Genome Sequences of Microbacterium foliorum Phages BouleyBill and Carostasia, isolated in France

TL;DR

This paper reports the genome sequences of two new bacteriophages found in France that infect a specific bacterial strain, expanding the known geographic range of these phage subclusters.

Contribution

The study expands the geographic distribution of phage subclusters EA4 and EA10 to Europe for the first time.

Findings

BouleyBill and Carostasia phages were isolated in France and infect Microbacterium foliorum strain NRRL B-24224.

The phages belong to subclusters EA4 and EA10, respectively, with genome sizes of 39,215-bp and 40,393-bp.

This is the first report of these subclusters being found in Europe.

Abstract

Bacteriophages BouleyBill and Carostasia, exhibiting siphovirus morphology, were isolated in France. Both infected
Microbacterium foliorum
strain NRRL B-24224. Their 39,215-bp and 40,393-bp genomes were assigned to subclusters EA4 and EA10, respectively, widening for the first time the known geographical distribution of these subclusters to Europe.

Figures

Tables

Phage name

Sample collection site (GPS coordinates)

Sample type

Isolation method

Approx. Shotgun coverage (fold)

No. of 150-bp single-end reads

Genome accession

SRA accession

Genome length (bp)

Genome ends

G + C content (%)

No. of protein-coding genes

No. of tRNAs

Sub-cluster

BouleyBill

Dourdan, FR (48.533211 N, 2.007825 E)

Garden compost

Direct

1,359

375,027

PQ114747

SRX24123901

39,215

Circularly Permuted

64.2

55

1

EA4

Carostasia

Palaiseau, FR (48.713604 N, 2.248526 E)

Sprigs of grass

Direct

1,673

476,899

PQ114742

SRX24123902

40,393

Circularly Permuted

63.8

63

0

EA10

Full text

Description

Bacteriophages are abundant and highly diverse viruses that infect bacteria. While approximately 600 different phages infecting the Gram-positive bacterium Microbacterium foliorum have been characterized, none have yet been isolated in Europe, leaving their geographic distribution and genetic diversity underexplored (Russell and Hatfull 2017; Jacobs-Sera et al. 2020; Milhaven et al. 2023)(see https://www.phagesdb.org, accessed on 1 December 2024).

Here, we report on the isolation of phages BouleyBill and Carostasia in France (Table 1). Compost or grass sprigs, respectively, were incubated for 1h in LB (5 g/L NaCl; 5 g/L yeast extract; 10 g/L tryptone), shaking at 30°C. Following centrifugation for 15 min at 4000xg, the supernatants were passed through a 0.22-µm filter and plated with top agar (LB 0.75% agar) supplemented with 1 mM CaCl2 and M . foliorum strain NRRL B-24224 (Russell et al. 2019). BouleyBill and Carostasia formed clear plaques with diameter ranges of 0.5-1 mm and 0.5-2 mm, respectively, after 24h at 30°C. They were further purified through two rounds of single plaque picking and plating (Fig. 1). Negative-staining transmission electron microscopy showed that both phages exhibit siphovirus morphology (Fig. 1). BouleyBill capsids were 55 (±3) nm in diameter and tails were 128 (±7) nm long (n = 13), while Carostasia capsids measured 60 (±1) nm and tails 134 (±5) nm (n = 24).

Phage DNA was extracted from high-titer lysates using the PCI/SDS protocol (https://phagesdb.org/media/workflow/protocols/pdfs/PCI_SDS_DNA_Extraction_2.2013.pdf). A library was prepared using the NEB Ultra II kit and sequenced using an Illumina Miseq instrument (v3 reagents). De novo assembly was performed with Newbler v2.9 as previously described (Russell 2018) and further checked with Consed v.29 (Gordon et al. 1998). The results are listed in Table 1. Based on gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesDB, both phages were assigned to cluster EA, but to different subclusters (Pope et al. 2017; Russell and Hatfull 2017), i.e. EA4 (containing 11 genomes with BouleyBill) and EA10 (6 genomes including Carostasia).

Gene prediction was performed using DNAmaster v5.23.6 (Pope and Jacobs-Sera 2018), which incorporates Glimmer v3.02 (Delcher et al. 2007) and Genmark v2.5p (Besemer and Borodovsky 2005). Auto-annotation was refined with Aragorn v1.2.41 (Laslett 2004), tRNAscan-SE v2.0 (Chan et al. 2021), Starterator v1.2 (http://phages.wustl.edu/starterator/), and Phamerator v579 (Cresawn et al. 2011). Start sites were selected based on gene length, minimal gaps or overlaps, RBS scores, and BlastP alignment, with Starterator used for further validation. Functional annotation was done with BlastP using NCBI nonredundant or actinobacteriophage databases (e values < 0.001) (Altschul et al. 1990; Russell and Hatfull 2017), and with HHpred (Soding et al. 2005) using databases PDB_mmCIF70_16_Aug, Pfam-A_v37, Uniprot-Swissprot-viral70_3_Nov_2021, and NCBI_Conserved_Domain(CD)_v3.19. Transmembrane domains were predicted using DeepTMHMM v1.0.24 (Hallgren et al. 2022). All software were used with default settings. BouleyBill and Carostasia contained 55 and 63 predicted protein-coding genes, with no putative functional prediction for 50% and 57% of identified genes, respectively.

As previously described for phages belonging to subcluster EA4 (Jacobs-Sera et al. 2020), phage BouleyBill carries a tRNA gene and its tail assembly chaperones are produced through a putative programmed translational -1 frameshift, features that were not identified in Carostasia. BouleyBill contains two genes downstream of the major capsid protein gene, while Carostasia has only one, which differs from the two in BouleyBill. Using Phamerator, a comparison of genomic regions 18,629–21,723 bp in BouleyBill (in particular genes 24, 28 and 29) and 18,678–21,406 bp in Carostasia (genes 23, 26 and 28) shows that the endolysin and two other genes are not conserved. From position 30,564 in BouleyBill and 30,625 in Carostasia to the genome ends, only six genes encode proteins that are homologous between the two phages. Taken together, this report constitutes the first description of Microbacterium foliorum phages discovered in Europe. The overall relatedness of BouleyBill and Carostasia with fifteen phages isolated in Northern America or the Caribbean suggests that phages from subclusters EA4 and EA10 have a wide geographical distribution.

Table 1. Isolation, sequencing and genome characteristics of phages BouleyBill and Carostasia

**: **