scholarly journals Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426

2021 ◽  
Vol 12 ◽  
Author(s):  
Hirokazu Suzuki ◽  
Tatsunari Taketani ◽  
Misaki Tanabiki ◽  
Misaki Ohara ◽  
Jyumpei Kobayashi ◽  
...  
Keyword(s):  
Stress Responses ◽  
Sigma Factor ◽  
Elevated Temperatures ◽  
Thermophilic Bacterium ◽  
Insertion Sequences ◽  
Is Elements ◽  
Evolutionary Diversification ◽  
Growth Inhibitory ◽  
Geobacillus Kaustophilus ◽  
Mutant Genes

Geobacillus kaustophilus HTA426 is a thermophilic bacterium whose genome harbors numerous insertion sequences (IS). This study was initially conducted to generate mutant genes for thermostable T7 RNA polymerase in G. kaustophilus; however, relevant experiments unexpectedly identified that the organism transposed multiple IS elements and produced derivative cells that expressed a silent gene via transposition. The transposed elements were diverse and included members of the IS4, IS701, IS1634, and ISLre2 families. The transposition was relatively active at elevated temperatures and generated 4–9 bp of direct repeats at insertion sites. Transposition was more frequent in proliferative cells than in stationary cells but was comparable between both cells when sigX, which encodes an extra-cytoplasmic function sigma factor, was forcibly expressed. Southern blot analysis indicated that IS transposition occurred under growth inhibitory conditions by diverse stressors; however, IS transposition was not detected in cells that were cultured under growth non-inhibitory conditions. These observations suggest that G. kaustophilus enhances IS transposition via sigX-dependent stress responses when proliferative cells were prevented from active propagation. Considering Geobacillus spp. are highly adaptive bacteria that are remarkably distributed in diverse niches, it is possible that these organisms employ IS transposition for environmental adaptation via genetic diversification. Thus, this study provides new insights into adaptation strategies of Geobacillus spp. along with implications for strong codependence between mobile genetic elements and highly adaptive bacteria for stable persistence and evolutionary diversification, respectively. This is also the first report to reveal active IS elements at elevated temperatures in thermophiles and to suggest a sigma factor that governs IS transposition.

scholarly journals Characterizing a thermostable Cas9 for bacterial genome editing and silencing

2017 ◽  
Author(s):  
Ioannis Mougiakos ◽  
Prarthana Mohanraju ◽  
Elleke F. Bosma ◽  
Valentijn Vrouwe ◽  
Max Finger Bou ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Elevated Temperatures ◽  
Bacterial Genome ◽  
Thermophilic Bacterium ◽  
Geobacillus Thermodenitrificans ◽  
Temperature Range ◽  
Fundamental Research

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

scholarly journals Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degPMutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses

2001 ◽  
Vol 183 (18) ◽  
pp. 5230-5238 ◽  
Author(s):  
Geoffrey R. Langen ◽  
Jill R. Harper ◽  
Thomas J. Silhavy ◽  
S. Peter Howard
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Stress Responses ◽  
Elevated Temperatures ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Temperature Sensitive ◽  
Outer Membrane Phospholipase A ◽  
Temperature Sensitive Phenotype

ABSTRACT DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons ofEscherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded indegP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degPcells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype ofdegP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.

The ECF sigma factor σTis involved in osmotic and oxidative stress responses in Caulobacter crescentus

2007 ◽  
Vol 66 (5) ◽  
pp. 1240-1255 ◽  
Author(s):  
Cristina E. Alvarez-Martinez ◽  
Rogério F. Lourenço ◽  
Regina L. Baldini ◽  
Michael T. Laub ◽  
Suely L. Gomes
Keyword(s):  
Oxidative Stress ◽  
Stress Responses ◽  
Sigma Factor ◽  
Caulobacter Crescentus ◽  
Ecf Sigma Factor ◽  
Oxidative Stress Responses ◽  
And Oxidative Stress

scholarly journals The Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor SigL Regulates Polyketide Synthases and Secreted or Membrane Proteins and Is Required for Virulence

2005 ◽  
Vol 187 (20) ◽  
pp. 7062-7071 ◽  
Author(s):  
Mi-Young Hahn ◽  
Sahadevan Raman ◽  
Mauricio Anaya ◽  
Robert N. Husson
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stress Responses ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Infection Model ◽  
Transcription Start Sites ◽  
Promoter Sequences ◽  
Extracytoplasmic Function Sigma Factor

ABSTRACT Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the −35 region and CGTgtc in the −10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.

scholarly journals Engineering Deinococcus geothermalis for Bioremediation of High-Temperature Radioactive Waste Environments

