Complete genome sequences of StopSmel and Aussie, two Mu-like bacteriophages of Sinorhizobium meliloti

ABSTRACT

We report the complete genome sequences of two bacteriophages, Aussie and StopSmel, isolated from soil using the host Sinorhizobium meliloti NRRL L-50. The genomes are similar in length and gene content and share 76% nucleotide identity. Comparative analysis of Aussie and StopSmel identified core functional modules associated with Mu-like bacteriophages.

ANNOUNCEMENT

Members of the Rhizobiales order (Alphaproteobacteria) are of broad interest for their importance in agriculture, as they include beneficial and pathogenic bacteria (1). Sinorhizobium meliloti belongs to Rhizobiales and is well characterized for its ability to form symbiotic nitrogen fixation nodules on leguminous plants (2–4). Though the study of the bacteriophages that infect nitrogen-fixing rhizobia has been low compared to enteric bacteria (5), there have been increasing numbers of reported S. meliloti phages and prophages (6, 7).

Phages Aussie and StopSmel were isolated from soil collected in Fort Pierce, FL, USA (Table 1). Enrichment cultures were prepared by incubating 10 grams of soil with 0.5 mL of overnight culture of host S. meliloti NRRL L-50 in 50 mL of tryptone yeast broth containing 0.45% CaCl₂ and incubated at 28°C for 48 h with shaking at 90 rpm (6, 8). The culture was then passed through a 0.22-µm filter, and the presence of phage was determined by plaque assay. Three rounds of plaque purifications were conducted, during which time both phages consistently formed clear, round plaques approximately 1–2 mm in diameter (Fig. 1A and B). High-titer lysates were prepared and used for DNA isolation using the Promega Wizard DNA cleanup kit. Nextera XT library preparation (Illumina, San Diego, CA, USA) and Illumina NextSeq 2000 DNA sequencing were conducted by Microbial Genome Sequencing Center (USA) generating 151-bp paired-end reads. The raw reads were merged and assembled using Geneious Prime de novo assembler (v2021.2.2), resulting in complete genomes for Phage Aussie, with a length of 39,025 bp and 526-fold coverage, and Phage StopSmel, with a length of 37,851 bp and 1,572-fold coverage (Table 1). Both Aussie and StopSmel were identified to have genome ends consistent with transposing phages, as the termini of both phage genomes were determined by the presence of heterogeneous host DNA sequencing reads flanking the genome ends, and the characteristic 5′-TG-CA-3′ conserved ends common to Mu-like phage (9, 10). The GC content for phages Aussie and StopSmel is 60.8% and 61.9%, respectively. The phage genomes share 76% nucleotide identity by BLASTn global pairwise alignment. Genomic characterization of both phages and negative staining electron microscopy of StopSmel showing a non-contractile tail of 123 nm and capsid of 54 nm support classifying these phages as having siphovirus-like morphology (Fig. 1C).

TABLE 1.

GenBank, SRA accession numbers, collection location, and genome sequencing results

Phage name	GenBank accession no.	SRA accession no.	Collection location (GPS coordinates)	Number of reads	Average coverage (×)	Genome length (bp)	GC content (%)	No. of genes
Aussie	OR786373	SRX17705902	27.4226N −80.3568W	111,835	526	39,025	60.8	53
StopSmel	OR786374	SRX17705903	27.4228N −80.3560W	360,650	1,572	37,851	61.9	52

Fig 1.

Plaque morphology for phages (A) Aussie and (B) StopSmel infecting Sinorhizobium meliloti NRRL L-50 after incubation on tryptone yeast agar plates at 28°C for 48 h. (C) Electron micrograph of StopSmel prepared by applying 10 µL of phage solution to the top of 200 mesh formvar-coated copper grids (Ted Pella, Redding, CA, USA) for 1 minute. Phage solutions were wicked and grids were then stained with a 1% uranyl acetate solution for 1 minute. TEM was performed to visualize the phage morphology using an accelerating voltage of 25 kV. StopSmel has siphovirus morphology with a non-contractile tail of 123 nm and capsid of 54 nm (scale bar = 200 nm). Samples viewed with a Hitachi 4800 STEM electron microscope (USDA, ARS, U.S. Horticultural Research Lab, Fort Pierce, FL, USA).

Genome annotation was performed using the default settings of PECAAN v20221109 (https://discover.kbrinsgd.org), DNAMaster v5.23.6 (http://cobamide2.bio.pitt.edu), GLIMMER v3.02 (11), GeneMark v2.5 (12), and Aragorn v1.2.41 (13). Functions were assigned using BLASTp (14) and HHpred (15). A total of 53 genes were predicted in Aussie, with 22 assigned protein functions. StopSmel has 52 genes, of which 21 were assigned function. No tRNAs were identified in either genome. The genomes of bacteriophage Mu and other Mu-like bacteriophages have well-characterized functional blocks (16–18), which were identified in both genomes. Orthologs of genes for the lysogenic and lytic repression, c and ner, were identified. Additional Mu-like gene functions were called for transposases A and B, GemA, and Gam. Genes encoding the transcriptional regulators Mor and C were also present in both genomes, along with late genes related to structural phage proteins and lysis.

DATA AVAILABILITY

GenBank and SRA accession numbers for Aussie are OR786373 and SRX17705902, respectively. The GeneBank accession and SRA accession numbers for StopSmel are OR786374 and SRX17705903, respectively.