SummarysigA encodes a factor of the 70 family, A , that is found in all mycobacterial species. As A shows high similarity to the primary factor in Streptomyces coelicolor, it was postulated that A has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that A was an alternative factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the ␤-galactosidase activity in a strain carrying a sigA-lacZ transcriptional fusion and monitored A levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary factor in M. smegmatis and, most likely, in all mycobacteria.

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A search for Mycobacterium smegmatis genes showing similarity to the conserved family encoding major sigma factors in diverse prokaryotes has identified two such determinants. Both genes are expressed in exponentially growing cells, as judged by Western immunoassays. A series of chromatographic steps was used to purify M. smegmatis RNA polymerase holoenzyme and it was shown that its ability to initiate in vitro transcription with a heterologous Bacillus subtilis promoter is dependent on the presence of these sigma factor(s). Reconstitution of specific in vitro transcription activity was obtained upon mixing of M. smegmatis core RNA polymerase with the major sigma factor of Bacillus subtilis. We also demonstrated in vitro transcription of the M. smegmatis rrnB promoter by the M. smegmatis RNA polymerase. Significantly, highly active B. subtilis RNA polymerase holoenzyme was unable to transcribe this gene.

Aim:The aim of this study is to test the potency of bovine serum albumin (BSA) conjugated ampicillin (AMP) and enrofloxacin (ENR) antigens in eliciting an immune response in rats using indirect competitive enzyme-linked immunosorbent assay (icELISA).Materials and Methods:AMP and ENR antibiotics were conjugated with BSA by carbodiimide reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linker. The successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sprague-Dawley rats were immunized with the conjugates and blood samples were collected serially at 15 days time interval after first immunization plus first booster, second booster, third booster, and the fourth sampling was done 1½ month after the third booster. The antibody titres in the antisera of each antibiotic in all the four immunization cycles (ICs) were determined by an icELISA at various serum dilutions ranging from 1/100 to 1/6400.Results:Analysis of antibiotic-BSA conjugates by sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining revealed high molecular weight bands of 85 kDa and 74 kDa for AMP-BSA and ENR-BSA respectively when compared to 68 kDa band of BSA. Both the antibiotic conjugates elicited a good immune response in rats but comparatively the response was more with AMP-BSA conjugate than ENR-BSA conjugate. Maximum optical density 450 value of 2.577 was recorded for AMP-BSA antisera, and 1.723 was recorded for ENR-BSA antisera at 1/100th antiserum dilution in third IC.Conclusion:AMP and ENR antibiotics proved to be good immunogens when conjugated to BSA by carbodiimide reaction with EDC as crosslinker. The polyclonal antibodies produced can be employed for detecting AMP and ENR residues in milk and urine samples.