A simple and unified protocol to purify all seven Escherichia coli RNA polymerase sigma factors.

RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene's promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors. In our approach, we took advantage of the His8-SUMO tag, known to increase protein solubilization. Sigma factors were first purified in N-terminal fusions with this tag, which was followed by tag removal with Ulp1 protease. This allowed to obtain proteins in their native form. In addition, the procedure is simple and requires only one resin type. With the general protocol we employed, we were able to successfully purify σD, σE, σS, and σN. Final step modification was required for σF, while for σH and σFecI, denaturing conditions had to be applied. All seven sigma factors were fully functional in forming an active holoenzyme with core RNA polymerase which we demonstrated with EMSA studies.

LIC_12757 from the pathogenic spirochaete Leptospira interrogans encodes an autoregulated ECF σE-type factor.

ECF (extracytoplasmic function) σ factors, members of the σ70-family, are the largest class of alternative σ factors which are stimulated in the presence of specific signals and direct RNA polymerase to transcribe a defined subset of genes. Thanks to them, bacterial pathogens can effectively reprogram their gene expression and, consequently, survive in the host and establish infection in a relatively short time. The number of ECF σ factors encoded within bacterial genomes is different depending on a given species and it reflects the likelihood that these bacteria will encounter harsh environmental conditions. The genome of L. interrogans, a zoonotic pathogen responsible for leptospirosis, is predicted to encode 11 ECF σE-type factors, but none of them have been characterized biochemically to date and their functions are still unknown. Here, we focused on one of the leptospiral ECF σ factors, namely LIC_12757, which was previously found to be up-regulated at elevated temperatures and may be related to the expression of clpB encoding an important L. interrogans virulence factor. We report cloning of the coding sequence of the LIC_12757 gene, its expression with the pET system and biochemical characterization of LIC_12757. By performing EMSA and in vitro transcription assays, we provide strong evidence that LIC_12757 indeed functions as a transcriptional factor that enables RNA polymerase to bind to the specific σE-type promoter and to initiate transcription. Interestingly, we demonstrate that LIC_12757 is autoregulated at the transcriptional level. Our study is a first step towards determining key aspects of LIC_12757 function in pathogenic Leptospira.

Bacterial Toxin-Antitoxin Systems' Cross-Interactions-Implications for Practical Use in Medicine and Biotechnology.

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes' expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage.

Minigene as a Novel Regulatory Element in Toxin-Antitoxin Systems.

The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the axe-txe operon influences translation of the Txe toxin. This is the first example to date of such a regulatory mechanism identified in the TA modules. Here, in a series of genetic studies, we employed translational reporter gene fusions to establish the molecular basis of this phenomenon. Our results show that translation of the two-codon mini-ORF displays an in cis mode of action, and positively affects the expression of txe, possibly by increasing its mRNA stability through protection from an endonuclease attack. Moreover, we established that the reading frame in which the two cistrons are encoded, as well as the distance between them, are critical parameters that affect the level of such regulation. In addition, by searching for two-codon ORFs we found sequences of several potential minigenes in the leader sequences of several other toxins belonging to the type II TA family. These findings suggest that this type of gene regulation may not only apply for the axe-txe cassette, but could be more widespread among other TA systems.

Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe.

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the -35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

Emerging Roles of Toxin-Antitoxin Modules in Bacterial Pathogenesis.

Toxin-antitoxin (TA) cassettes are encoded widely by bacteria. The modules typically comprise a protein toxin and protein or RNA antitoxin that sequesters the toxin factor. Toxin activation in response to environmental cues or other stresses promotes a dampening of metabolism, most notably protein translation, which permits survival until conditions improve. Emerging evidence also implicates TAs in bacterial pathogenicity. Bacterial persistence involves entry into a transient semi-dormant state in which cells survive unfavorable conditions including killing by antibiotics, which is a significant clinical problem. TA complexes play a fundamental role in inducing persistence by downregulating cellular metabolism. Bacterial biofilms are important in numerous chronic inflammatory and infectious diseases and cause serious therapeutic problems due to their multidrug tolerance and resistance to host immune system actions. Multiple TAs influence biofilm formation through a network of interactions with other factors that mediate biofilm production and maintenance. Moreover, in view of their emerging contributions to bacterial virulence, TAs are potential targets for novel prophylactic and therapeutic approaches that are required urgently in an era of expanding antibiotic resistance. This review summarizes the emerging evidence that implicates TAs in the virulence profiles of a diverse range of key bacterial pathogens that trigger serious human disease.

