Persister cells survive severe stresses, including antibiotics, starvation, heat, oxidative conditions, and phage attack, by entering a dormant physiological state. They arise without genetic change and can resume growth once the stress is removed and nutrients are available. Critically, upon resuscitation, disease-causing persister cells can reconstitute infections. Although it is known that persister cells resuscitate in proportion to their ribosome content, it has remained unclear whether ribosome levels also influence the formation of persister cells. Here, we used fluorescence-activated cell sorting (FACS) to fractionate exponentially growing Escherichia coli cells into four populations spanning low to high ribosome levels and demonstrated, via antibiotic survival assays with both ampicillin and ciprofloxacin, that cells with low ribosome content form persister cells approximately 80-fold (ampicillin) and 50-fold (ciprofloxacin) more frequently than cells with population-average ribosome levels. These findings show that persister cell formation is inversely proportional to ribosome abundance. Cells with low ribosome levels are less metabolically active and therefore less capable of initiating stress responses like most cells; instead, they enter a dormant state in which antibiotic target activity is reduced, thereby promoting persister formation.

Persister cells survive any severe stress including antibiotics, starvation, heat, oxidative conditions, and phage attack, by entering a dormant physiological state. They arise without genetic change and can resume growth once the stress is removed and nutrients are available. Critically, upon resuscitation, persister cells can reconstitute infections. Although it is known that persister cells resuscitate in proportion to their ribosome content, it has remained unclear whether ribosome levels also influence the formation of persister cells. Here, we used fluorescence-activated cell sorting (FACS) to fractionate exponentially growing cells into four populations spanning low to high ribosome levels and demonstrated that cells with low ribosome content form persister cells approximately 80-fold more frequently than cells with population-average ribosome levels. These findings show that persister cell formation is inversely proportional to ribosome abundance. Cells with low ribosome levels are less metabolically-active and therefore less capable of initiating a stress response like most cells; instead, they become dormant. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, https://ror.org/021nxhr62, 2515448

Recombinases catalyze site-specific integration, excision, and inversion of DNA and are found in myriad defense islands; however, their function in phage-defense is unknown as they are frequently dismissed as markers of prophages. Here, we characterize the physiological role of the previously-uncharacterized serine recombinase PinR of Escherichia coli cryptic prophage rac and discover that it inhibits T2 phage infection by inverting a 1,797 bp segment in a different cryptic prophage e14 to inhibit T2 infection; this inversion leads to the formation of a novel protein from two spliced genes, StfE2, that we find blocks phage adsorption. Modeling shows StfE2 inhibits T2 phage adsorption by preventing Gp38 binding to its primary receptors porins FadL and OmpF. Corroborating the receptor-blocking hypothesis, T2 escape mutants evolve resistance to PinR phage defense by mutating gp38 to remove 16 aa in the hyper variable region 3. Therefore, we discovered the first recombinase-activated phage inhibition system. ### Competing Interest Statement The authors have declared no competing interest.

Recombinases catalyze site-specific integration, excision, and inversion of DNA and are often found adjacent to anti-phage system genes clustered in defense islands; however, their function in phage defense is unknown, as they are frequently dismissed as markers of prophages. Here, we characterize the physiological role of the previously uncharacterized serine recombinase PinQ (P segment inversion by Qin) of Escherichia coli cryptic prophage Qin and discover that it inhibits T2 phage infection by inverting a 1797 bp segment in a different cryptic prophage e14; this inversion leads to the formation of a novel protein from two chimeric genes, StfE2, that we find blocks phage adsorption. Modeling shows StfE2 inhibits T2 phage adsorption by preventing Gp38 binding to its primary receptors, porins FadL and OmpF. Corroborating the receptor-blocking hypothesis, T2 escape mutants evolve resistance to PinQ anti-phage defense by mutating gp38 in the hypervariable region 3. Therefore, we discovered the first recombinase-activated phage inhibition system.

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.

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.

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.

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.

Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined that Escherichia coli persister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors that relay the signal to ribosomes via the secondary messenger CAMP, and persisters wake and utilize chemotaxis to acquire nutrients.

