Search PubMed⌕ Search

Biomedical subjects

Sangita Phadtare

Publications and source records attributed to Sangita Phadtare.

13 recordsLinked to original sources
PubMed2006

Analysis of Escherichia coli global gene expression profiles in response to overexpression and deletion of CspC and CspE.

The Escherichia coli cold shock protein CspA family consists of nine proteins (CspA to CspI), of which two, CspE and CspC, are constitutively produced at 37 degrees C and are involved in regulation of expression of genes encoding stress response proteins but can also perform an essential function during cold acclimation. In this study, we analyzed global transcript profiles of cells lacking cspE and cspC as well as cells individually overexpressing these proteins or a CspE mutant that is unable to melt nucleic acids and is defective in cold acclimation. The analysis reveals sets of genes whose expression (i) is regulated by CspC and CspE at physiological temperature or cold shock conditions and (ii) depends on the nucleic acid melting function of CspE. Bioinformatic analysis of the latter group reveals that many of those genes contain promoter-proximal sequences that can block transcript elongation and may be targeted by the nucleic acid melting function of CspE.

Escherichia coli↗
PubMed2005

Nucleic acid melting by Escherichia coli CspE.

Escherichia coli contains nine members of the CspA family. CspA and some of its homologues play critical role in cold acclimation of cells by acting as RNA chaperones, destabilizing nucleicacid secondary structures. Disruption of nucleic acid melting activity of CspE led to loss of its transcription antitermination activity and consequently its cold acclimation activity. To date, the melting activity of Csp proteins was studied using partially double-stranded model nucleic acids substrates forming stem-loop structures. Here, we studied the mechanism of nucleic acid melting by CspE. We show that CspE melts the stem region in two directions, that CspE-induced melting does not require the continuity of the substrate's loop region, and CspE can efficiently melt model substrates with single-stranded overhangs as short as 4 nt. We further show that preferential binding of CspE at the stem-loop junction site initiates melting; binding of additional CspE molecules that fully cover the single-stranded region of a melting substrate leads to complete melting of the stem.

Binding Sites↗
PubMed2005

Extended -10 motif is critical for activity of the cspA promoter but does not contribute to low-temperature transcription.

Bacterial promoters belonging to the extended -10 class contain a conserved TGn motif upstream of the -10 promoter consensus element. Open promoter complexes can be formed on some extended -10 Escherichia coli promoters at temperatures as low as 6 degrees C, when complexes on most promoters are closed. The promoter of cspA, a gene that codes for the major cold shock protein CspA of E. coli, contains an extended -10 motif. CspA is dramatically induced upon temperature downshift from 37 to 15 degrees C, and its cold shock induction has been attributed to transcription, translation, and mRNA stabilization effects. Here, we show that though the extended -10 motif is critical for high-level expression of cspA, it does not contribute to low-temperature expression. In fact, transcription from the wild-type cspA promoter is cold sensitive in vitro and in vivo. Thus, transcription appears to play little or no role in low-temperature induction of cspA expression.

Bacterial Proteins↗
PubMed2004

Cold-shock induced high-yield protein production in Escherichia coli.

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

Amino Acid Sequence↗
PubMed2004

Cold shock response and adaptation at near-freezing temperature in microorganisms.

Microorganisms that naturally encounter sharp temperature shifts must develop strategies for responding and adapting to these shifts. Escherichia coli, which are adapted to living at both warm temperatures inside animals and cooler ambient temperatures, respond to low temperatures (10 degrees to 15 degrees C) by adjusting membrane lipid composition and increasing the production of proteins that act as "RNA chaperones" required for transcription and translation and proteins that facilitate ribosomal assembly. In contrast, yeast, which are adapted to cooler temperatures, show a relatively minor cold shock response after temperature shifts from 30 degrees to 10 degrees C but respond with a dramatic increase in the synthesis of trehalose and a heat shock protein when exposed to freezing or near-freezing temperatures. This emphasizes the fact that different groups of microorganisms exhibit distinct types of cold shock responses.

Adaptation, Physiological↗
PubMed2004

The mechanism of nucleic acid melting by a CspA family protein.

Cold-shock proteins of the CspA family help bacterial cells to acclimate to low temperatures. Some Csps bind single-stranded nucleic acids and destabilize nucleic acid secondary structures in vitro, and act as transcription antiterminators in vivo and in vitro. Nucleic acid melting by Escherichia coli CspE is critical for its ability to support low-temperature survival of the cell. Here, we explore the molecular mechanism of nucleic acid melting using CspE mutants harboring substitutions in surface-exposed residues critical for this function. Analysis of the mutants identifies two intermediates of the melting pathway and shows that CspE Phe17 and Phe30 act at the earliest stages of melting, while His32 acts later and is necessary for the propagation of melting. The results allow us to orient a CspE molecule relative to the melting substrate and to put forward a mechanistic model of nucleic acid melting by Csps.

Cold Temperature↗
PubMed2004

Genome-wide transcriptional analysis of the cold shock response in wild-type and cold-sensitive, quadruple-csp-deletion strains of Escherichia coli.

A DNA microarray-based global transcript profiling of Escherichia coli in response to cold shock showed that in addition to the known cold shock-inducible genes, new genes such as the flagellar operon, those encoding proteins involved in sugar transport and metabolism, and remarkably, genes encoding certain heat shock proteins are induced by cold shock. In the light of strong reduction in metabolic activity of the cell after temperature downshift, the induction of sugar metabolism machinery is unexpected. The deletion of four csps (cspA, cspB, cspG, and cspE) affected cold shock induction of mostly those genes that are transiently induced in the acclimation phase, emphasizing that CspA homologues are essential in the acclimation phase. Relevance of these findings with respect to the known RNA chaperone function of CspA homologues is discussed.

