Machine-learning from Pseudomonas putida transcriptomes reveals its transcriptional regulatory network

Bacterial gene expression is orchestrated by numerous transcription factors (TFs). Elucidating how gene expression is regulated is fundamental to understanding bacterial physiology and engineering it for practical use. In this study, a machine-learning approach was applied to uncover the genome-scale transcriptional regulatory network (TRN) in Pseudomonas putida, an important organism for bioproduction. We performed independent component analysis of a compendium of 321 high-quality gene expression profiles, which were previously published or newly generated in this study. We identified 84 groups of independently modulated genes (iModulons) that explain 75.7% of the total variance in the compendium. With these iModulons, we (i) expand our understanding of the regulatory functions of 39 iModulon associated TFs (e.g., HexR, Zur) by systematic comparison with 1,993 previously reported TF-gene interactions; (ii) outline transcriptional changes after the transition from the exponential growth to stationary phases; (iii) capture group of genes required for utilizing diverse carbon sources and increased stationary response with slower growth rates; (iv) unveil multiple evolutionary strategies of transcriptome reallocation to achieve fast growth rates; and (v) define an osmotic stimulon, which includes the Type VI secretion system, as coordination of multiple iModulon activity changes. Taken together, this study provides the first quantitative genome-scale TRN for P. putida and a basis for a comprehensive understanding of its complex transcriptome changes in a variety of physiological states.

Ozonized biochar filtrate effects on the growth of Pseudomonas putida and cyanobacteria Synechococcus elongatus PCC 7942

Background Biochar ozonization was previously shown to dramatically increase its cation exchange capacity, thus improving its nutrient retention capacity. The potential soil application of ozonized biochar warrants the need for a toxicity study that investigates its effects on microorganisms. Results In the study presented here, we found that the filtrates collected from ozonized pine 400 biochar and ozonized rogue biochar did not have any inhibitory effects on the soil environmental bacteria Pseudomonas putida, even at high dissolved organic carbon (DOC) concentrations of 300 ppm. However, the growth of Synechococcus elongatus PCC 7942 was inhibited by the ozonized biochar filtrates at DOC concentrations greater than 75 ppm. Further tests showed the presence of some potential inhibitory compounds (terephthalic acid and p-toluic acid) in the filtrate of non-ozonized pine 400 biochar; these compounds were greatly reduced upon wet-ozonization of the biochar material. Nutrient detection tests also showed that dry-ozonization of rogue biochar enhanced the availability of nitrate and phosphate in its filtrate, a property that may be desirable for soil application. Conclusion Ozonized biochar substances can support soil environmental bacterium Pseudomonas putida growth, since ozonization detoxifies the potential inhibitory aromatic molecules. Graphical Abstract

Second messenger c-di-GMP modulates exopolysaccharide Pea-dependent phenotypes via regulating eppA expression in Pseudomonas putida

Exopolysaccharides (EPSs) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida . The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through unknown mechanism(s). Herein, we found that c-di-GMP modulated wrinkly colony morphology via regulating expression of eppA ( PP_5586 ), a small individually transcribed gene with 177 base pairs, and this gene was adjacent to the upstream of pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with the loss of the entire pea operon. EppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blot revealed that EppA was located on cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on transcriptomic profile of P. putida . Bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes, and that c-di-GMP modulates Pea-dependent phenotypes via regulating eppA expression in P. putida . IMPORTANCE Microbe-secreted EPSs are high molecular weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes, and provide possible pathway to construct genetically engineered strain for high Pea production.

Development of bio-chemical route to C5 plasticizer synthesis using glutaric acid produced by metabolically engineered Corynebacterium glutamicum

Corynebacterium glutamicum was engineered to produce glutaric acid by metabolic engineering approaches starting from the heterologous introduction of glutaric acid biosynthesis pathway by the expression of Pseudomonas putida davT, davD,...

