Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R.

The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.

A large family of anti-activators accompanying XylS/AraC family regulatory proteins.

AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

The importance of molecular analyses for understanding the genetic diversity of Histoplasma capsulatum: an overview / Importancia de los análisis moleculares en la comprensión de la diversidad genética de Histoplasma capsulatum: revisión

Advances in the classification of the human pathogen Histoplasma capsulatum (H. capsulatum) (ascomycete) are sustained by the results of several genetic analyses that support the high diversity of this dimorphic fungus. The present mini-review highlights the great genetic plasticity of H. capsulatum. Important records with different molecular tools, mainly single- or multi-locus sequence analyses developed with this fungus, are discussed. Recent phylogenetic data with a multi-locus sequence analysis using 5 polymorphic loci support a new clade and/or phylogenetic species of H. capsulatum for the Americas, which was associated with fungal isolates obtained from the migratory bat Tadarida brasiliensis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

Los resultados de diversos análisis genéticos que respaldan la alta diversidad de este hongo dimorfo confirman los progresos en la clasificación del patógeno humano Histoplasma capsulatum (H. capsulatum) (un ascomiceto). La presente revisión destaca la importante plasticidad genética de H. capsulatum. Se describen los datos importantes con los diferentes instrumentos moleculares, sobre todo, los análisis de las secuencias individuales o multi-loci establecidos con este hongo.Datos filogenéticos recientes con un análisis multi-loci de secuencias utilizando 5 loci polimorfos respaldan un nuevo clado y/o especie filogenética de H. capsulatum del continente americano, asociado a aislamientos fúngicos obtenidos del murciélago migratorio Tadarida brasiliensis.Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Salmonella enterica serovar typhimurium strains with regulated delayed attenuation in vivo.

Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P(BAD) cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.

Genome-wide prediction and annotation of Burkholderia pseudomallei AraC/XylS family transcription regulator.

Many members of the AraC/XylS family transcription regulator have been proven to play a critical role in regulating bacterial virulence factors in response to environmental stress. By using the Hidden Markov Model (HMM) profile built from the alignment of a 99 amino acid conserved domain sequence of 273 AraC/XylS family transcription regulators, we detected a total of 45 AraC/XylS family transcription regulators in the genome of the Gram-negative pathogen, Burkholderia pseudomallei. Further in silico analysis of each detected AraC/XylS family transcription regulatory protein and its neighboring genes allowed us to make a first-order guess on the role of some of these transcription regulators in regulating important virulence factors such as those involved in three type III secretion systems and biosynthesis of pyochelin, exopolysaccharide (EPS) and phospholipase C. This paper has demonstrated an efficient and systematic genome-wide scale prediction of the AraC/XylS family that can be applied to other protein families.

Mono and diterpene production in Escherichia coli.

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system.

The mxi-spa locus on the virulence plasmid of Shigella flexneri encodes components of the type III secretion system. mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined. We characterized the virulence phenotype of a nonpolar mxiE mutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-type Shigella at cell-to-cell spread. Despite its invasive properties in culture, the mxiE mutant was completely avirulent in an animal model. Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions. Six MxiE-regulated genes were discovered: ospB, ospC1, ospE2, ospF, virA, and ipaH(9.8). Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37 degrees C in liquid culture. Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors. Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence. Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose that Shigella evolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol.

AraC/XylS family members, HilC and HilD, directly bind and derepress the Salmonella typhimurium hilA promoter.

During infection, Salmonella enterica serovar Typhimurium colonizes the small intestine of its hosts. This process requires a type III secretion system encoded by several genes on Salmonella pathogenicity island 1 (SPI1), a 40 kb region of DNA near centisome 63 of the Salmonella chromosome. SPI1 gene expression is controlled by a complex regulatory cascade. HilA, a member of the OmpR/ToxR family of transcriptional regulators, directly activates the expression of two SPI1 operons encoding type III apparatus components. hilA transcription is repressed by many environmental conditions and regulatory mutations. This repression requires an upstream repressing sequence (URS) located between -314 and -68 relative to the hilA transcription start site. The repressing activity of the URS is counteracted by two AraC/XylS family members named HilC and HilD. We show that HilC and HilD bind directly to the hilA promoter region in vitro. We also provide evidence that HilC and HilD bind to the same or overlapping sites within the URS. Our data are consistent with a model in which HilC and HilD derepress hilA expression by binding directly to the URS and counteracting its repressing effect in vivo.

