PMID:12821484 Nonsense Mutations in the lsa-Like Gene in Enterococcus faecalis Isolates Susceptible to Lincosamides and Streptogramins A

[jingluodatacurator]

Curated by @jingluodatacurator

https://canto.phi-base.org/curs/144c8f8625c95e5e

Uniprot ID: Q7WT47 Strains: V583, ATCC29212 Main expt. for curaion: see section 'Selection of revertant mutants' and Table 1

[CuzickA]

Hi @jingluodatacurator, please could you email me your highlighted PDF of this paper so that I can check the session. Thanks.

[CuzickA]

Checking UniProt id - looks fine Text

Search UniProt for AY225127 gives https://www.uniprot.org/uniprotkb/Q7WT47/entry

[CuzickA]

Current annotations

[CuzickA]

Expt information

Some background info _'The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux.'

_'The lsa-like genes of two clinical isolates of E. faecalis susceptible to lincosamides and dalfopristin contained mutations that produced premature termination codons.'__ These isolates were called UCN32 and UCN33.

The lsa gene was sequenced in the two susceptible isolates UCN32 and UCN33 and sequence was compared to two intrinsically resistant isolates V583 and ATCC 29212 (reference). This is what was found...

'In E. faecalis UCN32, in addition to a few substitutions, deletion of an adenine was detected at position 618 (base relative to ATG), which generated a stop codon four nucleotides downstream. In E. faecalis UCN33, a G-T mutation at position 987 produced a stop codon.'

Note: this on its own can't be curated as it is just comparing isolates without an element of 'lab-engineering' to give us a clear control wt/parental strain for comparison. However, the following revertant expt can be curated.

Expt for curation

'Revertant mutants were obtained by selection on agar plates containing clindamycin.'

'Selection of revertant mutants. Approximately 108 cells of E. faecalis UCN32 and E. faecalis UCN33 were spread onto brain heart infusion agar plates containing 20 g of clindamycin/ ml and were incubated for 24 h at 37°C. Resistant mutants were obtained at frequencies approximately equal to 106. Three mutants derived from each parental strain were selected and were further studied. All displayed an LSA phenotype (coresistance to clindamycin, lincomycin, and dalfopristin) (Table 1). lsa sequence analysis in the three mutants, UCN32-1, UCN32-2, and UCN32-3, derived from E. faecalis UCN32 showed an insertion of an adenine in place of the deletion in the parent strain, which restored a sequence iden-tical to that of E. faecalis V583. In the mutants from E. faecalis UCN33, the stop codon (TAA) was changed to a glutamic acid codon (GAA) in mutant UCN33-1 or to a glutamine codon (CAA) in mutants UCN33-2 and UCN33-3. There were no differences between the MICs of lincosamides and dalfopristin for mutants (Table 1).'

[CuzickA]

Parental strain UCN32 susceptible clinical isolate Revertant mutants UCN32-1, UCN32-2, and UCN32-3 all 'showed an insertion of an adenine in place of the deletion in the parent strain, which restored a sequence identical to that of E. faecalis V583.' Therefore genotype would be 1) E. faecalis (strain UCN32) nucleotide insertion of adenine (A) at 618 (was deleted in parent UCN32, now inserted in revertant mutant) phenotype would be shift of susceptible to resistant

Parental strain UCN33 susceptible clinical isolate Revertant mutant UCN33-1 'the stop codon (TAA) was changed to a glutamic acid codon (GAA) in mutant UCN33-1' 'In E. faecalis UCN33, a G-T mutation at position 987 produced a stop codon.' Therefore in parent UCN33 thymine (T) was present at 987 and changed to guanine (G) in the revertant. Revertant mutants UCN32-2, and UCN32-3 'or to a glutamine codon (CAA) in mutants UCN33-2 and UCN33-3.' Therefore in parent UCN33 thymine (T) was present at 987 and changed to cytosine (C) in the revertants. Therefore genotypes would be

2) E. faecalis (strain UCN33) nucleotide substitution of thymine (T) at 987 for guanine (G) 3) E. faecalis (strain UCN33) nucleotide substitution of thymine (T) at 987 for cytosine (C)

phenotypes would be shift of susceptible to resistant

[CuzickA]

Next steps 1) @jingluodatacurator, please could you read through my notes on my understanding of the expt in this paper and see you agree. 2) @jingluodatacurator, the genotypes will need remaking please. Note that in this case the paper reports on 'nucleotide' changes rather than 'amino acid' changes. 3) AC I think that adding the AE alteration in archetype is N/A as NTSR, the authors state 'The Lsa protein shows similarities to members of a superfamily of transport-related proteins known as ABC transporters.' and 'The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux.'

