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• 1
Article
Language: English
In: Science (New York, N.Y.), 17 May 2013, Vol.340(6134), pp.837-41
Description: In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σ(E) stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σ(E) transcription factor, thereby modulating the expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.
Keywords: Signal Transduction ; Stress, Physiological ; Bacterial Outer Membrane Proteins -- Metabolism ; Cell Membrane -- Metabolism ; Escherichia Coli -- Metabolism ; Lipopolysaccharides -- Metabolism
ISSN: 00368075
E-ISSN: 1095-9203
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• 2
Article
National Academy of Sciences
Language: English
In: Proceedings of the National Academy of Sciences of the United States of America, 2011, Vol.108(31), pp.12875-12880
Description: The Escherichia coli ÏE envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The ÏE stress response was characterized as a single-tier activation network comprised of ; p. 12875-12880.
Keywords: Regulon ; Porins ; Messenger Rna ; Homeostasis ; Non-Coding Rna ; Stress Response ; Labor ; Viability ; Mica ; Gram-Negative Bacteria ; Escherichia Coli ; Gene Activation ; Transactivators
ISSN: 0027-8424
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• 3
Article
Language: English
In: Proceedings of the National Academy of Sciences of the United States of America, 01 February 2011, Vol.108(5), pp.2106-11
Description: In Escherichia coli, the σ(E) transcription factor monitors and maintains outer membrane (OM) integrity by activating genes required for assembly of its two key components, outer membrane proteins (OMPs) and lipopolysaccharide (LPS) and by transcribing small RNAs to down-regulate excess unassembled OMPs. σ(E) activity is governed by the rate of degradation of its membrane-spanning anti-σ factor, RseA. Importantly, the DegS protease can initiate RseA cleavage only when activated by binding to unassembled OMPs. The prevalent paradigm has been that the σ(E) response is controlled by the amount of activated DegS. Here we demonstrate that inactivation of a second negative regulator, the periplasmic protein RseB, is also required for σ(E) induction in vivo. Moreover, OMPs, previously known only to activate DegS, also generate a signal to antagonize RseB inhibition. This signal may be lipid related, as RseB is structurally similar to proteins that bind lipids. We propose that the use of an AND gate enables σ(E) to sense and integrate multivariate signals from the envelope.
Keywords: Signal Transduction ; Escherichia Coli -- Physiology ; Escherichia Coli Proteins -- Physiology ; Membrane Proteins -- Physiology
ISSN: 00278424
E-ISSN: 1091-6490
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• 4
Article
Language: English
In: Journal of the American Chemical Society, 31 October 2012, Vol.134(43), pp.18074-81
Description: The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis.
Keywords: Escherichia Coli Proteins -- Genetics ; Methyltransferases -- Genetics ; S-Adenosylmethionine -- Metabolism
ISSN: 00027863
E-ISSN: 1520-5126
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• 5
Article
Language: English
In: Proceedings of the National Academy of Sciences of the United States of America, 19 May 2015, Vol.112(20), pp.6437-42
Description: Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.
Keywords: Murj ; Flippase ; Lipid II ; Peptidoglycan ; Sigm ; Bacillus Subtilis -- Enzymology ; Bacterial Proteins -- Metabolism ; Cell Wall -- Physiology ; Gene Expression Regulation, Bacterial -- Physiology ; Morphogenesis -- Physiology ; Phospholipid Transfer Proteins -- Metabolism
ISSN: 00278424
E-ISSN: 1091-6490
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• 6
Article
Language: English
In: Proceedings of the National Academy of Sciences of the United States of America, 02 August 2011, Vol.108(31), pp.12875-80
Description: The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function.
Keywords: Escherichia Coli Proteins -- Genetics ; RNA, Small Untranslated -- Genetics ; Regulon -- Genetics ; Sigma Factor -- Genetics
ISSN: 00278424
E-ISSN: 1091-6490
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• 7
Article
Language: English
In: Current Biology, 19 May 2014, Vol.24(10), pp.R424-R434
Description: Microorganisms live in fluctuating environments, requiring stress response pathways to resist environmental insults and stress. These pathways dynamically monitor cellular status, and mediate adaptive changes by remodeling the proteome, largely accomplished by remodeling transcriptional networks and protein degradation. The complementarity of fast, specific proteolytic degradation and slower, broad transcriptomic changes gives cells the mechanistic repertoire to dynamically adjust cellular processes and optimize response behavior. Together, this enables cells to minimize the ‘cost’ of the response while maximizing the ability to survive environmental stress. Here we highlight recent progress in our understanding of transcriptional networks and proteolysis that illustrates the design principles used by bacteria to generate the complex behaviors required to resist stress. Microorganisms live in fluctuating environments, requiring stress response pathways to resist environmental insults and stress. Guo and Gross highlight recent progress in our understanding of transcriptional networks and proteolysis that illustrates the design principles used by bacteria to generate the complex behaviors required to resist stress.