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. formamide into formate and ammonia, hence fungal cultures generate the carboxylic acid product, but purified nitrilase does not. Analysis of the Genbank database indicates numerous variants that closely align with the archetypal Gloeocercospora sorghi sequence (M99044). The conservation at the protein level is generally greater than 80% identity across the entire protein length among the known or presumed cyanide hydratases (excluding the highly variant C-terminus), more than 60 such homologs are found in the database (BlastP against non-redundant protein database of December 2016). Homologs with somewhat lower sequence identity (between 70–80%) are also revealed by Blast searching although these have not been demonstrated to have activity on cyanide. Analyzing those matches with yet lower levels of identity reveal a variety of nitrilases with either known activity on other substrates or unknown activities. Not surprisingly the sequences are similarly conserved at the DNA level. The second member of this family, cyanide dihydratase (CynD), is of bacterial origin. It is frequently referred to as a cyanidase and converts cyanide into ammonia and formate: HCN+ 2H2O Æ NH3 + HCOOH (Jandhyala et al., 2003, 2005; Watanabe et al., 1998). Structural similarities between these two groups of cyanide-degrading nitrilases and other nitrilases are abundant. In addition to the abba fold motif and the conserved catalytic residues, their higher-order structure is that of a left-handed spiral oligomer, generally with 10 or more subunits. Hence, the native proteins have molecular masses ranging from 300 kDa to over 1 mDa depending upon species and conditions. The sequence conservation is generally quite high with a notable exception at the protein C-terminus that is highly variable (Jandhyala et al., 2005). Even among proteins from the same organism, strain types with otherwise Cyanide-degrading nitrilases in nature

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.

Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k(cat). However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein protein interfaces leading to the protein quaternary structure.

Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases.

Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from Bacillus pumilus, mutations in a predicted oligomeric surface region altered its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation. It was hypothesized that Y70 is playing an additional role necessary for CynD activity beyond influencing oligomerization. Here, we performed saturation mutagenesis at residue 70 and demonstrated that only tyrosine or phenylalanine is permissible for CynD activity. Furthermore, we show that other residues at this position are not only inactive, but have altered or disrupted oligomer conformations. These results suggest that Y70's essential role in activity is independent of its role in the formation of the spiral oligomer.

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/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin) is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.

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.

AimsTo investigate the impact of the highly variable C-terminal domain of cyanide dihydratase, a member of the nitrilase superfamily, on its activity and stability.Methods and ResultsGenerating and analysing the thermal stability and pH tolerance of chimeric cyanide dihydratase proteins has provided a platform to investigate domains within the C-terminus and their effect on quaternary structure of the protein. The protein oligomerization state was inferred from native protein size by gel exclusion chromatography.ConclusionsOur data indicates that the influence of the cyanide dihydratase C-terminus on thermal stability stems from its participation in oligomerization at the major C-surface interface. The formation of this surface is crucial for the activity and stability of CynD. Gel filtration chromatography of an N-terminal deletion mutant, CynD(pum) 303, revealed a defect in oligomerization, and another mutant R67C was suppressed by introduction of a heterologous C-terminus as a chimeric protein. This indicates that the C-terminus from Pseudomonas stutzeri stabilizes CynD by supporting oligomerization between dimers at the C-surface. The chimeric protein CynD(pum-stut) exhibited full activity at pH 9, a pH where the parent enzyme is nearly inactive, and retained 40% of its activity at pH 95 making it a unique pH tolerant mutant.Significance and Impact of the StudyThe study characterized a chimeric protein with remarkable thermal stability and tolerance to alkaline conditions, features essential for practical application as industrial cyanide solutions are maintained as highly alkaline solutions to prevent formation of hydrogen cyanide gas.

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 (CynD pum ) and P. stutzeri (CynD stut ) 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, CynD stut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynD stut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynD stut - 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 CynD pum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynD stut Δ302 or enhance the activity of full-length CynD stut and therefore does not act as a general stability motif.