Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella.

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.

CRISPR-Cas Controls Cryptic Prophages.

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

Cyanide bioremediation: the potential of engineered nitrilases.

The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes. The genetic approaches that have been used to date for engineering improved enzymes are described; however, recent structural insights provide a promising new approach.

RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli.

For toxin/antitoxin (TA) systems, no toxin has been identified that functions by cleaving DNA. Here, we demonstrate that RalR and RalA of the cryptic prophage rac form a type I TA pair in which the antitoxin RNA is a trans-encoded small RNA with 16 nucleotides of complementarity to the toxin mRNA. We suggest the newly discovered antitoxin gene be named ralA for RalR antitoxin. Toxin RalR functions as a non-specific endonuclease that cleaves methylated and unmethylated DNA. The RNA chaperone Hfq is required for RalA antitoxin activity and appears to stabilize RalA. Also, RalR/RalA is beneficial to the Escherichia coli host for responding to the antibiotic fosfomycin. Hence, our results indicate that cryptic prophage genes can be functionally divergent from their active phage counterparts after integration into the host genome.

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.

The MqsR/MqsA toxin/antitoxin system protects Escherichia coli during bile acid stress.

Toxin/antitoxin (TA) systems are ubiquitous within bacterial genomes, and the mechanisms of many TA systems are well characterized. As such, several roles for TA systems have been proposed, such as phage inhibition, gene regulation and persister cell formation. However, the significance of these roles is nebulous due to the subtle influence from individual TA systems. For example, a single TA system has only a minor contribution to persister cell formation. Hence, there is a lack of defining physiological roles for individual TA systems. In this study, phenotype assays were used to determine that the MqsR/MqsA type II TA system of Escherichia coli is important for cell growth and tolerance during stress from the bile salt deoxycholate. Using transcriptomics and purified MqsR, we determined that endoribonuclease toxin MqsR degrades YgiS mRNA, which encodes a periplasmic protein that promotes deoxycholate uptake and reduces tolerance to deoxycholate exposure. The importance of reducing YgiS mRNA by MqsR is evidenced by improved growth, reduced cell death and reduced membrane damage when cells without ygiS are stressed with deoxycholate. Therefore, we propose that MqsR/MqsA is physiologically important for E. coli to thrive in the gallbladder and upper intestinal tract, where high bile concentrations are prominent.

Toxin YafQ increases persister cell formation by reducing indole signalling.

Persister cells survive antibiotic and other environmental stresses by slowing metabolism. Since toxins of toxin/antitoxin (TA) systems have been postulated to be responsible for persister cell formation, we investigated the influence of toxin YafQ of the YafQ/DinJ Escherichia coli TA system on persister cell formation. Under stress, YafQ alters metabolism by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites. Production of YafQ increased persister cell formation with multiple antibiotics, and by investigating changes in protein expression, we found that YafQ reduced tryptophanase levels (TnaA mRNA has 16 putative YafQ cleavage sites). Consistently, TnaA mRNA levels were also reduced by YafQ. Tryptophanase is activated in the stationary phase by the stationary-phase sigma factor RpoS, which was also reduced dramatically upon production of YafQ. Tryptophanase converts tryptophan into indole, and as expected, indole levels were reduced by the production of YafQ. Corroborating the effect of YafQ on persistence, addition of indole reduced persistence. Furthermore, persistence increased upon deleting tnaA, and persistence decreased upon adding tryptophan to the medium to increase indole levels. Also, YafQ production had a much smaller effect on persistence in a strain unable to produce indole. Therefore, YafQ increases persistence by reducing indole, and TA systems are related to cell signalling.

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole.

Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c-di-GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c-di-GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that breakdown c-di-GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c-di-GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP.

Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein.

The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195-206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.

Catabolic plasmid specifying polychlorinated biphenyl degradation in Cupriavidus sp. strain SK-4: mobilization and expression in a pseudomonad.

Strain SK-4, a polychlorinated biphenyl (PCB) degrader previously reported to utilize di-ortho-substituted biphenyl, was genotypically re-characterized as a species of Cupriavidus. The bacterium harbored a single plasmid (pSK4), which resisted curing and which, after genetic marking by a transposon (SK4Tn5), could be mobilized into a pseudomonad. Analysis of pSK4 in both the transconjugant and the wild type revealed that it specifies the genes coding for 2-hydroxy-2,4-pentadienoate degradation in addition to those of the upper biphenyl pathway. Expression of the benzoate metabolic pathway in the transconjugant is evidence suggesting that the benzoate catabolic genes are also localized on the plasmid. This implies that pSK4 codes for all the genes involved in biphenyl mineralization. It is therefore reasonable to propose that the plasmid is the determinant for the unique metabolic capabilities known to exist in Cupriavidus sp. strain SK-4.

Toxin GhoT of the GhoT/GhoS toxin/antitoxin system damages the cell membrane to reduce adenosine triphosphate and to reduce growth under stress.

