Artificial micro heat engines are prototypical models to explore and elucidate the mechanisms of energy transduction in a regime that is dominated by fluctuations [1,2].Micro heat engines realized hitherto mimicked their macroscopic counterparts and operated between reservoirs that were effectively thermal [3][4][5][6][7]. For such reservoirs, temperature is a well-defined state variable and stochastic thermodynamics provides a precise framework for quantifying engine performance [8,9]. It remains unclear whether these concepts readily carry over to situations where the reservoirs are outof-equilibrium [10], a scenario of particular importance to the functioning of synthetic [11,12] and biological [13] micro engines and motors. Here we experimentally realized a micrometer-sized active Stirling engine by periodically cycling a colloidal particle in a time-varying harmonic optical potential across bacterial baths at different activities.Unlike in equilibrium thermal reservoirs, the displacement statistics of the trapped particle becomes increasingly non-Gaussian with activity. We show that as much as ≈ 85% of the total power output and ≈ 50% of the overall efficiency stems from large non-Gaussian particle displacements alone. Most remarkably, at the highest activities investigated, the efficiency of our quasi-static active heat engines surpasses the equilibrium saturation limit of Stirling efficiency -the maximum efficiency of a Stirling engine with the ratio of cold and hot reservoir temperatures T C T H → 0. Crucially, the failure of effective temperature descriptions [14-16] for active reservoirs highlights the dire need for theories that can better capture the physics of micro motors and heat engines that operate in strongly non-thermal environments.

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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 Rel Msm 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. Fulllength 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.Keywords: Rel protein; stringent response; ppGpp; multimerization; Rel domains Mycobacterium tuberculosis can be categorized as one of the most successful among human pathogens as several decades of research have not yet been able to completely eradicate tuberculosis (TB), the deadly disease caused by this organism. The major barrier toward complete cure from mycobacterial infection is the unique feature, termed ''latency,'' that these bacteria undergo on infection, leading to overexpression of genes that enable the survival of the pathogen within host organisms under oxygen-(Wayne and Hayes 1996) and nutrient-deprived (Nyka 1974) conditions. Such latent bacteria have been known to be confined in calcified lesions, termed ''granulomas,'' which enable the dormant bacteria to resist conventional antibiotics used against active bacilli. It had been proposed that the morphology and hydrophobicity of the in vivo persistors can be mimicked in laboratory cultures by starving bacteria in vitro (Nyka 1974). Under such stress conditions, adaptation to the Reprint requests to: Dipankar Chatterji, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; e-mail: dipankar@mbu.iisc.ernet.in; fax: +91-80-23600535.Abbreviations: pppGpp, guanosine 39-diphosphate 59-triphosphate; ppGpp, guanosine 39, 59-bis(diphosphate); IPTG, isopropyl b-D-1-thiogalactopyranoside; Ni-NTA, nickel-nitr...

Background: 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.

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-Dps in 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'smediated 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.Microorganisms have developed efficient mechanisms to adapt rapidly and to survive a variety of chemical and physical stress conditions (1). Generation of reactive oxygen species (ROS) 1 is one such stressful condition. ROS are potent cellular oxidizing agents that damage proteins, membrane lipids, and DNA (2-3). During aerobic growth, generation of ROS and of hydrogen peroxide (H 2 O 2 ) is unavoidable. Reaction of H 2 O 2 with free transition metals like ferrous iron can result in the formation of highly reactive hydroxyl radicals (OH ⅐ ) (4). To minimize damages through such ROS, microorganisms have evolved a number of protective ways that help in maintaining the biomolecules in native state. ROS scavenging enzymes such as superoxide dismutases, catalases, and peroxidases, oxidative damage repair enzymes (2, 3), and a nonspecific DNA binding and protecting protein, Dps, (DNA binding protein from stationary phase cells) (5) are a few examples in this category. Almost all the bacteria when exposed to ROS exhibit an adaptive response by switching on the expression of genes coding for these proteins (6). Such strategies are all the more important for pathogenic bacteria because production of reactive oxygen species is a major killing mechanism adopted by many hosts. These schemes also become important during the growth of the organism in stationary phase or during nutrient limiting condition. Thus, the regulation of gene expression upon the induction of starvation and during the stationary phase has been an area of intense research.In the stationary phase cultures of Escherichia coli, the existence of a novel protein Dps was discovered around a decade ago (5). It is a nonspecific DNA bind...

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.

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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.