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In JoVE (1)
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- Journal of Bacteriology
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- FEMS Microbiology Letters
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- Bioinformatics (Oxford, England)
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- Microbial Ecology
- Molecular Microbiology
- Molecular Plant-microbe Interactions : MPMI
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- Environmental Microbiology
- Canadian Journal of Microbiology
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Articles by Trevor C. Charles in JoVE
JoVE Bioengineering
Site-specific Engenharia cromossomo bacteriano: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
Biology, University of Waterloo
Um método rápido e eficiente para integrar DNA estranho de juros em cepas pré-fabricados receptoras, chamadas de tensões almofada de aterragem, é descrito. O método permite site-specific integração de uma cassete de ADN no locus engenharia aterragem almofada de uma estirpe dada, por meio de conjugação e expressão da integrase ΦC31.
Other articles by Trevor C. Charles on PubMed
Identification of an Acetoacetyl Coenzyme A Synthetase-dependent Pathway for Utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium Meliloti
D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.
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 alpha-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.
Application of Crossover-PCR-mediated Deletion-insertion Mutagenesis to Analysis of the BdhA-xdhA2-xdhB2 Mixed-function Operon of Sinorhizobium Meliloti
The bdhA-xdhA2-xdhB2 mixed-function operon was used to demonstrate the application of crossover PCR to the construction of in-frame, non-polar deletion-insertion mutations in Sinorhizobium meliloti. Replacement of a 474-bp internal portion of the 774-bp coding sequence of bdhA with a 21-bp in-frame synthetic sequence resulted in loss of the bdhA-encoded d-3-hydroxybutyrate dehydrogenase activity. Such mutants retained the xanthine oxidase activity encoded by xdhA2-xdhB2, thus illustrating the non-polar nature of the mutation. This method of constructing unmarked, in-frame deletions should be generally applicable to functional genomics studies in S. meliloti and other alpha-Proteobacteria.
Purification and Characterization of Homodimeric Methylmalonyl-CoA Mutase from Sinorhizobium Meliloti
High activity (>60 munit/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000+/-5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0+/-2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH(2)-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.
Expression of an Exogenous 1-aminocyclopropane-1-carboxylate Deaminase Gene in Sinorhizobium Meliloti Increases Its Ability to Nodulate Alfalfa
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.
Heterologous Complementation of the Exopolysaccharide Synthesis and Carbon Utilization Phenotypes of Sinorhizobium Meliloti Rm1021 Polyhydroxyalkanoate Synthesis Mutants
A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.
Characterization of BdhA, Encoding the Enzyme D-3-hydroxybutyrate Dehydrogenase, from Sinorhizobium Sp. Strain NGR234
A genomic library of Sinorhizobium sp. strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize D-3-hydroxybutyrate as a sole carbon source were isolated. Subcloning experiments identified a 2.3 kb EcoRI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment. The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA. Site-directed insertion mutagenesis was performed by introduction of a OmegaSmSp cassette at a unique EcoRV site within the bdhA coding region. A NGR234 bdhA mutant, NGRPA2, was generated by homogenotization, utilizing the sacB gene-based lethal selection procedure. This mutant was devoid of D-3-hydroxybutyrate dehydrogenase activity, and was unable to grow on D-3-hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants. Furthermore, the D-3-hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid-encoded multiple copies of the S. meliloti acsA2 gene. The glpK-bdhA-xdhA gene organization and the bdhA-xdhA operon arrangement observed in S. meliloti are also conserved in NGR234.
NodMutDB: a Database for Genes and Mutants Involved in Symbiosis
Functional genomics research is producing large amounts of data on the functions of individual genes related to symbiosis. We have developed a relational database, NodMutDB (Nodulation Mutant Database), to provide a comprehensive resource for depositing, organizing and retrieving information on symbiosis-related genes, mutants and published literature. NodMutDB brings together new studies and existing mutant-based literature to facilitate our understanding of how genes function in symbiotic processes in both Rhizobia and their host plants. AVAILABILITY: http://nodmutdb.vbi.vt.edu CONTACT: cmao@vbi.vt.edu SUPPLEMENTARY INFORMATION: Database schema and data curation model are available at http://nodmutdb.vbi.vt.edu.
Isolation of Poly-3-hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants
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.
The Role of PHB Metabolism in the Symbiosis of Rhizobia with Legumes
The carbon storage polymer poly-beta-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.
