Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L.

Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACXand GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue- and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis （DPA）, however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate （MeJA）, cold, and abscisic acid （ABA） treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism.

A novel and complete gene cluster involved in the degradation of aniline by Delftia sp. AN3

A recombinant strain, Escherichia coli JM109-AN1, was obtained by constructing of a genomic library of the total DNA of Delftia sp. AN3 in E. coli JM109 and screening for catechol 2,3-dioxygenase activity. This recombinant strain could grow on aniline as sole carbon, nitrogen and energy source. Enzymatic assays revealed that the exogenous genes including aniline dioxygenase （AD） and catechol 2,3-dioxygenase （C230） genes could well express in the recombinant strain with the activities of AD and C230 up to 0.31 U/mg wet cell and 1.92 U/mg crude proteins, respectively. The AD or C23O of strain AN3 could only catalyze aniline or catechol but not any other substituted substrates. This recombinant strain contained a recombinant plasmid, pKC505-AN1, in which a 29.7-kb DNA fragment from Delftia sp. AN3 was inserted. Sequencing and open reading frame （orfs） analysis of this 29.7 kb fragment revealed that it contained at least 27 orfs, among them a gene cluster （consisting of at least 16 genes, named danQTA1A2BRDCEFG1HIJKG2） was responsible for the complete metabolism of aniline to TCA-cycle intermediates. This gene cluster could be divided into two main parts, the upper sequences consisted of 7 genes （danQTA1A2BRD） were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, and the central genes （danCEFG1HIJKG2） were expected to encode meta-cleavage pathway enzymes for catechol degradation to TCA-cycle intermediates. Unlike clusters tad from Delftia tsuruhatensis AD9 and tdn from Pseudomonas putida UCC22, in this gene cluster, all the genes were in the same transcriptional direction. There was only one set of C230 gene （danC） and ferredoxin-like protein gene （danD）. The presence of only one set of these two genes and specificity of AD and C230 might be the reason for strain AN3 could only degrade aniline. The products of danQTA1A2BRDC showed 99%-100% identity to those from Delftia acidovorans 7N, and 50%-85% identity to those of tad cluster from D. tsuruhatensis AD9 in amino acid residues. Besides this dan cluster, the 29.7 kb fragment also contained genes encoding the trans-membrane transporter and transposases which might be needed for transposition of the gene cluster. Pulsed-field gel electrophoresis （PFGE） and plasmid curing experiments suggested that the dan cluster might be encoded on the chromosome of strain AN3. The GenBank accession number for the dan cluster of Delftia sp. AN3 is DQ661649.

Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The re- duced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope.