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Single zinc finger domain containing proteins are very few in number. Of numerous zinc finger proteins in eukaryotes, only three of them like GAGA, Superman and DNA binding by one finger (Dof) have single zinc finger domain. Although few zinc finger proteins have been described in eubacteria, no protein with single C4 zinc finger has been described in details in anyone of them. In this article, we are describing two novel C-terminal C4 zinc finger proteins-Msmeg_0118 and Msmeg_3613 from Mycobacterium smegmatis. We have named these proteins as Mszfp1 (Mycobacterial Single Zinc Finger Protein 1) and Mszfp2 (Mycobacterial Single Zinc Finger Protein 2). Both the proteins are expressed constitutively, can bind to DNA and regulate transcription. It appears that Mszfp1 and Mszfp2 may activate transcription by interacting with RNA polymerase. [ABSTRACT FROM AUTHOR]

The article discusses the Kishor Vaigyanic Protsahan Yojana (KVPY), administered by the Department of Science and Technology (DST), ceasing to exit from 2022. Topics include the growth and popularity of KVPY from the very beginning; reasons behind such a whimsical decision, and various reputed undergraduate science programs such as at IISc and IISERs.

(p)ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross-linking experiments suggested that the β-subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of β'/ω-subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra- and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo-labeled with 8-azido-ppGpp/8-azido-pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8-azido-ppGpp, of which two peptides correspond to the same pocket on β'-subunit as predicted by X-ray structural analysis, whereas the third peptide was mapped on the β-subunit. In the case of 8-azido-pppGpp-labeled RNA polymerase, we have found only one cross-linked peptide from the β'-subunit. However, we were unable to identify any binding site of pppGpp on the β-subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel-based assay as well as by a new method known as DRaCALA assay. [ABSTRACT FROM AUTHOR]

The Pup proteasome system (PPS) in bacteria is equivalent to the eukaryotic ubiquitin proteasome system (UPS) that allows controlled protein degradation. Unlike the UPS, however, the PPS machinery and regulation is still poorly understood. In this issue of The FEBS Journal, Gur and colleagues combine experimental and modelling analyses to show how the PPS maintains steady‐state levels of protein pupylation and consequently tightly controlled protein degradation. [ABSTRACT FROM AUTHOR]

The pathogenesis of Mycobacterium tuberculosis is associated with its ability to survive inside the human host and the bacteria use a variety of mechanism to evade the host's defence. A clearer understanding of the host-pathogen interaction is needed to follow the pathogenicity and virulence. Recent advances in the study of inter and intra-cellular communication in bacteria had prompted us to study the role of quorum sensing in bacterial survival and pathogenicity. The cell-cell communication in bacteria (quorum sensing) is mediated through the exchange of small molecules called as autoinducers that allow bacteria to modulate their gene expression in response to change in cellpopulation density. It is a coordinated response that confers multicellularity to a bacterial population in response to stress from external environment. Quorum sensing molecules are the global regulators and regulate a wide range of physiological processes including biofilm formation, motility, cell differentiation, long-term survival and many others. Many bacterial pathogens require quorum sensing to produce the virulence factors in response to host-pathogen interaction. Here, we summarize our current understanding on small molecule signalling and their role in the bacterial persistence. New discoveries in these areas have enriched our knowledge on intracellular signalling and their role in the long-term survival of mycobacteria under nutrient starvation. [ABSTRACT FROM AUTHOR]

Species of opportunistic mycobacteria are the major causative agent for disseminating pulmonary infections in immuno-compromised individuals. These naturally resistant strains recruit a unique type of glycolipid known as glycopeptidolipids (GPLs), noncovalently attached to the outer surface of their thick lipid rich cell envelope. Species specific GPLs constitute the chemical determinants of most nontuberculous mycobacterial serotypes, and their absence from the cell surface confers altered colony morphology, hydrophobicity, and inability to grow as biofilms. The objective of this review is to present a comprehensive account and highlight the renewed interest on this much neglected group of pleiotropic molecules with respect to their structural diversity and biosynthesis. In addition, the role of GPLs in mycobacterial survival, both intracellular and in the environment is also discussed. It also explores the possibility of identifying new targets for intervening Mycobacterium avium complex-related infections. These antigenic molecules have been considered to play a pivotal role in immune suppression and can also induce various cytokine mediated innate immune responses, the molecular mechanism of which remains obscure. © 2012 IUBMB IUBMB Life, 2012 [ABSTRACT FROM AUTHOR]

During active growth of Escherichia coli, majority of the transcriptional activity is carried out by the housekeeping sigma factor (σ70), 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 σ70 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 σ70 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 σ70 activity in E. coli. [ABSTRACT FROM AUTHOR]

In this article, the author discusses the challenges encountered following the initiation of the Kishore Vaigyanik Protsahan Yojana (KVPY) by the Department of Science and Technology (DST) in India. It notes that KVPY opens fellowships for medical and engineering students who have the ability to design an original research project based on science. It points out that it increases the number of examination centres across the country.

