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1.
FEBS J ; 287(9): 1865-1885, 2020 05.
Article in English | MEDLINE | ID: mdl-31679177

ABSTRACT

Rapid adaptation to environmental changes is crucial for bacterial survival. Almost all bacteria possess a conserved stringent response system to prompt transcriptional and metabolic responses toward stress. The adaptive process relies on alarmones, guanosine pentaphosphate (pppGpp), and tetraphosphate (ppGpp), to regulate global gene expression. The ppGpp is more potent than pppGpp in the regulatory activity, and pppGpp phosphohydrolase (GppA) plays a key role in (p)ppGpp homeostasis. Sharing a similar domain structure, GppA is indistinguishable from exopolyphosphatase (PPX), which mediates the metabolism of cellular inorganic polyphosphate. Here, our phylogenetic analysis of PPX/GppA homologs in bacteria shows a wide distribution with several distinct subfamilies, and our structural and functional analysis of Escherichia coli GppA and Helicobacter pylori PPX/GppA reveals unique properties of each homolog. These results explain how each homolog possesses its distinct functionality.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , Guanosine Pentaphosphate/metabolism , Helicobacter pylori/enzymology , Helicobacter pylori/metabolism , Amino Acid Sequence , Guanosine Pentaphosphate/chemistry , Models, Molecular , Molecular Structure , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Sequence Alignment
2.
Sci Rep ; 7(1): 8500, 2017 08 17.
Article in English | MEDLINE | ID: mdl-28819106

ABSTRACT

Chloride intracellular channels (CLIC) are non-classical ion channels lacking a signal sequence for membrane targeting. In eukaryotes, they are implicated in cell volume regulation, acidification, and cell cycle. CLICs resemble the omega class of Glutathione S-transferases (GST), yet differ from them in their ability to form ion channels. They are ubiquitously found in eukaryotes but no prokaryotic homolog has been characterized. We found that indanyloxyacetic acid-94 (IAA-94), a blocker of CLICs, delays the growth of Escherichia coli. In silico analysis showed that the E. coli stringent starvation protein A (SspA) shares sequence and structural homology with CLICs. Similar to CLICs, SspA lacks a signal sequence but contains an omega GST fold. Electrophysiological analysis revealed that SspA auto-inserts into lipid bilayers and forms IAA-94-sensitive ion channels. Substituting the ubiquitously conserved residue leucine 29 to alanine in the pore-forming region increased its single-channel conductance. SspA is essential for cell survival during acid-induced stress, and we found that acidic pH increases the open probability of SspA. Further, IAA-94 delayed the growth of wild-type but not sspA null mutant E. coli. Our results for the first time show that CLIC-like proteins exist in bacteria in the form of SspA, forming functional ion channels.


Subject(s)
Chloride Channels/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Amino Acid Substitution , Chloride Channels/genetics , Chlorides/metabolism , DNA Mutational Analysis , Enzyme Inhibitors/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Genes, Essential , Glycolates/metabolism , Hydrogen-Ion Concentration , Microbial Viability
3.
Genetics ; 206(1): 179-187, 2017 05.
Article in English | MEDLINE | ID: mdl-28341651

ABSTRACT

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.


Subject(s)
Escherichia coli Proteins/genetics , Genes, Reporter/genetics , Transcription Factors/genetics , Transcription, Genetic , Transcriptional Elongation Factors/genetics , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Peptide Elongation Factors , Promoter Regions, Genetic , RNA/biosynthesis , RNA/genetics
4.
Front Microbiol ; 6: 497, 2015.
Article in English | MEDLINE | ID: mdl-26052320

ABSTRACT

Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

5.
Nucleic Acids Res ; 42(9): 5823-9, 2014 May.
Article in English | MEDLINE | ID: mdl-24711367

ABSTRACT

Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5' end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination.


Subject(s)
Bacteriophage lambda , DNA-Directed RNA Polymerases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Viral Regulatory and Accessory Proteins/chemistry , Base Sequence , Escherichia coli/virology , Genes, Reporter , Transcription, Genetic , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
6.
Nucleic Acids Res ; 41(12): 6058-71, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23632166

ABSTRACT

To fit within the confines of the cell, bacterial chromosomes are highly condensed into a structure called the nucleoid. Despite the high degree of compaction in the nucleoid, the genome remains accessible to essential biological processes, such as replication and transcription. Here, we present the first high-resolution chromosome conformation capture-based molecular analysis of the spatial organization of the Escherichia coli nucleoid during rapid growth in rich medium and following an induced amino acid starvation that promotes the stringent response. Our analyses identify the presence of origin and terminus domains in exponentially growing cells. Moreover, we observe an increased number of interactions within the origin domain and significant clustering of SeqA-binding sequences, suggesting a role for SeqA in clustering of newly replicated chromosomes. By contrast, 'histone-like' protein (i.e. Fis, IHF and H-NS) -binding sites did not cluster, and their role in global nucleoid organization does not manifest through the mediation of chromosomal contacts. Finally, genes that were downregulated after induction of the stringent response were spatially clustered, indicating that transcription in E. coli occurs at transcription foci.


Subject(s)
Chromosomes, Bacterial/chemistry , DNA Replication , Escherichia coli/genetics , Transcription, Genetic , Bacterial Outer Membrane Proteins/metabolism , Binding Sites , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Genome, Bacterial , Replication Origin , Serine/analogs & derivatives , Serine/pharmacology
7.
J Bacteriol ; 189(14): 4984-93, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17513476

ABSTRACT

The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a DeltarhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.


