Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.
Biofilm is a predominant lifestyle of bacteria that comprises of cells as collectives enmeshed in a polymeric matrix. Biofilm formation is vital for bacterial species as it provides access to nutrients and protects the cells from environmental stresses. Here we show that interference in biofilm matrix production is a strategy by the competing bacterial species to reduce the ability of the other species to colonize a surface. Escherichia coli colonies that differ in matrix production display different morphologies on Congo red agar media, which we exploited for screening bacterial isolates capable of inhibiting the matrix. The cell-free supernatants from growth culture of the screened isolates impaired uropathogenic E. coli (UPEC) UTI89 strain's biofilm. A physicochemical analysis suggested that the compound could be a glycopeptide or a polysaccharide. Isolates that inhibited matrix production belonged to species of the family Enterobacteriaceae such as Shigella, Escherichia, Enterobacter, and Salmonella. Competition experiments between the isolates and the UPEC strain resulted in mutual inhibition, particularly during biofilm formation causing significant reduction in productivity and fitness. Furthermore, we show that Salmonella strains competitively excluded the UPEC strain in the biofilm by inhibiting its matrix production, highlighting the role of interference competition.
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