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You can see this happening here, with the bacteria receiving the plasmid (circled in red) stopping growth while everyone else is happily dividing.

We could see this happening under the microscope in the TacC lab. Cells expressing Namtar stop growing as soon as they receive the plasmid, that is visualized as bright green ParB-mChartreuse foci.

A functional Namtar homolog from E. coli induces a non-replicative state upon conjugative acquisition of R388

E. coli has a Namtar homolog and Julie Baltenneck in @francoisrousset.bsky.social lab showed that does the same thing. It kills cell that receive the R388 plasmid.

The two other genes encode the new Namtar and Attar immune systems, also widespread in bacteria. They are membrane-associated proteins defending against R388. Namtar depends on sensing VirB8, while Attar senses VirB2. Both trigger a non-growing, ATP-depleted state. Let's call this death.

Namtar and Attar defense systems induce loss of viability in R388-carrying cells

Two of them are part of the same family encoding the new Ishtar immune system that is widespread in Bacteria, and also found in Archaea. Ishtar carries a HEPN domain and specifically promotes loss of RP4 plasmid.

The Ishtar immune system induces plasmid loss in RP4-carrying cells

Which genes/function could limit conjugation? Tn mutagenesis revealed the culprits in 12 strains. Capsule is one of the barriers. Interestingly, H-NS expression can sometimes lift the barriers. Most importantly 4 genes of patchy distribution specifically block R388 or RP4 conjugation.

Random transposon mutagenesis identifies genetic elements conferring resistance to conjugation in a diverse subsample of A. baumannii strains.

Is conjugation really so efficient? Are there barriers? Do bacteria defend against it? Ludovic tested 364 A. baumannii strains as recipients of the R388 and RP4 plasmids. This revealed that resistance to conjugation is the norm and is a fast-evolving phenotype.

A high-throughput screening method reveals large variations in the susceptibility of diverse A. baumannii samples to receive plasmids R388 and RP4 by conjugation

Without the nuclease/helicase, the rare recombination events are very short. Hence, bacteria have evolved two distinct systems to efficiently resolve the D-loop and generate large recombination events by transformation

Cleaving the displaced strand might be critical to allow for the helicase-driven extension of the D-loop. In vivo, genetic assays show that CoiA assists D-loop extension by the DnaB-type RadA helicase. Similarly, YraN assists, and interacts with, the transformation-specific MCM-type ComM helicase

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Indeed, in an in vitro-generated transformation D-loop, CoiA specifically cleaves the displaced strand at the edge of the D-loop

Could the nucleases be helping resolve the D-loop? Biochemical analyses show that YraN is an endonuclease, cleaving ssDNA. CoiA is more specific, cleaving ssDNA if it's branching out of dsDNA. Both proteins might cleaved the parental ssDNA displaced by the transforming DNA molecule

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Our two labs separetely found that transformation requires a nuclease. CoiA in Streptococcus pneumoniae and a protein of unknown function in Legionella/Acinetobacter, YraN. CoiA and YraN are different proteins of the PD-(D/E)XK family of phosphodiesterases. Most bacteria possess one or the other

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As they actively import DNA, bacteria process it into ssDNA. Then, RecA-dependent homology search leads the ssDNA to invade the chromosomal DNA, generating a 3-strand intermediate called a D-loop. How this structure is resolved to stably integrate the transforming DNA is unknown