Bacterial Proteins↗
PubMed2004

Recent developments in bacterial cold-shock response.

In response to temperature downshift, a number of changes occur in cellular physiology such as, (i) decrease in membrane fluidity, (ii) stabilization of secondary structures of nucleic acids leading to reduced efficiency of mRNA translation and transcription, (iii) inefficient folding of some proteins, and (iv) hampered ribosome function. Cold-shock response and adaptation has been quite extensively studied in Escherichia coli and Bacillus subtilis. A number of cold shock proteins are induced to counteract these harmful effects of temperature downshift. General principles of cold-shock response along with recent findings on desaturase system, RNA chaperone and transcription antitermination function of CspA homologues, cold shock induction of chaperones and synthesis of trehalose, CspA homologues from hyperthermophilic bacteria and possible multiple roles of cold shock proteins in other stress responses of bacteria are discussed.

Bacillus↗
PubMed2003

CspB and CspL, thermostable cold-shock proteins from Thermotoga maritima.

BACKGROUND: Cold-shock proteins (Csps) are important for cellular adaptation to low temperature. Csps help cells adapt to low-temperature growth through their RNA-binding and nucleic acid melting abilities, which lead to anti-termination of transcription. RESULTS: We studied the two most thermostable Csps known to date, TmCspB and TmCspL from Thermotoga maritima, a hyperthermophilic eubacterium for which no cold-shock response has been demonstrated so far. For comparison, we used a well-characterized Escherichia coli CspE protein. TmCspB and TmCspL are able to bind RNA at both low and high temperatures. They are also able to 'melt' nucleic acids secondary structures and as a result decrease E. coli RNA polymerase transcription termination in vivo and E. coli and T. maritima RNA polymerases transcription termination in vitro. Over-expression of TmCsps allowed E. coli cold-sensitive mutant cells to acclimate to the low temperatures of 15 degrees C. CONCLUSIONS: TmCspB and TmCspL (i) are able to perform essential functions of E. coli Csps in vitro and in vivo, 50-65 degrees C below the temperature optimum of T. maritima and (ii) can anti-terminate transcription by T. maritima RNA polymerase at 55 degrees C, the lower limit of temperature range for growth of T. maritima. We propose that the observed properties of TmCsps are physiologically relevant and that TmCsps are important for adaptation of T. maritima to physiologically low temperatures.

Adaptation, Physiological↗
PubMed2002

Three amino acids in Escherichia coli CspE surface-exposed aromatic patch are critical for nucleic acid melting activity leading to transcription antitermination and cold acclimation of cells.

Cold-shock proteins of the CspA family of Escherichia coli help the cells to acclimate to low temperature conditions through an unknown mechanism. In vitro, these proteins bind to single-stranded nucleic acids and destabilize nucleic acid secondary structures. An unusual surface-exposed patch of 6 evolutionarily conserved aromatic amino acids is thought to be involved in RNA binding by the cold-shock proteins. Here we investigated the functional role of the aromatic patch in E. coli CspE by substituting individual aromatic residues with positively charged Arg residues. These substitutions do not affect the RNA binding activity of the CspE mutants. We show that substitutions of three centrally located aromatic patch amino acid residues, Phe(17), Phe(30), and His(32), abolish the ability of the mutant CspE to acclimatize cells to cold, antiterminate transcription and melt nucleic acids but have no effect on RNA binding. On the other hand, peripherally located Trp(10), Phe(19), and Phe(33) can be substituted with Arg without loss of any of the in vivo and in vitro CspE functions tested. The results thus indicate that these aromatic patch residues have clearly distinct functional roles and further extend the correlation between the essential function of CspA homologues in cold acclimation and their ability to antiterminate transcription.

Adaptation, Physiological↗
PubMed2002

DNA microarray analysis of the expression profile of Escherichia coli in response to treatment with 4,5-dihydroxy-2-cyclopenten-1-one.

We carried out DNA microarray-based global transcript profiling of Escherichia coli in response to 4,5-dihydroxy-2-cyclopenten-1-one to explore the manifestation of its antibacterial activity. We show that it has widespread effects in E. coli affecting genes encoding proteins involved in cell metabolism and membrane synthesis and functions. Genes belonging to the regulon involved in synthesis of Cys are upregulated. In addition, rpoS and RpoS-regulated genes responding to various stresses and a number of genes responding to oxidative stress are upregulated.

Cyclopentanes↗
PubMed2001

The nucleic acid melting activity of Escherichia coli CspE is critical for transcription antitermination and cold acclimation of cells.

Members of bacterial Csp (cold-shock protein) family promote cellular adaptation to low temperature and participate in many other aspects of gene expression regulation through mechanisms that are not yet fully elucidated. Csp proteins interact with single-stranded nucleic acids and destabilize nucleic acid secondary structures. Some Csp proteins also act as transcription antiterminators in vivo and in vitro. Here, we selected a mutation in the cloned cspE gene that abolished CspE-induced transcription antitermination. In vitro, mutant CspE showed RNA binding activity similar to that of the wild-type CspE but was unable to destabilize nucleic acid secondary structures. Thus, nucleic acid melting ability of CspE and its transcription antitermination activity are correlated. In vivo, mutant cspE was functional with respect to up-regulation of expression of rpoS, but, unlike the wild-type cspE, it did not complement the cold-sensitive phenotype of the quadruple DeltacspADeltacspBDeltacspGDeltacspE deletion strain. Thus, the nucleic acid-melting activity of Csp is critical for its prototypical function of supporting low temperature survival of the cell.

Arginine↗