Evidence for a Role of rpoE in Stressed and Unstressed Cells of Marine Vibrio angustumStrain S14

ABSTRACT We report the cloning, sequencing, and characterization of therpoE homolog in Vibrio angustum S14. TherpoE gene encodes a protein with a predicted molecular mass of 19.4 kDa and has been demonstrated to be present as a single-copy gene by Southern blot analysis. The deduced amino acid sequence of RpoE is most similar to that of the RpoE homolog of Sphingomonas aromaticivorans, ς24, displaying sequence similarity and identity of 63 and 43%, respectively. Northern blot analysis demonstrated the induction of rpoE 6, 12, and 40 min after a temperature shift to 40°C. An rpoE mutant was constructed by gene disruption. There was no difference in viability during logarithmic growth, stationary phase, or carbon starvation between the wild type and the rpoE mutant strain. In contrast, survival of the mutant was impaired following heat shock during exponential growth, as well as after oxidative stress at 24 h of carbon starvation. The mutant exhibited microcolony formation during optimal growth temperatures (22 to 30°C), and cell area measurements revealed an increase in cell volume of the mutant during growth at 30°C, compared to the wild-type strain. Moreover, outer membrane and periplasmic space protein analysis demonstrated many alterations in the protein profiles for the mutant during growth and carbon starvation, as well as following oxidative stress, in comparison with the wild-type strain. It is thereby concluded that RpoE has an extracytoplasmic function and mediates a range of specific responses in stressed as well as unstressed cells of V. angustum S14.

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Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

Applied and Environmental Microbiology ◽

10.1128/aem.02838-19 ◽

2020 ◽

Vol 86 (7) ◽

Author(s):

Rui Yao ◽

Pei Zhou ◽

Chengjin Wu ◽

Liming Liu ◽

Jing Wu

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Survival Rate ◽

Type Strain ◽

Mutation Frequency ◽

Wild Type Strain ◽

Yeast Cells ◽

Wild Type ◽

Content Type

ABSTRACT In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

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The Spirochete FlaA Periplasmic Flagellar Sheath Protein Impacts Flagellar Helicity

Journal of Bacteriology ◽

10.1128/jb.182.23.6698-6706.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6698-6706 ◽

Cited By ~ 49

Author(s):

Chunhao Li ◽

Linda Corum ◽

David Morgan ◽

Everett L. Rosey ◽

Thaddeus B. Stanton ◽

...

Keyword(s):

Blot Analysis ◽

Type Strain ◽

Wild Type Strain ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Dark Field ◽

Wild Type ◽

Brachyspira Hyodysenteriae ◽

Flagellar Filament ◽

Cognate Gene

ABSTRACT Spirochete periplasmic flagella (PFs), including those fromBrachyspira (Serpulina),Spirochaeta, Treponema, andLeptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructedflaA::cat,flaA::kan, andflaB1::kan mutants and newly constructed flaB2::cat andflaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found withflaA::cat,flaA::kan, andflaB1::kan mutants,flaB2::cat andflaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan,flaB2::cat, andflaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found thatflaA::cat andflaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.

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Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

Journal of Bacteriology ◽

10.1128/jb.182.23.6630-6637.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6630-6637 ◽

Cited By ~ 13

Author(s):

Chin Li ◽

Yi Ping Tao ◽

Lee D. Simon

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Type Strain ◽

Wild Type Strain ◽

Transcription Termination ◽

Carbon Starvation ◽

Lower Percentage ◽

Wild Type ◽

Lower Efficiency ◽

E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

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Contribution of YjbIH to Virulence Factor Expression and Host Colonization inStaphylococcus aureus

Infection and Immunity ◽

10.1128/iai.00155-19 ◽

2019 ◽

Vol 87 (6) ◽

Cited By ~ 2

Author(s):

Crystal M. Austin ◽

Siamak Garabaglu ◽

Christina N. Krute ◽

Miranda J. Ridder ◽

Nichole A. Seawell ◽

...

