Genome of Enterobacteriophage Lula/phi80 and insights into its ability to spread in the laboratory environment

Abstract

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80's genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80's DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage's damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion.

Fig 1

The layout of the Lula/phi80 genome versus that of the lambda genome. The two genomes, which are 46,150 bp and 48,502 bp in length, respectively, are shown in their virion forms, linearized at the cos site, with the terminase genes on the left. Black arrows, genes transcribed from left to right; gray arrows, genes transcribed from right to left. The names of the important/interesting genes are indicated. The regions of shared sequence identity (averaging 70%) are indicated by gray parallelograms. The genome region labels are as follows: Head, head assembly and DNA packaging (terminase); Tail, tail assembly; Legs, tail fibers; Att, attachment site and integration/excision; Rec, homologous recombination and host exonuclease inhibition; Reg-Rep, regulation/decision making and replication initiation; Lysis, host cell lysis.

Fig 2

Comparison of the control regions of Lula/phi80 and lambda. The region bracketed by kil on the left and Rz on the right is shown, with the (mostly) control genes indicated above the genome line and control sequence elements below the genome line. The gray parallelograms indicate the three regions to which the ∼70% sequence identity between the two genomes is limited.

Fig 3

Lula/phi80 virion DNA modification and the ΔdamL mutant. Ethidium bromide (EtBr)-stained gels are framed and inverted for clarity. (A) Virion Lula/phi80 DNA is not cuttable by many restriction enzymes that cut genomic DNA from lysogens. Left and central panels, inverted pictures of EtBr-stained gels; right panel, Southern blot of the genomic DNA digest, hybridized with whole-genome Lula probe. (B) Scheme of the damL (34/35) gene and its position in the Lula/phi80 genome. The genome of Lula/phi80 is shown in lambda genome notation, opened at the cos site, with the att site indicated in the middle. Gene 34 starts with two Phe residues, while gene 35 ends with two Phe residues, with a +1 nucleotide frameshift between the two genes. (C) The virion DNA from the ΔdamL phage is equally uncuttable by EcoRI and HindIII. (D) The Δdam host has a 100-fold-higher titer of Lula/phi80 in overnight cultures of lysogens. Strains: Host WT Lula WT, EL103; Host WT Lula damL, EL124; Host dam Lula WT, EL125; Host dam Lula damL, EL123.

Fig 4

Lack of phenotypes of the ΔdinL mutant of Lula/phi80. (A) UV survival curves. All the values are means of three independent measurements ± standard errors of the means (SEM). Strains: Nonlysogen, AB1157; WT Lula/phi80, EL103; ΔdinL Lula/phi80, EL113. (B) Phage titer at 60 min or 180 min postinduction with 0, 4, or 10 J/m² UV. All the values are means of four independent measurements ± SEM. Strains: WT phage, EL103; ΔdinL phage, EL113. (C) Cell titer at 60 or 180 min postinduction with 0, 4, or 10 J/m² UV. All the values are means of four independent measurements ± SEM. Strains are the same as in panel B. (D) UV sensitivity of the WT-Lula WT lysogen versus that of the ΔdinI-Lula ΔdinL lysogen. The WT and ΔdinI nonlysogens are shown for comparison. All the values are means of five independent measurements ± SEM. Strains: ΔdinI-no prophage, EL120; WT-no prophage, AB1157; WT-Lula WT, EL103; ΔdinI-Lula ΔdinL, EL122.

Fig 5

Lula/phi80 genes 39 and 40, coding for GamL and its modifier Agt. (A) Overexpressed gene 39 is toxic to cells, especially if they are recA mutants. Strains: recA⁺, ER119; recA⁻, DH5α Z1. (B) Toxicity of the overexpressed gene 39 is alleviated by overexpression of gene 40, which has no effect on its own. The cells are all recA mutants (DH5α Z1). Plasmids: vector, pLAC11; 40, pER17; 39, pER18; 39 + 40, pER19. (C) Expression of gene 39, alone or together with gene 40, makes E. coli cells ExoV⁻ phenocopies, as shown by spotting of T4 2 mutant phages as a test for ExoV status of E. coli. Top, wild-type AB1157 cells are ExoV⁺ and therefore are nonpermissive for the T4 2 mutant, while the recBC mutants are ExoV⁻ and therefore allow T4 2 mutant plating. Bottom, plasmids are as in panel B. The lawn is wild-type strain ALS225. (D) Expression of GamL or Agt, alone or together, has little influence on the recombinational repair capacity of E. coli, as tested by sensitivity to UV. Plasmids are as in panel B, while the strain is ALS225.

Fig 6

The Lula/phi80 lysogen is an ExoV⁻ SbcCD⁻ phenocopy. (A) The Lula/phi80 lysogen allows T4 2 mutant plating but restricts T4 WT phage. Strains: WT nonlysogen, AB1157; WT lysogen, EL103; recBC nonlysogen, SK129; recBC lysogen, ER114. (B) Gene 39 (Lula/phi80 gamL), is the only gene required for plating of lambda-pal. On the right is a scheme of the truncated phi80 DNA in DH5α and the constructs we built. Lawns were made with the following strains: (i) recBC(Ts), SK129; (ii) DH5α Δ(attP-46), L-264; (iii) L-264/pTOPO:2; (iv) L-264/pER17; (v) L-264/pER18; and (vi) L-264/pER19. The phage is lambda-pal, MMS1632. (C) Titer of lambda-pal on a recA mutant in the AB1157 background, carrying various additional mutations or ExoV-inactivating plasmids. Strains according to bar number on the right: 1. DH5α; 2, DH5α/pER19; 3, JC10287; 4, AK10; 5, L-293; 6, JC10287 Lula/phi80 lysogen; 7, JC10287/pER19; 8, JC10287/pKM590; 9, JC10287/pKM590.