rec-2 weirdness

The protein encoded by the H. influenzae rec-2 locus is needed to transport DNA across the cell's inner membrane, from the periplasm to the cytoplasm. Rec-2 homologs play similar roles in other gram-negative bacteria and in gram-positive bacteria, where they are need for transport across the homologous cytoplasmic membrane. However a H. influenzae rec-2 knockout also has unexpected pleiotropic effects; before considering the old evidence for these we need to consider the bizarre history of this mutation. The original isolation is reported in a 1971 paper by Ken Beattie and Jane Setlow (Nature New Biology 231:177-179)

Setlow had originally isolated the rec-1 mutant of H. influenzae. This strain has a mutation in the recA homolog; it takes up DNA normally but is unable to recombine it into its chromosome. Like other recA mutants it is also very sensitive to DNA damage. I think the mutant was isolated after nitrosoguanidine treatment of Rd cells and was identified by its sensitivity to UV irradiation; its strain name is DB117.

She wanted to find other mutants unable to transform. Because she had recently discovered that many H. influenzae cells died after taking up DNA from the related H. parainfluenzae, she decided to use this as a way to select for cells that couldn't take up DNA. So she and Ken Beattie repeatedly gave competent H. influenzae DNA from H. parainfluenzae, isolated the survivors, made them competent and gave them more of the same toxic DNA. (She later discovered that the 'toxicity' arises because heterologous DNA induces the SOS response to DNA damage, which induces a resident prophage that kills the cells.) However, even 20 repetitions of this treatment produced a population of cells with only a slight transformation defect.

So (I don't know why) they tried again, this time pretreating the cells by exposing them once to DNA from her rec-1 mutant, followed by 20 cycles of exposure to H. parainfluenzae DNA. Surprisingly (to me), this treatment gave populations with 1000-fold reductions in transformation. The pretreatment with rec-1 DNA was almost as effective as mutagenesis with nitrosoguanidine to 60% survival.

They then tested single colony isolates from these populations for DNA uptake and transformation. Almost all of them did not take up detectable amounts of DNA, but a few took up as much DNA as normal cells but did not produce any transformants. None of the 8 mutants isolated from the population treated with rec-1 mutant DNA had the DNA repair defects of the rec-1 mutant. (Note that these mutants are very likely to be multiple descendants of a single original mutant.)

Because of this they erroneously but only temporarily concluded that the rec-1 mutant's two defects (in transformational recombination and in DNA repair) were due to different mutations. They seem to have also invoked another mutation, mex, causing sensitivity to the DNA-damaging chemical MMS. They suggested that the new mutant (one of the 8) had this mex mutation as well as another mutation that prevented recombination, perhaps acquired from the H. parainfluenzae DNA it had been repeatedly exposed to. They initially called this mutant Rd(DB117)^rec but later simply called it rec-2.

They and others did a lot of work on the phenotype of this rec-2 mutant. They found that the mutant was a bit sensitive to MMS (attributed to the somewhat-hypothetical mex mutation). It took up DNA into a state that was resistant to externally added DNaseI and to the restriction enzymes known to be in the cytoplasm. This state was originally thought to be inside the vesicles then called 'transformasomes' (see this post about these) but we're now pretty sure it's just the periplasm. Making the mutant competent for DNA uptake across the outer membrane did not increase the ability of the cells to support phage recombination (see this post) as it did for wildtype cells. Competent cells of the rec-2 strain did not develop the single-strand DNA gaps detected in wildtype cells.

However interpretations were always confounded by uncertainty about its genotype. Did it carry a mex mutation (whatever that might be)? Did it contain any other DNA from the rec-1 strain? Did it contain any segments of H. parainfluenzae DNA? Did it have any loss-of-function mutations?

In 1989 Dave McCarthy tried to sort this mess out (McCarthy Gene 75:135-143). He isolated a transformation-preventing miniTn10kan insertion into a H. influenzae gene, and showed that a plasmid carrying the wild-type version of this gene restored transformability to Setlow's rec-2 mutant. By probing Southern blots with the cloned gene he showed that Setlow's rec-2 mutant contains a large rearrangement (later identified as a ~80kb insertion) in this gene. I'll call his rec-2:: miniTn10kan mutant rec-2*. This mutant had the same DNA uptake defect as Setlow's mutant.

With Doris Kupfer he then characterized the phenotype of the rec-2* rec-2, it took up DNA but could not mutant. Like Setlow'stranslocate it across the inner membrane. It was also just as defective in phage recombination, and examination of its DNA by electron microscopy showed that competence induction did not cause the increase in single-strand gaps or tails seen in wildtype cells.

The DNA translocation defect is consistent with the phenotypes of rec-2 homolog mutants in other bacteria. But the phage recombination and single-strand gap differences make no sense to me.