Molecular basis for lethal cross-talk between two unrelated bacterial transcription factors - the regulatory protein of a restriction-modification system and the repressor of a defective prophage.

Bacterial gene expression depends on the efficient functioning of global transcriptional networks, however their interconnectivity and orchestration rely mainly on the action of individual DNA binding proteins called transcription factors (TFs). TFs interact not only with their specific target sites, but also with secondary (off-target) sites, and vary in their promiscuity. It is not clear yet what mechanisms govern the interactions with secondary sites, and how such rewiring affects the overall regulatory network, but this could clearly constrain horizontal gene transfer. Here, we show the molecular mechanism of one such off-target interaction between two unrelated TFs in Escherichia coli: the C regulatory protein of a Type II restriction-modification system, and the RacR repressor of a defective prophage. We reveal that the C protein interferes with RacR repressor expression, resulting in derepression of the toxic YdaT protein. These results also provide novel insights into regulation of the racR-ydaST operon. We mapped the C regulator interaction to a specific off-target site, and also visualized C protein dynamics, revealing intriguing differences in single molecule dynamics in different genetic contexts. Our results demonstrate an apparent example of horizontal gene transfer leading to adventitious TF cross-talk with negative effects on the recipient's viability. More broadly, this study represents an experimentally-accessible model of a regulatory constraint on horizontal gene transfer.

Increased Levels of (p)ppGpp Correlate with Virulence and Biofilm Formation, but Not with Growth, in Strains of Uropathogenic Escherichia coli.

Urinary tract infections are one of the most frequent bacterial diseases worldwide. UPECs are the most prominent group of bacterial strains among pathogens responsible for prompting such infections. As a group, these extra-intestinal infection-causing bacteria have developed specific features that allow them to sustain and develop in their inhabited niche of the urinary tract. In this study, we examined 118 UPEC isolates to determine their genetic background and antibiotic resistance. Moreover, we investigated correlations of these characteristics with the ability to form biofilm and to induce a general stress response. We showed that this strain collection expressed unique UPEC attributes, with the highest representation of FimH, SitA, Aer, and Sfa factors (100%, 92.5%, 75%, and 70%, respectively). According to CRA (Congo red agar) analysis, the strains particularly predisposed to biofilm formation represented 32.5% of the isolates. Those biofilm forming strains presented a significant ability to accumulate multi-resistance traits. Most notably, these strains presented a puzzling metabolic phenotype-they showed elevated basal levels of (p)ppGpp in the planktonic phase and simultaneously exhibited a shorter generation time when compared to non-biofilm-forming strains. Moreover, our virulence analysis showed these phenotypes to be crucial for the development of severe infections in the Galleria mellonella model.

Three Microbial Musketeers of the Seas: Shewanella baltica, Aliivibrio fischeri and Vibrio harveyi, and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.

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.

Virus-Host Interaction Gets Curiouser and Curiouser. PART II: Functional Transcriptomics of the E. coli DksA-Deficient Cell upon Phage P1vir Infection.

The virus-host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage-host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA- hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.

Virus-Host Interaction Gets Curiouser and Curiouser. PART I: Phage P1vir Enhanced Development in an E. coli DksA-Deficient Cell.

Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an E. coli global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1vir biology in an attempt to understand the basis of this phenomenon. We found several minor improvements in phage development in the dksA mutant host, including more efficient adsorption to bacterial cell and phage DNA replication. In addition, gene expression of the main repressor of lysogeny C1, the late promoter activator Lpa, and lysozyme are downregulated in the dksA mutant. We also found nucleotide substitutions located in the phage immunity region immI, which may be responsible for permanent virulence of phage P1vir. We suggest that downregulation of C1 may lead to a less effective repression of lysogeny maintaining genes and that P1vir may be balancing between lysis and lysogeny, although finally it is able to enter the lytic pathway only. The mentioned improvements, such as more efficient replication and more "gentle" cell lysis, while considered minor individually, together may account for the phenomenon of a more efficient P1 phage development in a DksA-deficient host.

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.

Identification of σE-Dependent Promoter Upstream of clpB from the Pathogenic Spirochaete Leptospira interrogans by Applying an E. coli Two-Plasmid System.

