Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis

Cyclic di-GMP (c-di-GMP) plays an important role in bacterial adaptation to enable survival in changing environments. It orchestrates various pathways involved in biofilm formation, changes in the cell surface, host colonization and virulence. In this article, we report the presence of c-di- GMP in Mycobacterium smegmatis, and its role in the long-term survival of the organism. M. smegmatis has a single bifunctional protein with both GGDEF and EAL domains, which show diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activity, respectively, in vitro. We named this protein MSDGC-1. Deletion of the gene encoding MSDGC-1 did not affect growth and biofilm formation in M. smegmatis, but long-term survival under conditions of nutritional starvation was affected. Most of the proteins that contain GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. To gain further insight into the regulation of the protein, we cloned the individual domains, and tested their respective activities. MSDGC-1, the full-length protein, is required for activity, as its GGDEF and EAL domains are inactive when separated

Direct observation of large temperature fluctuations during DNA thermal denaturation

In this paper we report direct measurement of large low frequency temperature fluctuations in double stranded (ds) DNA when it undergoes thermal denaturation transition. The fluctuation, which occurs only in the temperature range where the denaturation occurs, is several orders more than the expected equilibrium fluctuation. It is absent in single stranded (ss) DNA of the same sequence. The fluctuation at a given temperature also depends on the wait time and vanishes in a scale of few hours. It is suggested that the large fluctuation occurs due to coexisting denaturated and closed base pairs that are in dynamic equilibrium due to transition through a potential barrier in the scale of 25-30k_{B}T_{0}(T_{0}=300K).Comment: 4 pages, 5 figures, Replaced with revised versio

Understanding protein-protein interactions by genetic suppression

Protein-protein interactions influence many cellular processes and it is increasingly being felt that even a weak and remote interplay between two subunits of a protein or between two proteins in a complex may govern the fate of a particular biochemical pathway. In a bacterial system where the complete genome sequence is available, it is an arduous task to assign function to a large number of proteins. It is possible that many of them are peripherally associated with a cellular event and it is very difficult to probe such interaction. However, mutations in the genes that encode such proteins (primary mutations) are useful in these studies. Isolation of a suppressor or a second-site mutation that restores the phenotype abolished by the primary mutation could be an elegant yet simple way to follow a set of interacting proteins. Such a reversion site need not necessarily be geometrically close to the primary mutation site

Evaluation of the role of sigma B in Mycobacterium smegmatis

The alternate sigma factor, sigB, is known to play a crucial role in maintaining the stationary phase in mycobacteria. In this communication, we have studied the proteomics of Mycobacterium smegmatis mc2155 and its two derivatives, one of which has a disrupted sigB gene and the other, PMVSigB, which contains a multicopy plasmid containing sigB. We have identified by two-dimensional gel analyses, several proteins that are over-expressed in PMVSigB compared to mc2155. These proteins are either stress proteins or participate actively in different metabolic pathways of the organisms. On the other hand, when sigB deleted mycobacteria were grown until the stationary phase and its two-dimensional protein profile was compared to that of mc2155, few DNA binding proteins were found to be up-regulated. We have shown recently that upon over-expressing sigB, the cell surface glycopeptidolipids of M. smegmatis are hyperglycosylated, a situation similar to what was observed for nutritionally starved bacteria. Gene expression profile through quantitative PCR presented here identified a Rhamnosyltransferase responsible for this hyperglycosylation

Bimodal protection of DNA by Mycobacterium smegmatis DNA-binding protein from stationary phase cells

Some members of the DNA-binding protein from stationary phase cells (Dps) family of proteins have been shown to play an important role in protecting microorganisms from oxidative or nutritional stress. Dps homologs have been identified in various bacteria such as Escherichia coli, Bacillus subtilis, and Listeria innocua. Recently we have reported the presence of a Dps homolog, Ms-Dps, in Mycobacterium smegmatis. Ms-Dps was found to have a nonspecific DNA binding ability. Here we have detected two stable oligomeric forms of Ms-Dpsin vitro, a trimeric and a dodecameric form. Interestingly, the conversion of Dps from a trimeric to a dodecameric form takes place upon incubation at 37° C for 12 h. These two oligomeric forms differ in their DNA binding properties. The dodecameric form is capable of DNA binding and forming large crystalline arrays with DNA, whereas the trimeric form cannot do so. However, even in the absence of DNA binding, the trimeric form has the capacity to protect the DNA against Fenton's-mediated damage. The protection is afforded by the ferroxidase activity of the trimer. However, the trimeric form cannot protect DNA from DNaseI attack, for which a direct physical shielding of DNA by the dodecamer is required. Thus we suggest that Ms-Dps provides a bimodal protection of DNA by its two different oligomeric forms

