Lethal perturbation of an Escherichia coli regulatory network is triggered by a restriction-modification system's regulator and can be mitigated by excision of the cryptic prophage Rac.

Bacterial gene regulatory networks orchestrate responses to environmental challenges. Horizontal gene transfer can bring in genes with regulatory potential, such as new transcription factors (TFs), and this can disrupt existing networks. Serious regulatory perturbations may even result in cell death. Here, we show the impact on Escherichia coli of importing a promiscuous TF that has adventitious transcriptional effects within the cryptic Rac prophage. A cascade of regulatory network perturbations occurred on a global level. The TF, a C regulatory protein, normally controls a Type II restriction-modification system, but in E. coli K-12 interferes with expression of the RacR repressor gene, resulting in de-repression of the normally-silent Rac ydaT gene. YdaT is a prophage-encoded TF with pleiotropic effects on E. coli physiology. In turn, YdaT alters expression of a variety of bacterial regulons normally controlled by the RcsA TF, resulting in deficient lipopolysaccharide biosynthesis and cell division. At the same time, insufficient RacR repressor results in Rac DNA excision, halting Rac gene expression due to loss of the replication-defective Rac prophage. Overall, Rac induction appears to counteract the lethal toxicity of YdaT. We show here that E. coli rewires its regulatory network, so as to minimize the adverse regulatory effects of the imported C TF. This complex set of interactions may reflect the ability of bacteria to protect themselves by having robust mechanisms to maintain their regulatory networks, and/or suggest that regulatory C proteins from mobile operons are under selection to manipulate their host's regulatory networks for their own benefit.

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.

Use of transcriptional slippage for diverse gene expression.

We present here an alternative for two-promoter systems ensuring highly diverse expression of several genes from a single promoter. This approach assumes an introduction of a deletion mutation into an A/T homopolymeric run in a gene's proximal part, and employs the transcriptional slippage mechanism for insertion-dependent reinstatement of the proper reading frame by the T7 RNA polymerase.

Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies.

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression.

Non-programmed transcriptional frameshifting is common and highly RNA polymerase type-dependent.

BACKGROUND: The viral or host systems for a gene expression assume repeatability of the process and high quality of the protein product. Since level and fidelity of transcription primarily determines the overall efficiency, all factors contributing to their decrease should be identified and optimized. Among many observed processes, non-programmed insertion/deletion (indel) of nucleotide during transcription (slippage) occurring at homopolymeric A/T sequences within a gene can considerably impact its expression. To date, no comparative study of the most utilized Escherichia coli and T7 bacteriophage RNA polymerases (RNAP) propensity for this type of erroneous mRNA synthesis has been reported. To address this issue we evaluated the influence of shift-prone A/T sequences by assessing indel-dependent phenotypic changes. RNAP-specific expression profile was examined using two of the most potent promoters, ParaBAD of E. coli and φ10 of phage T7. RESULTS: Here we report on the first systematic study on requirements for efficient transcriptional slippage by T7 phage and cellular RNAPs considering three parameters: homopolymer length, template type, and frameshift directionality preferences. Using a series of out-of-frame gfp reporter genes fused to a variety of A/T homopolymeric sequences we show that T7 RNAP has an exceptional potential for generating frameshifts and is capable of slipping on as few as three adenine or four thymidine residues in a row, in a flanking sequence-dependent manner. In contrast, bacterial RNAP exhibits a relatively low ability to baypass indel mutations and requires a run of at least 7 tymidine and even more adenine residues. This difference comes from involvement of various intrinsic proofreading properties. Our studies demonstrate distinct preference towards a specific homopolymer in slippage induction. Whereas insertion slippage performed by T7 RNAP (but not deletion) occurs tendentiously on poly(A) rather than on poly(T) runs, strong bias towards poly(T) for the host RNAP is observed. CONCLUSIONS: Intrinsic RNAP slippage properties involve trade-offs between accuracy, speed and processivity of transcription. Viral T7 RNAP manifests far greater inclinations to the transcriptional slippage than E. coli RNAP. This possibly plays an important role in driving bacteriophage adaptation and therefore could be considered as beneficial. However, from biotechnological and experimental viewpoint, this might create some problems, and strongly argues for employing bacterial expression systems, stocked with proofreading mechanisms.

Evaluation of GFP reporter utility for analysis of transcriptional slippage during gene expression.

BACKGROUND: Epimutations arising from transcriptional slippage seem to have more important role in regulating gene expression than earlier though. Since the level and the fidelity of transcription primarily determine the overall efficiency of gene expression, all factors contributing to their decrease should be identified and optimized. RESULTS: To examine the influence of A/T homopolymeric sequences on introduction of erroneous nucleotides by slippage mechanism green fluorescence protein (GFP) reporter was chosen. The in- or out-of-frame gfp gene was fused to upstream fragment with variable number of adenine or thymine stretches resulting in several hybrid GFP proteins with diverse amino acids at N-terminus. Here, by using T7 phage expression system we showed that the intensity of GFP fluorescence mainly depends on the number of the retained natural amino acids. While the lack of serine (S2) residue results in negligible effects, the lack of serine and lysine (S2K3) contributed to a significant reduction in fluorescence by 2.7-fold for polyA-based in-frame controls and twofold for polyTs. What is more, N-terminal tails amino acid composition was rather of secondary importance, since the whole-cell fluorescence differed in a range of 9-18% between corresponding polyA- and polyT-based constructs. CONCLUSIONS: Here we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP hybrid fluorescence mainly depends on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. Moreover, only in case of GFP hybrids with relatively short N-terminal tags the fluorescence level solely reflects production yield, what further indicates the impact of an individual slippage sequence. Our results demonstrate that in contrast to the E. coli enzyme, T7 RNA polymerase exhibits extremely high propensity to slippage even on runs as short as 3 adenine or 4 thymine residues.