There is limited information on gene expression in the pathogenic spirochaete Leptospira interrogans and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of Leptospira to mammalian hosts. Thus, promoter and transcriptional start site identification are crucial for determining gene expression regulation and for the understanding of genetic regulatory mechanisms existing in Leptospira. Here, we characterized the promoter region of the L. interrogans clpB gene (clpBLi) encoding an AAA+ molecular chaperone ClpB essential for the survival of this spirochaete under thermal and oxidative stresses, and also during infection of the host. Primer extension analysis demonstrated that transcription of clpB in L. interrogans initiates at a cytidine located 41 bp upstream of the ATG initiation codon, and, to a lesser extent, at an adenine located 2 bp downstream of the identified site. Transcription of both transcripts was heat-inducible. Determination of clpBLi transcription start site, combined with promoter transcriptional activity assays using a modified two-plasmid system in E. coli, revealed that clpBLi transcription is controlled by the ECF σE factor. Of the ten L. interrogans ECF σ factors, the factor encoded by LIC_12757 (LA0876) is most likely to be the key regulator of clpB gene expression in Leptospira cells, especially under thermal stress. Furthermore, clpB expression may be mediated by ppGpp in Leptospira.

Autoregulation of greA Expression Relies on GraL Rather than on greA Promoter Region.

GreA is a well-characterized transcriptional factor that acts primarily by rescuing stalled RNA polymerase complexes, but has also been shown to be the major transcriptional fidelity and proofreading factor, while it inhibits DNA break repair. Regulation of greA gene expression itself is still not well understood. So far, it has been shown that its expression is driven by two overlapping promoters and that greA leader encodes a small RNA (GraL) that is acting in trans on nudE mRNA. It has been also shown that GreA autoinhibits its own expression in vivo. Here, we decided to investigate the inner workings of this autoregulatory loop. Transcriptional fusions with lacZ reporter carrying different modifications (made both to the greA promoter and leader regions) were made to pinpoint the sequences responsible for this autoregulation, while GraL levels were also monitored. Our data indicate that GreA mediated regulation of its own gene expression is dependent on GraL acting in cis (a rare example of dual-action sRNA), rather than on the promoter region. However, a yet unidentified, additional factor seems to participate in this regulation as well. Overall, the GreA/GraL regulatory loop seems to have unique but hard to classify properties.

[50th anniversary of discovering "magic spots" ­ the latest advances in (p)ppGpp research]. / 50-ta rocznica odkrycia "magicznych plamek" - najnowsze osiagniecia w badaniach nad (p)ppGpp

About 50 years ago, "magic spots" - mediators of the bacterial stringent response, were discovered and were later identified as guanosine tetra- and pentaphosphate (ppGpp and pppGpp, jointly referred to as (p)ppGpp). At first, it seemed that stringent response is associated only with bacterial response to amino acid starvation, however, it soon turned out that (p)ppGpp is synthesized in response to other stresses as well. The mentioned alarmones are found to exist in all known bacterial species, as well as in plants. In recent years, a significant progress has been made in research on (p)ppGpp metabolism. It is also known that the stringent response affects many cellular processes, among which its effect on transcription is the best characterized. Moreover, (p)ppGpp is involved in the DNA repair pathway associated with transcription. In addition, the stringent response inhibits cell division, mainly by hindering DNA replication. (p)ppGpp is also of significant medical importance - it is necessary for virulence of many bacterial species and for turning them into persisters, i.e. cells which have elevated tolerance to many antibiotics.

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis.

UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Differential regulation by ppGpp versus pppGpp in Escherichia coli.

Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In addition, expression of E. coli GppA, a pppGpp 5'-gamma phosphate hydrolase that converts pppGpp to ppGpp, is manipulated to fine tune differential accumulation of ppGpp and pppGpp. In vivo and in vitro experiments show that pppGpp is less potent than ppGpp with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter transcription inhibition, threonine operon promoter activation and RpoS induction. To provide further insights into regulation by (p)ppGpp, we have also determined crystal structures of E. coli RNA polymerase-σ(70) holoenzyme with ppGpp and pppGpp. We find that both nucleotides bind to a site at the interface between ß' and ω subunits.