The proximate aim of this review is to investigate the specific interaction between two macromolecules, either two complementary strands of DNA or the binding of DNA with a protein. Although a lot of experiments have been done to address these issues, our aim here is different. We either create a dense brush of DNA chains at the air-water interface or orient a large protein, like RNA polymerase, such that they are amenable for specific interaction at the surface. The advantage of our system is that the macromolecules are stretched, oriented parallel to each other, and their concentrations can be made similar to these encountered in real nuclei. In this way we plan to construct an 'artificial nucleus'. Other methods adopted so far can check for the possibility of collective behaviour and the effect of chain elongation or compaction. We have used Langmuir-Blodgett technique for the same and extensively performed FTIR and AFM experiments to monitor the L-B surface. Each macromolecule has been attached by one of its extremities to a hydrophobic buoy to keep it at the interface. Detailed thermodynamic analysis results in some interesting conclusions. [ABSTRACT FROM AUTHOR]

The article presents a study which analyses the presence of cyclic di-GMP (c-di-GMP) in Mycobacterium smegmatis, and its physiological role in the long-term survival of the organism. It notes that c-di-GMP plays an important role in biofilm formation and adaptation in several bacterial species which enable such organisms survive in changing environments. With bioinformatics analysis, it was found out that Mycobacterium smegmatis has a bifunctional single protein with GGDEF and EAL domains, having both the diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activities. It also found out that the identified bifunctional protein did not affect growth and biofilm formation of the mycobacterium, however, long-term survival under conditions of nutritional starvation was affected.

Abstract: Rapid emergence of multi-drug resistance in Mycobacterium tuberculosis has necessitated the development of newer candidate drugs which can selectively inhibit the growth of the organism. Among the best targets available today, transcription machinery is, by far, the most important one and the antibiotic rifampicin binds to a specific site on the enzyme RNA polymerase. However, it is not very effective towards the stationary phase of the organism or the persistors. In order to address this problem, we report here a protocol for generating an affinity tagged RNA polymerase, which can be purified easily from different phases of growth of the organism. It allows exploring RNAP associated proteins, which may confer resistance to rifampicin, using the approach of functional proteomics. [Copyright &y& Elsevier]

Abstract: Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the ω subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc2155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting. [Copyright &y& Elsevier]

Abstract: Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster''s distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone. [Copyright &y& Elsevier]

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level. [ABSTRACT FROM AUTHOR]

Omega (ω) is the smallest subunit of bacterial RNA polymerase (RNAP). Although identified early in RNAP research, its function remained ambiguous and shrouded by controversy for a considerable period. It has subsequently been shown that the protein has a structural role in maintenance of the conformation of the largest subunit, β′, and recruitment of β′ to the enzyme assembly. Conservation of this function across all forms of life indicates the importance of its role. Several recent observations have suggested additional functional roles for this protein and have settled some long-standing controversies surrounding it. In this context, revisiting the ω subunit story is especially interesting; here, we review the progress of ω research since its discovery and highlight the importance of these recent observations. [Copyright &y& Elsevier]

Abstract: The T4 AsiA is an anti-sigma factor encoded by one of the early genes of Bacteriophage T4. It has been shown that AsiA inhibits transcription from promoters containing −10 and −35 consensus sequence by binding to σ70 of E. coli. Binding of AsiA to σ70 in vivo, in E. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. By using various in vitro methods, the region of σ70 binding to AsiA have been mapped to domain 4.2. Additionally, mutational analysis of σ70 has also identified amino acid residues in domain 4.1 which are critical for interaction with AsiA. Based on NMR studies it has been suggested that either of these regions can bind to AsiA, a conclusion which was supported by high degree of amino acid homology between domain 4.1 and 4.2. However, it is not clear whether under in vivo conditions, AsiA exerts its transcription inhibitory effect by binding to one of these regions or both the regions together. In order to understand the mechanism of AsiA mediated inhibition of E. coli transcription in vivo, in terms of specific binding requirements to region 4.1 and/or 4.2, we have studied the interaction of these sub-domains with AsiA by Yeast two hybrid system as well as by co-expressing and affinity purification of the interacting partners in vivo in E. coli. It was observed that minimum fragment of σ70 showing observable binding to AsiA, must possess sub-domains 4.1 and 4.2 together. No binding could be detected in σ70 fragments lacking a part of either domain 4.1 or 4.2, in any of the assays. This data was also supported by in vitro binding studies wherein only σ70 fragments carrying both region 4.1 and 4.2 showed binding to AsiA. Co-expression of region 4.1 and 4.2 fragments together also did not show any interaction with AsiA. The results presented here suggest that binding of AsiA to σ70, in vivo, requires the presence of both sub-domains of region 4 of σ70. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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 [ABSTRACT FROM AUTHOR]