Charge transport through a semiconductor quantum dot-ring nanostructure.

Transport properties of a gated nanostructure depend crucially on the coupling of its states to the states of electrodes. In the case of a single quantum dot the coupling, for a given quantum state, is constant or can be slightly modified by additional gating. In this paper we consider a concentric dot-ring nanostructure (DRN) and show that its transport properties can be drastically modified due to the unique geometry. We calculate the dc current through a DRN in the Coulomb blockade regime and show that it can efficiently work as a single-electron transistor (SET) or a current rectifier. In both cases the transport characteristics strongly depend on the details of the confinement potential. The calculations are carried out for low and high bias regime, the latter being especially interesting in the context of current rectification due to fast relaxation processes.

Regulating toxin-antitoxin expression: controlled detonation of intracellular molecular timebombs.

Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.

Amino acid residues crucial for specificity of toxin-antitoxin interactions in the homologous Axe-Txe and YefM-YoeB complexes.

Toxin-antitoxin complexes are ubiquitous in bacteria. The specificity of interactions between toxins and antitoxins from homologous but non-interacting systems was investigated. Based on molecular modeling, selected amino acid residues were changed to assess which positions were crucial in the specificity of toxin-antitoxin interaction in the related Axe-Txe and YefM-YoeB complexes. No cross-interactions between wild-type proteins were detected. However, a single amino acid substitution that converts a Txe-specific residue to a YoeB-specific residue reduced, but did not abolish, Txe interaction with the Axe antitoxin. Interestingly, this alteration (Txe-Asp83Tyr) promoted functional interactions between Txe and the YefM antitoxin. The interactions between Txe-Asp83Tyr and YefM were sufficiently strong to abolish Txe toxicity and to allow effective corepression by YefM-Txe-Asp83Tyr of the promoter from which yefM-yoeB is expressed. We conclude that Asp83 in Txe is crucial for the specificity of toxin-antitoxin interactions in the Axe-Txe complex and that swapping this residue for the equivalent residue in YoeB relaxes the specificity of the toxin-antitoxin interaction.

The axe-txe complex of Enterococcus faecium presents a multilayered mode of toxin-antitoxin gene expression regulation.

Multidrug-resistant variants of human pathogens from the genus Enterococcus represent a significant health threat as leading agents of nosocomial infections. The easy acquisition of plasmid-borne genes is intimately involved in the spread of antibiotic resistance in enterococci. Toxin-antitoxin (TA) systems play a major role in both maintenance of mobile genetic elements that specify antibiotic resistance, and in bacterial persistence and virulence. Expression of toxin and antitoxin genes must be in balance as inappropriate levels of toxin can be dangerous to the host. The controlled production of toxin and antitoxin is usually achieved by transcriptional autoregulation of TA operons. One of the most prevalent TA modules in enterococcal species is axe-txe which is detected in a majority of clinical isolates. Here, we demonstrate that the axe-txe cassette presents a complex pattern of gene expression regulation. Axe-Txe cooperatively autorepress expression from a major promoter upstream of the cassette. However, an internal promoter that drives the production of a newly discovered transcript from within axe gene combined with a possible modulation in mRNA stability play important roles in the modulation of Axe:Txe ratio to ensure controlled release of the toxin.

Role of heat-shock proteins and cobalamine in maintaining methionine synthase activity.

Atheromatous plaque is one of the most common cardiovascular-related diseases. Reports show a connection between its development and the levels of homocysteine. In pathological states high levels of homocysteine in the organism can be caused by the malfunction of the methionine synthase pathway. Bacterial methionine synthase (MetH) is a homologue of the human methionine syntase (MS). In this study we aimed to investigate the functional relations between MetH and its cofactor--cobalamine--under stress conditions. We have demonstrated that heat shock proteins (Hsp 70/100 system or HtpG) can protect MetH activity under stress conditions. Moreover, in the presence of cobalamine they can restore the activity of partially denatured methionine synthase.

Centromere anatomy in the multidrug-resistant pathogen Enterococcus faecium.

Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.

The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit plays a role in the CI-dependent activation of the bacteriophage lambda pM promoter.