Effects of Nitrogen and Phosphorus Limitation on the Activated Sludge Biomass in a Kraft Mill Biotreatment System
Unlike wastewater, pulp and paper mill effluents are generally severely deficient in bioavailable nitrogen and phosphorus. The influence of nitrogen and phosphorus limitations on steady-state or typical pulp and paper mill activated sludge floc properties and performance was studied using a bioreactor-fed synthetic raw mill effluent and seeded with mill activated sludge. Limitation of either nitrogen or phosphorus decreased growth, five-day biochemical oxygen demand, and suspended solids removal. Nitrogen limitation greatly enhanced activated sludge floc poly-beta3-hydroxybutyrate (PHB), but not carbohydrate or extracellular polymeric substances (EPS). In contrast, phosphorus limitation increased total floc carbohydrate and EPS, but not PHB. The flocs showed little ability to store either nitrogen or phosphorus. Nitrogen limitation, but not phosphorus limitation, produced much more negative net floc surface charge, increasing fines, while phosphorus limitation, but not nitrogen limitation, increased the floc bound water content and surface hydrophobicity and decreased fines.
Roles of Poly-3-hydroxybutyrate (PHB) and Glycogen in Symbiosis of Sinorhizobium Meliloti with Medicago Sp
Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia-legume symbiosis are not fully understood. Glycogen synthase mutations were constructed by in-frame deletion (glgA1) or insertion (glgA2). These mutations were combined with a phbC mutation to make all combinations of double and triple mutants. PHB was not detectable in any of the mutants containing the phbC mutation; glycogen was not detectable in any of the mutants containing the glgA1 mutation. PHB levels were significantly lower in the glgA1 mutant, while glycogen levels were increased in the phbC mutant. Exopolysaccharide (EPS) was not detected in any of the phbC mutants, while the glgA1 and glgA2 mutants produced levels of EPS similar to the wild-type. Symbiotic properties of these strains were investigated on Medicago truncatula and Medicago sativa. The results indicated that the strains unable to synthesize PHB, or glycogen, were still able to form nodules and fix nitrogen. However, phbC mutations caused greater nodule formation delay on M. truncatula than on M. sativa. Time-course studies showed that (1) the ability to synthesize PHB is important for N(2) fixation in M. truncatula nodules and younger M. sativa nodules, and (2) the blocking of glycogen synthesis resulted in lower levels of N(2) fixation on M. truncatula and older nodules on M. sativa. These data have important implications for understanding how PHB and glycogen function in the interactions of S. meliloti with Medicago spp.
Influence of the Poly-3-hydroxybutyrate (PHB) Granule-associated Proteins (PhaP1 and PhaP2) on PHB Accumulation and Symbiotic Nitrogen Fixation in Sinorhizobium Meliloti Rm1021
Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.
ACC Deaminase from Plant Growth-promoting Bacteria Affects Crown Gall Development
In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.
Presence of a Novel 16S-23S RRNA Gene Intergenic Spacer Insert in Bradyrhizobium Canariense Strains
Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.
Mutational Analysis of the Sinorhizobium Meliloti Short-chain Dehydrogenase/reductase Family Reveals Substantial Contribution to Symbiosis and Catabolic Diversity
The short-chain dehydrogenase/reductase (SDR) family is one of the largest and most ubiquitous protein families in bacterial genomes. Despite there being a few well-characterized examples, the substrate specificities or functions of most members of the family are unknown. In this study, we carried out a large-scale mutagenesis of the SDR gene family in the alfalfa root nodule symbiont Sinorhizobium meliloti. Subsequent phenotypic analysis revealed phenotypes for mutants of 21 of the SDR-encoding genes. This brings the total number of S. meliloti SDR-encoding genes with known function or associated phenotype to 25. Several of the mutants were deficient in the utilization of specific carbon sources, while others exhibited symbiotic deficiencies on alfalfa (Medicago sativa), ranging from partial ineffectiveness to complete inability to form root nodules. Five of the mutants had both symbiotic and carbon utilization phenotypes. These results clearly demonstrate the importance of the SDR family in both symbiosis and saprotrophy, and reinforce the complex nature of the interaction of S. meliloti with its plant hosts. Further analysis of the genes identified in this study will contribute to the overall understanding of the biology and metabolism of S. meliloti in relation to its interaction with alfalfa.
1-aminocyclopropane-1-carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan
A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain reaction (PCR) was used to rapidly isolate ACC deaminase structural genes from the above-mentioned 27 strains; 17 of them have 99% identities with the previously characterized ACC deaminase structural gene (acdS) from R. leguminosarum bv. viciae 128C53K, whereas the other ten strains are 84% identical (864-866/1,020 bp) compared to the acdS from strain 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 27 strains, and 17 of these strains were shown to encode a leucine-responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase in R. leguminosarum 128C53K.
