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.T he laboratory setting is, by design, an inhospitable environment, as very few species are capable of overcoming the researcher-imposed barriers for growth, multiplication, and spread. The well-known examples of organisms successfully propagating in the laboratory environment are mycoplasmas (76) and HeLa cells (39), both of which cross-contaminate mammalian cell cultures and spread through the laboratories around the world with appalling ease (48). Part of their success is their rapid growth and hardiness, giving them the ability to outgrow and outlive any laboratory strain competitors. Until recently, however, no such creature was known for cultures of rapidly growing microbes, which, precisely because of their speed of growth, effectively deny such chances to a contaminating competitor. This has changed with a description of a temperate phage, Lula, that infects Escherichia coli, converts it into a lysogen, and efficiently spreads in the laboratory setting, stealthily contaminating laboratory strains without researchers' knowledge (75). Lula contamination is apparently widespread yet not broadly recognized, and, because of this combination, it can be a scourge in shared facilities that handle both contaminated cultures (lysogens) and noncontaminated cultures (nonlysogens), resulting in a frustratingly high frequency of lysis and growth inhibition of the latter "for no ap...

Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells

“…Unlike the lambda prophage, the ϕ80 prophages seemed complete and thus likely active. This observation caught our attention, because ϕ80 is known as a highly infectious laboratory contaminant ( 30 , 31 ) and because we had widely distributed the strains of our TA module deletion series to other research groups. We therefore performed plaque assays on regular overnight cultures of various strains to determine the infectivity of ϕ80 present in our lysogens.…”

The Genomes, Proteomes, and Structures of Three Novel Phages That Infect the Bacillus cereus Group and Carry Putative Virulence Factors

“…This ATP-dependent double-strand DNA exonuclease is present in many bacteria and has been shown to eliminate or repair DNA secondary structures, preventing propagation of palindromic phage sequences (72)(73)(74). Phage Lula/phi80 encodes an inhibitor of the host SbcCD that facilitates its replication (75), suggesting the SbcCD encoded by JL/Shanette may inhibit replication of other phages or may act as a decoy/inhibitor complex to allow replication of JL/Shanette.…”

Cryptic prophage-encoded small protein DicB protectsEscherichia colifrom phage infection by inhibiting inner membrane receptor proteins

“…The genomes of λ phage and ϕ80 (a lambdoid phage) have strikingly similar organization, allowing for easy construction of recombinant phage (48, 49). One prominent difference between λ and ϕ80 is their use of different outer and inner membrane receptors.…”