Bacterial consortia constructed for the decomposition of Agave biomass
Research has shown that a greater variety of enzymes, as well as variety of microorganisms producing enzymes, can have an overall synergistic effect on the decomposition of lignocellulosic biomass for the production of value-added bio-products. Here, 8 cellulase-degrading bacterial isolates were selected to develop co-, tri-, and tetra- cultures for the decomposition of lignocellulosic biomass. Glucose and xylose equivalents released from imitation biomass media containing 0.5% (w/v) beechwood xylan and 0.5% (w/v) Avicel was measured using di-nitrosalicylic acid for all consortia; along with cell growth and survival. Thereafter, 6 co- and 2 tri-cultures with greatest decomposition were examined for ability to degrade Agave Americana fiber. Interestingly, when strains were paired up in co-culture, four pairs: G+5, G+A, C+A1, and G+A1 produced high reducing sugars in 24 h: 6 µM, 8 µM, 8 µM, and finally, 6 µM, respectively. From 4 co-cultures with highest reducing sugar equivalents, tri- and tetra-cultures were produced. The bacterial consortia which had the highest reducing sugars detected were 2 tri-cultures: G+A1+A4 and G+A1+5, displaying levels as high as 9 µM and 5 µM in day 1, respectively. All co- and tri- cultures maintained high cell survival for 14 days with 0.5 g ground Agave. Upon evaluating Agave dry weight after treatment, it was evident that almost half the biomass could be decomposed in 14 days. Scanning electron microscopy of treated Agave supported decomposition when compared with the control. These bacterial consortia have potential for further study of value-added by-product production during metabolism of lignocellulosic biomasses.
Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3
Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor/bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection/activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.
Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system
Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world’s residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS.
Regulation of pH attenuates toxicity of a byproduct produced by an ethanologenic strain of Sphingomonas sp. A1 during ethanol fermentation from alginate
Marine macroalgae is a promising carbon source that contains alginate and mannitol as major carbohydrates. A bioengineered ethanologenic strain of the bacterium Sphingomonas sp. A1 can produce ethanol from alginate, but not mannitol, whereas the yeast Saccharomyces paradoxus NBRC 0259–3 can produce ethanol from mannitol, but not alginate. Thus, one practical approach for converting both alginate and mannitol into ethanol would involve two-step fermentation, in which the ethanologenic bacterium initially converts alginate into ethanol, and then the yeast produces ethanol from mannitol. In this study, we found that, during fermentation from alginate, the ethanologenic bacterium lost viability and secreted toxic byproducts into the medium. These toxic byproducts inhibited bacterial growth and killed bacterial cells and also inhibited growth of S. paradoxus NBRC 0259–3. We discovered that adjusting the pH of the culture supernatant or the culture medium containing the toxic byproducts to 6.0 attenuated the toxicity toward both bacteria and yeast, and also extended the period of viability of the bacterium. Although continuous adjustment of pH to 6.0 failed to improve the ethanol productivity of this ethanologenic bacterium, this pH adjustment worked very well in the two-step fermentation due to the attenuation of toxicity toward S. paradoxus NBRC 0259–3. These findings provide information critical for establishment of a practical system for ethanol production from brown macroalgae.
Addition of poly (propylene glycol) to multiblock copolymer to optimize siRNA delivery
Previous studies have examined different strategies for siRNA delivery with varying degrees of success. These include use of viral vectors, cationic liposomes, and polymers. Several copolymers were designed and synthesized based on blocks of poly(ethylene glycol) PEG, poly(propylene glycol) PPG, and poly(l-lysine). These were designated as P1, P2, and P3. We studied the copolymer self-assembly, siRNA binding, particle size, surface potential, architecture of the complexes, and siRNA delivery. Silencing of GFP using copolymer P3 to deliver GFP-specific siRNA to Neuro-2a cells expressing GFP was almost as effective as using Lipofectamine 2000, with minimal cytotoxicity. Thus, we have provided a new copolymer platform for siRNA delivery that we can continue to modify for improved delivery of siRNA in vitro and eventually in vivo.
Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii
Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters, resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.