A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice.

As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 µM for PEP and 83 µM for S3P, and a significantly higher Ki value for glyphosate (373 µM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system.

Cloning and molecular characterization of a novel rolling-circle replicating plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1.

Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki Kl which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pKlS-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pKlS-1, ORF2 (MobKl) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8-25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pKlS-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pKlS-1, seven subclones were contructed in the B. thuringiensis ori-negative pHTIK vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (ReplK), dso and ORF4, exhibited replication ability. These findings identified pKlS-1 as a new RCR group VII plasmid, and determined its replication region.

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions.

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.

[The analysis of Bacillus thuringiensis vegetative insecticical protein gene cloning and expression].

Three kinds of Bacillus thuringiensis serotype-subsp. Leesis(H33) strain YBT-833, subsp. Aizawai(H7) strain YBT-1416 and subsp. Kurstaki(H3ab) strain YBT-1535, which were isolated by our lab, are chosen as original strain to clone vegetative insecticidal protein gene. Southern hybridization showed that vip genes are all localized at roughly 4-5 kb size-fractionated XbaI fragments of total DNA from YBT-833, YBT-1416 and YBT-1535. Three subgenomic libraries containing the vip gene fragment, were constructed with pUC19 as vector. Then, three vegetative insecticidal protein gene vip83, vip14 and vip15 are obtained from the libraries through the methods of colony-blot-in-situ screening and enzyme-cut detection. Comparision of DNA sequence made out that only vip83 gene exist five different base pairs with known vip genes. Because the sequences of vip14 and vip15 are the same, two of the three genes, vip83 and vip14, were subcloned to shuttle vehicle pHT315 to get recombinant plasmids pBMB8901 and pBMB8902 in turn. The plasmids were separately transformed into vip Bt. receptors BMB171 and 4Q7 to obtain four engineered strains BMB8901-171, BMB8902-171, BMB8901-4Q7 and BMB8902-4Q7. SDS-PAGE results indicated that all recombinant strains express 88 kD vegetative insecticidal protein. Bioassay also showed that the proteins of genes vip83 and vip14 both have certain toxicity to Lepidopteran insect larvae such as Heliochis armigera, Spodotera exigua and Plutella xylostella. While the toxicity of vip protein from four engineered strains to Plutella xylostellas are highest, whose LC50 value is 28.6, 31.6, 45.4 and 37.6 microL/mL respectively. This study will contributed to construct high efficacy and wide spectrum engineered strains on theory and reality.