Keywords: Biology
ISSN: 0960-9822
E-ISSN: 1879-0445
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• 8
Article
Language: English
In: Proceedings of the National Academy of Sciences of the United States of America, 1 February 2010, Vol.107(7), pp.2854-2859
Description: Sequenced bacterial genomes provide a wealth of information but little understanding of transcriptional regulatory circuits largely because accurate prediction of promoters is difficult. We examined two important issues for accurate promoter prediction: (1) the ability to predict promoter strength and (2) the sequence properties that distinguish between active and weak/inactive promoters. We addressed promoter prediction using natural core promoters recognized by the well-studied alternative sigma factor, Escherichia coli $\sigma ^E$ , as a representative of group 4 σs, the largest σ group. To evaluate the contribution of sequence to promoter strength and function, we used modular position weight matrix models comprised of each promoter motif and a penalty score for suboptimal motif location. We find that a combination of select modules is moderately predictive of promoter strength and that imposing minimal motif scores distinguished active from weak/inactive promoters. The combined -35/-10 score is the most important predictor of activity. Our models also identified key sequence features associated with active promoters. A conserved "AAC" motif in the -35 region is likely to be a general predictor of function for promoters recognized by group 4 σs. These results provide valuable insights into sequences that govern promoter strength, distinguish active and inactive promoters for the first time, and are applicable to both in vivo and in vitro measures of promoter strength.
Keywords: Biological sciences -- Biology -- Microbiology ; Biological sciences -- Biology -- Genetics ; Applied sciences -- Research methods -- Modeling ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Applied sciences -- Research methods -- Modeling ; Physical sciences -- Chemistry -- Chemical compounds ; Economics -- Economic disciplines -- Consumer economics ; Biological sciences -- Biochemistry -- Enzymology ; Physical sciences -- Physics -- Energy ; Biological sciences -- Biology -- Microbiology ; Biological sciences -- Biology -- Genetics ; Applied sciences -- Research methods -- Modeling ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Applied sciences -- Research methods -- Modeling ; Physical sciences -- Chemistry -- Chemical compounds ; Economics -- Economic disciplines -- Consumer economics ; Biological sciences -- Biochemistry -- Enzymology ; Physical sciences -- Physics -- Energy
ISSN: 00278424
E-ISSN: 10916490
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• 9
Article
Language: English
In: Issues in Teacher Education, 2011, Vol.20(1), p.85
Description: When the author adopted her son, she was an early childhood educator with a Master's Degree who had taught young children for eight years and directed a child care and infant-toddler center for seven. She thought she knew something about children. Actually, she had a great deal to learn. It took years for her to learn about his needs. He slowly teaches her, but it took time to understand what his behavior was telling her, how to listen to the underlying messages and filter out her own wishes. Perhaps even more than these typical parenting questions, it took time for her to recognize the signs of a learning disability. In this article, the author offers the story of this journey from the perspective of a parent who is also a teacher, in the hope of evoking in other teachers new, perhaps deeper, understanding for the child and the family who are dealing with learning disabilities.
Keywords: Learning Disabilities ; Child Rearing ; Young Children ; Student Needs ; Student Behavior ; Symptoms (Individual Disorders) ; Disability Identification ; Teachers ; Personal Narratives ; Education
ISSN: 1536-3031
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• 10
Article
Language: English
In: Proceedings of the National Academy of Sciences of the United States of America, 15 December 2015, Vol.112(50), pp.E6862-71
Description: Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3',5'-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo-cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.
Keywords: Lineage-Specific Insertions ; Molecular Modeling ; Protein Cross-Linking ; Stringent Response ; Transcription Regulation ; DNA-Directed RNA Polymerases -- Metabolism ; Escherichia Coli -- Enzymology ; Escherichia Coli Proteins -- Physiology
ISSN: 00278424
E-ISSN: 1091-6490
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