdtS system, is significantly different from the first two types. The identification of HdtS is verylimited, and there is no report about its regulator. The gene hdtS encoding an AHL synthase was firstlycloned from Pseudomonas fluorescens in2000, which has20%identity with the plsC encodinglysophosphatidic acid acyl transferase enzyme in Escherichia coli. HdtS can produce three kinds ofAHLs:3-OH-C14:1, C10:1and C6:1. Another HdtS-type AHL synthase, Act, was accordingly clonedfrom Acidithiobacillus ferrooxidan and identified, which has37%identity with HdtS and also forms acluster with glyQ and glyS and gph encoding a phospholipase. This gene cluster is highly conserved inProteobacteria, and the gene sequence is also very consistent. Act was confirmed to encode a C14:1AHL, and regulate the Lux QS system in A. ferrooxidans.The genome of Oxalobacter formigenes OXCC13and HOxBLS has been sequenced. In this study,we blasted them with the QS-related hdtS genes in Pseudomonas fluorescens, luxI/luxR in Vibriofischeri and luxM/luxN in V. fischeri and its homologues AinS/AinR in V. fischeri. We found that bothgenes with the Accession No. OFBG₀1243（O. formigenes OXCC13） and OFAG₀1224（O.formigenes HOxBLS） encoding putative1-acyl-sn-glycerol-3-phosphate acyltransferase showed thehighest identities with hdtS. In addition to hdtS gene in P. fluorences, these two genes showed higheridentity with the act gene in A. ferrooxidans which has been identified to encode HdtS-type AHLsynthetase. Moreover, in O. formigenes, hdtS form a cluster with glyQ and glyS encoding the glycinetRNA synthetase alpha, beta subunit, respectively, and gph encoding a phospholipase, which is highlyconsistent with the gene order of act, glyQ, glyS and gph cluster in A. ferrooxidans. Therefore, the hdtSgene is hypothesized to be the candidate gene encoding the HdtS-type AHL synthetase. Moreover,another gene was found to be predicted to encode the putative1-acyl-sn-glycerol3-phosphatetransferase, which also has identity with the hdtS above in P. fluorences and Act in A. ferrooxidans.Since AGPA and hdtS are presumed to encode1-acyl-sn-glycerol-3-phosphate aminotransferase in O.formigenes, and have a certain identity with each other, AGPA was also hypothesized to be anothercandidate gene encoding an AHL synthase.With O. formigenes HOxBLS genomic DNA as a template, hdtS and AGPA genes was cloned by PCRand constructed into the pEASY-T3vector, and then the positive clones were tested with PCR anddouble digestionby restriction enzymes NdeI and BamHI, and one of them was chosen for sequencing,and the correct positive clone for hdtS and AGPA genes was finally obtained. To further study whetherhdtS and AGPA encode an AHL synthetase, hdtS and AGPA were constructed into the expression vectorpET19, respectively. Both genes were found to be able to be expressed in E. coli BL21, and most ofthem were also soluble, and the length of the protein is consistent with the predicted protein. Thisprovides a strong theoretical basis for the next verification of the function of these two genes. Finally, strain report method is used to preliminarily identify if either gene has the AHL synthetase function. Asa result, hdtS is found to encode an AHL synthetase, but AGPA not. To further test whether hdtS encodesan AHL synthetase, GC-MS should be used to make it sure.So far, there is no report about the QS-related gene in O. formigenes. Since O. formigenes is anextremely important probiotic to the treatment and prevention of kidney stones disease, cloning andfunctional analysis of the QS-related gene is very helpful for understanding of the quorum sensingsystem of O. formigenes, which is very important for the improvement of its colonization in humanintestines.