Monday, September 3, 2018

BacterioFiles 354 - Prokaryote Protection Promotes Protein Passing

Pectobacterium cells
By: The James Hutton Institute
This episode: The bacterial immune system, CRISPR-Cas, can enhance gene transfer via transduction (phages carrying bacteria DNA) despite preventing it via conjugation!

Thanks to Bridget Watson for her contribution!

Download Episode (14.9 MB, 16.3 minutes)

Show notes:
Microbe of the episode: Human polyomavirus 8

Journal Paper:
Watson BNJ, Staals RHJ, Fineran PC. 2018. CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction. mBio 9:e02406-17.

Other interesting stories:

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Episode outline:
  • Background: CRISPR-Cas systems in bacteria and archaea amazing and powerful
  • Here's Bridget Watson, first author on today's paper: statement 1
  • Kinda like facial recognition, except genome recognition
    • Cas protein goes around with CRISPRs, trying to find match
    • If found, chop chop chop, no more genome
  • Talked a lot about using this system for gene editing purposes
    • But also important for research and in nature
    • Bacteria with system can be hard to engineer: new stuff rejected
    • And presence in microbes in nature can affect transfer of genes between species
      • Here's Bridget again: statement 2
  • What’s new: Now, Bridget Watson, Raymond Staals, and Peter Fineran, publishing in mBio, have discovered that CRISPR-Cas systems can affect transduction, in unexpected ways!
  • Methods: Studying bacteria Pectobacterium atrosepticum
    • Plant pathogen, infects potatoes and causes blackleg (stem blackening and decay)
    • Has 3 CRISPR arrays with targeting sequences, spacers
  • Previously found could inhibit plasmid uptake by transformation or conjugation
    • But what about transduction? Interferes with it?
  • Phage is phiTE, occasionally packages bits of host DNA
    • Grew on bacteria with either control plasmid or plasmid with targeted gene
    • Some bacteria had CRISPR spacer targeting the targeted gene
      • This reduced transduction of targeted plasmid ~1000x relative to control plasmid or cells
  • Also tried with main chromosome, see if CRISPR interferes with transducing bits
    • Inserted kanamycin resistance in various places, see if it transfers
    • If CRISPR present targeting resistance gene, transduction is inhibited
    • Otherwise, not
    • Phage could still infect though
  • Also showed that transduction could transfer a pathogenicity island
    • Or section of genomic DNA that usually moves around as a chunk
    • Encodes things that allow bacteria to cause plant disease
    • CRISPR could prevent the transfer of this island, and cause it to be lost from genome
  • But what about CRISPR targeting normal phages with their own genome?
    • Here's Bridget explaining the unexpected transduction outcome in this case: statement 3
    • So when no spacers target transduced DNA, CRISPR-Cas doesn't interfere—not surprising
    • But when targeting normal phage, more transductants survive
    • Like if 100 virus particles, 95 normal and 5 transducing
      • Without CRISPRs, 5 transducing mutate 5 bacteria, but other phages kill 4, see 1
      • With CRISPRs, protects the 5 mutants so more survive
    • Confirmed effect with another phage too, phiM1
    • From 5-60x higher rate
  • Even more effect if only some bacteria have CRISPR defense
    • Then susceptible hosts get infected and create more phages and transducing particles
    • These can make more mutants out of resistant bacteria, without reducing their population
    • But only transfer from sensitive to resistant bacteria this way
    • Other direction is still possible though, if mutant phages can avoid CRISPR detection
    • Phages can also carry CRISPR resistance with them to new hosts
    • Gets complicated
  • Summary: Here's Bridget again: statement 4
  • Applications and implications: statement 5
  • Overall CRISPR helpful or preventive in microbe gene transfer? Hard to say
    • Can prevent transfer by conjugation or transformation or transduction if targeted
    • But apparently can enhance transfer by transduction too
    • Phages then can enhance spread of resistance; risk of phage therapy
  • What do I think: Interesting: CRISPR definitely benefits in resisting infection
    • But can be harmful if inhibits uptake of useful genes
    • Sometimes even best to get rid of it to acquire new abilities
  • Human takeway: Sometimes isolating oneself from others can be helpful to heal
  • But at some point, important to interact to get new perspectives on the world
Author Transcript:
CRISPR-Cas systems protect bacteria from invading genetic elements, such as bacteriophages and plasmids. By acquiring small bits of DNA from the invaders and putting this into a memory bank in the chromosome, the cell will use this to recognize if the invader infects again, and if it does, the plasmid or bacteriophage DNA will be degraded.

So there are three main ways that cells can gain extra chromosome from the environment or from other cells, which allows them to gain traits, like antimicrobial resistance. These are transformation, transduction, and conjugation. Transduction is when phages transfer part of the bacterial chromosome from one bacterium to another. It occurs as an error of phage replication. And while most of the phages have phage DNA, a tiny proportion will have packaged part of the bacterial chromosome, and these are called transducing particles, and will carry out transduction. We were interested in transduction because it is predicted to be an important way that bacteria acquire new genetic material. We were also interested to look at transduction because while CRISPR-Cas systems have been shown to inhibit the other ways that bacteria acquire new material, transformation and conjugation, no one had really looked at whether CRISPR-Cas could stop transduction.

However, since transducing particles, which carry out transduction, are rare, and most of the time the CRISPR-Cas system will encounter normal phages, we looked at the effect of having CRISPR-Cas system that matched and targeted phages on transduction. So we saw that when the strains had CRISPR-Cas immunity against phages, transduction was enhanced. We show that this is because, during the experiments, there are normal phages present with the small number of transducing particles, and the normal phages can infect and kill the transductants. But the strains that are resistant to phages were able to survive the infection, and hence we detected more of these transductants.

In summary, we showed that when the CRISPR-Cas sequences target the DNA that is being transduced, transduction is inhibited. However, in strains with CRISPR-Cas sequences targeting phages, transduction is enhanced, because more transductants survive and are detected.

This paper shows that the systems that protect bacteria from bacteriophage infection influence the ability of bacteria to acquire genetic material in ways we had not thought of before, and so CRISPR-Cas may influence the ability of bacteria to acquire antimicrobial resistance and other important traits.

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