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.

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.

Effects on growth by changes of the balance between GreA, GreB, and DksA suggest mutual competition and functional redundancy in Escherichia coli.

It is well known that ppGpp and DksA interact with bacterial RNA polymerase (RNAP) to alter promoter activity. This study suggests that GreA plays a major role and GreB plays a minor role in the ppGpp-DksA regulatory network. We present evidence that DksA and GreA/GreB are redundant and/or share similar functions: (i) on minimal medium GreA overproduction suppresses the growth defects of a dksA mutant; (ii) GreA and DksA overexpression partially suppresses the auxotrophy of a ppGpp-deficient strain; (iii) microarrays show that many genes are regulated similarly by GreA and DksA. We also find instances where GreA and DksA seem to act in opposition: (i) complete suppression of auxotrophy occurs by overexpression of GreA or DksA only in the absence of the other protein; (ii) PgadA and PgadE promoter fusions, along with many other genes, are dramatically affected in vivo by GreA overproduction only when DksA is absent; (iii) GreA and DksA show opposite regulation of a subset of genes. Mutations in key acidic residues of GreA and DksA suggest that properties seen here probably are not explained by known biochemical activities of these proteins. Our results indicate that the general pattern of gene expression and, in turn, the ability of Escherichia coli to grow under a defined condition are the result of a complex interplay between GreA, GreB, and DksA that also involves mutual control of their gene expression, competition for RNA polymerase binding, and similar or opposite action on RNA polymerase activity.

Circuitry linking the Csr and stringent response global regulatory systems.

CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

Imprecise transcription termination within Escherichia coli greA leader gives rise to an array of short transcripts, GraL.

We report that greA expression is driven by two strong, overlapping P1 and P2 promoters. The P1 promoter is sigma(70)-dependent and P2 is sigma(E)-dependent. Two-thirds of transcripts terminate within the leader region and the remaining third comprises greA mRNA. Termination efficiency seems to be unaffected by growth phase. Two collections of small 40-50 (initiating from P2) and 50-60 nt (from P1) RNA chains, termed GraL, are demonstrable in vivo and in vitro. We document that GraL arrays arise from an intrinsic terminator with an 11 bp stem followed by an AU(7)GCU(2) sequence. Atypical chain termination occurs at multiple sites; the 3'-ends differ by 1 nt over a range of 10 nt. Transcripts observed are shown to be insensitive to Gre factors and physically released from RNAP-DNA complexes. The abundance of individual chains within each cluster displays a characteristic pattern, which can be differentially altered by oligonucleotide probes. Multiple termination sites are particularly sensitive to changes at the bottom of the stem. Evolutionarily conserved GraL stem structures and fitness assays suggest a biological function for the RNA clusters themselves. Although GraL overexpression induces >/=3-fold transcriptional changes of over 100 genes, a direct target remains elusive.

TraR, a homolog of a RNAP secondary channel interactor, modulates transcription.

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.

ppGpp is the major source of growth rate control in E. coli.

It is widely accepted that the DNA, RNA and protein content of Enterobacteriaceae is regulated as a function of exponential growth rates; macromolecular content increases with faster growth regardless of specific composition of the growth medium. This phenomenon, called growth rate control, primarily involves regulation of ribosomal RNA and ribosomal protein synthesis. However, it was uncertain whether the global regulator ppGpp is the major determinant for growth rate control. Therefore, here we re-evaluate the effect of ppGpp on macromolecular content for different balanced growth rates in defined media. We find that when ppGpp is absent, RNA/protein and RNA/DNA ratios are equivalent in fast and slow growing cells. Moreover, slow growing ppGpp-deficient cells with increased RNA content, display a normal ribosomal subunit composition although polysome content is reduced when compared with fast growing wild-type cells. From this we conclude that growth rate control does not occur in the absence of ppGpp. Also, artificial elevation of ppGpp or introduction of stringent RNA polymerase mutants in ppGpp-deficient cells restores this control. We believe these findings strongly argue in favour of ppGpp and against redundant regulation of growth rate control by other factors in Escherichia coli and other enteric bacteria.