Toxin/antitoxin (TA) systems perhaps enable cells to reduce their metabolism to weather environmental challenges although there is little evidence to support this hypothesis. Escherichia coli GhoT/GhoS is a TA system in which toxin GhoT expression is reduced by cleavage of its messenger RNA (mRNA) by antitoxin GhoS, and TA system MqsR/MqsA controls GhoT/GhoS through differential mRNA decay. However, the physiological role of GhoT has not been determined. We show here through transmission electron microscopy, confocal microscopy and fluorescent stains that GhoT reduces metabolism by damaging the membrane and that toxin MqsR (a 5'-GCU-specific endoribonuclease) causes membrane damage in a GhoT-dependent manner. This membrane damage results in reduced cellular levels of ATP and the disruption of proton motive force (PMF). Normally, GhoT is localized to the pole and does not cause cell lysis under physiological conditions. Introduction of an F38R substitution results in loss of GhoT toxicity, ghost cell production and membrane damage while retaining the pole localization. Also, deletion of ghoST or ghoT results in significantly greater initial growth in the presence of antimicrobials. Collectively, these results demonstrate that GhoT reduces metabolism by reducing ATP and PMF and that this reduction in metabolism is important for growth with various antimicrobials.

A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS.

Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

Phages produce persisters.

Arguably, the greatest threat to bacteria is phages. It is often assumed that those bacteria that escape phage infection have mutated or utilized phage-defence systems; however, another possibility is that a subpopulation forms the dormant persister state in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defence systems more time to clear the phage infection. Here, we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages and sequencing their genomes. We found that bacteria in plaques can escape phage attack both by mutation (i.e. become resistant) and without mutation (i.e. become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g. causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially growing cells. Furthermore, our results seem general in that phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

Arrested protein synthesis increases persister-like cell formation.

Biofilms are associated with a wide variety of bacterial infections and pose a serious problem in clinical medicine due to their inherent resilience to antibiotic treatment. Within biofilms, persister cells comprise a small bacterial subpopulation that exhibits multidrug tolerance to antibiotics without undergoing genetic change. The low frequency of persister cell formation makes it difficult to isolate and study persisters, and bacterial persistence is often attributed to a quiescent metabolic state induced by toxins that are regulated through toxin-antitoxin systems. Here we mimic toxins via chemical pretreatments to induce high levels of persistence (10 to 100%) from an initial population of 0.01%. Pretreatment of Escherichia coli with (i) rifampin, which halts transcription, (ii) tetracycline, which halts translation, and (iii) carbonyl cyanide m-chlorophenylhydrazone, which halts ATP synthesis, all increased persistence dramatically. Using these compounds, we demonstrate that bacterial persistence results from halted protein synthesis and from environmental cues.

Type II toxin/antitoxin MqsR/MqsA controls type V toxin/antitoxin GhoT/GhoS.

Toxin endoribonucleases of toxin/antitoxin (TA) systems regulate protein production by selectively degrading mRNAs but have never been shown to control other TA systems. Here we demonstrate that toxin MqsR of the MqsR/MqsA system enriches toxin ghoT mRNA in vivo and in vitro, since this transcript lacks the primary MqsR cleavage site 5'-GCU. GhoT is a membrane toxin that causes the ghost cell phenotype, and is part of a type V TA system with antitoxin GhoS that cleaves specifically ghoT mRNA. Introduction of MqsR primary 5'-GCU cleavage sites into ghoT mRNA reduces ghost cell production and cell death likely due to increased degradation of the altered ghoT mRNA by MqsR. GhoT also prevents cell elongation upon the addition of low levels of ampicillin. Therefore, during stress, antitoxin GhoS mRNA is degraded by toxin MqsR allowing ghoT mRNA translation to yield another free toxin that forms ghost cells and increases persistence. Hence, we show that GhoT/GhoS is the first TA system regulated by another TA system.

Antitoxin MqsA helps mediate the bacterial general stress response.

Although it is well recognized that bacteria respond to environmental stress through global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin-antitoxin systems are vital in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease, resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin-antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).

Draft genome sequence of the cyanide-utilizing bacterium Pseudomonas fluorescens strain NCIMB 11764.

We report here the 6.97-Mb draft genome sequence of Pseudomonas fluorescens strain NCIMB 11764, which is capable of growth on cyanide as the sole nitrogen source. The draft genome sequence allowed the discovery of several genes implicated in enzymatic cyanide turnover and provided additional information contributing to a better understanding of this organism's unique cyanotrophic ability. This is the first sequenced genome of a cyanide-assimilating bacterium.

Antitoxin DinJ influences the general stress response through transcript stabilizer CspE.

Antitoxins are becoming recognized as proteins that regulate more than their own synthesis; for example, we found previously that antitoxin MqsA of the Escherichia coli toxin/antitoxin (TA) pair MqsR/MqsA directly represses the gene encoding the stationary-phase sigma factor RpoS. Here, we investigated the physiological role of antitoxin DinJ of the YafQ/DinJ TA pair and found DinJ also affects the general stress response by decreasing RpoS levels. Corroborating the reduced RpoS levels upon producing DinJ, the RpoS-regulated phenotypes of catalase activity, cell adhesins and cyclic diguanylate decreased while swimming increased. Using a transcriptome search and DNA-binding assays, we determined that the mechanism by which DinJ reduces RpoS is by repressing cspE at the LexA palindrome; cold-shock protein CspE enhances translation of rpoS mRNA. Inactivation of CspE abolishes the ability of DinJ to influence RpoS. Hence, DinJ influences the general stress response indirectly by regulating cspE.