Project description:Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.
Project description:Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNA-Tyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNA-Tyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNA-Lys, to counteract a tRNA anticodon defense system (PrrC170).
Project description:During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.
Project description:Bacteriophages (phages) are widespread in Streptococcus pneumoniae, with most strains carrying phage genomes integrated into the chromosome. RNA sequencing was utilised to explore whether phage gene expression could be detected. The pneumococcal reference strain PMEN3 (Spain9V-3), which contained two full-length phages and one partial phage, was grown in broth culture and mitomycin C was added to facilitate phage induction. PMEN3 culture samples were taken at sequential time points and RNA was extracted and sequenced.
Project description:By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to contend with suboptimal conditions that would otherwise reduce their fitness. In addition, certain types of dormancy like sporulation, can serve as a refuge from parasitic infections. Phages are unable to attach to spores, but their genomes can be entrapped in the resting structures and are able to resume infection upon host germination. Thus, dormancy has the potential to affect both the reproductive and survival components of phage fitness. Here, we characterized the distribution and diversity of sigma factors in nearly 3,500 phage genomes. Homologs of bacterial sigma factors that are responsible for directing transcription during sporulation were preferentially recovered in phages that infect spore-forming hosts. While non-essential for lytic infection, when expressed in Bacillus subtilis, we demonstrate that phage-encoded sigma factors activated sporulation gene networks and reduced spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate the expression of complex traits, like the transitions involved in bacterial dormancy.
Project description:Klebsiella pneumoniae has risen to prominence as a major threat to human health, with hypervirulent and drug-resistant lineages spreading globally. Given their antimicrobial resistant phenotypes, new therapies are required for the treatment of these infections, and bacteriophages (phages) that kill Klebsiella are being identified for use in phage therapy. In order to circumvent the evolution of phage-resistance taking hold the way that drug-resistance has, clear and considered actions are needed in selecting the phages that would be used in therapeutic cocktails. It is known that annotation of phage genomes is poor, potentially obscuring those phages with the most therapeutic potential. Here we show that phages isolated from infrequently sampled environments have features of therapeutic potential and developed a computational tool called STEP3 to understand the evolutionary features that distinguish the component parts of diverse phages, features that proved particularly suitable to detection of virion proteins with only distantly related homologies. These features were integrated into an ensemble framework to achieve a stable and robust prediction performance by STEP3. Proteomics-based analysis of two phages validated the prediction accuracy of STEP3 and revealed the virions contain component parts that include DNA-binding factors, otherwise unrecognizable capsule degradation enzymes and membrane translocation factors.
Project description:Several members of the Bacillus cereus group of bacteria carry lysogenic phages of the family Tectiviridae. The Bacilus cereus reference strain (ATCC 14579) harbor a linear plasmid (pBClin15) that is highly similar to the genomes of the Tectiviruses. Production of active phages has however never been reported for pBClin15 and the role of pBClin15 in B. cereus physiology has been enigmatic. We have for the first time demonstrated an effect of pBClin15 on the physiology of B. cereus ATCC 14579 whereby the wild type is more sensitive to DNA damaging antibiotics compared to a pBClin15 cured strain. The microarray experiments in this study were designed to highlight transcriptional differences between the B. cereus ATCC 14579 wild type and a plasmid cured variant that potentially could explain the impact of pBClin15 on the B. cereus physiology. Microarray experiments were carried out under non-stressful conditions (growth in logarithmic phase in LB medium) under which there were no phenotypic difference between the wild type and pBClin15 cured strain as well as under stressful conditions under which there were phenotypic differences between the two strain (growth in LB medium supplemented with 0.5M-5g norfloxacin per ml).