Demonstrates that only the dodecameric species of the Ms-Dps homolog of DNA-binding protein from stationary phase cells is capable of complex formation with DNA. Bimodal type of DNA protection by Ms-Dps; Role of Ms-Dps as a link between ferritins and Dps.

BackgroundInhibition 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. ResultsHere, the azido-derivative of ppGpp was synthesized from azido-GDP and [γ-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the β-subunit of RNA polymerase. ConclusionIt was observed that both the N-terminal and C-terminal domains of the β-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp. [ABSTRACT FROM AUTHOR]

Escherichia coli RNA polymerase has two sites, the i and i + 1, for the binding of the first two substrates. The i site is template- and Mg2+-independent and purine-nucleotide-specific, whereas the i + 1 site is template- and Mg2+-dependent and shows no nucleotide preference. The specificity of the i site for purine nucleotides is well in accord with the fact that most promoters initiate with a purine nucleotide. But there are a few promoters that initiate with a pyrimidine nucleotide. Dinucleotide synthesis at these promoters is completely inhibited by rifampicin. Earlier studies have failed to identify an i site for pyrimidine nucleotides. In this paper, using a fluorescent analog of UTP, namely uridine 5'-[γ-(5-sulfonic acid)naphthylamidate]-triphosphate, abbreviated as UTP[AmNS], we are able to show its binding to RNA polymerase, with a Kd of 0.8 μM, in the absence of Mg2+ and template. This suggests the presence of an i pyrimidine nucleotide site. The fact that UTP-[AmNS] is capable of initiating RNA synthesis from the i site is further evidenced by the abortive transcription analyses at the lac promoter. Fluorescence titration studies performed in the presence and absence of purine initiator molecules indicate that this site is different from the i purine site. Scatchard analysis of the above data indicates the presence of a single binding site for UTP[AmNS] in the absence of Mg2+. Moreover UTP[AmNS] binds to the core enzyme with a Kd of 3.0 μM implying that, unlike the i purine nucleotide site, the sigma protein confers a tighter binding of UTP-[AmNS] to the low-Kd site. Forster's energy transfer measurements using UTP[AmNS] as the donor and rifampicin as the acceptor have been used for estimation of the distance of the i pyrimidine nucleotide site from the rifampicin site. From these measurements, we infer that there is no direct interference of rifampicin with the first [ABSTRACT FROM AUTHOR]

Highly purified Escherichia coli RNA polymerase contains a small subunit termed ω that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. To elucidate the function of ω, whose role is as yet undefined, the subunit was purified to over 95% purity from an overproducing strain [BL21 (pGP1-2, pE3C-2)]. Purified ω was then reconstituted with RNA polymerase isolated from an ω-less mutant. Externally added ω inhibited promoter-specific transcriptional activity at all promoters tested. Renaturation of fully denatured ω-less RNA polymerase in the presence of excess ω yielded maximum recovery of activity suggesting a structural rather than functional role for ω. [ABSTRACT FROM AUTHOR]

Among various group 1 sigma factors, two amino acids. Va155 and A1a59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]σ70 and [Gly59)σ70, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type σ70 has an apparent molecular mass of 87 kDa. However, [Gly434]σ70 which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]σ70 and [Gly59]σ70 have reduced helicity (20%) compared to wild-type σ70 (50%). Binding of sigma factors with the hydrophobic, surface active probe I-anilinonapthalene-8-sulphonate, has shown that more hydrophobic suites are available/exposed in [Gly55]σ70. [Gly59]σ70 as well as in [Gly434]σ70 in comparison to wild-type σ70. Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (α2ββ') have shown reduced trunscnptional activity in comparison to the enzyme containing wild-type sigma factor. However; another mutation (Val→Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83] σ70, has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in σ70 may interact hydrophobically with the 2.3/2.4 DNA-binding region. [ABSTRACT FROM AUTHOR]

The article focuses on online teaching is a hot topic of discussion in print and visual media. Topics include the arguments in favour highlight the necessity to engage kids in meaningful, constructive ways to avoid the colossal waste of their precious time otherwise, and growing up in Covid situation means no outdoor activities, spending the whole day in limited, defined space mostly with elders.

