scholarly journals Characterization of the SOS Regulon of Caulobacter crescentus

2007 ◽  
Vol 190 (4) ◽  
pp. 1209-1218 ◽  
Author(s):  
Raquel Paes da Rocha ◽  
Apuã César de Miranda Paquola ◽  
Marilis do Valle Marques ◽  
Carlos Frederico Martins Menck ◽  
Rodrigo S. Galhardo
Keyword(s):  
Dna Damage ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Direct Repeat ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Wild Type ◽  
Promoter Regions ◽  
Potential Promoter

ABSTRACT The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Isolation and Characterization of NaCl-Sensitive Mutants of Caulobacter crescentus

2003 ◽  
Vol 69 (6) ◽  
pp. 3029-3035 ◽  
Author(s):  
Luiz Fernando G. Zuleta ◽  
Val�ria C. S. Italiani ◽  
Marilis V. Marques
Keyword(s):  
Caulobacter Crescentus ◽  
Osmotic Shock ◽  
Promoter Regions ◽  
Isolation And Characterization ◽  
Lipopolysaccharide Biosynthesis ◽  
Severe Reduction ◽  
Mutant Strains ◽  
High Concentrations ◽  
Severe Growth

ABSTRACT An attempt to characterize Caulobacter crescentus genes important for the response to high concentrations of NaCl was initiated by the isolation of mutants defective in survival in the presence of 85 mM NaCl. A transposon Tn5 library was screened, and five strains which contained different genes disrupted by the transposon were isolated. Three of the mutants had the Tn5 in genes involved in lipopolysaccharide biosynthesis, one had the Tn5 in the nhaA gene, which encodes a Na+/H+ antiporter, and one had the Tn5 in the ppiD gene, which encodes a peptidyl-prolyl cis-trans isomerase. All the mutant strains showed severe growth arrest in the presence of 85 mM NaCl, but only the nhaA mutant showed decreased viability under these conditions. All the mutants except the nhaA mutant showed a slightly reduced viability in the presence of 40 mM KCl, but all the strains showed a more severe reduction in viability in the presence of 150 mM sucrose, suggesting that they are defective in responding to osmotic shock. The promoter regions of each disrupted gene were cloned in lacZ reporter vectors, and the pattern of expression in response to NaCl and sucrose was determined; this showed that both agents induced ppiD and nhaA gene expression but did not induce the other genes. Furthermore, the ppiD gene was not induced by heat shock, indicating that it does not belong to the σ32 regulon, as opposed to what was observed for its Escherichia coli homolog.

scholarly journals A Helicobacter hepaticus catalase mutant is hypersensitive to oxidative stress and suffers increased DNA damage

2007 ◽  
Vol 56 (4) ◽  
pp. 557-562 ◽  
Author(s):  
Yang Hong ◽  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Catalase Activity ◽  
Insertional Mutagenesis ◽  
Specific Activity ◽  
Bacterial Species ◽  
Helicobacter Hepaticus ◽  
Wild Type ◽  
Crude Cell Extract ◽  
H Pylori

Catalase (KatA) is known to play an important role in oxidative stress resistance in many bacterial species and a homologue exists in Helicobacter hepaticus, a member of the enterohepatic Helicobacter species. Here, a katA mutant was constructed by insertional mutagenesis and its oxidative stress phenotype was investigated. Catalase activity was readily detected [196 units (mg protein crude cell extract)−1] in the wild-type, whereas the mutant strain was deficient in, but not devoid of, activity. In contrast, Helicobacter pylori katA strains lack detectable catalase activity and wild-type H. pylori generally contains higher specific activity than H. hepaticus. Wild-type H. hepaticus cells tolerated 6 % O2 for growth, whilst the katA mutant could not survive at this oxygen level. Even at the optimal O2 level, the growth of the H. hepaticus katA strain was severely inhibited, which is also in contrast to H. pylori katA strains. Wild-type H. hepaticus cells withstood exposure to 100 mM H2O2 but the katA mutant cells were killed by the same treatment. Wild-type cells suffered no significant DNA damage by H2O2 treatment (100 mM for 6 min), whilst the same treatment resulted in severe DNA fragmentation in the katA mutant. Thus H. hepaticus KatA plays an important role as an antioxidant protein.

scholarly journals Identification and Characterization of the Potential Promoter Regions of 1031 Kinds of Human Genes

2001 ◽  
Vol 11 (5) ◽  
pp. 677-684 ◽  
Author(s):  
Y. Suzuki ◽  
T. Tsunoda ◽  
J. Sese ◽  
H. Taira ◽  
J. Mizushima-Sugano ◽  
...  
Keyword(s):  
Promoter Regions ◽  
Human Genes ◽  
Potential Promoter ◽  
Identification And Characterization

scholarly journals Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7347-7356 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Michel Popielarz ◽  
Georges Hildwein ◽  
James Stevenin
Keyword(s):  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Adenovirus E1a ◽  
Differential Involvement ◽  
Splicing Enhancer

ABSTRACT The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 186-186
Author(s):  
Natalie B. Collins ◽  
Andrei Tomashevski ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Damage Response ◽  
And Function ◽  
Mutant Cells

Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.