Keyword(s):

Oxidative Stress ◽

Virulence Factor ◽

Type Strain ◽

Wild Type Strain ◽

Acute Infection ◽

Minor Role ◽

Wild Type ◽

Truncated Hemoglobin ◽

Factor Expression ◽

And Oxidative Stress

ABSTRACTTo persist within the host and cause disease,Staphylococcus aureusrelies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon,yjbIH, resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observedyjbIHmutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression ofcrtOPQMNandaur. Previous studies found that YjbH targets the disulfide- and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence ofyjbHoryjbIresulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in theyjbHandyjbImutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in theyjbHmutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of theyjbIHdeletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.

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Global Analysis of Cellular Factors and Responses Involved in Pseudomonas aeruginosa Resistance to Arsenite

Journal of Bacteriology ◽

10.1128/jb.187.14.4853-4864.2005 ◽

2005 ◽

Vol 187 (14) ◽

pp. 4853-4864 ◽

Cited By ~ 43

Author(s):

Kislay Parvatiyar ◽

Eyad M. Alsabbagh ◽

Urs A. Ochsner ◽

Michelle A. Stegemeyer ◽

Alan G. Smulian ◽

...

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Glutathione Reductase ◽

Type Strain ◽

Wild Type Strain ◽

Storage Proteins ◽

Global Analysis ◽

Wild Type ◽

Isogenic Mutants ◽

The Impact

ABSTRACT The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, crc, and gor mutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization-time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RplY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.

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Functional Analysis of preA in Aeromonas veronii TH0426 Reveals a Key Role in the Regulation of Virulence and Resistance to Oxidative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms21010098 ◽

2019 ◽

Vol 21 (1) ◽

pp. 98

Author(s):

Bintong Yang ◽

Haichao Song ◽

Dingjie An ◽

Dongxing Zhang ◽

Sayed Haidar Abbas Raza ◽

...

Keyword(s):

Oxidative Stress ◽

Type Strain ◽

Wild Type Strain ◽

Lethal Dose ◽

Tolerance Test ◽

Economic Losses ◽

Cytotoxicity Test ◽

Test Results ◽

Wild Type ◽

Aeromonas Veronii

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. This bacterium has caused serious economic losses in the aquaculture industry worldwide, and it has become an important zoonotic and aquatic agent. However, little is known about the molecular mechanism of pathogenesis of A. veronii. In this study, we first constructed an unmarked mutant strain (ΔpreA) by generating an in-frame deletion of the preA gene, which encodes a periplasmic binding protein, to investigate its role in A. veronii TH0426. Our results showed that the motility and biofilm formation ability of ΔpreA were similar to those of the wild-type strain. However, the adhesion and invasion ability in epithelioma papulosum cyprini (EPC) cells were significantly enhanced (2.0-fold). Furthermore, the median lethal dose (LD50) of ΔpreA was 7.6-fold higher than that of the wild-type strain, which illustrates that the virulence of the mutant was significantly enhanced. This finding is also supported by the cytotoxicity test results, which showed that the toxicity of ΔpreA to EPC cells was enhanced 1.3-fold relative to the wild type. Conversely, tolerance test results showed that oxidative stress resistance of ΔpreA decreased 5.9-fold compared to with the wild-type strain. The results suggest that preA may negatively regulate the virulence of A. veronii TH0426 through the regulation of resistance to oxidative stress. These insights will help to further elucidate the function of preA and understand the pathogenesis of A. veronii.

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Crp-Like Protein Plays Both Positive and Negative Roles in Regulating the Pathogenicity of Bacterial Pustule Pathogen Xanthomonas axonopodis pv. glycines

Phytopathology ◽

10.1094/phyto-07-18-0225-r ◽

2019 ◽

Vol 109 (7) ◽

pp. 1171-1183 ◽

Cited By ~ 3

Author(s):

Wei Guo ◽

Jie Gao ◽

Qingshan Chen ◽

Bojun Ma ◽

Yuan Fang ◽

...

