Dipankar Chatterji

Revisiting the stringent response, ppGpp and starvation signaling.

Microbial adaptation to environmental stress plays an important role in survival. It is necessary to understand the mechanisms underlying the survival of microbes under stress, as they may eventually aid in the successful control of the growth and persistence of these organisms. During nutrient starvation, Escherichia coli elicits a stringent response to conserve energy. The hallmark of the stringent response is the accumulation of guanosine tetra- (ppGpp) and pentaphosphates (pppGpp), which probably bind RNA polymerase to regulate gene expression at certain promoters. Recently, there has been renewed interest in the stringent responses of other microbes, with a view to correlating it with sporulation, virulence and long-term persistence.

The mediator for stringent control, ppGpp, binds to the β‐subunit of Escherichia coli RNA polymerase

Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure.

Transcriptional switching in Escherichia coli during stress and starvation by modulation of σ70 activity

During active growth of Escherichia coli, majority of the transcriptional activity is carried out by the housekeeping sigma factor (sigma(70)), whose association with core RNAP is generally favoured because of its higher intracellular level and higher affinity to core RNAP. In order to facilitate transcription by alternative sigma factors during nutrient starvation, the bacterial cell uses multiple strategies by which the transcriptional ability of sigma(70) is diminished in a reversible manner. The facilitators of shifting the balance in favour of alternative sigma factors happen to be as diverse as a small molecule (p)ppGpp (represents ppGpp or pppGpp), proteins (DksA, Rsd) and a species of RNA (6S RNA). Although 6S RNA and (p)ppGpp were known in literature for a long time, their role in transcriptional switching has been understood only in recent years. With the elucidation of function of DksA, a new dimension has been added to the phenomenon of stringent response. As the final outcome of actions of (p)ppGpp, DksA, 6S RNA and Rsd is similar, there is a need to analyse these mechanisms in a collective manner. We review the recent trends in understanding the regulation of sigma(70) by (p)ppGpp, DksA, Rsd and 6S RNA and present a case for evolving a unified model of RNAP redistribution during starvation by modulation of sigma(70) activity in E. coli.

High Intracellular Level of Guanosine Tetraphosphate in Mycobacterium smegmatis Changes the Morphology of the Bacterium

ABSTRACT Almost one-third of the world population today harbors the tubercle bacillus asymptomatically. It is postulated that the morphology and staining pattern of the long-term persistors are different from those of actively growing culture. Interestingly, it has been found that the morphology and staining pattern of the starved in vitro population of mycobacteria is similar to the persistors obtained from the lung lesions. In order to delineate the biochemical characteristics of starved mycobacteria, Mycobacteria smegmatis was grown in 0.2% glucose as a sole carbon source along with an enriched culture in 2% glucose. Accumulation of the stringent factor guanosine tetraphosphate (ppGpp) with a concomitant change in morphology was observed for M. smegmatis under carbon-deprived conditions. In addition, M. smegmatis assumed a coccoid morphology when ppGpp was ectopically produced by overexpressing Escherichia coli relA, even in an enriched medium. The Mycobacterium tuberculosis relA and spoT homologue, when induced inM. smegmatis, also resulted in the overproduction of ppGpp with a change in the bacteriums growth characteristics.

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 Fentons-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.

Evidence for a ppGpp‐binding site on Escherichia coli RNA polymerase: proximity relationship with the rifampicin‐binding domain

On amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of PpGpp with a fluorescent probe, 1‐aminonapthalene‐5‐sulphonate (AmNS), at the terminal phosphates. AmNS‐ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titration of the tryptophan emission of RNA polymerase by AmNS‐ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS‐ppGpp with RNA polymerase. Forsters distance measurement was carried out which placed AmNS‐ppGpp 27Å away from the rifampicin‐binding domain of RNA polymerase.

A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

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.

Molecular dissection of the mycobacterial stringent response protein Rel.

Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram‐negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of RelMsm is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full‐length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full‐length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full‐length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA‐mediated synthesis of ppGpp.

Stress responses in mycobacteria

Mycobacterium tuberculosis is a successful pathogen that overcomes numerous challenges presented by the immune system of the host. This bacterium usually establishes a chronic infection in the host where it may silently persist inside a granuloma until, a failure in host defenses, leads to manifestation of the disease. None of the conventional anti‐tuberculosis drugs are able to target these persisting bacilli. Development of drugs against such persisting bacilli is a constant challenge since the physiology of these dormant bacteria is still not understood at the molecular level. Some evidence suggests that the in vivo environment encountered by the persisting bacteria is anoxic and nutritionally starved. Based on these assumptions, anaerobic and starved cultures are used as models to study the molecular basis of dormancy. This review outlines the problem of persistence of M. tuberculosis and the various in vitro models used to study mycobacterial latency. The basis of selecting the nutritional starvation model has been outlined here. Also, the choice of M. smegmatis as a model suitable for studying mycobacterial latency is discussed. Lastly, general issues related to oxidative stress and bacterial responses to it have been elaborated. We have also discussed general control of OxyR‐mediated regulation and emphasized the processes which manifest in the absence of functional OxyR in the bacteria. Lastly, a new class of protein called Dps has been reviewed for its important role in protecting DNA under stress.IUBMB Life, 57: 149‐159, 2005