Characterization of Pph3-mediated dephosphorylation of Rad53 during methyl methanesulfonate-induced DNA damage repair in Candida albicans

2017 ◽  
Vol 474 (7) ◽  
pp. 1293-1306 ◽  
Author(s):  
Guangyin Yao ◽  
Junhua Wan ◽  
Qizheng Liu ◽  
Chunhua Mu ◽  
Yue Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Pathogenic Fungus ◽  
Genotoxic Stress ◽  
Methyl Methanesulfonate ◽  
Wild Type ◽  
Cycle Arrest ◽  
Damage Repair ◽  
Damage Response

Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus Candida albicans. The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in C. albicans. We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in pph3Δ/Δ cells. Phosphomimetic mutation of the three residues (rad53-3D) impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the rad53-3D mutant also occurred in the pph3Δ/Δ mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in C. albicans.

scholarly journals Chk2 Activation Dependence on Nbs1 after DNA Damage

2001 ◽  
Vol 21 (15) ◽  
pp. 5214-5222 ◽  
Author(s):  
Giacomo Buscemi ◽  
Camilla Savio ◽  
Laura Zannini ◽  
Francesca Miccichè ◽  
Debora Masnada ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Nijmegen Breakage Syndrome ◽  
Mutant Form ◽  
Dependent Manner ◽  
Wild Type ◽  
Normal Cells ◽  
Chromosome Fragility ◽  
Carboxy Terminal

ABSTRACT The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G1 arrest. Here we show that the ATM-dependent activation of Chk2 by γ- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.

scholarly journals Systematic Analysis of c-di-GMP Signaling Mechanisms and Biological Functions in Dickeya zeae EC1

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Yufan Chen ◽  
Jianuan Zhou ◽  
Mingfa Lv ◽  
Zhibin Liang ◽  
Matthew R. Parsek ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Economic Losses ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Content Type ◽  
Bacterial Physiology ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Rice Plantations

ABSTRACT Dickeya zeae is an important and aggressive bacterial phytopathogen that can cause substantial economic losses in banana and rice plantations. We previously showed that c-di-GMP signaling proteins (cyclases/phosphodiesterases) in D. zeae strain EC1 play a significant role in the bacterial sessile-to-motile transition. To determine whether there is any synergistic effect among these c-di-GMP signaling proteins, we prepared a series of mutant strains by generating consecutive in-frame deletions of the genes encoding diguanylate cyclases (which make c-di-GMP) and phosphodiesterases (which break down c-di-GMP), respectively, using EC1 as a parental strain. The results showed that the complete deletion of all the putative diguanylate cyclases resulted in significantly increased bacterial motility and abrogated biofilm formation but did not appear to affect pathogenicity and virulence factor production. In contrast, the deletion of all the c-di-GMP phosphodiesterase genes disabled motility and prevented the invasion of EC1 into rice seeds. By measuring the c-di-GMP concentrations and swimming motility of all the mutants, we propose that c-di-GMP controlled swimming behavior through a multitiered program in a c-di-GMP concentration-dependent manner, which could be described as an L-shaped regression curve. These features are quite different from those that have been shown for other bacterial species such as Salmonella and Caulobacter crescentus. Further analysis identified three c-di-GMP signaling proteins, i.e., PDE10355, DGC14945, and PDE14950, that play dominant roles in influencing the global c-di-GMP pool of strain EC1. The findings from this study highlight the complexity and plasticity of c-di-GMP regulatory circuits in different bacterial species. IMPORTANCE Dickeya zeae is the etiological agent of bacterial foot rot disease, which can cause massive economic losses in banana and rice plantations. Genome sequence analysis showed that D. zeae strain EC1 contains multiple c-di-GMP turnover genes, but their roles and regulatory mechanisms in bacterial physiology and virulence remain vague. By generating consecutive in-frame deletion mutants of the genes encoding c-di-GMP biosynthesis and degradation, respectively, we analyzed the individual and collective impacts of these c-di-GMP metabolic genes on the c-di-GMP global pool, bacterial physiology, and virulence. The significance of our study is in identifying the mechanism of c-di-GMP signaling in strain EC1 more clearly, which expands the c-di-GMP regulating patterns in Gram-negative species. The methods and experimental designs in this research will provide a valuable reference for the exploration of the complex c-di-GMP regulation mechanisms in other bacteria.

scholarly journals Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 1890-1900 ◽  
Author(s):  
Lisha Ding ◽  
Yao Wang ◽  
Yangbo Hu ◽  
Steve Atkinson ◽  
Paul Williams ◽  
...  
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Functional Characterization ◽  
Direct Interaction ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
C Terminus ◽  
In Trans ◽  
Differential Gene ◽  
Flagellar Biogenesis

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ 28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ 28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ 28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ 28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ 28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ 28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

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