Keyword(s):

Cell Motility ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Extracellular Polysaccharide ◽

Polymerase Chain Reaction Analysis ◽

Single Copy ◽

Wild Type ◽

Cell Wall Degrading Enzymes ◽

In The Wild

The global regulator Crp-like protein (Clp) is positively involved in the production of virulence factors in some of the Xanthomonas spp. However, the functional importance of Clp in X. axonopodis pv. glycines has not been investigated previously. Here, we showed that deletion of clp led to significant reduction in the virulence of X. axonopodis pv. glycines in soybean, which was highly correlated with the drastic reductions in carbohydrates utilization, extracellular polysaccharide (EPS) production, biofilm formation, cell motility, and synthesis of cell wall degrading enzymes (CWDEs). These significantly impaired properties in the clp mutant were completely rescued by a single-copy integration of the wild-type clp into the mutant chromosome via homologous recombination. Interestingly, overexpression of clp in the wild-type strain resulted in significant increases in cell motility and synthesis of the CWDEs. To our surprise, significant reductions in carbohydrates utilization, EPS production, biofilm formation, and the protease activity were observed in the wild-type strain overexpressing clp, suggesting that Clp also plays a negative role in these properties. Furthermore, quantitative reverse transcription polymerase chain reaction analysis suggested that clp was positively regulated by the diffusible signal factor-mediated quorum-sensing system and the HrpG/HrpX cascade. Taken together, our results reveal that Clp functions as both activator and repressor in multiple biological processes in X. axonopodis pv. glycines that are essential for its full virulence.

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The chvH Locus ofAgrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis

Journal of Bacteriology ◽

10.1128/jb.183.1.36-45.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 36-45 ◽

Cited By ~ 32

Author(s):

Wen-Tao Peng ◽

Lois M. Banta ◽

Trevor C. Charles ◽

Eugene W. Nester

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Elongation Factor ◽

Peptide Bond ◽

Single Copy ◽

Tumor Formation ◽

Wild Type ◽

Chromosomal Locus ◽

Gene Encoding ◽

Elongation Factor P

ABSTRACT The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis inEscherichia coli. The analysis of vir-lacZ andvir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH andefp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed achvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.

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Transcriptional and Proteomic Analysis of a Ferric Uptake Regulator (Fur) Mutant of Shewanella oneidensis: Possible Involvement of Fur in Energy Metabolism, Transcriptional Regulation, and Oxidative Stress

Applied and Environmental Microbiology ◽

10.1128/aem.68.2.881-892.2002 ◽

2002 ◽

Vol 68 (2) ◽

pp. 881-892 ◽

Cited By ~ 127

Author(s):

Dorothea K. Thompson ◽

Alexander S. Beliaev ◽

Carol S. Giometti ◽

Sandra L. Tollaksen ◽

Tripti Khare ◽

...

Keyword(s):

Oxidative Stress ◽

Transcriptional Regulation ◽

Energy Metabolism ◽

Type Strain ◽

Wild Type Strain ◽

Shewanella Oneidensis ◽

Anaerobic Growth ◽

Ferric Uptake Regulator ◽

Wild Type ◽

And Oxidative Stress

ABSTRACT The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems.

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Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp. yingchengensis Strain 10–22

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/36.8.519 ◽

2004 ◽

Vol 36 (8) ◽

pp. 519-528 ◽

Cited By ~ 1

Author(s):

Lei Qin ◽

Heng-An Wang ◽

Zhong-Qin Wu ◽

Xiao-Feng Zhang ◽

Mei-Lei Jin ◽

...

Keyword(s):

Multidrug Resistance ◽

Type Strain ◽

Wild Type Strain ◽

Sequence Similarity ◽

Gene Replacement ◽

Major Facilitator Superfamily ◽

Streptomyces Hygroscopicus ◽

Wild Type ◽

Gene Encoding ◽

Major Facilitator

Abstract The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10–22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane α-helical spanners, belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5–20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

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