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In order to develop an efficient system for deleting genomic segment in Agrobacterium tumefaciens to analyze gene functions and construct marker gene-free recombinant strains, a Cre recombinase expression plasmid was constructed by placing its encoding gene under the control of Ptet promoter and cloning into the plasmid replicable in both A. tumefaciens and E. coli. Triple recombineering was applied to efficiently construct integrative vectors which were used to introduce loxP sites and selection markers into the chromosome of A. tumefaciens. Cre recombinase could be properly induced by anhydrotetracycline in A. tumefaciens, which was revealed by the fact that kanamycin resistance gene flanked by two parallel loxP sites was excised from the genome of A. tumefaciens with virtually 100% efficiency. And what is more, an A. tumefaciens mutant carrying large-deletion (~85 kb) in genome was successfully constructed by Cre/loxP system. Here, we described the application of combination of Cre/loxP system and triple recombineering to efficiently excise genomic segment in A. tumefaciens, which also would facilitate efficient construction of multiple gene disruptions in A. tumefaciens.

Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation.

Agrobacterium tumefaciens has a chemtaxis operon, which includes orf1, orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, and orf10. In-frame deletions of cheY1 and cheY2 were constructed and the chemosensory behavior of the mutants was examined on swarm plates and in a chemotaxis assay toward acetosyringone. The cheY2 mutant (C1/delY2) showed impaired chemotactic capabilities in both swarming and chemotaxis assays. The effect of lacking CheY1 on chemotaxis is less severe than that of CheY2, under the conditions studied.

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H2O2 induced adaptive protection to lethal concentrations of H2O2. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an inducing concentration of menadione conferred cross-protection against H2O2, but not to tert-butyl hydroperoxide (tBOOH), killing. The menadione induced cross-protection to H2O2 was due to the compound's ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacterium's ability to survive H2O2 treatment. Some aspects of the oxidative stress response of A. tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions.