Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.IMPORTANCEThe roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

The dicBF operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. In this study, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon, and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to those of CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes carried on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. IMPORTANCE Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host, or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examined the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.

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.

Bacterial Rho-dependent transcription termination regulates many physiological processes. Here, we report that it controls the expression of toxin-antitoxin (TA) modules of cryptic prophages in E. coli. Microarray profiles of Rho mutants showed upregulation of genes of the CP4-6 and CP4-44 prophages, including their TA modules, that were validated by RT-qPCR. Analysis of the in vivo termination efficiency and the mRNA sequences of these prophages revealed the presence of many Rho-dependent terminators. The prophage TA modules exhibited synthetic lethality with the Rho mutants, indicating functional involvement of Rho-dependent termination in controlling these modules. Rho-dependent termination does not regulate most of the chromosomal TA modules. We conclude that Rho-dependent termination specifically silences the TA modules of prophages, thereby augmenting bacterial innate immunity.

Outer membrane vesicles (OMVs) are nanostructures mostly produced by blebbing of the outer membrane in Gram negative bacteria. They contain biologically active proteins and perform a variety of processes. OMV production is also a typical response to events inducing stress in the bacterial envelope. In these cases, hypervesiculation is regarded as a strategy to avoid the dangerous accumulation of undesired products within the periplasm. Several housekeeping genes influence the biogenesis of OMVs, including those correlated with peptidoglycan and cell wall dynamics. In this work, we have investigated the relationship between OMV production and the lysis module of the E. coli DLP12 cryptic prophage. This module is an operon encoding a holin, an endolysin and two spannins, and is known to be involved in cell wall maintenance. We find that deleting the lysis module increases OMV production, suggesting that during evolution this operon has been domesticated to regulate vesiculation, likely through the elimination of non-recyclable peptidoglycan fragments. We also show that the expression of the lysis module is negatively regulated by environmental stress stimuli as high osmolarity, low pH and low temperature. Our data further highlight how defective prophages finely contribute to bacterial host fitness.

In this study, we investigated the effect of cryptic prophage regions in a blaNDM-1-bearing plasmid, which was identified in a patient from South Korea, on the survival of bacteria against adverse environmental conditions. First, we conjugated the intact plasmid and plasmids with deleted cryptic prophages into Escherichia coli DH5α. The E. coli transconjugants carrying the plasmid with intact cryptic prophages showed increased survival during treatment with a high concentration of NaCl, high and low temperatures, an oxidative stressor (H2O2), and an immunological stressor (human serum). By contrast, the transconjugants carrying the plasmid with a single-cryptic prophage knockout did not show any change in survival rates. mRNA expression analyses revealed that the genes encoding sigma factor proteins were highly upregulated by the tested stressors and affected the expression of various proteins (antioxidant, cell osmosis-related, heat shock, cold shock, and universal stress proteins) associated with the specific defense against each stress. These findings indicate that a bacterial strain carrying a plasmid with intact carbapenemase gene and cryptic prophage regions exhibited an increased resistance against simulated environmental stresses, and cryptic prophages in the plasmid might contribute to this enhanced stress resistance. Our study indicated that the coselection of antibiotic resistance and resistance to other stresses may help bacteria to increase survival rates against adverse environments and disseminate.

Cells are social, and self-recognition is a conserved aspect of group behavior where cells assist kin and antagonize non-kin. However, the role of phage in self-recognition is unexplored. Here we find that a demarcation line is formed between different swimming Escherichia coli strains but not between identical clones; hence, motile cells discriminate between self and non-self. The basis for this self-recognition is a 49 kb, T1-type, lytic phage of the family Siphoviridae (named here SW1) that controls formation of the demarcation line by utilizing one of the host\'s cryptic prophage proteins, YfdM of CPS-53, to propagate. Critically, SW1 provides a conditional benefit to E. coli K-12 compared with the identical strain that lacks the phage. A demarcation line is also formed when strains harbor either the lysogenic phage ϕ80 or lambda and encounter siblings that lack the lysogen. In summary, bacteria can use phage to distinguish siblings that lack phage.

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.

The genome of Escherichia coli K-12 contains ten cryptic phages, altogether constituting about 3.6% of the genome in sequence. Among more than 200 predicted genes in these cryptic phages, 14 putative transcription factor (TF) genes exist, but their regulatory functions remain unidentified. As an initial attempt to make a breakthrough for understanding the regulatory roles of cryptic phage-encoded TFs, we tried to identify the regulatory function of CP4-6 cryptic prophage-encoded YagI with unknown function. After SELEX screening, YagI was found to bind mainly at a single site within the spacer of bidirectional transcription units, yagA (encoding another uncharacterized TF) and yagEF (encoding 2-keto-3-deoxy gluconate aldolase, and dehydratase, respectively) within this prophage region. YagEF enzymes are involved in the catabolism of xylose downstream from xylonate. We then designated YagI as XynR (regulator of xylonate catabolism), one of the rare single-target TFs. In agreement with this predicted regulatory function, the activity of XynR was suggested to be controlled by xylonate. Even though low-affinity binding sites of XynR were identified in the E. coli K-12 genome, they all were inside open reading frames, implying that the regulation network of XynR is still fixed within the CR4-6 prophage without significant influence over the host E. coli K-12.

Hundreds of small RNAs (sRNAs) have been identified in diverse bacterial species, and while the functions of most remain unknown, some regulate key processes, particularly stress responses. The sRNA DicF was identified over 25 years ago as an inhibitor of cell division but since then has remained uncharacterized. DicF consists of 53 nucleotides and is encoded by a gene carried on a prophage (Qin) in the genomes of many Escherichia coli strains. We demonstrated that DicF inhibits cell division via direct base pairing with ftsZ mRNA to repress translation and prevent new synthesis of the bacterial tubulin homolog FtsZ. Systems analysis using computational and experimental methods identified additional mRNA targets of DicF: xylR and pykA mRNAs, encoding the xylose uptake and catabolism regulator and pyruvate kinase, respectively. Genetic analyses showed that DicF directly base pairs with and represses translation of these targets. Phenotypes of cells expressing DicF variants demonstrated that DicF-associated growth inhibition is not solely due to repression of ftsZ, indicating that the physiological consequences of DicF-mediated regulation extend beyond effects on cell division caused by reduced FtsZ synthesis. IMPORTANCE sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages.