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Methods:Dealing with a possible T1 infection

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It is very difficult to get rid of phage T1 since it is highly dessication resistant.

Contents

General T1 Information

  • Phage T1 requires FhuA (Ferric Heme Uptake), also known as TonA (T One), as a receptor for adsorption to its host.
    • Phage T1 can infect fhuA+ E. coli, including B, C & K strains [1], along with some Salmonella strains [2].
    • FhuA (TonA) is non-essential. A number of strains currently being offered for cloning, or protein overexpresssion that are already TonA-.
    • A number of antibiotic resistance cassettes have been inserted into fhuA (tonA) and are readily available to researchers.
  • Phages T5 and phi80, as well as colicin M, also require FhuA (TonA) for adsorption.
  • T1 also requires the presence of a second protein, TonB. However, a fhuA deletion strain was shown to partially restored sensitivity of the strain to phage T1 [3].



Here are some suggestions to identify if you have a T1 infection in your lab

  1. Prepare a sensitive (fhuA+, also known as tonA+) lawn and a resistant lawn (fhuA+/tonA-) of bacteria on rich media. Leave the plates open in the contaminated area for approximately 1 hour and incubate overnight at 37oC. Look for large (almost the size of a nickel after an overnight incubation with MG1655 as the host E. coli K-12 tonA+ strain) plaques (clear zones where the bacterial cells have been lysed after successive rounds of infection) on the fhuA+ strain, but not the fhuA- plate.
  2. Titer any liquids you think might also be contaminated (LB media, water) using serial dilutions and spot titering on the same strains described above. Again, look for large plaques on the fhuA+ strain, but not the fhuA- plate.
  3. Practice sterile technique, parafilm the plates before incubation and bleach everything you can after you are done.


Need an example of plaque size? Have a look at Figure 1 from 'On the problem of a common receptor of colicin M and T1 and T5 bacteriophages' [4].

If you still think you have a T1 infection

Here are some approaches:

  1. DO NOT GROW T1-SENSITIVE STRAINS. This is often difficult, requiring significant labor to P1 transduce necessary strains to a TonA- phenotype, but it is effective. Alternatively, numerous strains are available to researchers ( see Mutants available from CGSC) or are sold by various companies. If this is not an option, prepare for lots of cleaning and bleaching...
  2. Decontaminate everything. Bleach and UV irradiate everything you can. Remember that T1 will exist for WEEKS TO MONTHS as an aerosol, so this means that pipetmen, water baths, spectrophotometers, glassware, growth chambers, etc will need to be treated. The most likely result will be that T1 will stay around somewhere in the lab!
  3. Use of formaldehyde to effectively decontaminate laminar flow hoods after introduction of phage T1 has been described [5].
  4. Identify any areas not contaminated with T1 and carefully manage these areas (sterile technique, parafilm plates in case of contamination, etc). If you can keep a growth area free of T1 with careful sterile technique, bleach and UV, you can keep your experiments going.
  5. Bleach anything and everything in contact or suspetced to be in contact with T1.


Speaking from experience, a lab I worked in managed to keep T1 out of our large, dry incubators by using sterile technique, parafilming plates, etc, and general good luck. However, our growth room, containing multiple waterbaths, was contaminated. We knew that phage T1 was surviving in our sipping spectrophotometer, among other places, even after multiple rounds of bleaching and months of nightly UV treatment. Other labs were nice enough to provide me with a T1-free workspace to grow larger cultures in T1-free waterbaths when I was required to grow a tonA+ strain.
Eventually, after multiple rounds of bleaching, UV light treatment every night for 6 months after the initial contamination, conversion to T1-resistant strains, and about 2 years, T1 is not detectable in the lab.

References

  1. Wagner, EF et al. (1979) Development of Escherichia coli virus T1: escape from host restriction. J. Virol. 29 1229-31 PubMed EcoliWiki page
  2. Graham, AC & Stocker, BA (1977) Genetics of sensitivity of Salmonella species to colicin M and bacteriophages T5, T1, and ES18. J. Bacteriol. 130 1214-23 PubMed EcoliWiki page
  3. Langenscheid, J et al. (2004) A FhuA mutant of Escherichia coli is infected by phage T1-independent of TonB. FEMS Microbiol. Lett. 234 133-7 PubMed EcoliWiki page
  4. Smarda, J (1967) On the problem of a common receptor of colicin M and T1 and T5 bacteriophages. Folia Microbiol. (Praha) 12 492-3 PubMed EcoliWiki page
  5. Jones, KE et al. (1981) T1 bacteriophage as an indicator for decontamination of laminar-flow biological safety cabinets. Appl. Environ. Microbiol. 41 1072-3 PubMed EcoliWiki page