A new member of the ribbon-helix-helix transcription factor superfamily from the plant pathogen Xanthomonas axonopodis pv. citri

XACb0070 is an uncharacterized protein coded by the two large plasmids isolated from Xanthomonas axonopodis pv. cirri, the agent of citrus canker and responsible for important economical losses in citrus world production. XACb0070 presents sequence homology only with other hypothetical proteins belonging to plant pathogens, none of which have their structure determined. The NMR-derived solution structure reveals this protein is a homodimer in which each monomer presents two domains with different structural and dynamic properties: a folded N-terminal domain with beta alpha alpha topology which mediates dimerization and a long disordered C-terminal tail. The folded domain shows high structural similarity to the ribbon-helix-helix transcriptional repressors, a family of DNA-binding proteins of conserved 3D fold but low sequence homology: indeed XACb0070 binds DNA. Primary sequence and fold comparison of XACb0070 with other proteins of the ribbon-helix-helix family together with examination of the genes in the vicinity of xacb0070 suggest the protein might be the component of a toxin-antitoxin system. (C) 2010 Elsevier Inc. All rights reserved.

Prospecção de genes com interesse biotecnológico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Control of the master virulence regulatory gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

Epitope-specific tolerance induction with an engineered immunoglobulin.

Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years. The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors. The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides. To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein. This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system. We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26. Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes. This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity.

Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex.

The human immunodeficiency virus type 1 transactivator protein, Tat, stimulates transcriptional elongation from the viral long terminal repeat. To test whether Tat associates directly with activated transcription complexes, we have used the lac repressor protein (LacR) to "trap" elongating RNA polymerases. The arrested transcription complexes were purified by binding biotinylated templates to streptaviridin-coated magnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II. The Tat protein copurified with RNA polymerase bound to wild-type templates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response element (TAR) RNA. We conclude that Tat and cellular cofactors become attached to the transcription complex during its transit through TAR.

Análise funcional das proteínas HrcA, GroES/GroEL e DnaK/DnaJ em Caulobacter crescentus

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc.

Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons

BACKGROUND Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. RESULTS Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. CONCLUSIONS Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain.

BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.

Approximately 40% of diffuse large cell lymphoma are associated with chromosomal translocations that deregulate the expression of the BCL6 gene by juxtaposing heterologous promoters to the BCL-6 coding domain. The BCL6 gene encodes a 95-kDa protein containing six C-terminal zinc-finger motifs and an N-terminal POZ domain, suggesting that it may function as a transcription factor. By using a DNA sequence selected for its ability to bind recombinant BCL-6 in vitro, we show here that BCL-6 is present in DNA-binding complexes in nuclear extracts from various B-cell lines. In transient transfectin experiments, BCL6 can repress transcription from promoters linked to its DNA target sequence and this activity is dependent upon specific DNA-binding and the presence of an intact N-terminal half of the protein. We demonstrate that this part of the BCL6 molecule contains an autonomous transrepressor domain and that two noncontiguous regions, including the POZ motif, mediate maximum transrepressive activity. These results indicate that the BCL-6 protein can function as a sequence-specific transcriptional repressor and have implications for the role of BCL6 in normal lymphoid development and lymphomagenesis.

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

The copper-responsive repressor CopR of Lactococcus lactis is a 'winged helix' protein

CopR of Lactococcus lactis is a copper-responsive repressor involved in copper homoeostasis. It controls the expression of a total of 11 genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N-terminal domain to a 'cop box' of consensus TACANNTGTA, which is conserved in Firmicutes. We have solved the NMR solution structure of the N-terminal DNA-binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors, and the present structure represents a novel family of copper regulators, which we propose to call the CopY family.

The STAR protein QKI-6 is a translational repressor

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3′ untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.