2003 ◽  
Vol 69 (8) ◽  
pp. 4575-4582 ◽  
Author(s):  
Hassan Brim ◽  
Amudhan Venkateswaran ◽  
Heather M. Kostandarithes ◽  
James K. Fredrickson ◽  
Michael J. Daly
Keyword(s):  
High Temperature ◽  
Radioactive Waste ◽  
Elevated Temperatures ◽  
Deinococcus Radiodurans ◽  
Nitrilotriacetic Acid ◽  
Thermophilic Bacterium ◽  
Prospective Development ◽  
Deinococcus Geothermalis ◽  
Radiation Resistant

ABSTRACT Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55°C.

scholarly journals A Role for Bradyrhizobium japonicum ECF16 Sigma Factor EcfS in the Formation of a Functional Symbiosis with Soybean

2012 ◽  
Vol 25 (1) ◽  
pp. 119-128 ◽  
Author(s):  
S. B. Stockwell ◽  
L. Reutimann ◽  
M. L. Guerinot
Keyword(s):  
Stress Responses ◽  
Sigma Factor ◽  
Bradyrhizobium Japonicum ◽  
Critical Role ◽  
Negative Regulator ◽  
In Planta ◽  
Cellular Processes ◽  
Core Enzyme ◽  
Σ Factor ◽  
Target Promoters

Alternative sigma (σ) factors, proteins that recruit RNA polymerase core enzyme to target promoters, are one mechanism by which bacteria transcriptionally regulate groups of genes in response to environmental stimuli. A class of σ70 proteins, termed extracytoplasmic function (ECF) σ factors, are involved in cellular processes such as bacterial stress responses and virulence. Here, we describe an ECF16 σ factor, EcfS (Blr4928) from the gram-negative soil bacterium Bradyrhizobium japonicum USDA110, that plays a critical role in the establishment of a functional symbiosis with soybean. Nonpolar insertional mutants of ecfS form immature nodules that do not fix nitrogen, a defect that can be successfully complemented by expression of ecfS. Overexpression of the cocistronic gene, tmrS (blr4929), phenocopies the ecfS mutant in planta and, therefore, we propose that TmrS is a negative regulator of EcfS, a determination consistent with the prediction that it encodes an anti-σ factor. Microarray analysis of the ecfS mutant and tmrS overexpressor was used to identify 40 transcripts misregulated in both strains. These transcripts primarily encode proteins of unknown and transport-related functions and may provide insights into the symbiotic defect in these strains.

scholarly journals PsrA, the Pseudomonas Sigma Regulator, Controls Regulators of Epiphytic Fitness, Quorum-Sensing Signals, and Plant Interactions in Pseudomonas syringae pv. tomato Strain DC3000

2007 ◽  
Vol 73 (11) ◽  
pp. 3684-3694 ◽  
Author(s):  
Asita Chatterjee ◽  
Yaya Cui ◽  
Hiroaki Hasegawa ◽  
Arun K. Chatterjee
Keyword(s):  
Quorum Sensing ◽  
Pseudomonas Syringae ◽  
Stress Responses ◽  
Sigma Factor ◽  
Rna Binding ◽  
Negative Regulator ◽  
Insertion Mutant ◽  
Plant Interactions ◽  
Mobility Shift ◽  
Quorum Sensing Signals

ABSTRACT Pseudomonas syringae pv. tomato strain DC3000, a pathogen of tomato and Arabidopsis, occurs as an epiphyte. It produces N-acyl homoserine lactones (AHLs) which apparently function as quorum-sensing signals. A Tn5 insertion mutant of DC3000, designated PsrA− (Psr is for Pseudomonas sigma regulator), overexpresses psyR (a LuxR-type regulator of psyI) and psyI (the gene for AHL synthase), and it produces a ca. 8-fold-higher level of AHL than does DC3000. The mutant is impaired in its ability to elicit the hypersensitive reaction and is attenuated in its virulence in tomato. These phenotypes correlate with reduced expression of hrpL, the gene for an alternate sigma factor, as well as several hrp and hop genes during early stages of incubation in a Hrp-inducing medium. PsrA also positively controls rpoS, the gene for an alternate sigma factor known to control various stress responses. By contrast, PsrA negatively regulates rsmA1, an RNA-binding protein gene known to function as negative regulator, and aefR, a tetR-like gene known to control AHL production and epiphytic fitness in P. syringae pv. syringae. Gel mobility shift assays and other lines of evidence demonstrate a direct interaction of PsrA protein with rpoS promoter DNA and aefR operator DNA. In addition, PsrA negatively autoregulates and binds the psrA operator. In an AefR− mutant, the expression of psyR and psyI and AHL production are lower than those in DC3000, the AefR+ parent. In an RpoS− mutant, on the other hand, the levels of AHL and transcripts of psyR and psyI are much higher than those in the RpoS+ parent, DC3000. We present evidence, albeit indirect, that the RpoS effect occurs via psyR. Thus, AefR positively regulates AHL production, whereas RpoS has a strong negative effect. We show that AefR and RpoS do not regulate PsrA and that the PsrA effect on AHL production is exerted via its cumulative, but independent, effects on both AefR and RpoS.