In eubacteria, cyclic di-GMP (c-di-GMP) signaling is involved in virulence, persistence, motility and generally orchestrates multicellular behavior in bacterial biofilms. Intracellular c-di-GMP levels are maintained by the opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDEs). The c-di-GMP homeostasis in Mycobacterium smegmatis is supported by DcpA, a conserved, bifunctional protein with both DGC and PDE activities. DcpA is a multidomain protein whose GAF-GGDEF-EAL domains are arranged in tandem and are required for these two activities. To gain insight into how interactions among these three domains affect DcpA activity, here we studied its domain dynamics using real-time FRET. We demonstrate that substrate binding in DcpA results in domain movement that prompts a switch from an "open" to a "closed" conformation and alters its catalytic activity. We found that a single point mutation in the conserved EAL motif (E384A) results in complete loss of the PDE activity of the EAL domain and in a significant decrease in the DGC activity of the GGDEF domain. Structural analyses revealed multiple hydrophobic and aromatic residues around Cys579 that are necessary for proper DcpA folding and maintenance of the active conformation. On the basis of these observations and taking into account additional bioinformatics analysis of EAL domain-containing proteins, we identified a critical putatively conserved motif, GCXXXQGF, that plays an important role in c-di-GMP turnover. We conclude that a substrate-induced conformational switch involving movement of a loop containing a conserved motif in the bifunctional diguanylate cyclase-phosphodiesterase DcpA controls c-di-GMP turnover in M. smegmatis. [ABSTRACT FROM AUTHOR]

The author comments on the challenges being faced by college administrators in India on budget spending. He explains that the process for spending the budget for campus development or purchase of equipment must go through certain rules and controls. He calls on financial experts to modify the rule of annual submission of the utilization certificate which is necessary in claiming release of funds. He emphasizes that institutions are seemed to be in a hurry to spend their budget.

Abstract: ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the β′‐ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross‐linking experiments using 32P azido‐labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω. Furthermore, we followed the binding kinetics of ppGpp to RNAP with and without ω by isothermal titration calorimetry and found it to be concurrent with the cross‐linking results. Native ω is intrinsically disordered, and we have used a previously characterized structured mutant of ω, which affects the plasticity of the active site of RNAP. Results show that the flexibility conferred by the unstructured ω is a prerequisite for ppGpp binding to RNAP. We have analyzed the stress‐associated phenotypes in an E. coli strain devoid of ω (∆rpoZ). ppGpp levels in ∆rpoZ strain were found to be similar to that of the wild‐type strain. Interestingly, when the ∆rpoZ strain of E. coli was transferred after nutritional stress to an enriched media, the recovery of growth was compromised. We have identified a new phenotype of ∆rpoZ strain corresponding to defect in biofilm formation in minimal media. [ABSTRACT FROM AUTHOR]

Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using RelMSM enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to RelMSM by isothermal titration calorimetry. Enzyme kinetics was followed where K0.5 was found to be increased with the concomitant reduction of Vmax value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here. [ABSTRACT FROM AUTHOR]

The article presents information on the augmentation of teaching biology at the undergraduate level in Indian Institute of Technology (IIT) along with other core subjects and informs that due to immense challenge and potential, engineering graduates are opting for research in biology.

A response to a letter to the editor about a news item in "The Hindu" in the September 11, 2010 issue regarding the Indian Institute of Technology (IIT) is presented.

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. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra- and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response. [ABSTRACT FROM AUTHOR]

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (ΔrelMsm) or c-di-GMP synthesis (ΔdcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the ΔrelMsm and ΔdcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis. [ABSTRACT FROM AUTHOR]

Bacteria have evolved to survive the ever-changing environment using intriguing mechanisms of quorum sensing (QS). Very often, QS facilitates formation of biofilm to help bacteria to persist longer and the formation of such biofilms is regulated by c-di-GMP. It is a well-known second messenger also found in mycobacteria. Several methods have been developed to study c-di-GMP signaling pathways in a variety of bacteria. In this review, we have attempted to highlight a connection between c-di-GMP and biofilm formation and QS in mycobacteria and several methods that have helped in better understanding of c-di-GMP signaling. © 2014 IUBMB Life, 66(12):823-834, 2014 [ABSTRACT FROM AUTHOR]

Abstract: C-di-GMP [Bis-(3′-5′)-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the +1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are ~10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density. [Copyright &y& Elsevier]

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon--comprising the ori, RNAI, RNAII, and Rom--with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses the study which examines the role and function of an RNA polymerase interacting protein called MsRbpA which is derived from Mycobacterium smegmatis in phenotypic tolerance to rifampicin. It notes that rifampicin is leading the standard chemotherapeutic for active tuberculosis which inhibit the transcription activity of prokaryotic RNA polymerase. It implies that M. smegmatis interacts with RNA polymerase and increases the refampicin tolerance levels.