Subject(s)
DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Transcriptional Activation , AraC Transcription Factor/genetics , AraC Transcription Factor/metabolism , Base Sequence , Binding Sites/genetics , Blotting, Western , Cyclic AMP Receptor Protein/genetics , Cyclic AMP Receptor Protein/metabolism , DNA Footprinting/methods , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Molecular Sequence Data , Plasmids/genetics , Protein Binding , Regulon/genetics , Rhamnose/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism
8.
J Bacteriol ; 188(11): 4007-14, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16707692

ABSTRACT

In contrast to eukaryotes, bacteria such as Escherichia coli contain only one form of RNA polymerase (RNAP), which is responsible for all cellular transcription. Using an RNAP-green fluorescent protein fusion protein, we showed previously that E. coli RNAP is partitioned exclusively in the nucleoid and that stable RNA synthesis, particularly rRNA transcription, is critical for concentrating a significant fraction of RNAP in transcription foci during exponential growth. The extent of focus formation varies under different physiological conditions, supporting the proposition that RNAP redistribution is an important element for global gene regulation. Here we show that extra, plasmid-borne copies of an rRNA operon recruit RNAP from the nucleoid into the cytoplasmic space and that this is accompanied by a reduction in the growth rate. Transcription of an intact rRNA operon is not necessary, although a minimal transcript length is required for this phenotype. Replacement of the ribosomal promoters with another strong promoter, Ptac, abolished the effect. These results demonstrate that active synthesis from rRNA promoters is a major driving force for the distribution of RNAP in bacteria. The implications of our results for the regulation of rRNA synthesis and cell growth are discussed.


Subject(s)
Bacteria/genetics , DNA-Directed RNA Polymerases/metabolism , RNA, Ribosomal/genetics , Transcription, Genetic , rRNA Operon/genetics , Bacteria/enzymology , Base Sequence , Chromosomes, Bacterial/genetics , Genes, Reporter , Green Fluorescent Proteins/genetics , Molecular Sequence Data , Plasmids , Recombinant Fusion Proteins/metabolism
9.
J Biol Chem ; 280(16): 15921-7, 2005 Apr 22.
Article in English | MEDLINE | ID: mdl-15705577

ABSTRACT

By exploring global gene expression of Escherichia coli growing on six different carbon sources, we discovered a striking genome transcription pattern: as carbon substrate quality declines, cells systematically increase the number of genes expressed. Gene induction occurs in a hierarchical manner and includes many factors for uptake and metabolism of better but currently unavailable carbon sources. Concomitantly, cells also increase their motility. Thus, as the growth potential of the environment decreases, cells appear to devote progressively more energy on the mere possibility of improving conditions. This adaptation is not what would be predicated by classic regulatory models alone. We also observe an inverse correlation between gene activation and rRNA synthesis suggesting that reapportioning RNA polymerase (RNAP) contributes to the expanded genome activation. Significant differences in RNAP distribution in vivo, monitored using an RNAP-green fluorescent protein fusion, from energy-rich and energy-poor carbon source cultures support this hypothesis. Together, these findings represent the integration of both substrate-specific and global regulatory systems, and may be a bacterial approximation to metazoan risk-prone foraging behavior.


Subject(s)
Carbon/metabolism , Escherichia coli/metabolism , Transcription, Genetic , Acetic Acid/metabolism , Alanine/metabolism , Computer Simulation , Escherichia coli/genetics , Gene Expression Profiling , Glucose/metabolism , Glycerol/metabolism , Models, Biological , Proline/metabolism , Promoter Regions, Genetic , RNA, Ribosomal/metabolism , Succinic Acid/metabolism , Up-Regulation
10.
Mol Microbiol ; 50(5): 1493-505, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14651633

ABSTRACT

Despite extensive genetic, biochemical and structural studies on Escherichia coli RNA polymerase (RNAP), little is known about its location and distribution in response to environmental changes. To visualize the RNAP by fluorescence microscopy in E. coli under different physiological conditions, we constructed a functional rpoC-gfp gene fusion on the chromosome. We show that, although RNAP is located in the nucleoid and at its periphery, the distribution of RNAP is dynamic and dramatically influenced by cell growth conditions, nutrient starvation and overall transcription activity inside the cell. Moreover, mutational analysis suggests that the stable RNA synthesis plays an important role in nucleoid condensation.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Escherichia coli/growth & development , Gene Expression Regulation, Bacterial , Heat-Shock Response , Cell Nucleus/metabolism , Culture Media , DNA-Directed RNA Polymerases/genetics , Escherichia coli/physiology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
11.
Biochem Biophys Res Commun ; 290(4): 1183-7, 2002 Feb 01.
Article in English | MEDLINE | ID: mdl-11811987

ABSTRACT

It is well known that the GC-rich discriminator region between the -10 region and the transcription start site is important for the stringent control of the transcription. However, the discriminator activity is influenced by flanking regions, in particular in conjunction with the promoter -35 and -10 sequences. In this study, we changed the sequence in the -35 region of the rnpB P-1 promoter to see how such changes affect the stringent control. The sequence variation in the -35 region changed the stringent signal. The change to the consensus TTGACA sequence caused the most prominent relieving effect on stringent repression of the rnpB transcription. The spacing between the -35 and -10 regions is also significant because the relieving effect of the TTGACA was offset by the change of the spacing from 17 to 16 bp. The nucleotide just upstream of the -35 region contributes toward generating stringent signals as well.


Subject(s)
Endoribonucleases/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genes, Bacterial , Mutation , RNA, Catalytic/genetics , Base Sequence , DNA, Bacterial/genetics , Escherichia coli/enzymology , Genetic Variation , Molecular Sequence Data , Plasmids/genetics , Promoter Regions, Genetic , Ribonuclease P
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