Project description:Microbes play a critical role in the global arsenic biogeocycle. Most studies have focused on redox cycling of inorganic arsenic in bacteria and archaea. The parallel cycles of organoarsenical biotransformations are less well characterized. Here we describe organoarsenical biotransformations in the environmental microbe Shewanella putrefaciens. Under aerobic growth conditions, S. putrefaciens reduced the herbicide MSMA (methylarsenate or MAs(V)) to methylarsenite (MAs(III)). Even though it does not contain an arsI gene, which encodes the ArsI C-As lyase, S. putrefaciens demethylated MAs(III) to As(III). It cleaved the C-As bond in aromatic arsenicals such as the trivalent forms of the antimicrobial agents roxarsone (Rox(III)), nitarsone (Nit(III)) and phenylarsenite (PhAs(III)), which have been used as growth promoters for poultry and swine. S. putrefaciens thiolated methylated arsenicals, converting MAs(V) into the more toxic metabolite monomethyl monothioarsenate (MMMTAs(V)), and transformed dimethylarsenate (DMAs(V)) into dimethylmonothioarsenate (DMMTAs(V)). It also reduced the nitro groups of Nit(V), forming p-aminophenyl arsenate (p-arsanilic acid or p-AsA(V)), and Rox(III), forming 3-amino-4-hydroxybenzylarsonate (3A4HBzAs(V)). Elucidation of organoarsenical biotransformations by S. putrefaciens provides a holistic appreciation of how these environmental pollutants are degraded.
Project description:5' RNASeq of mRNA from Shewanella putrefaciens CN-32 grown aerobically in Luria-Bertani broth (LB) and defined lactate minimal medium 5'-end mRNA profiles of mid-log phase bacterial cells growing in LB or lactate medium were generated by next-generation sequencing.
Project description:Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S.?putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S.?putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.