Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6-to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCEThe present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravitybased settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. Bioremediation strategies, such as bioreduction (1-3), biosorption (4-8), bioaccumulation (9, 10), and bioprecipitation (5, 11, 12, 13), have been studied for their potential to immobilize U from solutions. There is also a large body of work on microbial interactions with uranium relevant to environmental in situ bioremediation. The...

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The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug.

23The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for 24 various applications. In case of the latter, a couple of previous studies had shown Cas9 protein 25 expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, 26 Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and 27 Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for 28 Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results 29 showed differential levels of Cas9 toxicity among the bacteria and lower transformation 30 efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated 31 lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate 32 Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, 33 Cas9/dCas9 associated toxicity was acute. 34 35 36 37 38 39 40 41 42 43 44 3 | P a g e Introduction: 45 Recombinant DNA technology together with High Throughput Sequencing in recent 46times, has allowed us to harvest a large amount of genetic information from the microbial 47 world. The technologies have been used extensively to find out which genes determine how 48 microbes, grow, travel, starve, cause diseases, ward of predators and even die. This information 49 is especially important for studying pathogenic bacteria, bacteria of industrial importance and 50 ones with special stress tolerance abilities. Perturbing the normal functioning of the genome 51 has emerged as the best method to probe function and dynamics of individual genes. 52 Discovery of the CRISPR -Cas viral defence systems opened up another novel and 53 efficient tool box for genome editing, gene silencing, targeted gene methylation, etc. in all 54 kinds of organisms from bacteria to humans [1][2][3][4]. The ease and efficiency of the system 55 has made it an extremely popular go-to system for various applications. The system has further 56 had widespread applications in metabolic engineering of bacteria as it allows easy 57 programming and multiplexing [5][6]. Among the CRISPR-Cas systems, the Cas9 system from 58 Streptomyces pyogens has gained popularity on account of being one of the earliest systems to 59 be discovered and its simplicity of usage [7][8][9]. The system comprises a single protein, Cas9 60 and the sgRNA , which together can be easily employed to bring about a host of desired changes 61 inside the cell of virtually any living being [10]. While the nucleoprotein, Cas9 itself has been 62 extensively used for genome editing [7][8][11][9], its nuclease deficient variant, dCas9 has 63 been useful for regulation of gene expression [3][1]. Systems employing the Cas9 variants have 64 shown great promise for use in eukaryotic, particularly mammalian systems [11]. Attempts to 65 use them in microbes have met with mixed success. 66 The Cas9/dCas9 and also Cas9 nickase systems were used successfully to probe gene 67 function...