Complete Genome Sequence of Pseudomonas Stutzeri S116 Provides Insights into the Mechanism of Microbial Fuel Cells

To identify suitable biocatalysts applied in microbial fuel cells (MFCs), Pseudomonas stutzeri S116 isolated from marine sludge was investigated, which possessed excellcent bioelectricity generation ability (BGA). Herein, P. stutzeri as a bioanode and biocathode achieved maximum output voltage (254.2 mV and 226.0 mV), and power density of (765 mW/m2 and 656.6 mW/m2). Complete genome sequencing of P. stutzeri was performed to reveal its potential microbial functions. The results exhibited that the strain was the ecologically dominant Pseudomonas, and its primary annotations were associated with energy production and conversion (6.84%), amino acid transport and metabolism (6.82%) and inorganic ion transport and metabolism (6.77%). The thirty-six genes involved in oxidative phosphorylation indicate that strain possesses an integrated electron transport chain. Moreover, many genes encoding redox mediators (mainly riboflavin and phenazine) were detected in the databases. Simultaneously, thiosulfate oxidization and dissimilatory nitrate reduction were annotated in the sulfur metabolism and nitrogen metabolism pathway. Gene function and cyclic voltammetry (CV) analysis indicated BGA of P. stutzeri probably was attributed to its cytochrome c and redox mediators, which enhance extracellular electron transfer (EET) rate.

Comparative Genomics of Pseudomonas stutzeri Complex: Taxonomic Assignments and Genetic Diversity

Pseudomonas stutzeri is a species complex with extremely broad phenotypic and genotypic diversity. However, very little is known about its diversity, taxonomy and phylogeny at the genomic scale. To address these issues, we systematically and comprehensively defined the taxonomy and nomenclature for this species complex and explored its genetic diversity using hundreds of sequenced genomes. By combining average nucleotide identity (ANI) evaluation and phylogenetic inference approaches, we identified 123 P. stutzeri complex genomes covering at least six well-defined species among all sequenced Pseudomonas genomes; of these, 25 genomes represented novel members of this species complex. ANI values of ≥∼95% and digital DNA-DNA hybridization (dDDH) values of ≥∼60% in combination with phylogenomic analysis consistently and robustly supported the division of these strains into 27 genomovars (most likely species to some extent), comprising 16 known and 11 unknown genomovars. We revealed that 12 strains had mistaken taxonomic assignments, while 16 strains without species names can be assigned to the species level within the species complex. We observed an open pan-genome of the P. stutzeri complex comprising 13,261 gene families, among which approximately 45% gene families do not match any sequence present in the COG database, and a large proportion of accessory genes. The genome contents experienced extensive genetic gain and loss events, which may be one of the major mechanisms driving diversification within this species complex. Surprisingly, we found that the ectoine biosynthesis gene cluster (ect) was present in all genomes of P. stutzeri species complex strains but distributed at very low frequency (43 out of 9548) in other Pseudomonas genomes, suggesting a possible origin of the ancestors of P. stutzeri species complex in high-osmolarity environments. Collectively, our study highlights the potential of using whole-genome sequences to re-evaluate the current definition of the P. stutzeri complex, shedding new light on its genomic diversity and evolutionary history.

Biodegradation of Alprazolam in Pharmaceutical Wastewater Using Mesoporous Nanoparticles-Adhered Pseudomonas stutzeri

The release of pharmaceutical wastewaters in the environment is of great concern due to the presence of persistent organic pollutants with toxic effects on environment and human health. Treatment of these wastewaters with microorganisms has gained increasing attention, as they can efficiently biodegrade and remove contaminants from the aqueous environments. In this respect, bacterial immobilization with inorganic nanoparticles provides a number of advantages, in terms of ease of processing, increased concentration of the pollutant in proximity of the cell surface, and long-term reusability. In the present study, MCM-41 mesoporous silica nanoparticles (MSN) were immobilized on a selected bacterial strain to remove alprazolam, a persistent pharmaceutical compound, from contaminated water. First, biodegrading microorganisms were collected from pharmaceutical wastewater, and Pseudomonas stutzeri was isolated as a bacterial strain showing high ability to tolerate and consume alprazolam as the only source for carbon and energy. Then, the ability of MSN-adhered Pseudomonas stutzeri bacteria was assessed to biodegrade alprazolam using quantitative HPLC analysis. The results indicated that after 20 days in optimum conditions, MSN-adhered bacterial cells achieved 96% biodegradation efficiency in comparison to the 87% biodegradation ability of Pseudomonas stutzeri freely suspended cells. Kinetic study showed that the degradation process obeys a first order reaction. In addition, the kinetic constants for the MSN-adhered bacteria were higher than those of the bacteria alone.