Null Mutations in Sinorhizobium Meliloti ExoS and ChvI Demonstrate the Importance of This Two-component Regulatory System for Symbiosis
Exopolysaccharides, either succinoglycan or galactoglucan, are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid-facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. Other studied alpha-proteobacteria ExoS/ChvI orthologues are required for the bacteria to invade or persist in host cells and thus we present more evidence that this two-component regulatory system is essential for alpha-proteobacterial host interaction.
Sinorhizobium Meliloti 1021 Loss-of-function Deletion Mutation in ChvI and Its Phenotypic Characteristics
Bacterial two-component regulatory systems (TCS) are common components of complex regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI controls exopolysaccharide succinoglycan production and flagellum biosynthesis. Although this system plays a crucial role in establishing the symbiosis between S. meliloti and its host plant, it is not well characterized. Attempts to generate complete loss-of-function mutations in either exoS or chvI in S. meliloti have been unsuccessful; thus, it was previously suggested that exoS or chvI are essential genes for bacterial cell growth. We constructed a chvI mutant by completely deleting the open reading frame encoding this gene. The mutant strain failed to grow on complex medium, exhibited lower tolerance to acidic condition, produced significantly less poly-3-hydroxybutyrate than the wild type, was hypermotile, and exhibited an altered lipopolysaccharide profile. In addition, this mutant was defective in symbiosis with Medicago truncatula and M. sativa (alfalfa), although it induced root hair deformation as efficiently as the wild type. Together, our results demonstrate that ChvI is intimately involved in regulatory networks involving the cell envelope and metabolism; however, its precise role within the regulatory network remains to be determined.
Identification and Characterization of the Intracellular Poly-3-hydroxybutyrate Depolymerase Enzyme PhaZ of Sinorhizobium Meliloti
S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized.
ACC Deaminase Increases the Agrobacterium Tumefaciens-mediated Transformation Frequency of Commercial Canola Cultivars
The plant hormone ethylene has been reported to inhibit the Agrobacterium tumefaciens-mediated transformation efficiency of many plants. In this study, an acdS gene that encodes 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, an enzyme that breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, was introduced into A. tumefaciens GV3101::pMP90. It was found that the presence of active ACC deaminase in A. tumefaciens reduced ethylene levels produced by plant tissues during the process of infection and cocultivation, and significantly increased the transformation efficiency of three commercial canola cultivars: Brassica napus cv. Westar, B. napus cv. Hyola 401 and B. napus cv. 4414RR.
Methods for the Isolation of Genes Encoding Novel PHB Cycle Enzymes from Complex Microbial Communities
Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.
Identification and Characterization of New LuxR/LuxI-type Quorum Sensing Systems from Metagenomic Libraries
Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, metagenomic libraries constructed using DNA from activated sludge and soil were screened, using an Agrobacterium biosensor strain, for novel QS synthase genes. Three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homologue proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxI(QS6-1) directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxI(QS10-1) directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxI(QS10-2) directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxI(QS6-1).
ACC Deaminase Activity in Avirulent Agrobacterium Tumefaciens D3
Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58.
The Fluorescence Theatre: a Cost-effective Device Using Theatre Gels for Fluorescent Protein and Dye Screening
Here we report a simple cost-effective device for screening colonies on plates for expression of the monomeric red fluorescent protein mRFP1 and the fluorescent dye Nile red. This device can be built from any simple light source, in our case a Quebec Colony Counter, and cost-effective theatre gels. The device can be assembled in as little as 20 min, and it produces excellent results when screening a large number of colonies.
Harvesting of Novel Polyhydroxyalkanaote (PHA) Synthase Encoding Genes from a Soil Metagenome Library Using Phenotypic Screening
We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages.
Nonlinear Electrophoresis for Purification of Soil DNA for Metagenomics
Purification of microbial DNA from soil is challenging due to the co-extraction of humic acids and associated phenolic compounds that inhibit subsequent cloning, amplification or sequencing. Removal of these contaminants is critical for the success of metagenomic library construction and high-throughput sequencing of extracted DNA. Using three different composite soil samples, we compared a novel DNA purification technique using nonlinear electrophoresis on the synchronous coefficient of drag alteration (SCODA) instrument with alternate purification methods such as direct current (DC) agarose gel electrophoresis followed by gel filtration or anion exchange chromatography, Wizard DNA Clean-Up System, and the PowerSoil DNA Isolation kit. Both nonlinear and DC electrophoresis were effective at retrieving high-molecular weight DNA with high purity, suitable for construction of large-insert libraries. The PowerSoil DNA Isolation kit and the nonlinear electrophoresis had high recovery of high purity DNA suitable for sequencing purposes. All methods demonstrated high consistency in the bacterial community profiles generated from the DNA extracts. Nonlinear electrophoresis using the SCODA instrument was the ideal methodology for the preparation of soil DNA samples suitable for both high-throughput sequencing and large-insert cloning applications.
