Alexander Schenk

Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids

Ionic liquids (ILs) are novel and chemically inert solvents for a wide range of reactions in organic synthesis and biocatalysis, and at least one of them is known to dissolve cellulose. ILs would provide novel options for cellulose degradation in homogenous catalysis if cellulases were sufficiently stable and active. By screening metagenomic libraries 24 novel cellulase clones were identified and tested for their performance in the presence of ILs. Most enzyme clones showed only very poor or no activities. Three enzyme clones (i.e. pCosJP10, pCosJP20 and pCosJP24) were moderately active and stable in the presence of 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfonate. The corresponding genes of these environment-derived cosmids were similar to known cellulases from Cellvibrio japonicus and a salt-tolerant cellulase from an uncultured microorganism, S. Voget, H. L. Steele and W. R. Streit, J. Biotechnol., 2006, 126, 26-36.1 The most active protein (CelA10) belonged to GH5 family cellulases and was active at IL concentrations of up to 30% (v/v). Recombinant CelA10 was extremely tolerant to 4 M NaCl and KCl. Furthermore improved cellulase variants of CelA10 were isolated in a directed evolution experiment employing SeSaM-technology. Analysis of these variants revealed that the N-terminal cellulose binding domain plays a pivotal role for IL resistance.

Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques.

SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR.

Generating high‐quality mutant libraries in which each amino acid is equally targeted and substituted in a chemically diverse manner is crucial to obtain improved variants in small mutant libraries. The sequence saturation mutagenesis method (SeSaM‐Tv+) offers the opportunity to generate such high‐quality mutant libraries by introducing consecutive mutations and by enriching transversions. In this study, automated gel electrophoresis, real‐time quantitative PCR, and a phosphorimager quantification system were developed and employed to optimize each step of previously reported SeSaM‐Tv+ method. Advancements of the SeSaM‐Tv+ protocol and the use of a novel DNA polymerase quadrupled the number of transversions, by doubling the fraction of consecutive mutations (from 16.7 to 37.1 %). About 33 % of all amino acid substitutions observed in a model library are rarely introduced by epPCR methods, and around 10 % of all clones carried amino acid substitutions that are unobtainable by epPCR.

Extraction of high‐quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization

Plant pathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. In planta gene expression analysis is important to understand the impact of these genes on bacterial virulence. A new mRNA-based approach using multiplexed Northern hybridization was developed. High-quality bacterial and plant total RNA was successfully isolated from leaf tissue infiltrated with Pseudomonas syringae. The procedure employs a new extraction buffer formulation containing glycine, sodium dodecylsulphate, cetyltrimethylammonium bromide, high-molecular-weight polyethylene glycol and beta-mercaptoethanol. Cell lysis and classical acid-phenol extraction steps followed by LiCl precipitation yielded large amounts of total RNA of high purity and integrity. Multiplexing of DIG and chemically fluorescently labelled RNA probes was developed and expression data were normalized using the 23S rRNA gene as reference. The method was validated by studying in planta expression of the P. syringae genes mucD, cmaA, cfl, corR, corS and corP comprising a selection of highly expressed biosynthetic and low-expressed regulatory genes. The method was assessed regarding its sensitivity and might by useful for studying a variety of plant-microbe interactions.

Impact of temperature on in planta expression of genes involved in synthesis of the Pseudomonas syringae phytotoxin coronatine.

Coronatine (COR) is a chlorosis-inducing phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Confocal laser scanning microscopy was used to investigate in vitro and in planta expression of COR genes by two model organisms, P. syringae pv. glycinea PG4180, a pathogen of soybean, and P syringae pv. tomato DC3000, a pathogen of tomato and crucifers. Previously, it was shown in vitro that the cma operon involved in COR synthesis in PG4180 is expressed in a temperature-dependent manner, with maximal rates at 18 degrees C and low activity at 28 degrees C. However, nothing was known about the influence of temperature on the expression of COR biosynthetic genes in planta. Therefore, transcriptional fusions of the PG4180 and DC3000 cma promoter regions to a promoterless egfp gene were constructed and expressed in both P. syringae strains. The fluorescence patterns in response to temperature during growth of a strain in vitro were consistent with its COR production and the cma transcript abundance as revealed by RNA dot blot hybridization. Quantification of fluorescence indicated that cma promoter activity was dependent on the genetic background of the host strain. Expression of cma::egfp in PG4180 was temperature-dependent in minimal medium as well as inside the plant tissue. In contrast, transcription of the cma operon was not significantly affected by temperature in DC3000. However, cells of DC3000 harboring the cma::egfp fusions showed higher levels of fluorescence when recovered from infected host plants compared with cells grown in minimal medium. These results indicate that the signals for induction of COR biosynthesis differ significantly in PG4180 and DC3000.

The alternative sigma factor AlgT, but not alginate synthesis, promotes in planta multiplication of Pseudomonas syringae pv. glycinea.