[CuzickA]

Note to self: I might need to add new strains to PHI-Canto.

[jingluodatacurator]

Next steps

1. @jingluodatacurator, please could you read through my notes on my understanding of the expt in this paper and see you agree.
2. @jingluodatacurator, the genotypes will need remaking please. Note that in this case the paper reports on 'nucleotide' changes rather than 'amino acid' changes.
3. AC I think that adding the AE alteration in archetype is N/A as NTSR, the authors state 'The Lsa protein shows similarities to members of a superfamily of transport-related proteins known as ABC transporters.' and 'The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux.'

Hi @CuzickA, can we discuss points 1 & 2 in our meeting tomorrow for some further guidance please thanks.

[jingluodatacurator]

Next steps

1. @jingluodatacurator, please could you read through my notes on my understanding of the expt in this paper and see you agree.
2. @jingluodatacurator, the genotypes will need remaking please. Note that in this case the paper reports on 'nucleotide' changes rather than 'amino acid' changes.
3. AC I think that adding the AE alteration in archetype is N/A as NTSR, the authors state 'The Lsa protein shows similarities to members of a superfamily of transport-related proteins known as ABC transporters.' and 'The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux.'

Hi @CuzickA, thanks for the feedback. I have gone through the paper again and summarized key steps of expt., and agree with your notes above. I have amended the genotypes. Please let me know any further actions thanks.

[CuzickA]

Copy of updated annotations for checking

[CuzickA]

Hi @jingluodatacurator, In your updated annotation, I don't see the 3 genotypes described in my comment above. Please could you check? Thanks

[jingluodatacurator]

Hi @jingluodatacurator, In your updated annotation, I don't see the 3 genotypes described in my comment above. Please could you check? Thanks

Hi @CuzickA, I have double checked, in the updated curation above, one genotype is nucleotide insertion lsa(618-A) and the other two genotypes are nucleotide substitutions lsa(ntT987G, T987C). Maybe I have misunderstood something here? I'd like to talk it through in our meeting tomorrow please.

[CuzickA]

Hi @jingluodatacurator, In your updated annotation, I don't see the 3 genotypes described in my comment above. Please could you check? Thanks

Hi @CuzickA, I have double checked, in the updated curation above, one genotype is nucleotide insertion lsa(618-A) and the other two genotypes are nucleotide substitutions lsa(ntT987G, T987C). Maybe I have misunderstood something here? I'd like to talk it through in our meeting tomorrow please.

Hi @jingluodatacurator, 'two genotypes are nucleotide substitutions lsa(ntT987G, T987C)' This should be two separate genotypes, currently it is one genotype with the nt at the same position 987 changing from T to either G or C.

Also the genotype strains need changing as outlined in the 3 suggested genotypes described in the comment above.

[jingluodatacurator]

Hi @jingluodatacurator, In your updated annotation, I don't see the 3 genotypes described in my comment above. Please could you check? Thanks

Hi @CuzickA, I have double checked, in the updated curation above, one genotype is nucleotide insertion lsa(618-A) and the other two genotypes are nucleotide substitutions lsa(ntT987G, T987C). Maybe I have misunderstood something here? I'd like to talk it through in our meeting tomorrow please.

Hi @jingluodatacurator, 'two genotypes are nucleotide substitutions lsa(ntT987G, T987C)' This should be two separate genotypes, currently it is one genotype with the nt at the same position 987 changing from T to either G or C.

Also the genotype strains need changing as outlined in the 3 suggested genotypes described in the comment above.

Hi @CuzickA, I have updated genotypes as well as genotype strains. Please check thanks.

https://canto.phi-base.org/curs/144c8f8625c95e5e

[CuzickA]

Looks good @jingluodatacurator

[CuzickA]

Next steps 1) AC to add new strains 'UCN32' and 'UCN33' to Enterococcus faecalis strain list DONE 31_01_2024 b97a12e 2) Alteration in archetype N/A as NTSR

[CuzickA]

Session approved, closing ticket.

[CuzickA]

This is a bacterial species and not a fungal species - also see https://github.com/PHI-base/curation/issues/159

Note: this paper was identified for curation by MP.