Differential mechanisms of binding of anti-sigma factors Escherichia coli Rsd and bacteriophage T4 Asia to E. coli RNA polymerase lead to diverse physiological consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70

Promoter search and strength of a promoter: two important means for regulation of gene expression in Escherichia coli

Search for a promoter element by RNA polymerase from the extremely large DNA base sequence is thought to be the slowest and rate-determining for the regulation of transcription process. Few direct experiments we described here which have tried to follow the mechanistic implications of this promoter search. However, once the promoter is located, transcription complex, constituting mainly the RNA polymerase molecule and few transcription factors has to unidirectionally clear the promoter and elongate the RNA chain through a series of steps which altogether define the initiation of transcription process. Thus, it appears that the promoter sequence acts as a trap for RNA polymerase associated with a large binding constant, although to clear the promoter and to elongate the transcript such energy barrier has to be overcome. Topological state of the DNA, particularly in the neighbourhood of the promoter plays an important role in the energetics of the whole process

Identification, Activity and Disulfide Connectivity of C-di-GMP Regulating Proteins in Mycobacterium tuberculosis

C-di-GMP, a bacterial second messenger plays a key role in survival and adaptation of bacteria under different environmental conditions. The level of c-di-GMP is regulated by two opposing activities, namely diguanylate cyclase (DGC) and phosphodiesterase (PDE-A) exhibited by GGDEF and EAL domain, respectively in the same protein. Previously, we reported a bifunctional GGDEF–EAL domain protein, MSDGC-1 from Mycobacterium smegmatis showing both these activities (Kumar and Chatterji, 2008). In this current report, we have identified and characterized the homologous protein from Mycobacterium tuberculosis (Rv 1354c) named as MtbDGC. MtbDGC is also a bifunctional protein, which can synthesize and degrade c-di-GMP in vitro. Further we expressed Mtbdgc in M. smegmatis and it was able to complement the MSDGC-1 knock out strain by restoring the long term survival of M. smegmatis. Another protein Rv 1357c, named as MtbPDE, is an EAL domain protein and degrades c-di-GMP to pGpG in vitro. Rv1354c and 1357c have seven cysteine amino acids in their sequence, distributed along the full length of the protein. Disulfide bonds play an important role in stabilizing protein structure and regulating protein function. By proteolytic digestion and mass spectrometric analysis of MtbDGC, connectivity between cysteine pairs Cys94-Cys584, Cys2-Cys479 and Cys429-Cys614 was determined, whereas the third cysteine (Cys406) from N terminal was found to be free in MtbDGC protein, which was further confirmed by alkylation with iodoacetamide labeling. Bioinformatics modeling investigations also supported the pattern of disulfide connectivity obtained by Mass spectrometric analysis. Cys406 was mutated to serine by site directed mutagenesis and the mutant MtbC406S was not found to be active and was not able to synthesize or degrade c-di-GMP. The disulfide connectivity established here would help further in understanding the structure – function relationship in MtbDGC

Mycobacterial stress regulation: the Dps "twin sister" defense mechanism and structure-function relationship

In this work, we have tried to emphasize the connection between mycobacterial growth and regulation of gene expression. Utilization of multiple carbon sources and diauxic growth helps bacteria to regulate gene expression at an optimum level so that the inhospitable conditions encountered during nutrient depletion can be circumvented. These aspects will be discussed with respect to mycobacterial growth in subsequent sections. Identification and characterization of genes induced under such conditions is helpful to understand the physiology of the bacterium. Although it is necessary to compare the total expression profile of proteins as they transit from vegetative growth to stationary phase, at times a lot of insights can be deciphered from the expression pattern of one or two proteins. We have compared the protein expression and sigma factor selectivity of two such proteins in M. smegmatis to understand the differential regulation of genes playing diverse function in the same species. Some newer insights on the structure and function of one of the Dps proteins are also explained

Unveiling the role of Dps in the organization of mycobacterial nucleoid

In order to preserve genetic information in stress conditions, bacterial DNA is organized into higher order nucleoid structure. In this paper, with the help of Atomic Force Microscopy, we show the different structural changes in mycobacterial nucleoid at different points of growth in the presence of different concentrations of glucose in the medium. We also observe that in Mycobacterium smegmatis, two different Dps proteins (Dps1 and Dps2) promote two types of nucleoid organizations. At the late stationary phase, under low glucose availability, Dps1 binds to DNA to form a very stable toroid structure. On the other hand, under the same condition, Dps2-DNA complex forms an incompletely condensed toroid and finally forms a further stable coral reef structure in the presence of RNA. This coral reef structure is stable in high concentration of bivalent ion like Mg2+