Uranium biomineralization by immobilized Chryseobacterium sp. strain PMSZPI cells for efficient uranium removal.

Uranium (U) contamination is hazardous to human health and the environment owing to its radiotoxicity and chemical toxicity and needs immediate attention. In this study, the immobilized biomass of Chryseobacterium sp. strain PMSZPI isolated from U enriched site, was investigated for U(VI) biomineralization in batch and column set-up. Under batch mode, the fresh or lyophilized cells successfully entrapped in calcium alginate beads demonstrated effectual U precipitation under acid and alkaline conditions. The maximum removal was detected at pH 7 wherein ∼98-99% of uranium was precipitated from 1 mM uranyl carbonate solution loading ∼350 mg U/g of biomass within 24 h in the presence of organic phosphate substrate. The resulting uranyl phosphate precipitates within immobilized biomass loaded beads were observed by SEM-EDX and TEM while the formation of U biomineral was confirmed by FTIR and XRD. Retention of phosphatase activity without any loss of uranium precipitation ability was observed for alginate beads with lyophilized biomass stored for 90 d at 4 °C. Continuous flow through experiment with PMSZPI biomass immobilized in polyacrylamide gel exhibited U loading of 0.8 g U/g of biomass at pH 7 using 1 l of 1 mM uranyl solution. This investigation established the feasibility for the application of immobilized PMSZPI biomass for field studies. ENVIRONMENTAL IMPLICATION: Uranium contamination is currently a serious environmental concern owing to anthropogenic activities and needs immediate attention. We have developed here a biotechnological method for successful uranium removal using immobilized cells of a uranium tolerant environmental bacterium, Chryseobacterium sp. strain PMSZPI isolated from U ore deposit via phosphatase enzyme mediated uranium precipitation. The ability of immobilized PMSZPI cells to precipitate U(VI) as long-term stable U phosphates under environmental conditions relevant for contaminated waters containing high concentrations of U that exerts toxicity for biological systems is explored here. The long term stability of the immobilized biomass without compromising its U removal capacity shows the relevance of the bioremediation strategy for uranium contamination proposed in this work.

T9GPred: A Comprehensive Computational Tool for the Prediction of Type 9 Secretion System, Gliding Motility, and the Associated Secreted Proteins.

Type 9 secretion system (T9SS) is one of the least characterized secretion systems exclusively found in the Bacteroidetes phylum, which comprises various environmental and economically relevant bacteria. While T9SS plays a central role in bacterial movement termed gliding motility, survival, and pathogenicity, there is an unmet need for a comprehensive tool that predicts T9SS, gliding motility, and proteins secreted via T9SS. In this study, we develop such a computational tool, Type 9 secretion system and Gliding motility Prediction (T9GPred). To build this tool, we manually curated published experimental evidence and identified mandatory components for T9SS and gliding motility prediction. We also compiled experimentally characterized proteins secreted via T9SS and determined the presence of three unique types of C-terminal domain signals, and these insights were leveraged to predict proteins secreted via T9SS. Notably, using recently published experimental evidence, we show that T9GPred has high predictive power. Thus, we used T9GPred to predict the presence of T9SS, gliding motility, and associated secreted proteins across 693 completely sequenced Bacteroidetes strains. T9GPred predicted 402 strains to have T9SS, of which 327 strains are also predicted to exhibit gliding motility. Further, T9GPred also predicted putative secreted proteins for the 402 strains. In a nutshell, T9GPred is a novel computational tool for systems-level prediction of T9SS and streamlining future experimentation. The source code of the computational tool is available in our GitHub repository: https://github.com/asamallab/T9GPred. The tool and its predicted results are compiled in a web server available at: https://cb.imsc.res.in/t9gpred/.

Structural Analysis of Gliding Motility of a Bacteroidetes Bacterium by Correlative Light and Scanning Electron Microscopy (CLSEM).

The members of the Bacteroidetes phylum move on surfaces by gliding motility in the absence of external motility appendages, leading to the formation of spreading colonies. Here, the structural features of the spreading colony were assessed in a uranium-tolerant Bacteroidetes bacterium, Chryseobacterium sp. strain PMSZPI, by using correlative light and scanning electron microscopy (CLSEM). We developed a simple and convenient workflow for CLSEM using a shuttle and find software module and a correlative sample holding slide designed to transport samples between the light/fluorescence microscope (LM/FM) and the scanning electron microscope (SEM) to image spreading colony edges. The datasets from the CLSEM studies allowed convenient examination of the colonial organization by LM/FM followed by ultrastructural analysis by SEM. The regions of interest (ROIs) of the spreading colony edges that were observed in LM/FM in the absence and presence of uranium could be re-identified in the SEM quickly without prolonged searching. Perfect correlation between LM and SEM could be achieved with minimum preparation steps. Subsequently, imaging of the correlated regions was done at higher resolution in SEM to obtain more comprehensive information. We further showed the association of uranium with the gliding PMSZPI cells by energy-dispersive X-ray spectroscopy (EDS) attached to SEM.