The bacteriophage lambda p(M) promoter is required for maintenance of the lambda prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the lambda repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of p(M). CI activates p(M) through cooperative binding to the O(R)1 and O(R)2 sites within the O(R) operator, with the O(R)2-bound CI dimer making contact with domain 4 of the RNA polymerase sigma subunit (sigma(4)). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of p(M). We also show that the location of alphaCTD at the p(M) promoter changes in the presence of CI. Thus, in the absence of CI, one alphaCTD is located on the DNA at position -44 relative to the transcription start site, whereas in the presence of CI, alphaCTD is located at position -54, between the CI-binding sites at O(R)1 and O(R)2. These results suggest that contacts between CI and both alphaCTD and sigma are required for efficient CI-dependent activation of p(M).

Toxin-antitoxin regulation: bimodal interaction of YefM-YoeB with paired DNA palindromes exerts transcriptional autorepression.

Toxin-antitoxin (TA) complexes function in programmed cell death or stress response mechanisms in bacteria. The YefM-YoeB TA complex of Escherichia coli consists of YoeB toxin that is counteracted by YefM antitoxin. When liberated from the complex, YoeB acts as an endoribonuclease, preferentially cleaving 3' of purine nucleotides. Here we demonstrate that yefM-yoeB is transcriptionally autoregulated. YefM, a dimeric protein with extensive secondary structure revealed by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, is the primary repressor, whereas YoeB is a repression enhancer. The operator site 5' of yefM-yoeB comprises adjacent long and short palindromes with core 5'-TGTACA-3' motifs. YefM binds the long palindrome, followed sequentially by short palindrome recognition. In contrast, the repressor-corepressor complex recognizes both motifs more avidly, impyling that YefM within the complex has an enhanced DNA-binding affinity compared to free YefM. Operator interaction by YefM and YefM-YoeB is accompanied by structural transitions in the proteins. Paired 5'-TGTACA-3' motifs are common in yefM-yoeB regulatory regions in diverse genomes suggesting that interaction of YefM-YoeB with these motifs is a conserved mechanism of operon autoregulation. Artificial perturbation of transcriptional autorepression could elicit inappropriate YoeB toxin production and induction of bacterial cell suicide, a potentially novel antibacterial strategy.

Role of the RNA polymerase alpha subunits in CII-dependent activation of the bacteriophage lambda pE promoter: identification of important residues and positioning of the alpha C-terminal domains.

The bacteriophage lambda CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the -35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) to demonstrate that one alphaCTD binds near position -41 at p(E), whilst the other alphaCTD binds further upstream. The alphaCTD bound near position -41 is oriented such that its 261 determinant is in close proximity to sigma(70). The location of alphaCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for alphaCTD at promoters where activators bind sites overlapping the -35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of alphaCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in alphaCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of alphaCTD at the three lysogenic promoters may be different.

Toxicity of the bacteriophage lambda cII gene product to Escherichia coli arises from inhibition of host cell DNA replication.

The bacteriophage lambda cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the "lysis-versus-lysogeny" decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase alpha subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA(+) or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII.

SeqA-mediated stimulation of a promoter activity by facilitating functions of a transcription activator.

It was demonstrated recently that the SeqA protein, a main negative regulator of Escherichia coli chromosome replication initiation, is also a specific transcription factor. SeqA specifically activates the bacteriophage lambda pR promoter while revealing no significant effect on the activity of another lambda promoter, pL. Here, we demonstrate that lysogenization by bacteriophage lambda is impaired in E. coli seqA mutants. Genetic analysis demonstrated that CII-mediated activation of the phage pI and paQ promoters, which are required for efficient lysogenization, is less efficient in the absence of seqA function. This was confirmed in in vitro transcription assays. Interestingly, SeqA stimulated CII-dependent transcription from pI and paQ when it was added to the reaction mixture before CII, although having little effect if added after a preincubation of CII with the DNA template. This SeqA-mediated stimulation was absolutely dependent on DNA methylation, as no effects of this protein were observed when using unmethylated DNA templates. Also, no effects of SeqA on transcription from pI and paQ were observed in the absence of CII. Binding of SeqA to templates containing the tested promoters occurs at GATC sequences located downstream of promoters, as revealed by electron microscopic studies. In contrast to pI and paQ, the activity of the third CII-dependent promoter, pE, devoid of neighbouring downstream GATC sequences, was not affected by SeqA both in vivo and in vitro. We conclude that SeqA stimulates transcription from pI and paQ promoters in co-operation with CII by facilitating functions of this transcription activator, most probably by allowing more efficient binding of CII to the promoter region.