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. © 2009 IUBMB IUBMB Life, 62(1):67–77, 2010 [ABSTRACT FROM AUTHOR]

Abstract: A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps–DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis. [Copyright &y& Elsevier]

Abstract: Bacteria respond to starvation by synthesizing a polyphosphate derivative of guanosine, (p)ppGpp, that helps the bacteria in surviving during stress. The protein in Gram-positive organisms required for (p)ppGpp synthesis is Rel, a bifunctional enzyme that carries out both synthesis and hydrolysis of this molecule. Rel shows increased pppGpp synthesis in the presence of uncharged tRNA, the effect of which is regulated by the C-terminal of Rel. We show by fluorescence resonance energy transfer that the distance between the N-terminus cysteine residue at the catalytic domain and C692 at the C-terminus increases upon the addition of uncharged tRNA. In apparent anomaly, the steady state anisotropy of the Rel protein decreases upon tRNA binding suggesting “compact conformation” vis-à-vis “open conformation” of the free Rel. We propose that the interaction between C692 and the residues present in the pppGpp synthesis site results in the regulated activity and this interaction is abrogated upon addition of uncharged tRNA. We also report here the binding of pppGpp to the C-terminal part of the protein that leads to more unfolding in this region. [Copyright &y& Elsevier]

A deletion mutation in the gene rpoZ of Mycobacterium smegmatis causes reduced growth rate and a change in colony morphology. During purification of RNA polymerase from the mutant strain, the β′ subunit undergoes fragmentation but the fragments remain associated with the enzyme and maintain it in an active state until the whole destabilized assembly breaks down in the final step of purification. Complementation of the mutant strain with an integrated copy of the wild-type rpoZ brings back the wild-type colony morphology and improves the growth rate and activity of the enzyme, and the integrity of the β′ subunit remains unaffected. [ABSTRACT FROM AUTHOR]

Omega (ω), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of ω in which it helps to maintain β′ in a correct conformation and recruit it to the α2β subassembly to form a functional core enzyme (α2ββ′ω). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch onto the N-terminal and C-terminal ends of the β′-subunit. In the present study we have monitored the conformational changes in β′ as the denatured protein is refolded in the presence and absence of ω using tryptophan fluorescence emission of β′ as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of ω is helpful in β′ refolding. We have also monitored the behavior of the C-terminal tail of ω by engineering three cysteine residues at three different sites in ω and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of ω is mobile in the free protein and gets restrained in the presence of β′. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the β′ subunit whereas the end of the C-terminal of ω, although it is restrained in the presence of β′, has no interacting partner within a 4-A˚ radius. [Copyright &y& Elsevier]

Examines the synthesis of polar glycopeptidolipid in glucose-limited culture of Mycobacterium smegmatis. Analysis of hyperglycosylated species; Emphasis on the starvation physiology of Mycobacteria to provide insights on the long-term persistence of bacilli; Mechanism on the environmental regulation of cell wall biosynthesis.

The ω subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (α2ββ′). The presence of ω is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an ω-less strain of E. coli, GroEL appears to substitute for ω in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch on to β′ at its N-terminus and C-terminus. We show here that ω binds only the intact β′ subunit and not the β′ N-terminal domain or β′ C-terminal domain, implying that ω binding requires both these regions of β′. We further show that ω can prevent the aggregation of β′ during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of ω is sufficient for binding and renaturation of β′. CD and functional assays show that this N-terminal fragment retains the structure of native ω and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps α + α → α2 + β → α2β + β′ → α2ββ′. It is shown here that ω participates during the last stage of enzyme assembly when β′ associates with the α2β subassembly. [ABSTRACT FROM AUTHOR]

We have purified DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc. [ABSTRACT FROM AUTHOR]

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ' subassembly. We observed that the interaction between α2ββ' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β' subunit, and it is the same for both types of mutants. [ABSTRACT FROM AUTHOR]