scholarly journals A Caulobacter crescentus Extracytoplasmic Function Sigma Factor Mediating the Response to Oxidative Stress in Stationary Phase

2006 ◽  
Vol 188 (5) ◽  
pp. 1835-1846 ◽  
Author(s):  
Cristina E. Alvarez-Martinez ◽  
Regina L. Baldini ◽  
Suely L. Gomes
Keyword(s):  
Oxidative Stress ◽  
Stationary Phase ◽  
Stress Responses ◽  
Sigma Factor ◽  
Caulobacter Crescentus ◽  
Mrna Levels ◽  
Posttranscriptional Control ◽  
Hydrogen Peroxide Treatment ◽  
Extracytoplasmic Function Sigma Factor ◽  
Null Strain

ABSTRACT Alternative sigma factors of the extracytoplasmic function (ECF) subfamily are important regulators of stress responses in bacteria and have been implicated in the control of homeostasis of the extracytoplasmic compartment of the cell. This work describes the characterization of sigF, encoding 1 of the 13 members of this subfamily identified in Caulobacter crescentus. A sigF-null strain was obtained and shown to be severely impaired in resistance to oxidative stress, caused by hydrogen peroxide treatment, exclusively during the stationary phase. Although sigF mRNA levels decrease in stationary-phase cells, the amount of σF protein is greatly increased at this stage, indicating a posttranscriptional control. Data obtained indicate that the FtsH protease is either directly or indirectly involved in the control of σF levels, as cells lacking this enzyme present larger amounts of the sigma factor. Increased stability of σF protein in stationary-phase cells of the parental strain and in exponential-phase cells of the ftsH-null strain is also demonstrated. Transcriptome analysis of the sigF-null strain led to the identification of eight genes regulated by σF during the stationary phase, including sodA and msrA, which are known to be involved in oxidative stress response.

scholarly journals Identification and characterization of heat-responsive microRNAs at the booting stage in two rice varieties 9311 and Nagina 22

Genome ◽  
2021 ◽  
Author(s):  
Ying Luo ◽  
Tao Wang ◽  
Dan Yang ◽  
Biao Luo ◽  
Weiping Wang ◽  
...  
Keyword(s):  
Stress Responses ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Elevated Temperatures ◽  
Differentially Expressed ◽  
Control Group ◽  
Microrna Target ◽  
Rice Varieties ◽  
Booting Stage ◽  
Differentially Expressed Mirnas

Abstract: MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs that play important roles in abiotic stress responses in plants. but their regulatory roles in the adaptive response to heat stress at the booting stage in two rice varieties 9311 and Nagina 22, remain largely unknown. In this study, 464 known miRNAs and 123 potential novel miRNAs were identified. Of these miRNAs, a total of 90 differential expressed miRNAs were obtained with 9311 libraries as control group, of which 54 upregulated and 36 downregulated miRNAs. To gain insight into functional significance, 2773 potential target genes of these 90 differentially expressed miRNAs were predicted. GO enrichment showed that the predicted target genes of differentially expressed miRNAs including NACs, LACs, CSD, and Hsp40. KEGG pathway analysis showed that target genes of these differentially expressed miRNAs were significantly enriched in plant hormone signal transduction pathway. The expression levels of ten differentially expressed miRNAs and their target genes obtained by qRT-PCR were largely consistent with the sequencing results. This study lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in rice at elevated temperatures. Key words: rice, heat-responsive, microRNA, target gene, booting stage, high-throughput sequencing

scholarly journals The genome-wide transcriptional response to varying RpoS levels inEscherichia coliK-12

2016 ◽  
Author(s):  
Garrett T. Wong ◽  
Richard P. Bonocora ◽  
Alicia N. Schep ◽  
Suzannah M. Beeler ◽  
Anna J. Lee Fong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Stress Responses ◽  
Sigma Factor ◽  
Core Promoter ◽  
Transcriptional Response ◽  
Transcriptional Responses ◽  
Cellular Processes ◽  
Functional Classes ◽  
Sigma Factor Rpos

AbstractThe alternative sigma factor RpoS is a central regulator of a many stress responses inEscherichia coli.The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We show that 23% of genes in theE. coligenome are regulated by RpoS level, and we identify many RpoS-transcribed genes and promoters. We observe three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (“sensitive” genes), and genes whose expression changes very little with the production of a little RpoS (“insensitive”). We show that sequences outside the core promoter region determine whether a RpoS-regulated gene in sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes, and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase, and may also contribute to the diversity of stress responses directed by RpoS.ImportanceThe sigma factor RpoS is a global regulator that controls the response to many stresses inEscherichia coli.Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase, and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes in response to stresses.

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