The phytopathogen Pseudomonas syringae pv. glycinea produces the exopolysaccharide (EPS) alginate, which is thought to function in epiphytic fitness and virulence. A key regulator for alginate biosynthesis in Pseudomonas aeruginosa and P. syringae is the alternative sigma factor AlgT (sigma(22)). In this study, the contribution of alginate synthesis and AlgT to in planta epiphytic fitness and virulence of P. syringae was examined. Alginate biosynthesis mutants were generated for the P. syringae pv. glycinea strains PG4180 and PG4180.muc, representing a comprehensive set of alginate- and AlgT-positive or -negative derivatives. Analysis of in vitro and in planta phenotypes revealed that AlgT strongly promoted in planta growth, survival and symptom development, but decreased the ability to grow in vitro. In contrast, alginate biosynthesis had only marginal impact. Quantitative in vitro and in planta gene expression analyses for alginate biosynthesis and algT were carried out at two temperatures in AlgT-negative and -positive backgrounds. algT as well as algD gene expression was AlgT-dependent, plant-inducible and temperature-dependent, with higher expression at 18 compared to 28 degrees C; however, no temperature dependence was observed in vitro. Our data suggest that AlgT may act as a global regulator for virulence and in planta fitness traits of P. syringae independent of its role in EPS biosynthesis.

The algT Gene of Pseudomonas syringae pv. glycinea and New Insights into the Transcriptional Organization of the algT-muc Gene Cluster

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae, the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d-mannuronic and l-guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii. These genes encode the alternative sigma factor AlgT (sigma(22)), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae, and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli. We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT, mucA, mucB, mucD, and algD, which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT, mucA, and mucB are cotranscribed as an operon in P. syringae. Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae.

Component and protein domain exchange analysis of a thermoresponsive, two-component regulatory system of Pseudomonas syringae

Two closely related phytopathogenic bacterial strains, Pseudomonas syringae pv. glycinea PG4180 and P. syringae pv. tomato DC3000, produce the chlorosis-inducing phytotoxin coronatine (COR) in a remarkably divergent manner. PG4180 produces COR at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C. In contrast, temperature has no effect on COR synthesis in DC3000. A modified two-component system consisting of the histidine protein kinase (HPK), CorS, the response regulator (RR), CorR, and a third component, CorP, governs COR biosynthesis in both strains. A plasmid-based component and domain swapping approach was used to introduce different combinations of RRs, HPKs and hybrid HPKs into corS mutants of both strains. Subsequently, expression levels of the COR biosynthetic cma operon were determined using RNA dot-blot analysis, suggesting that CorRSP of PG4180 mediates a thermoresponsive phenotype dependent on the genomic background of each strain. The reciprocal experiment demonstrated a loss of temperature dependence in the corS mutant of PG4180. The presence of corR from PG4180 led to more pronounced cma expression in DC3000 and was associated with thermoresponsiveness, while corS of PG4180 did not mediate a temperature-dependent phenotype in the DC3000 mutant containing native corR and corP. These findings were substantiated by RT-PCR experiments. The C-terminal domain of CorS of PG4180 mediated thermosensing, while the N terminus did not respond to temperature changes, suggesting cytosolic perception of the temperature signal.

A temperature-sensing histidine kinase: function, genetics, and membrane topology.

Two-component systems provide a means for bacteria to sense and adapt to environmental signals in order to survive in a continuously changing environment. Understanding of the mechanism by which these systems function is important in combating bacterial infections because many bacterial two-component systems are associated with virulence. The plant pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180 synthesizes high levels of the phytotoxin coronatine at the virulence-promoting temperature of 18 degrees , but not at 28 degrees , the optimal growth temperature. Temperature-dependent coronatine biosynthesis is regulated by a modified two-component system, consisting of the response regulator, CorR, the histidine protein kinase CorS, and a third component, CorP. To elucidate the mechanism by which CorRSP functions, genetic, transcriptional, and biochemical analyses were applied, including in vitro and in planta reporter gene analysis, mRNA quantification, protein expression, mutagenesis, and membrane topology analysis. A combination of these techniques helped to elucidate, to a considerable extent, the temperature-sensing activity of CorS, which seems to act as a membrane-bound molecular thermometer.

Coronatine Gene Expression In Vitro and In Planta, and Protein Accumulation During Temperature Downshift in Pseudomonas syringae

The plant pathogenic bacterium Pseudomonas syringae PG4180 synthesizes high levels of the phytotoxin coronatine (COR) at the virulence-promoting temperature of 18 °C, but negligible amounts at 28 °C. Temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of a response regulator, CorR, the histidine protein kinase CorS, and a third component, termed CorP. We analyzed at transcriptional and translational levels the expression of corS and the cma operon involved in COR biosynthesis after a temperature downshift from 28 to 18 °C. Expression of cma was induced within 20 min and increased steadily whereas corS expression was only slightly temperature-dependent. Accumulation of CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis. A transcriptional fusion of the cma promoter to a promoterless egfp gene was used to monitor the cma expression in vitro and in planta. A steady induction of cma::egfp by temperature downshift was observed in both environments. The results indicate that PG4180 responds to a temperature decrease with COR gene expression. However, COR gene expression and protein biosynthesis increased steadily, possibly reflecting adaptation to long-term rather than rapid temperature changes.