Nitrogen metabolism has been studied in detail in industrially important organisms such as Streptomyces and Corynebacteria but there have been very few reports on nitrogen metabolism of mycobacterial species. Earlier, several studies have reported that glnA1 gene is up-regulated in nitrogen starvation in M. tuberculosis and M. smegmatis[5, 12] but this study emphasizes on behaviour of glnA1 locus of M. bovis at both transcriptional and translational levels by altering nitrogen concentration in the medium. Also nitrogen conditions modulate the cell wall properties by altering synthesis of PLG layer in mycobacteria.
The conversion of glutamate to glutamine demands high energy consumption inside the cell. Because of this reason the expression of glnA1 gene is tightly regulated in most mycobacterial species. The transcription of glnA1 gene is regulated in M. tuberculosis by dual promoters . The P1 promoter, present just upstream to glnA1 gene is low nitrogen responding promoter while the P2 promoter, upstream to P1 is high nitrogen responding promoter . Further regulation is driven by GlnR protein which has putative binding site in the P1 promoter. GlnR binds to the P1 promoter and activates transcription during nitrogen starvation . In this study, we have studied the expression level of glnA1 gene of M. bovis in response to nitrogen availability, when the two promoters P1 and P2, are present independently or together. The real time data observed are in accordance with the earlier findings about the regulation of glnA1 gene at transcription level in response to nitrogen availability [11, 12]. The results clearly showed up-regulation of glnA1 expression in M. bovis and MSFP strains in low nitrogen conditions as compared to high nitrogen conditions. MSFP, MSP1 and M. bovis strains have P1 promoter upstream to the glnA1 gene and P1 promoter has binding site for GlnR protein. GlnR binds to the P1 promoter and activates transcription in low nitrogen conditions . This may be the reason for the differences observed in the expression level of the gene in low nitrogen and high nitrogen conditions in these strains. While, on the other hand in MSP2 strain there was no difference in glnA1 expression level in low and high nitrogen conditions. This may be due to lack of P1 promoter and hence GlnR binding site. Also, it can be observed that the difference in gene expression in low and high nitrogen conditions are higher in MSFP and M. bovis strains that have both the promoters upstream to the glnA1 gene. This difference is somewhat reduced in MSP1 and completely lost in MSP2 strain. It has been reported earlier that P1 promoter in M. tuberculosis is σ 60 type promoter . σ 60 is expressed in nitrogen limiting conditions, it recognizes the P1 promoter and transcription starts from P1 promoter.
In addition to regulation at the transcriptional level, GS enzyme encounters a second regulation at post translational level. GlnE protein adenylylate the GS protein in high nitrogen condition and thus makes it inactive [13, 22]. In all the strains, the difference in GS activity in ammonium starvation to ammonium pulse was significantly higher than the difference in expression at mRNA level. Hence, this marked difference observed in GS activity with change in nitrogen conditions in M. bovis, MSFP and MSP1 may be because of two possible reasons. First, there is a stringent regulatory mechanism exhibited by GlnR protein at the transcriptional level because of which the transcript of glnA1 gene itself, is significantly low in high nitrogen conditions. Secondly, after translation, GlnE protein comes into play and modifies the GS enzyme in high nitrogen conditions which makes GS enzyme inactive [13, 22]. MSP2 strain showed low expression of glnA1 gene as compared to the expression in other strains in low nitrogen condition because there was no regulation at transcriptional level due to lack of P1 promoter hence lack of GlnR binding motif also.
PLG layer has been known to be present in the cell wall of only virulent strains of mycobacteria [16, 23]. Harth and colleagues indicated that extracellular GS of pathogenic mycobacteria is involved in synthesis of this layer [10, 24, 25]. There has also been reports stating the involvement of PLG layer of M. bovis in cell wall strength and in providing resistance to various physical and chemical stress factors . The absence of PLG layer from the cell wall of mycobacteria grown in high nitrogen condition indirectly suggest that PLG layer may be a form of nitrogen assimilation in pathogenic mycobacteria. In macrophages, mycobacteria encounter nitrogen stress which leads to high GS expression and PLG layer synthesis in the cell wall. Immunogold localization and PLG isolation studies further validated the finding of no detectable PLG in the cell wall of M. bovis, MSFP, MSP1 and MSP2 strains when grown in high nitrogen conditions.
The ability of the pathogenic mycobacteria to form biofilm adds on to their virulence potential . Biofilm formed at air liquid interface are popularly known as pellicle. Additionally, mycolic acids are the major component of the biofilms formed by mycobacterial species [26, 27] but it is not clearly known whether mycolic acid synthesis or its amount in cell wall is affected by PLG layer. However, there are few reports that suggest the involvement of PLG layer in biofilm formation . A ∆glnA1 strain of M. bovis that lack PLG layer in the cell wall was found to be defective in biofilm formation . Additionally, our results showed that the biofilm and pellicle forming capability of M. smegmatis strain complemented with M. bovis glnA1 was enhanced than the wild type. This is due to the fact that higher expression of M. bovis glnA1 leads to the synthesis of PLG layer in the M. smegmatis complemented with M. bovis glnA1. There are reports also suggesting that microbial amyloids play a significant role in biofilms of actinobacteria [28, 29]. Additionally, it was observed that biofilm was formed significantly much better in low nitrogen conditions which added to the involvement of PLG layer in biofilm formation.
There is a gap in our understanding of the exact mechanisms and enzymes involved in the synthesis of PLG layer till date. In addition to it, characterization of PLG layer, can further help in our understanding of complex mycobacterial cell wall. Because of high molecular weight and inert nature of the polymer it may also act as an adjuvant. This needs further investigation. Establishment of the pathways involved in PLG synthesis will further help in identification of new drug targets against tuberculosis. The study of nitrogen metabolism can provide an insight in the survival of these pathogens in adverse conditions for long duration of time. Also this can help us to understand the mechanisms by which bacteria are able to survive and replicate in macrophages.