Screening of microorganisms which are able to accelerate the process of microbial transformation of bird droppings

The purpose of the research. Screening of collection strains of microorganisms with enzymatic properties to accelerate the processes of microbial biodegradation of bird droppings. Research methods. The proteolytic activity of the grown cultures was studied according to GOST 20264.2-88, the total microbial number in the chicken droppings (CFU/ml) was analyzed, and the ammonium nitrogen was determined. Research results. As a result of the experiments, it was found that the highest proteolytic activity was demonstrated by the strain Pseudomonas putida 90 biovar A (171), which amounted to 74.6 units/g. When analyzing the effect of the studied collection strains on the decomposition processes of droppings, it was revealed that the largest number of microbial cells in bird droppings was achieved using Pseudomonas putida 90 biovar A (171), which was 104 CFU/ml at the beginning of the researches, and was the maximum and amounted to 1011 CFU/ml by the 15th day. The content of ammonium nitrogen in droppings treated with this culture decreased from 340 mg/l from the beginning of the experiment to 174 (15th day) and 169 mg/l (20th day) and it was the best indicator. When selecting the dose and concentration of the strain-producer Pseudomonas putida 90 biovar A (171) under introduction to bird droppings, it was found that to accelerate the process of biodegradation of bird droppings, the optimal dose for applying the studied culture is 4.0 % of organic waste mass with preliminary dilution by 2 times with water. At the same time, the optimal time of droppings keeping and the studied culture is 15 days. Scientific novelty. It was established for the first time that the treatment of chicken manure with the collection strain Pseudomonas putida 90 biovar A (171) accelerates the process of its microbial transformation.

Removal of Acetone Vapor from Air Using a Biotrickling Filter Packed with Polymeric Bioballs

Acetone released into the atmosphere can adversely affect human health and the environment. The aim of this work was to evaluate the performance of a laboratory-scale biotrickling filter (BTF) with bioball packing material to remove acetone vapor from contaminated air. The acetone removal efficiency was investigated in two different scenarios: with and without the inoculation of microorganisms. Three strains of bacteria, Pseudomonas putida, Rhodococcus aerolatus, and Aquaspirillum annulus, were used in the BTF. In both cases, the filter units were simultaneously operated for 100 days under three different inlet acetone concentrations (0.18 ± 0.01 g/m3, 0.25 ± 0.01 g/m3, and 0.40 ± 0.02 g/m3) and two different gas flow rates (2.54 and 5.09 m3/h). The results showed that acetone removal was greater in the filter with the inoculated bacteria. In the filter operated without inoculum, the acetone removal efficiency gradually decreased with filtration time from 90.1% to 6.1%. While employing three types of bacteria in the BTF, the efficiency of acetone removal remained relatively stable and varied between 70.2% and 97.6%. The study also revealed that bioballs can be successfully used as a packing material in air biofiltration systems designed for acetone removal from the air.

Rational Protein Engineering of Bacterial N-Demethylases to Create Biocatalysts for the Production of Methylxanthines

Methylxanthines have a rich history as therapeutics and pharmaceuticals. However, natural dimethyl- and monomethylxanthines are difficult to produce synthetically, which has limited further exploration of these compounds in medicinal applications. A biosynthetic method for production of methylxanthines from whole cell biocatalysts is an attractive alternative. The bacterium Pseudomonas putida CBB5 contains a set of five enzymes, NdmABCDE, which are responsible for methylxanthine metabolism via N-demethylation to xanthine. The recent elucidation of the crystal structures of NdmA and NdmB, which remove the N1- and N3- methyl groups of caffeine, respectively, has opened new avenues to create biocatalysts for methylxanthine production. We have created a set of fifteen N-demethylase mutants and expressed them in E. coli BL21(DE3) as whole cell biocatalysts. The activity of each mutant was characterized for their affinity towards caffeine, theobromine, and theophylline. Two mutant enzymes in particular, labeled NdmA3 and NdmA4, both exhibited selectivity towards the N3-methyl group instead of the N1-methyl group. We also discovered that specific point mutations in NdmD resulted in the ability to tune the rate of the N-demethylase reaction. These new enzymes provide the capability of producing high-value methylxanthines, such as paraxanthine and 1-methylxanthine, through a biocatalytic route.