Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system.

A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.

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.

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.

A Case of Adaptive Laboratory Evolution (ALE): Biodegradation of Furfural by Pseudomonas pseudoalcaligenes CECT 5344.

Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a nitrogen source at alkaline pH. Genome sequencing of this strain allowed the detection of genes related to the utilization of furfurals as a carbon and energy source. Furfural and 5-(hydroxymethyl) furfural (HMF) are byproducts of sugars production during the hydrolysis of lignocellulosic biomass. Since they inhibit the yeast fermentation to obtain bioethanol from sugars, the biodegradation of these compounds has attracted certain scientific interest. P. pseudoalcaligenes was able to use furfuryl alcohol, furfural and furoic acid as carbon sources, but after a lag period of several days. Once adapted, the evolved strain (R1D) did not show any more prolonged lag phases. The transcriptomic analysis (RNA-seq) of R1D revealed a non-conservative punctual mutation (L261R) in BN5_2307, a member of the AraC family of activators, modifying the charge of the HTH region of the protein. The inactivation of the mutated gene in the evolved strain by double recombination reverted to the original phenotype. Although the bacterium did not assimilate HMF, it transformed it into value-added building blocks for the chemical industry. These results could be used to improve the production of cost-effective second-generation biofuels from agricultural wastes.

Arac/XylS family of transcriptional regulators.

The ArC/XylS family of prokaryotic positive transcriptional regulators includes more than 100 proteins and polypeptides derived from open reading frames translated from DNA sequences. Members of this family are widely distributed and have been found in the gamma subgroup of the proteobacteria, low- and high-G + C-content gram-positive bacteria, and cyanobacteria. These proteins are defined by a profile that can be accessed from PROSITE PS01124. Members of the family are about 300 amino acids long and have three main regulatory functions in common: carbon metabolism, stress response, and pathogenesis. Multiple alignments of the proteins of the family define a conserved stretch of 99 amino acids usually located at the C-terminal region of the regulator and connected to a nonconserved region via a linker. The conserved stretch contains all the elements required to bind DNA target sequences and to activate transcription from cognate promoters. Secondary analysis of the conserved region suggests that it contains two potential alpha-helix-turn-alpha-helix DNA binding motifs. The first, and better-fitting motif is supported by biochemical data, whereas existing biochemical data neither support nor refute the proposal that the second region possesses this structure. The phylogenetic relationship suggests that members of the family have recruited the nonconserved domain(s) into a series of existing domains involved in DNA recognition and transcription stimulation and that this recruited domain governs the role that the regulator carries out. For some regulators, it has been demonstrated that the nonconserved region contains the dimerization domain. For the regulators involved in carbon metabolism, the effector binding determinants are also in this region. Most regulators belonging to the AraC/XylS family recognize multiple binding sites in the regulated promoters. One of the motifs usually overlaps or is adjacent to the -35 region of the cognate promoters. Footprinting assays have suggested that these regulators protect a stretch of up to 20 bp in the target promoters, and multiple alignments of binding sites for a number of regulators have shown that the proteins recognize short motifs within the protected region.

Ram locus is a key regulator to trigger multidrug resistance in Enterobacter aerogenes.

PURPOSE: Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of E. aerogenes due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance. METHODOLOGY: Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of E. aerogenes. Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed. RESULTS: In the majority of the MDR isolates tested, ramR and a region upstream of ramA were mutated but marR or marA were unchanged. Expression and cloning experiments highlighted the involvement of the ram locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects. CONCLUSION: Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.

The AraC transcriptional activators.

The AraC family of bacterial transcriptional activators regulate diverse genetic systems. Recent X-ray diffraction studies show that the monomeric MarA and Rob activators bind to their asymmetric degenerate DNA sites via two different helix-turn-helix elements. Activation by MarA, SoxS or Rob requires a particular orientation of the asymmetric binding sequence (and hence the activator), depending on its distance from the -10 RNAP signal. Genetic studies are beginning to clarify how the activators interact with RNAP. Growing evidence suggests that for the sugar metabolism activators, multiple binding sites upstream of the promoter anchor the activator in a repressing or nonactivating configuration. By interaction with the sugar and/or CRP, the activator is allosterically altered so it can bind a new set of sites that enable it to activate the promoter. Surprisingly, the virulence activator, Rns, must bind to both upstream and downstream sites in order to activate the rns promoter.

Molecular mechanism for sphingosine-induced Pseudomonas ceramidase expression through the transcriptional regulator SphR.