Kinetics Modelling of Pseudomonas stutzeri strain DN2 Growth Behaviour in Tributyltin Chloride

A predictive model was performed to describe Pseudomonas stutzeri strain DN2 growth behaviour in tributyltin chloride, using primary Modelling and a polynomial model as a secondary predictive model. In this investigation, data predicted using the modified Logistic (ML) was the most accurate. The Bias Factor (Bf) and Accuracy Factor (Af) values for the (ML) model were 1.39 and 1.51, indicating that the predictions were within a reliable range. The low RMSE value of 0.14, R2 and adj R2 (0.99) value closer to 1, showing that modified logistics is better than the other models at describing the growth behaviour of Pseudomonas stutzeri strain DN2 in toxic tributyltin chloride. Both the Aiba and Haldane models on the other hand, among the secondary model best fit the behaviours having low RMSE and MSE values and adjR2 value closer to 1. In this study, the primary and secondary kinetics of Pseudomonas stutzeri strain DN2 growth behaviour in tributyltin chloride was explored and it was shown in this study that the modified logistic and the Haldane models better suit the growth behavior of Pseudomonas stutzeri strain DN2 in tributyltin chloride. The parameters obtained from the modelling exercise will be very valuable in transferring the laboratory results to the field.

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL SCREENING OF LEAF EXTRACTS OF SENNA TORA (L.) ROXB

Plants have always been an important source of medicines since ancient times and seventy percent of the worldwide population still relies on one or other forms of traditional plant based medicine. Plant items have been essential for phytomedicines since days of yore. These can be derived from any part of the plants like bark, leaves, flowers, roots, fruits, seeds, etc. The present exploration has been conducted in the leaf of Senna tora performing various phytochemical tests to identify the secondary metabolites present in it such as alkaloids, flavonoids, sugars, glycosides, saponins, steroids, tannins, phenolic compounds, Vitamin C, proteins, amino acids and carbohydrates. The maximum phenolic content was presented in methanol solvents 1.41 ± 0.44 and lowest content was presented in petroleum ether extract 0.17 ± 0.21. Antibacterial activity were estimated and evaluated by using different types of extract against Escherichia coli, Klebsiella pneumoniae, Pseudomonas stutzeri, Bacillus thuriengensis and Staphylococcus. Among these the maximum antibacterial activity (Zone of inhibition 19.0 mm) shown against Klebsiella pneumoniae in the extract of Petroleum ether. The minimum antibacterial activity observed (Zone of inhibition 11.0 mm) against Staphylococcus ceureus in extract of Ethanol extract of Senna tora (L.) Roxb. Keywords: Senna tora (L.), phytochemical analysis, antibacterial activity, bacterial strains

Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Pseudomonas Co-culture Biofilms

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia-based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm-based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition, and invasion) between colony-forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and 13 other species of pseudomonas isolated from non-contaminated soil. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling of a DNA-barcoded mutant library; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and mass spectrometry based metabolomics.

Changes in natural transformation after salt adaptation

The exchange of genes between potentially unrelated bacteria is termed horizontal gene transfer (HGT) and is a driving force in bacterial evolution. Natural transformation is one mechanism of HGT where extracellular DNA (eDNA) from the environment is recombined into a host genome. The widespread conservation of transformation in bacterial lineages implies there is a fitness benefit. However, the nature of these benefits and the evolutionary origins of transformation are still unknown. Here, I examine how ∼330 generations or 100 days of serial passage in either constant or increasing salinities impacts the growth rate and transformation efficiency of Pseudomonas stutzeri. While the growth rate generally improved in response to serial transfer, the transformation efficiency of the evolved lineages varied extensively, with only 39-64% of populations undergoing transformation at the end of adaptive evolution. In comparison, 100% of the ancestral populations were able to undergo natural transformation. I also found that evolving P. stutzeri with different cell lysates (or populations of dead cells) minimally affected the growth rate and transformation efficiency, especially in comparison to the pervasiveness with which transformation capacity was lost across the evolved populations. Taken together, I show that the efficiency of eDNA uptake changes over relatively rapid timescales, suggesting that transformation is an adaptive and selectable trait that could be lost in environments where it is not beneficial.