Complete Genome Sequences of Lambdoid Phages 21, 434, and 434B and Several Lambda Hybrids

ABSTRACT

Recombinational hybrids between phage λ and its relatives were instrumental in the beginnings of molecular biology. Here, we report the complete genome sequences of lambdoid phages 21 and 434 and three of their λ hybrids. In addition, we describe 434B, where the entire lysis gene region was replaced by cryptic prophage sequences.

ANNOUNCEMENT

Escherichia coli phages 21 and 434, isolated by Wollman and Jacob in 1961 (1), were used to form genetic hybrids with the canonical phage λ. As they are close natural relatives, an analysis of these lambdoid phages and their hybrids was foundational to originating modern molecular genetics. Here, we report the complete genome sequences of phages 21 and 434; their λ recombinants λ imm434, λ imm21, and λ h434 imm21; and 434B, a clear plaque mutant of 434.

Phages were sourced as follows: phage 21, A. Campbell and R. Young; 434 wild type, S. Adhya; 434B clear mutant, C. Georgopoulos; λ imm21 and λ imm434, M. Feiss (original source A. Campbell); and λ h434 imm21, C. Georgopoulos (original source of the h434 host range allele, E. Signer). Phages were propagated on E. coli SKB-178 (2) by liquid infection in LB broth at 37°C. Virions were pelleted and purified by CsCl density step gradient centrifugation (3). Genomic DNA (gDNA) was isolated from purified virions with the phage DNA isolation kit (Norgen Biotek Corp., Thorold, Ontario) and sequenced individually by the Illumina MiSeq 150-bp paired-end run methodology with a 350-bp insert library prepared from a TruSeq DNA Nano kit at the University of Utah Sequencing Facility. Quality-controlled trimmed reads assembled into single >20-fold coverage contigs with Geneious 9.0.5 at default parameters and circular assemblies were reopened at known or homologous sticky ends (4–6). The phage 21 genome gave an identical assembly when sequenced by the dideoxy-nucleotide methodology at the Pittsburgh Bacteriophage Institute (7).

The genomes were annotated using the Center for Phage Technology Galaxy-Apollo phage annotation platform (8) with default parameters as follows: structural annotation, GLIMMER v3.0 and MetaGeneAnnotator v1.0 (9, 10); tRNA detection, ARAGORN v2.36 and tRNAscan-SE v2.0 (11, 12); gene function prediction, InterProScan v5.48, BLAST v2.9.0, TMHMM v2.0, LipoP v1.0, SignalP v5.0, and GenBank and Swiss-Prot databases, as well as HHPred using their HHSuite v3.0 Web server (13–20).

The phage 21 genome is 42,931 bp long with 73 protein-encoding genes and 2 tRNA genes. The 434 genome has 47,075 bp and 77 protein-encoding genes. The 434B clear plaque isolate was found to have a missense mutation in the cI repressor gene and a replacement of ∼5.5 kb, including the Q late activator, late promoter, and lysis gene region by a syntenic segment of the E. coli K-12 DLP12 cryptic prophage (21, 22). These sequences correct numerous single-base errors in previously reported segment sequences (Table 1). Essential genes in 21 and 434 are syntenic with phage λ, but the morons, including multigene loci encoding bacterial virulence factors, are different.

TABLE 1.

List of data for the phages in this study

Phage in BioProject PRJNA222858	Genome length (bp)	GC content (%)	GenBank accession no.	GenBank accession no. of sequence fragments deposited previously	BioSample accession no.	Sequence Read Archive accession no.	Total no. of reads
21	42,931	51	OL657228	M81255, M23775, AJ237660, DQ372054, M58702, M65239, AH001308, AH007390, M61865, AF017628, EU078592	SAMN20971088	SRR15608961	662,329
434	47,993	50	OL657226	M12904, Y00118, M12803, X73093, J02460, V00635, M60848	SAMN20971089	SRR15608960	698,456
434B	47,075	50	OL657227		SAMN20971090	SRR15608959	229,839
λ imm21	46,148	50	OM418625		SAMN20971091	SRR15608958	241,494
λ imm434	47,326	50	OM418626		SAMN20971092	SRR15608957	299,503
λ h434 imm21	43,452	51	OM418627		SAMN20971093	SRR15608956	341,536

The sequences of the hybrid phages λ imm21, λ imm434, and λ h434 imm21 confirm the genetically mapped locations of the nonhomologous 21 and 434 immunity segments, which confer host immunity to the same phage. The λ imm21 and λ imm434 hybrid sequences reveal additional 21 and 434 sequences, respectively, outside of the immunity regions; however, the interpretation of important early experiments using these phages is unaffected by these sequences (23, 24). The hybrid phage sequences identified 9 differences from the originally published λ sequence present in many extant laboratory λ strains (25).

Data availability.

The genome sequences and associated data for the reported genomes are available in GenBank under accession no. OL657226 to OL657228 and OM418625 to OM418627, and sequence reads are available under BioProject PRJNA222858 (Table 1).