Genome Sequence of Corynebacterium glutamicum Phage MicyPS

TL;DR

This paper reports the genome sequence of a new bacteriophage, MicyPS, which infects Corynebacterium glutamicum and was found in soil in France.

Contribution

The paper introduces a new phage cluster EQ based on gene-content similarity with another phage.

Findings

MicyPS has a 78,208-bp genome encoding 115 protein-encoding genes and 5 tRNAs.

MicyPS was classified into a new phage cluster EQ based on gene-content similarity with PSonyx.

Abstract

MicyPS is a bacteriophage with siphovirus morphology infecting
Corynebacterium glutamicum
strain MB001. It was isolated from soil near a henhouse in Villiers-sur-Marne, France. Its 78,208-bp genome encodes 115 predicted protein-encoding genes and 5 tRNAs. Based on gene-content similarity with actinobacteriophage PSonyx, MicyPS was assigned to the new cluster EQ.

Figures

A](#f1) ). Negative-staining electron microscopy revealed a siphovirus morphology with capsids 75 (±4) nm in diameter and tails 320 (±26) nm long (n=20).

Full text

Description

Despite the industrial significance of *Corynebacterium glutamicum * in the production of glutamate and lysine, only seven bacteriophages infecting this species have been fully characterized to date with respect to both genome sequence and virion morphology (Moreau et al. 1995; Bukovska et al. 2006; Chen et al. 2008; Lobanova et al. 2017; Yomantas et al. 2018; Hünnefeld et al. 2021; Rossier et al. 2024). This limited number underlines an important gap in our understanding of phage diversity, biology, and their potential impact on fermentation processes involving C. glutamicum .

Phage MicyPS was isolated from soil near a henhouse in Villiers-sur-Marne, France (GPS 48.829132 N, 2.537859 E). Soil was incubated for 1 hour with 25 mL LB broth (5 g/L NaCl; 5 g/L yeast extract; 10 g/L tryptone), whilst shaking at 30° C. Following centrifugation at 4000 xg for 15 minutes, the supernatant was passed through a 0.45-μm filter and supplemented with 1 mM CaCl 2 and inoculated with C . glutamicum strain MB001, a derivative of the reference strain ATCC 13032 in which the prophages CGP1, CGP2, and CGP3 were deleted (Hünnefeld et al. 2021). After shaking for 24 hours at 30° C, enrichment cultures were centrifuged and the supernatant was filtered through a 0.22-μm filter. Five mL of the supernatant were then spotted on double agar plate supplemented with 1 mM CaCl 2 and strain MB001. After 24 hours of incubation at 30° C, individual plaques were visible and further purified through three rounds of single plaque picking and plating. Plaques were clear and between 0.5 and 3-mm in diameter ( Figure 1A ). Negative-staining electron microscopy revealed a siphovirus morphology with capsids 75 (±4) nm in diameter and tails 320 (±26) nm long (n=20).

Viral DNA was extracted using the PCI/SDS protocol (see Methods). NEB Ultra II FS Kit was used to prepare the library that was sequenced using Illumina NextSeq 1000, yielding 1,078,713 single-end 100-base reads. Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with Skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly (Martin 2011; Jiang et al. 2014). De novo assembly was performed with Newbler v2.9 as previously described (Russell 2018) and further checked with Consed v.29 and Unicycler (Gordon et al. 1998; Wick et al. 2017). The resulting genome was 78,208-bp in length, with a G+C content of 53.2% and circularly permutated ends. Based on gene content similarity of 76.2% with the previously singleton phage PSonyx (Rossier et al. 2024), phage MicyPS was assigned to the new cluster EQ in the Actinophage database PhagesDB (Russell and Hatfull 2017).

Genome annotation identified 120 putative genes including 5 tRNAs with the following tools: DNA master v5.23.6 (Pope and Jacobs-Sera 2018) which incorporates Glimmer v3.02 (Delcher et al. 2007) and GeneMark v2.5p (Besemer and Borodovsky 2005), Aragorn v1.2.40 (Laslett 2004), tRNAscan-SE v2.0 (Chan et al. 2021) and Phamerator (Cresawn et al. 2011). Start sites were selected based on gene length, minimal gaps or overlaps, RBS scores, and BLASTP alignment. Functional annotation was performed with BLASTP (Altschul et al. 1990) using PhagesDB and NCBI non-redundant databases (e values < 0.001) and with HHpred (Soding et al. 2005) using databases PDB_mmCIF70_30_Mar, Pfam-A_v37, Uniprot-Swissprot-viral70_3_Nov_2021, and NCBIConservedDomain(CD)_v3.19. Using DeepTMHMM v1.0.42 (Hallgren et al. 2022), nine proteins were predicted to localize to the membrane. Only 35% of putative genes had a predicted function. One region (representing 40% of the genome) is transcribed in the rightward direction and encodes proteins involved in virion assembly and host lysis. The next 40%, transcribed leftwards, contains tRNAs and genes likely involved in DNA replication. Located in this region, nine (of a total of 15) predicted genes have no significant similarity with any other predicted genes in the Actinophage database. These genes are termed orphams.  The final 20%, transcribed rightwards, end with a large putative gene which is predicted to encode a 2,873-amino-acid protein of unknown function. Both phages in cluster EQ (MicyPS and PSonyx) have no identifiable immunity repressor or integrase functions suggesting the use of the lytic cycle for replication.

Nucleotide sequence accession numbers

MicyPS is available at GenBank with Accession No. PV876931 and Sequence Read Archive (SRA) No. SRX28943171.

Methods

Viral DNA was extracted using the PCI/SDS protocol ( https://phagesdb.org/media/workflow/protocols/pdfs/PCI_SDS_DNA_Extraction_2.2013.pdf ).