The complete hydrolysis of lignocellulose requires the actions of a variety of enzymes, including those that cleave the linkage between lignin and hemicellulose. The enzyme glucuronoyl esterase (GE) that constitutes a novel family of carbohydrate esterases, CE15, has been shown to display a unique ability to cleave the ester linkage between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid of hemicellulose. We herein report identification, expression, and functional characterization of a new GE, NcGE, from the filamentous fungus Neurospora crassa. C-terminally c-myc and hexahistidine-tagged NcGE was heterologously expressed in the methylotrophic yeast Pichia pastoris. NcGE purified from the culture supernatant through Ni-NTA and anion exchange chromatographies showed the ability to hydrolyze the substrate 3-(4-methoxyphenyl) propyl methyl 4-O-methyl-α-D-glucopyranosiduronate, which mimics the ester linkage of 4-O-methyl-D-glucuronic acid in lignin-carbohydrate complexes (LCCs). This esterase showed the characteristic of a mesophilic enzyme with the temperature optimum at 40–50°C, and displayed the optimal activity at pH 7 and broad pH stability. Based on the alignment of NcGE with other GEs so far characterized, we propose novel consensus sequences for GEs containing the catalytic triad.
Recombineering has been used to facilitate the development of in vivo cloning methods. However, the method relies heavily on PCR, which still generates a much higher error rate than DNA replication in vivo, even when amplifying large DNA inserts. Here, a precise technique is reported in Salmonella enterica that enables the cloning of up to at least 19 kb target chromosomal DNA segments that had been marked by FRTs, which were derived from two consecutive lambda Red-mediated recombination events. P22 phage was utilized to transduce the target DNA segments from donor strains to recipient strains harboring a derivative of bacterial artificial chromosome (BAC) containing a FRT and a plasmid expressing Flp recombinase. This method was successful in cloning a gene cluster responsible for lipopolysaccharide (LPS) modifications that confer polymyxin B resistance and in complementing its mutant. Further optimized procedures should be widely applicable because large insert fragments are precise clones of the wild-type genome.
In this paper, rhamnolipids are investigated, for the first time, for their feasibility for inhibiting dimorphic fungi. Rhamnolipids were found to effectively inhibit a dimorphic fungus isolated from tomato plants which was identified as Mucor circinelloides according to characterizations by morphologies as well as 28S rDNA sequences. Rhamnolipids markedly reduced growth of this fungus in both the yeast-like form and the filamentous form. Such an inhibitive effect was similarly obtained with Verticillium dahliae, a representative member of dimorphic fungi, confirming the effectiveness of rhamnolipids in the two growth forms of dimorphic fungi. Interestingly, rhamnolipids showed a greater inhibitive function in the case of the pathogenic growth mode of dimorphic fungi, such as the mycelium growth for M. circinelloides and the yeast-like growth for V. dahliae, than their non-pathogenic modes. The use of rhamnolipids might greatly reduce the frequently-reported drugresistance to the common anti-fungal agents by deterring the possible switch between the two modes of dimorphic fungi. Overall, rhamnolipids as environment-friendly biocontrol agents have a potential use in protecting plants from dimorphic fungi infections, and could also offer guidance toward future research into controlling dimorphic disease infection in humans.
LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to the signaling molecules acyl-homoserine lactones (acyl-HSLs). The function of the LuxR homologs is remarkably plastic, and naturally occurring acyl-HSLs are structurally diverse. To investigate the molecular basis of the functional plasticity of Vibrio fischeri LuxR, we directed the evolution of LuxR toward three different specificities in the laboratory. We found an orthogonal pair of LuxR mutants specific either to 3-oxo-hexanoyl homoserine lactone or to 3-oxo-octanoyl homoserine lactone. Interestingly, the majority of the specificity changes did not arise from modulating the recognition event but rather from changing the efficiency of the transition from the inactive form to the active form upon signal binding. This finding explains how quorum sensing systems can rapidly diverge in nature and in the laboratory and how signal orthogonality and mutual inhibition frequently occur among closely related diverging systems.
Fluorescent Pseudomonas are ubiquitous soil bacteria that usually establish mutualistic associations with plants, promoting their growth and health by several mechanisms. This makes them interesting candidates for the development of crop bio-inoculants. In this work, we isolated phosphate-solubilizing fluorescent Pseudomonas from the rhizosphere and inner tissues of different plant species growing in red soil from Misiones, Argentina. Seven isolates displaying strong phosphate solubilization were selected for further studies. Molecular identification by rpoD genotyping indicated that they belong to different species within the P. fluorescens and P. putida phylogenetic groups. Screening for in vitro traits such as phosphate solubilization, growth regulators synthesis or degradation, motility and antagonism against phytopathogens or other bacteria, revealed a unique profile of characteristics for each strain. Their plant growth-promoting potential was assayed using lettuce as a model for inoculation under controlled and greenhouse conditions. Five of the strains increased the growth of lettuce plants. Overall, the strongest lettuce growth promoter under both conditions was strain ZME4, isolated from inner tissues of maize. No clear association between lettuce growth promotion and in vitro beneficial traits was detected. In conclusion, several phosphate solubilizing pseudomonads from red soil were isolated that display a rich array of plant growth promotion traits, thus showing a potential for the development of new inoculants.
Although many bacteria are tolerant to heavy metals and play important roles in the immobilization of heavy metals, they cannot always be dependably reproduced under field conditions. In this work, a cadmium (Cd)-resistant bacterium was isolated from a Cd-contaminated oil field and identified as Pseudomonas aeruginosa (Pse-w). We then determined various plant growth promoting features such as the solubilization of phosphate, and the production of indole-3-acetic acid and siderophores. Lastly, we engineered the strain Pse-w-MT by targeting metallothioneins to the cell surface of Pse-w to immobilize Cd2+ and promote plant growth. Our data revealed that Pse-w exhibited high levels of resistance to Cd2+ (4 mM) and showed various plant growth promoting features. The engineered strain Pse-w-MT was found to adsorb Cd2+ mainly via extracellular deposition, and had an enhanced ability for immobilizing Cd2+ ions from the external media. Furthermore, the inoculation of Cd-polluted soil with Pse-w-MT significantly elevated the shoot and root biomass and leaf chlorophyll content. Similarly, plants inoculated with Pse-w-MT demonstrated markedly lower Cd2+ accumulation in the root and shoot system. It was concluded that plant growth promoting rhizobacteria with a high Cd2+ tolerance was an ideal candidate to be engineered for bioremediation and plant growth promotion against Cd-induced stress.