Unauthorized Horizontal Spread in the Laboratory Environment: The Tactics of Lula, a Temperate Lambdoid Bacteriophage of Escherichia coli

We investigated the characteristics of a lambdoid prophage, nicknamed Lula, contaminating E. coli strains from several sources, that allowed it to spread horizontally in the laboratory environment. We found that new Lula infections are inconspicuous; at the same time, Lula lysogens carry unusually high titers of the phage in their cultures, making them extremely infectious. In addition, Lula prophage interferes with P1 phage development and induces its own lytic development in response to P1 infection, turning P1 transduction into an efficient vehicle of Lula spread. Thus, using Lula prophage as a model, we reveal the following principles of survival and reproduction in the laboratory environment: 1) stealth (via laboratory material commensality), 2) stability (via resistance to specific protocols), 3) infectivity (via covert yet aggressive productivity and laboratory protocol hitchhiking). Lula, which turned out to be identical to bacteriophage phi80, also provides an insight into a surprising persistence of T1-like contamination in BAC libraries

Chromosomal Fragmentation in Escherichia Coli: Its Absence in mutT Mutants and Its Mechanisms in seqA Mutants

202 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.While studying mutT and seqA mutants, we discovered a contaminating bacteriophage We studied, sequenced, and identified the phage as phi80. We looked at three interesting phenotypes: modification of the phage DNA to protect against restriction enzymes, sensitivity of lysogens to UV damage, and the toxicity when the gam gene was overexpressed in the cell.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

The mutT Defect Does Not Elevate Chromosomal Fragmentation in Escherichia coli Because of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications▿

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT→CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our ΔmutT allele elevates the AT→CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT→CG transversions in our mutT+ progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in ΔmutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the ΔmutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation

The DNA-damage sensitivity of the “Δ<i>ligB</i>” mutants and the assay for Lula presence.

<p><b>A.</b> MMS treatment. Strains: wild type, GR523; “Δ<i>ligB</i>”, LAP1. <b>B.</b> Nalidixic acid treatment. Strains as in “A”. <b>C.</b> Hydrogen peroxide treatment. Strains as in “A”. <b>D.</b> Assay for the presence of Lula. Supernatants of saturated cultures were spotted by 10 µl onto a lawn of uninfected cells (AB1157), and the plates were incubated at 30°C for 20 hours. <b>E.</b> SDS-sensitivity of Lula. 10 µl of the first, second and third dilution of a supernatant of saturated lysogen culture were spotted on a lawn of uninfected cells (AB1157). <b>F.</b> An inverted image of ethidium bromide-stained gel showing Lula virion DNA digested with BamHI.</p

Characteristics of Lambda and Lula/phi80 contributing to their different levels of spread in laboratory environment.

<p>Characteristics of Lambda and Lula/phi80 contributing to their different levels of spread in laboratory environment.</p

Interaction with Lambda, T4 and P1 phages.

<p><b>A.</b> Plating of Lula and various Lambdas on each other's lysogens. Strains are: non-lysogen, AB1157; Lambda i21 lysogen, MO (λi²¹); Lula lysogen, EL103. <b>B.</b> The lysogeny test. First, fresh colonies of a non-lysogen (AB1157), a Lula single lysogen (EL103) and a Lula/lambda double lysogen (MO (λi²¹)(phi80)(λ <i>vir</i>^R)) are streaked horizontally from left to right across two vertical phage lines — the left one made with a high-titer stock of Lula, the right one made with a high-titer stock of Lambda. The next day, since the non-lysogen grew equally well both before and after crossing the Lula streak, we took cells from indicated locations, streaked them to single colonies and passed these clones through “Lula contamination” test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011106#pone-0011106-g001" target="_blank">Fig. 1D</a>). The test confirmed that, although no lysis is apparent, cells become Lula lysogens after crossing the Lula line. <b>C.</b> Lula lysogens do not plate T4. Serial dilutions of T4 stock were spotted by 10 µl on lawns of either a non-lysogen (AB1157), Lula lysogen (EL103) or Lambda lysogen (EL104). <b>D.</b> Interaction of Lula and Lambda lysogens with P1. The P1 columns: P1 lysate was prepared in parallel on the two cultures of the same density, and the resulting P1 phage titer was determined either at 42°C (to inhibit Lula) or on a Lambda lysogen (to inhibit Lambda). “Lula” or “Lambda” columns: either mock-infected or P1-infected corresponding lysogen was taken through the “preparation of P1 lysate” procedure, and the titer of the phage was determined in the resulting lysate by plating in the presence of 20 mM Sodium Citrate (to inhibit P1). The values are averages of two measurements. Strains are: non-lysogen, AB1157; Lula lysogen, EL103; Lambda lysogen, EL104.</p

Epistatic analysis of Lula prophage versus DNA repair mutants sensitivity to UV irradiation.

<p><b>A.</b> Interaction of Lula prophage with the <i>recA</i> and <i>uvrA</i> defects. Strains: LAP2, 3, 11, 12 and 15. <b>B.</b> A scheme of the recombinational repair pathways. <b>C.</b> Interaction of Lula prophage with the <i>recBCD</i> defect. Strains: GR523, AK147, LAP1 and LAP4. <b>D.</b> Interaction of Lula prophage with the <i>uvrD</i> and <i>ruvC</i> defects. Strains: GR523, LAP1, 7, 8, 13 and 14. <b>E.</b> Interaction of Lula prophage with the <i>recF</i> defect. Strains: GR523, LAP1, 9 and 10. <b>F.</b> Interaction of Lula prophage with the <i>recG</i> defect. Strains: GR523, LAP1, 5 and 6.</p

Translational Control of Tetracycline Resistance and Conjugation in the Bacteroides Conjugative Transposon CTnDOT

The tetQ-rteA-rteB operon of the Bacteroides conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of tetQ and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for tetQ. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of tetQ. Finally, comparison of the CTnDOT tetQ leader regions with upstream regions of five tetQ genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well