Gliding motility of a uranium-tolerant Bacteroidetes bacterium Chryseobacterium sp. strain PMSZPI: insights into the architecture of spreading colonies.

Uranium-tolerant soil bacterium Chryseobacterium sp. strain PMSZPI moved over solid agar surfaces by gliding motility thereby forming spreading colonies which is a hallmark of members of Bacteroidetes phylum. PMSZPI genome harboured orthologs of all the gld and spr genes considered as core bacteroidetes gliding motility genes of which gldK, gldL, gldM and gldN were co-transcribed. Here, we present the intriguing interplay between gliding motility and cellular organization in PMSZPI spreading colonies. While nutrient deficiency enhanced colony spreading, high agar concentrations and presence of motility inhibitor like 5-hydroxyindole reduced the spreading. A detailed in situ structural analysis of spreading colonies revealed closely packed cells forming multiple layers at centre of colony while the edges showed clusters of cells periodically arranged in hexagonal lattices interconnected with each other. The cell migration within colony was visualized as branched structures wherein the cells were buried within extracellular matrix. PMSZPI colonies exhibited strong iridescence possibly as a result of periodicity within the cell population achieved through gliding motility. Presence of uranium reduced motility and iridescence and induced biofilm formation. The coordinated study of gliding motility and iridescence apparently influenced by uranium provides unique insights into the lifestyle of PMSZPI residing in uranium enriched environment.

Genomic and functional insights into the adaptation and survival of Chryseobacterium sp. strain PMSZPI in uranium enriched environment.

Metal enriched areas represent important and dynamic microbiological ecosystems. In this study, the draft genome of a uranium (U) tolerant bacterium, Chryseobacterium sp. strain PMSZPI, isolated from the subsurface soil of Domiasiat uranium ore deposit in Northeast India, was analyzed. The strain revealed a genome size of 3.8 Mb comprising of 3346 predicted protein-coding genes. The analysis indicated high abundance of genes associated with metal resistance and efflux, transporters, phosphatases, antibiotic resistance, polysaccharide synthesis, motility, protein secretion systems, oxidoreductases and DNA repair. Comparative genomics with other closely related Chryseobacterium strains led to the identification of unique inventory of genes which were of adaptive significance in PMSZPI. Consistent with the genome analysis, PMSZPI showed superior tolerance to uranium and other heavy metals. The metal exposed cells exhibited transcriptional induction of metal translocating PIB ATPases suggestive of their involvement in metal resistance. Efficient U binding (~90% of 100 µM U) and U bioprecipitation (~93-94% of 1 mM U at pH 5, 7 and 9) could be attributed as uranium tolerance strategies in PMSZPI. The strain demonstrated resistance to a large number of antibiotics which was in agreement with in silico prediction. Reduced gliding motility in the presence of cadmium and uranium, enhanced biofilm formation on uranium exposure and tolerance to 1.5 kGy of 60Co gamma radiation were perceived as adaptive responses in PMSZPI. Overall, the positive correlation observed between uranium/metal tolerance abilities predicted using genome analysis and the functional characterization reinforced the multifaceted adaptation strategies employed by PMSZPI for its survival in the soil of uranium ore deposit comprising of high concentrations of uranium and other heavy metals.

Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis.

BACKGROUND: Small RNAs (sRNAs) are important modulators of gene expression in bacteria. Regulation by sRNAs is yet to be established in Bacillus anthracis. Here, regulation and RNA-binding properties of Hfq-like RNA chaperones in B. anthracis are investigated. METHODS: Transcript levels were measured by RT-PCR. Proteins were cloned, purified, and their ability to bind sRNA was seen by EMSA. Regulators of Hfq1 were identified by in silico analysis and validated by EMSA. Conserved sRNAs were identified by homology search and their ability to bind Hfq1 was seen by EMSA. Residues crucial for sRNA binding were identified by mutational studies. RESULTS: hfq1 and hfq3 showed expression during exponential phase on BHI medium, while hfq2 showed no expression. Hfq1 and Hfq3 formed hexamer and sRNA-Hfq complex, not Hfq2. In silico prediction and EMSA confirmed AbrB binding to the promoter of Hfq1. Homology search identified 7 sRNAs in B. anthracis. The sRNA CsfG showed binding to Hfq1 via residues Y24, F29, Q30, R32, K56, and H57. CONCLUSIONS: Hfq1 and Hfq3 can function as RNA chaperones in B. anthracis. The transition phase repressor AbrB might be responsible for the growth-dependent expression of Hfq1. The sporulation-specific sRNA CsfG binds to Hfq1 via its distal surface and requires an intact hexameric structure for forming CsfG-Hfq1 complex. GENERAL SIGNIFICANCE: This is the first report demonstrating the regulation and functional properties of Hfq-like RNA chaperones in B. anthracis and paves the path towards establishing sRNA-based regulation in B. anthracis.