Tumefaciens A136 Research Articles

Evidence of autoinducer activity in naturally occurring biofilms.

N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria. Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens. In nature, bacteria often grow as surface-adherent biofilm communities. As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology. However, proof of AHL production within biofilms has heretofore been lacking. In this study we have employed a cross-feeding assay, using A, tumefaciens A136 (traI::lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones. AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm. This represents the first report of AHL activity in naturally occurring biofilms.

The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation.

Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.

Production of N-acyl homoserine lactones by gram-negative bacteria isolated from contact lens wearers.

The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules in ocular gram-negative bacteria. A total of 91 ocular strains isolated from contact lens adverse response patients and asymptomatic subjects were used in the study. These included Acinetobacter, Aeromonas hydrophila, Escherichia coli, Haemophilus influenzae, Klebsiella oxytoca, Pseudomonas aeruginosa, Serratia liquefaciens, Serratia marcescens, and Stenotrophomonas maltophilia. The biosensor strains Chromobacterium violaceum mutant CV026 and Agrobacterium tumefaciens A136 were used for detection of AHL signal molecules. The majority of A. hydrophila, P. aeruginosa, and S. liquefaciens strains produced more than one AHL molecule. Serratia marcescens strains were AHL positive only under detection of A136. The rest of the test species did not show any AHL production under the current detection system. These findings indicate that AHL-mediated quorum-sensing systems are present in some of the ocular bacteria, and the different signal molecules may be involved with the quorum-sensing pathway in the other bacterial species.

Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics

In the process of evaluating the role of acylated homoserine lactones (AHLs) in food-spoiling Gram-negative bacteria, we have combined a range of bacterial AHL monitor systems to determine the AHL-profile and the kinetics of AHL-production. AHL production from 148 strains of Enterobacteriaceae isolated from foods was tested using Escherichia coli pSB403 (LuxR), Agrobacterium tumefaciens A136 (TraR) and both induction and inhibition of Chromobacterium violaceum CV026 (CviR). All strains except one was found to produce AHL(s). In no case could a single monitor system identify more than 64% of the Enterobacteriaceae as AHL-producers, showing that the simultaneous use of monitor strains is required in the process of screening bacterial populations for AHL-production. AHLs from 20 selected strains were profiled by thin layer chromatography. Most strains produced more than one AHL with 3- N-oxo-hexanoyl homoserine lactone being the most prominent. It was found that the simultaneous use of monitor strains in the top-layer was necessary for the detection of (presumably) all the AHLs. An agar well-diffusion assay based on A. tumefaciens pDZLR4 was used for quantifying AHLs from bacterial supernatants and enabled an assessment of the kinetics of AHL-production of 3 strains ( Serratia proteamaculans strain B5a, Erwinia carotovora ATCC 39048 and V. fischeri strain MJ-1). As expected, the production of AHL (OHHL) and luminescence in Vibrio fischeri strain MJ-1 increased faster than growth indicating up-regulation of the AHL regulated phenotype and auto-induction of AHL production. In contrast, production kinetics of AHL (OHHL) in the two Enterobacteriaceae indicated lack of auto-induction.

STUDIES TO IMPROVE TRANSIENT GUSA EXPRESSION IN A. TUMEFACIENS-MEDIATED TRANSFORMATION OF RICE

The direct-DNA uptake methods has been routinely used in transforming rice. The recent development of rice genetic transformation using A. tumefaciens as a natural vector necessitate the investigation of factors that will promote optimum gene expression and T-DNA transfer in the recalcitrant and economically important indica rice varieties. Activities of three different promoters were investigated for transient expression using gusA after particle bombardment of suspension cells. Actin and ubiquitin promoters were found to induce higher GUS activity compared to the chimeric promoter of mannopine synthethase and a truncated cauliflower mosaic virus 35S promoter. A binary vector pCGN1558 was modified to contain these promoters in front of the gusA-intron. A. tumefaciens strain EHA101 and A136 were transformed with the derivatives of the binary vector and transformed colonies were tested for infectivity on coleoptile bases of IR54 and IR72 seedlings. GUS activity of the infected coleoptile bases showed that the A. tumefaciens colonies were comparable to but not better than the positive control At656. Possible reasons for the obtained results were explained with emphasis on the importance of the compatibility of the A. tumefaciens strains with the binary vector, the border sequences, and the Ti-plasmid present.

Transfer of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii to Agrobacterium tumefaciens.

This study examined the symbiotic properties of Agrobacterium transconjugants isolated by transferring a Tn5-mob-marked derivative of the 315 kb megaplasmid pRt4Sa from Rhizobium leguminosarum bv. trifolii 4S (wild-type strain) to Agrobacterium tumefaciens A136 as the recipient. The genetic characteristics of the AT4S transconjugant strains were ascertained by random amplified polymorphic DNA (RAPD) analyses and Southern hybridization using Tn5-mob and nod genes as probes. Several of these AT4S transconjugants carrying pRt4Sa were able to nodulate roots of the normal legume host, white clover. In addition, some AT4S transconjugant strains were able to induce nodules on other leguminous plants, including alfalfa and hairy vetch. A characteristic bacteroid differentiation was observed in clover and alfalfa nodules induced by the AT4S-series strains, although nitrogen-fixing activity (acetylene reduction) was not found. Furthermore, strain H1R1, obtained by retracing transfer of the pRt4Sa::Tn5-mob from strain AT4Sa to strain H1 (pRt4Sa cured derivative of 4S), induced Fix(+) nodules on clover roots. These results indicate the evidence that only nod genes can be expressed in the Agrobacterium background.

Evidence of autoinducer activity in naturally occurring biofilms

N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria. Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens. In nature, bacteria often grow as surface-adherent biofilm communities. As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology. However, proof of AHL production within biofilms has heretofore been lacking. In this study we have employed a cross-feeding assay, using A. tumefaciens A136 ( traI:: lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones. AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm. This represents the first report of AHL activity in naturally occurring biofilms.

Transport of nonmetabolizable opines by Agrobacterium tumefaciens

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.

PicA, a novel plant-inducible locus on the Agrobacterium tumefaciens chromosome

We used the transposon Mu dI1681 to identify genes on the Agrobacterium tumefaciens chromosome that are inducible by extracts from carrot roots. One such locus (picA, for plant inducible chromosomal), harbored by A. tumefaciens At156, was inducible 10- to 50-fold by these extracts. Mutation of picA had no detectable effect upon bacterial growth or virulence under laboratory assay conditions. However, A. tumefaciens cells harboring a mutated picA locus aggregated into long "ropes" when incubated with pea root tip cells. Such aggregation was not displayed by the parental strain A. tumefaciens A136. A preliminary characterization of the inducing compound in the carrot root extract suggests that the active substance is an acidic polysaccharide that is most likely derived from the pectic portion of the plant cell wall.