Trevor C. Charles

An ACC deaminase minus mutant of Enterobacter cloacae UW4 no longer promotes root elongation.

The ACC deaminase gene (acdS) from Enterobacter cloacae UW4 was replaced by homologous recombination with the acdS gene with a tetracycline resistance gene inserted within the coding region. Upon characterization of this AcdS minus mutant, it was determined that both ACC deaminase activity and the ability to promote the elongation of canola roots under gnotobiotic conditions were greatly diminished. This result is consistent with a previously postulated model that suggests that a major mechanism utilized by plant growth-promoting bacteria involves the lowering of plant ethylene levels, and hence ethylene inhibition of root elongation, by bacterial ACC deaminase.

Amelioration of high salinity stress damage by plant growth- promoting bacterial endophytes that contain ACC deaminase

Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity). Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase containing bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a greater number of flowers and buds than the other treatments. Since the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not normally suitable for the majority of crops due to their high salt contents.

Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in Sinorhizobium meliloti Increases Its Ability To Nodulate Alfalfa

ABSTRACT 1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.

Analysis of C4-dicarboxylate transport genes in Rhizobium meliloti

A 5.1 kbp DNA fragment was isolated which complemented C4‐dicarboxylate transport mutants (dct) of Rhizobium meliloti. Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD. TnphoA‐generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription. Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression. The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants. This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD. Three independent second‐site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus. Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible. These results are consistent with the direct requirement of the ntrA gene product for dctA expression. Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen‐fixing activity. Nodules induced by dctA mutants failed to fix nitrogen. These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.

The role of PHB metabolism in the symbiosis of rhizobia with legumes.

The carbon storage polymer poly-β-hydroxybutyrate (PHB) is a potential biodegradable alternative to plastics, which plays a key role in the cellular metabolism of many bacterial species. Most species of rhizobia synthesize PHB but not all species accumulate it during symbiosis with legumes; the reason for this remains unclear, although it was recently shown that a metabolic mutant of a nonaccumulating species retains the capacity to store PHB in symbiosis. Although the precise roles of PHB metabolism in these bacteria during infection, nodulation, and nitrogen fixation are not determined, the elucidation of these roles will influence our understanding of the metabolic nature of the symbiotic relationship. This review explores the progress that was made in determining the biochemistry and genetics of PHB metabolism. This includes the elucidation of the PHB cycle, variations in PHB metabolism among rhizobial species, and the implications of these variations, while proposing a model for the role of PHB metabolism and storage in symbiosis.

A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification.

Aims:  The aim of this study was to identify and characterize a compound produced by the plant growth promoting bacterium, Bacillus thuringiensis non‐Bradyrhizobium Endophytic Bacterium 17.

A global pH sensor: Agrobacterium sensor protein ChvG regulates acid-inducible genes on its two chromosomes and Ti plasmid.

A sensor protein ChvG is part of a chromosomally encoded two-component regulatory system ChvG/ChvI that is important for the virulence of Agrobacterium tumefaciens. However, it is not clear what genes ChvG regulates or what signal(s) it senses. In this communication, we demonstrate that ChvG is involved in the regulation of acid-inducible genes, including aopB and katA, residing on the circular and linear chromosomes, respectively, and the tumor-inducing (Ti)-plasmid-harbored vir genes, virB and virE. ChvG was absolutely required for the expression of aopB and very important for the expression of virB and virE. However, it was responsible only for the responsiveness of katA and, to a limited extent, the vir genes to acidic pH. ChvG appears to play a role in katA expression by repressing katA at neutral pH. ChvG had no effect on the expression of two genes that were not acid-inducible. Because ChvG regulates unlinked acid-inducible genes encoding different functions in different ways, we hypothesize that ChvG is a global sensor protein that can directly or indirectly sense extracellular acidity. We also analyzed the re-sequenced chvG and found that ChvG is more homologous to its Sinorhizobium meliloti counterpart ExoS than was previously thought. Full-length ChvG is conserved in members of the α-proteobacteria, whereas only the C-terminal kinase domain is conserved in other bacteria. Sensing acidity appears to enable Agrobacterium to coordinate its coping with the environment of wounded plants to cause tumors.

Isolation of Poly-3-Hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants

ABSTRACT The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate d-3-hydroxybutyrate due to absence of the enzyme d-3-hydroxybutyrate dehydrogenase activity. Clones that conferred d-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored d-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.

Cellobiose dehydrogenase is essential for wood invasion and nonessential for kraft pulp delignification by Trametes versicolor

Abstract Cellobiose dehydrogenase (CDH)-deficient strains of the basidiomycete Trametes versicolor were produced by transforming protoplasts of strain 52J with a plasmid carrying the T. versicolor cdh gene disrupted with a phleomycin resistance cassette. Of 143 phleomycin-resistant colonies analyzed, 3 did not produce measurable CDH during two successive two-week culture periods. Two of these mutants were shown to lack functional CDH when grown in CDH induction medium. They biobleached and delignified industrial unbleached kraft pulp as efficiently as did wild-type T. versicolor, indicating that CDH is not required for the degradation and biobleaching of kraft lignin. The ability to degrade 14C-guaiacyl dehydrogenative polymerizate (synthetic lignin) also appeared to be unaffected. However, compared to the parent strain, all three mutants grew poorly on minimal agar with highly crystalline cellulose as the sole carbon source. This difference was not observed on non-crystalline carbohydrates. All three mutants had a greatly decreased ability to colonize and degrade both seasoned and fresh native white birch wood, a natural substrate of T. versicolor. The dramatic decrease in T. versicolor 52J’s ability to invade and grow on birch wood caused by the loss of its secreted CDH strongly suggests that this enzyme is essential to its wood invading and degrading niche in the forest ecosystem.

CLONING AND SEQUENCING OF A GENE ENCODING CELLOBIOSE DEHYDROGENASE FROM TRAMETES VERSICOLOR

Cellobiose dehydrogenase (CDH) is an enzyme produced under lignocellulose-degrading conditions by Trametes versicolor strain 52J (Tv) and several other wood-degrading fungi, including Phanerochaete chrysosporium (Pc). In order to understand better the nature and properties of this enzyme, we isolated a genomic clone of Tv cdh using heterologous probes derived from the sequence of Pc cdh. DNA sequence analysis revealed that Tv cdh consists of 3091 bp of coding sequence interrupted by 14 introns. Southern blotting showed that the gene was present in a single copy in the strain of Tv analyzed. Tv cdh was shown by Northern blot analysis to be expressed as a single transcript under cellulolytic conditions. RT-PCR of poly(A)+ RNA isolated under cellulolytic conditions was used to generate a near full-length cDNA copy of the cdh mRNA. The deduced protein encoded by Tv cdh consists of 768 amino acids (aa), including a predicted 19 aa signal peptide. The protein had 73% identity to the corresponding protein from Pc, which is the only other CDH-encoding gene that has been cloned. Based upon its deduced primary structure and alignment to similar sequences, Tv CDH shares a general structural organization with Pc CDH and other hemoflavoenzymes. Amino acid residues H-109 and M-61 in the N-terminal heme domain are hypothesized to function in heme binding; the C-terminal flavin domain contained a consensus sequence for flavin binding between residues 217-222. Although the protein is known to bind to cellulose, no obvious homology to bacterial or fungal cellulose binding domains was observed. However, a strong homology was observed to a region of Pc CDH that is hypothesized to be involved in cellulose binding.