Genome Sequence of the Bacteriophage CL31 and Interaction with the Host Strain Corynebacterium glutamicum ATCC 13032

Abstract

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4%). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the ∆resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope

“…As with the Tn-Seq analysis, the majority of the isolates had mutations in genes required for mycolic acid synthesis ( Figure 7—Table supplement 1 ), such as fadD2, pccB, and otsA or genes known to be associated with defects in mycolic acid synthesis, such as deoC (41). These findings were consistent with a recent report in which pks and pccB mutations were also found to promote CL31 resistance (17). In addition to mutations in well-characterized genes, we also isolated phage resistant alleles in a gene of unknown function, cgp_0475, including a five-codon duplication (+EVLPL) within the reading frame.…”

“…Most of the plaques were large and turbid, but a small percentage were small and clear. A similar heterogeneity in plaque morphology for lab-adapted CL31 was recently reported, but the cause of the phenomenon was not investigated (17). Phage purified from the clear plaques retained this phenotype and gave rise to only clear plaques on the MB001 host (Figure 1B).…”

“…Whole genome sequencing of the clear plaque CL31 isolate ( CP CL31) identified single nucleotide polymorphisms (SNPs) in the 3’ end of clg55 , which encodes a predicted tail protein in the structural protein locus of the genome ( Figure 1—table supplement 1 ). Notably, previous work has identified a putative variable region within Clg55, independent of the region in which these mutations occur (17), suggesting that clg55 is under selection, possible due to its role in receptor recognition. Four additional CP CL31 variants were isolated, and Sangar sequencing revealed that all also had mutations in clg55 , further implicating this structural gene in the in the altered plaque morphology phenotype ( Figure 1— table supplement 1 ).…”

Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope

“…Most of the plaques were large and turbid, but a small percentage were small and clear. A similar heterogeneity in plaque morphology for lab-adapted CL31 was recently reported, but the cause of the phenomenon was not investigated ( Hünnefeld et al, 2021 ). Phage purified from the clear plaques retained this phenotype and gave rise to only clear plaques on the MB001 host ( Figure 1B ).…”

“…Whole-genome sequencing of the clear plaque CL31 isolate ( CP CL31) identified single nucleotide polymorphisms (SNPs) in the 3’ end of clg55 , which encodes a predicted tail protein in the structural protein locus of the genome ( Figure 1—source data 1 ). Notably, previous work has identified a putative variable region within Clg55, independent of the region in which these mutations occur ( Hünnefeld et al, 2021 ), suggesting that clg55 is under selection, possibly due to its role in receptor recognition. Four additional CP CL31 variants were isolated, and Sanger sequencing revealed that all also had mutations in clg55 , further implicating this structural gene in the altered plaque morphology phenotype ( Figure 1—source data 1 ).…”