A HilA-independent pathway to Salmonella typhimurium invasion gene transcription.

Salmonella typhimurium invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci, sirB and sirC, which contribute to SPI1 gene transcription were defined. sirC is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype. sirB is required for maximal expression of sirC and consists of two open reading frames located near kdsA, a gene involved in lipopolysaccharide biosynthesis. sirC expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of sirC or sirA restores expression of a subset of SPI1 genes, including invF and sspC, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of inv expression is important for efficient assembly and function of the SPI1 TTSS.

Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator.

We describe the development and analysis of broad-host-range (BHR) cloning vectors that carry the araC-PBAD controlled expression cassette from Escherichia coli. These plasmids are designed to facilitate l-arabinose-responsive control of target genes in a variety of Gram-negative bacterial hosts. BHR PBAD::lacZ fusions were used to analyze the utility of this controlled expression system in the plant pathogen Agrobacterium tumefaciens. In A. tumefaciens, the level of control afforded is significant, although less stringent than that observed in E. coli. The BHR PBAD vectors offer a useful alternative to currently used controlled expression systems, and can be employed in conjunction with other regulated promoters to simultaneously regulate expression of multiple genes. Addition of a variety of carbon sources, namely C4 acids and the anti-inducer d-fucose, allows modulation of l-arabinose induction. Activation of PBAD expression in A. tumefaciens requires a plasmid-borne copy of araC, and is not affected by endogenous regulators.

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells.

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene.

Genotoxic effects of p-aminophenol in Chinese hamster ovary and mouse lymphoma cells: results of a multiple endpoint test.

p-Aminophenol (PAP), a metabolite of aniline and acetaminophen, has been reported to be mutagenic in the L5178Y mouse lymphoma assay, but not in the CHO/HGPRT assay. In the present study, the effects of PAP in these two cells lines were examined to determine if the difference in activity is related to an intrinsic difference in the cell lines. CHO and L5178Y tk +/- mouse lymphoma cells were treated with PAP for 4 hr and assayed for 3 genetic endpoints: gene mutation at the HGPRT or TK locus, respectively; chromosomal aberrations at approximately 20 hr after initiation of treatment; and single-strand DNA breaks as detected by the single cell electrophoresis assay immediately after treatment. All treatments were conducted in the absence of S9. There was a dose-related, significant increase in TFT-resistant mouse lymphoma cells at dose levels that reduced survival to < or = 50% of concurrent controls. In CHO cells, however, there was no increase in thioguanine-resistant cells at dose levels that reduced cell survival to < 20%. These results are consistent with published reports on PAP. While the CHO cells were slightly more resistant to the toxic effects of PAP, the dose levels used in the two cell lines did not differ by more than 2-fold. At equivalent survival levels, PAP induced a significant (up to 20% aberrant cells) number of aberrations, primarily complex rearrangements, in both cell lines. In the single cell electrophoresis assay, there was a reproducible dose-related increase in cells with single-strand DNA breaks with both the L5178Y cells and the CHO cells. The induction of single-strand breaks and chromosome aberrations by PAP suggests that, mechanistically, PAP produces similar genetic damage in both CHO and L5178Y cell lines, but intrinsic differences between assay systems are responsible for the divergent gene mutation results.

The occurrence of heritable Mu excisions in starving cells of Escherichia coli.

A strain of Escherichia coli constructed by Shapiro has a segment of Mu bacteriophage DNA inserted between the araC and lacZ genes. Excision events that produce an in-frame fusion of lacZ to araB result in a cell (here designated Ara-Lac+) that can grow on lactose if arabinose is present as an inducer. Whether or not these excision events occur in the absence of selection for the Ara-Lac+ phenotype has figured prominently in the debate of the phenomenon known as 'directed' or 'adaptive' mutation. In an attempt to settle the issue, we have used classic fluctuation tests to show that cells capable of producing a clone of descendants that are phenotypically Ara-Lac+ do, indeed, arise in stationary phase cultures kept starving in depleted minimal medium. We found that Ara-Lac+ progenitors arise rapidly under these conditions, in contrast to the delayed appearance of Ara-Lac+ mutants when cells are incubated on lactose-arabinose minimal plates. Similar results are reported in the accompanying paper by Maenhaut-Michel and Shapiro, who used indirect selection to isolate Ara-Lac+ cells in the absence of selection. However, their sequencing data have introduced a new unexpected complication to the interpretation of all such experiments, and it is no longer clear exactly when the fusions arise.