Pseudomonas aeruginosa, an opportunistic, but serious multidrug-resistant pathogen, secretes a ceramidase capable of cleaving the N-acyl linkage of ceramide to generate fatty acids and sphingosine. We previously reported that the secretion of P. aeruginosa ceramidase was induced by host-derived sphingolipids, through which phospholipase C-induced hemolysis was significantly enhanced. We herein investigated the gene(s) regulating sphingolipid-induced ceramidase expression and identified SphR, which encodes a putative AraC family transcriptional regulator. Disruption of the sphR gene in P. aeruginosa markedly decreased the sphingomyelin-induced secretion of ceramidase, reduced hemolytic activity, and resulted in the loss of sphingomyelin-induced ceramidase expression. A microarray analysis confirmed that sphingomyelin significantly induced ceramidase expression in P. aeruginosa. Furthermore, an electrophoretic mobility shift assay revealed that SphR specifically bound free sphingoid bases such as sphingosine, dihydrosphingosine, and phytosphingosine, but not sphingomyelin or ceramide. A ß-galactosidase-assisted promoter assay showed that sphingosine activated ceramidase expression through SphR at a concentration of 100 nM. Collectively, these results demonstrated that sphingosine induces the secretion of ceramidase by promoting the mRNA expression of ceramidase through SphR, thereby enhancing hemolytic phospholipase C-induced cytotoxicity. These results facilitate understanding of the physiological role of bacterial ceramidase in host cells.

Determining residue-base interactions between AraC protein and araI DNA.

Depurination/depyrimidation binding-interference experiments (missing contact probing) identified specific candidate residue-base interactions lost by mutants of Escherichia coli L-arabinose operon regulatory protein, AraC, to one of its binding sites, araI. These candidates were then checked more rigorously by comparing the affinities of wild-type and alanine-substituted AraC protein to variants of araI with alterations in the candidate contacted positions. Residues 208 and 212 apparently contact DNA and support, but do not prove the existence of a helix-turn-helix structure in this region of AraC protein whereas contacts by mutants with alterations at positions 256, 257 and 261 which are within another potential helix-turn-helix region do not support the existence of such a structure there. The missing contacts displayed by three AraC mutants are found within two major groove regions of the DNA and are spaced 21 base-pairs apart in a pattern indicating a direct repeat orientation for the subunits of AraC.

The araC gene of Citrobacter freundii.

The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.

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.

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.

Mutagenicity testing in Salmonella typhimurium strains possessing both the His reversion and Ara forward mutation systems and different levels of classical nitroreductase or O-acetyltransferase activities.

The induction of forward mutations to L-arabinose resistance (AraR) and of reversions to histidine prototrophy (His+) can be quantitatively compared in Salmonella typhimurium BA strains. The BA bacteria carry the araD531 allele required for the Ara assay and a his auxotrophy (hisD3052 or hisG46) required for the His assay. In this study, 2 new sets of BA indicator strains have been constructed in order to combine the Ara forward and the His reverse mutation assays of S. typhimurium with deficiency, or over-production, in either classical nitroreductase or O-acetyltransferase for mutagenicity testing of nitro-containing chemicals. Nine mutagens with different chemical structures were tested to compare the specific mutagenic sensitivities of the new constructions with those of the parental and of the conventional TA indicator bacteria. The Ara test, which responded with high sensitivity to all chemicals tested, revealed important differences between the standard tester strains TA98 and TA100 with respect to the activation of mutagens considered to be dependent on classical nitroreductase activity. Total correspondence was found between the specific mutagenic sensitivities of the defective and the overproducing bacteria in the genetic background of TA98 but not in that of TA100. In the genetic background of TA100, chemicals such as nitrofurantoin and nitrofurazone displayed 10-fold reduced mutagenicity to the "classical nitroreductase" defective strain without increasing mutagenicity to the corresponding overproducing bacteria. This discrepancy might be attributed to the greater nitroreduction capability of strain TA100 (68.12 nmole/min/mg protein) as compared to TA98 (24.42 nmole/min/mg protein), by assuming that nitrofurantoin and nitrofurazone are such good substrates for classical nitroreductase that the additional enzyme activity produced from the corresponding overexpressing plasmid when present in TA100 no longer affected their metabolic activation. We propose that the Ara forward mutation test carried out in the set of over-producing bacteria constructed in the genetic background of TA98 might play a role for routine testing of large number of samples. The isogenic defective strains could be used in cases of uncertain results with the corresponding over-producing bacteria. Finally, the reversion of the his alleles accompanying the Ara assay in the BA strains could play a role in assessing the presence of mixtures of chemicals with different mutagenic specificity in samples of environmental relevance such as urban air, foods, and water.

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.

Entrainment of a population of synthetic genetic oscillators.

Biological clocks are self-sustained oscillators that adjust their phase to the daily environmental cycles in a process known as entrainment. Molecular dissection and mathematical modeling of biological oscillators have progressed quite far, but quantitative insights on the entrainment of clocks are relatively sparse. We simultaneously tracked the phases of hundreds of synthetic genetic oscillators relative to a common external stimulus to map the entrainment regions predicted by a detailed model of the clock. Synthetic oscillators were frequency-locked in wide intervals of the external period and showed higher-order resonance. Computational simulations indicated that natural oscillators may contain a positive-feedback loop to robustly adapt to environmental cycles.

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.