A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3691-3701 ◽  
Author(s):  
Zhanna I. Budarina ◽  
Dmitri V. Nikitin ◽  
Nikolay Zenkin ◽  
Marina Zakharova ◽  
Ekaterina Semenova ◽  
...  
Keyword(s):  
Dna Binding ◽  
Bacillus Cereus ◽  
Transcription Initiation ◽  
Food Poisoning ◽  
Haemolytic Activity ◽  
Activity Levels ◽  
Initiation Point ◽  
Dna Repeat ◽  
Opportunistic Human Pathogen

Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.

scholarly journals Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

2015 ◽  
Vol 82 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Varvara Tsilia ◽  
Frederiek-Maarten Kerckhof ◽  
Andreja Rajkovic ◽  
Marc Heyndrickx ◽  
Tom Van de Wiele
Keyword(s):  
Bacillus Cereus ◽  
Food Poisoning ◽  
Intestinal Bacteria ◽  
Low Ph ◽  
Final Concentration ◽  
Adhesion Assay ◽  
Content Type ◽  
The Stability ◽  
Bacterial Concentrations

ABSTRACTAdhesion to the intestinal epithelium could constitute an essential mechanism ofBacillus cereuspathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacteriumB. cereusNVH 0500/00 was exposedin vitroto gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherentB. cereusafter simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated.B. cereuscould grow and simultaneously adhere to mucin duringin vitroileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration ofB. cereusin the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml−1. At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retainB. cereusand to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.

scholarly journals Loss of Pseudomonas aeruginosa PhpA Aminopeptidase Activity Results in Increased algDTranscription

2001 ◽  
Vol 183 (15) ◽  
pp. 4674-4679 ◽  
Author(s):  
Samuel C. Woolwine ◽  
April B. Sprinkle ◽  
Daniel J. Wozniak
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Dna Binding ◽  
Transcription Initiation ◽  
Leucine Aminopeptidase ◽  
Initiation Site ◽  
Aminopeptidase Activity ◽  
Homologous Protein ◽  
Relevant Property

ABSTRACT Inactivation of Pseudomonas aeruginosa phpA, encoding a putative leucine aminopeptidase, results in increased transcription ofalgD. The homologous protein in Escherichia coli, PepA, is multifunctional, possessing independent aminopeptidase and DNA-binding activities. Here we provide in vitro evidence that PhpA is an aminopeptidase and show that this activity is the relevant property with regard to algD expression. This regulation occurred at the previously mapped algDtranscription initiation site and was not due to activation of an alternative promoter.

scholarly journals A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

1999 ◽  
Vol 19 (11) ◽  
pp. 7610-7620 ◽  
Author(s):  
Paul A. Moore ◽  
Josef Ozer ◽  
Moreh Salunek ◽  
Gwenael Jan ◽  
Dennis Zerby ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Related Protein ◽  
Multiple Promoters ◽  
Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

scholarly journals The Pseudomonas Quorum-Sensing Regulator RsaL Belongs to the Tetrahelical Superclass of H-T-H Proteins

2006 ◽  
Vol 189 (5) ◽  
pp. 1922-1930 ◽  
Author(s):  
Giordano Rampioni ◽  
Fabio Polticelli ◽  
Iris Bertani ◽  
Karima Righetti ◽  
Vittorio Venturi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Quorum Sensing ◽  
In Silico ◽  
Mobility Shift ◽  
Specific Domain ◽  
Signal Molecule ◽  
In Silico Model ◽  
Opportunistic Human Pathogen

ABSTRACT In the opportunistic human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of lasI, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the N-terminal domain of the lambda cI repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the lasI promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.

scholarly journals Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945 ◽  
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

scholarly journals The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0259386
Author(s):  
Devon W. Kavanaugh ◽  
Constance Porrini ◽  
Rozenn Dervyn ◽  
Nalini Ramarao
Keyword(s):  
Alcohol Dehydrogenase ◽  
Bacillus Cereus ◽  
Large Strain ◽  
Bacterial Species ◽  
Gene Encoding ◽  
Pathogenic Potential ◽  
Strain Collection ◽  
Opportunistic Human Pathogen

Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

scholarly journals Domain analysis of the plant DNA-binding protein GT1a: requirement of four putative alpha-helices for DNA binding and identification of a novel oligomerization region.

1995 ◽  
Vol 15 (2) ◽  
pp. 1014-1020 ◽  
Author(s):  
E Lam
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Cdna Clones ◽  
Protein Oligomerization ◽  
Gene Promoters ◽  
Alpha Helices

Light is an important environmental signal that can influence diverse developmental processes in plants. Many plant nuclear genes respond to light at the level of transcription initiation. GT-1 and GT2 are nuclear factors which interact with DNA sequences in many light-responsive gene promoters. cDNA clones which encode proteins with sequence binding specificities similar to those of these two factors have been isolated. They show significant amino acid sequence similarities within three closely spaced, putative alpha-helices that were predicted by secondary structure analysis but do not show significant homologies with any other reported DNA-binding protein. In this work, N- and C-terminal deletions of tobacco GT1a were generated by in vitro transcription and translation, and their DNA-binding activities and subunit structures were studied. The results suggest that the C-terminal domain of GT1a is critical for protein oligomerization, while a region predicted to contain four closely spaced alpha-helices is required for DNA binding. Direct chemical cross-linking and gel filtration analyses of full-length and truncated derivatives of GT1a suggest that this factor can exist in solution as a homotetramer and that oligomerization is independent of DNA binding. This study thus establishes two independent functional domains in this class of eukaryotic trans-acting factors. Possible implications of the multimeric nature of GT1a in relation to the known characteristics of light-responsive promoter architecture are discussed.

scholarly journals orf4 of the Bacillus cereus sigB Gene Cluster Encodes a General Stress-Inducible Dps-Like Bacterioferritin

2009 ◽  
Vol 191 (14) ◽  
pp. 4522-4533 ◽  
Author(s):  
Shin-Wei Wang ◽  
Chien-Yen Chen ◽  
Joseph T. Tseng ◽  
Shih-Hsiung Liang ◽  
Ssu-Ching Chen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Oxidative Damage ◽  
Environmental Stress ◽  
Bacillus Cereus ◽  
Size Exclusion ◽  
Null Mutant ◽  
Cell Protection ◽  
Ferroxidase Activity

ABSTRACT The function of orf4 in the sigB cluster in Bacillus cereus ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an orf4-null mutant and produced recombinant protein rOrf4 to establish the role of orf4. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl2. Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of orf4 had no effect on activation of the alternative sigma factor σB, and therefore, orf4 is not associated with σB regulation; however, orf4 can be significantly upregulated upon environmental stress but not H2O2 treatment. B. cereus strains with constitutive Orf4 expression exhibited a viability higher than that of the orf4-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding.

scholarly journals An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites

2009 ◽  
Vol 191 (17) ◽  
pp. 5489-5498 ◽  
Author(s):  
Steve D. Seredick ◽  
Barbara M. Seredick ◽  
David Baker ◽  
George B. Spiegelman
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Molecular Basis ◽  
Response Regulator ◽  
Mutant Protein ◽  
Wild Type ◽  
Dna Binding Sites

ABSTRACT In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

scholarly journals Structure and expression of the human immunoglobulin lambda genes.

1990 ◽  
Vol 172 (2) ◽  
pp. 609-620 ◽  
Author(s):  
T J Vasicek ◽  
P Leder
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Rna Splicing ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Enhancer Activity ◽  
Initiation Point ◽  
Reading Frame ◽  
Duplication Events

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

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