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates.

Here, we report results on systematic analysis of DNA substrate preferences of three N6-adenine ß-class DNA methyltransferases that are part of the type II restriction-modification systems. The studied enzymes were: M.EcoVIII, M.HindIII and M.LlaCI, which although found in phylogenetically distant bacteria (γ-proteobacteria and low-GC Gram-positive bacteria), recognize the same palindromic specific sequence 5'-AAGCTT-3' and catalyze formation of N6-methyladenine at the first A-residue. As expected overall the enzymes share the most analyzed features, but they show also some distinct differences in substrate recognition. Therefore DNA methylation reactions were carried out not only under standard, but also under relaxed conditions using DMSO or glycerol. We found that all of these enzymes preferred DNA containing a hemimethylated target site, but differ in modification of ssDNA, especially more pronounced for M.EcoVIII under relaxed conditions. In these conditions they also have shown varied preferences toward secondary sites, which differ by one nucleotide from specific sequence. They preferred sequences with substitutions at the 1st (A1 → G/C) and at the 2nd position (A2 → C), while sites with substitutions at the 3rd position (G3 → A/C) were modified less efficiently. Kinetic parameters of the methylation reaction carried out by M.EcoVIII were determined. Methylation efficiency (kcat/Km) of secondary sites was 4.5-10 times lower when compared to the unmethylated specific sequences, whilst efficiency observed for the hemimethylated substrate was almost 4.5 times greater. We also observed a distinct effect of analyzed enzymes on unspecific interaction with DNA phosphate backbone. We concluded that for all three enzymes the most critical is the phosphodiester bond between G3-C4 nucleotides at the center of the target site.

RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes.

DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential.

Relaxed specificity of prokaryotic DNA methyltransferases results in DNA site-specific modification of RNA/DNA heteroduplexes.

RNA/DNA hybrid duplexes regularly occur in nature, for example in transcriptional R loops. Their susceptibility to modification by DNA-specific or RNA-specific enzymes is, thus, a biologically relevant question, which, in addition, has possible biotechnological implications. In this study, we investigated the activity of four isospecific DNA methyltransferases (M.EcoVIII, M.LlaCI, M.HindIII, M.BstZ1II) toward an RNA/DNA duplex carrying one 5'-AAGCUU-3'/3'-TTCGAA-5' target sequence. The analyzed enzymes belong to the ß-group of adenine N6-methyltransferases and recognize the palindromic DNA sequence 5'-AAGCTT-3'/3'-TTCGAA-5'. Under standard conditions, none of these isospecific enzymes could detectibly methylate the RNA/DNA duplex. However, the addition of agents that generally relax specificity, such as dimethyl sulfoxide (DMSO) and glycerol, resulted in substantial methylation of the RNA/DNA duplex by M.EcoVIII and M.LlaCI. Only the DNA strand of the RNA/DNA duplex was methylated. The same was not observed for M.HindIII or M.BstZ1II. This is, to our knowledge, the first report that demonstrates such activity by prokaryotic DNA methyltransferases. Possible applications of these findings in a laboratory practice are also discussed.

Molecular characterization of the DNA methyltransferase M1.NcuI from Neisseria cuniculi ATCC 14688.

The methyltransferase M1.NcuI is a member of the restriction-modification system in Neisseria cuniculi ATCC14688 and recognizes the asymmetric pentanucleotide sequence 5'-GAAGA-3'/3'-CTTCT-5'. We purified M1.NcuI to electrophoretic homogeneity using a four-step chromatographic procedure. M1.NcuI is a protein with M(r)=32,000+/-1000 under denaturing conditions. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine to the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. M1.NcuI, like many other methyltransferases, occurs as a monomer in solution, as determined by gel filtration. Divalent cations inhibit the methylation activity of M1.NcuI. Optimal enzyme activity was observed at a pH of 8.0. M1.NcuI cross-reacted with anti-M1.MboII serum which reflects the similarity of M1.NcuI with M1.MboII at the amino acid level. The gene coding for the enzyme, designated ncuIM1, was cloned, sequenced and overexpressed in Escherichia coli. The structural gene is 780 nucleotides in length coding for a protein of 259 amino acids (M(r) 30,098). The presence and distribution of nine highly conserved amino acid sequence motifs and a putative target recognition domain in the enzyme structure suggest that M1.NcuI, similar to M1.MboII and M1.HpyAII, belongs to N(6)-adenine beta-class DNA methyltransferases.