The Role of the Novel Fem Protein VanK in Vancomycin Resistance in Streptomyces coelicolor

Abstract

The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high level resistance to vancomycin. The vanK gene has no counterpart in previously characterized vancomycin resistance clusters, yet vanK is required for vancomycin resistance in S. coelicolor. VanK belongs to the Fem family of enzymes, which add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. Upon exposure to vancomycin, the VanRS two-component system switches on expression of all seven van genes, and the VanHAX enzymes reprogram the cell wall such that precursors terminate d-Ala-d-lactate (Lac) rather than d-Ala-d-Ala, thus conferring resistance to vancomycin, which only binds d-Ala-d-Ala-containing precursors. Here we provide biochemical and genetic evidence that VanK is required for vancomycin resistance because the constitutively expressed FemX enzyme, encoded elsewhere on the chromosome, cannot recognize d-Lac-containing precursors as a substrate, whereas VanK can. Consistent with this view, d-Lac-containing precursors carrying the Gly branch are present in the wild type transiently exposed to vancomycin but are undetectable in a vanK mutant treated in the same way. Further, femX null mutants are viable in the presence of vancomycin but die in its absence. only VanK recognize d-Lac-containing expression of VanHAX in a vanK mutant is and vanK is required for vancomycin The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high level resistance to vancomycin. The vanK gene has no counterpart in previously characterized vancomycin resistance clusters, yet vanK is required for vancomycin resistance in S. coelicolor. VanK belongs to the Fem family of enzymes, which add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. Upon exposure to vancomycin, the VanRS two-component system switches on expression of all seven van genes, and the VanHAX enzymes reprogram the cell wall such that precursors terminate d-Ala-d-lactate (Lac) rather than d-Ala-d-Ala, thus conferring resistance to vancomycin, which only binds d-Ala-d-Ala-containing precursors. Here we provide biochemical and genetic evidence that VanK is required for vancomycin resistance because the constitutively expressed FemX enzyme, encoded elsewhere on the chromosome, cannot recognize d-Lac-containing precursors as a substrate, whereas VanK can. Consistent with this view, d-Lac-containing precursors carrying the Gly branch are present in the wild type transiently exposed to vancomycin but are undetectable in a vanK mutant treated in the same way. Further, femX null mutants are viable in the presence of vancomycin but die in its absence. only VanK recognize d-Lac-containing expression of VanHAX in a vanK mutant is and vanK is required for vancomycin The of vancomycin resistance is of and in the the of vancomycin resistance to a in has a with the of of S. high S. high in the S. S. S. and cell wall in but in because cannot the the of peptidoglycan precursors on the of the and this the of to its substrate, thus of the that the cell wall its of the of of vancomycin, in cannot to this that resistance to vancomycin because to the peptidoglycan the the of and to reprogram cell wall such that the pentapeptide of peptidoglycan precursors in rather than in S. The of vancomycin for precursors in is than for precursors S. the of cell wall precursors which a and a that that peptidoglycan precursors terminate in previously that the Streptomyces coelicolor carries a gene cluster conferring inducible, resistance to vancomycin S. coelicolor is the of a of for the of of the S. coelicolor a but all of the are the family to which the in the that S. coelicolor and that the van gene cluster confers a Further, is that all resistance genes are actinomycete which genes to Consistent with this the S. coelicolor resistance genes are with a S. coelicolor cluster of seven genes, are to the genes in and a two-component system that of the seven van genes in to vancomycin. and vanK are because are genes with no counterpart in previously characterized which a is required for vancomycin but vanK to for vancomycin resistance is a of the Fem family of enzymes, which are that add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. S. has a branch and Fem and are in its S. S. S. that femX is gene in S. and that of its expression in the of peptidoglycan precursors. and mutants are of to of the branch to whereas of the branch to a that FemX the the and and the and S. S. S. has in a in and FemX has to as for the in Streptomyces the branch is a yet S. coelicolor has and of the peptidoglycan enzymes VanHAX and the VanRS two-component system the that VanK for resistance in S. coelicolor the of vanK in the of VanK is a of the Fem family of we to vanK is required for vancomycin resistance that S. coelicolor to the of the peptidoglycan branch to in the of the branch to in of resistance in The that the FemX of S. coelicolor recognize only precursors that terminate as a substrate, and VanK required for vancomycin resistance because is the only that add the Gly branch to precursors of precursors a branch is in because of the peptidoglycan to cell Here we that the and and are in cell wall S. coelicolor in of for and with of and with for Streptomyces The with Streptomyces The with and the to of the of peptidoglycan to in and exposed to vancomycin for and for as previously and in this S. coelicolor Streptomyces The S. the resistance gene and the of S. S. of and a carrying a vanK with and with in for the of to Streptomyces femX femX femX femX in a of and vanK and null in which the with a carrying the resistance gene and of of and S. The S. carrying S. and the gene of the with the the femX and femX for and and for The and carrying and S. coelicolor for the of and the and and a mutant the of no on that of on vancomycin in to and the of femX and femX mutant The vanK mutant the the of S. of vanK in the femX expression of the gene the of the carrying a and a and with to vanK and and of the gene and to vanK as a and with to the femX mutant and with of peptidoglycan precursors a of the of and in The with for and the on a The and in a of on a S. S. a in in and the of precursors and in a in but the of a in precursors with and the of and and the a of we in which to precursors to with that the of a the of its a the of that that this which is is for the of the in to the of vancomycin on the stem peptidoglycan S. coelicolor in the presence of vancomycin, and the of and characterized The branch with a in all and exposure to vancomycin. and VanK the stem that VanK to the Gly to the mutant is to vancomycin, to the of vancomycin on its cell wall because cell wall in to vancomycin to the of vancomycin on in the S. coelicolor and the mutant to in Streptomyces The the of peptidoglycan exposed to vancomycin for precursors and The of the are in in the of vancomycin, the van genes no the and the mutant the precursors with and the branch with a of a that the of and a of that the of and The of its of peptidoglycan precursors S. coelicolor and a mutant with exposure to vancomycin. the of peptidoglycan to and exposed to vancomycin for are the for and to and in The the to vancomycin that the of the are than of the because of the of of cell wall precursors. pentapeptide and are of the than of the because of the of of and in a with of and to The with a of and the same to The of is as are of that of the in is but that of the in is The of the are than of the because of the of to vancomycin the of the and the in and a with a of the and the a to and the wild a of to and the d-Lac-containing with the Gly in the mutant The of its femX that present in the wild type exposed to vancomycin but undetectable in the mutant treated in the same we that the of VanK to add the Gly branch to d-Lac-containing precursors and that the constitutively expressed FemX cannot recognize precursors as a the Gly branch is to for peptidoglycan and cell VanK required for vancomycin this is that gene in S. S. S. in S. coelicolor. VanK for FemX the cell FemX expression of the van genes is vancomycin this we to femX in the presence and of vancomycin, the of S. the of this S. coelicolor genes on in are with a this the that the of genes because the gene is in rather than in S. for the of femX is constitutively but vanK expression is vancomycin. FemX the branch to the stem pentapeptide of S. coelicolor cell wall precursors of this branch is for cell wall femX is gene the van cluster a FemX which recognize precursors as a a femX is that the van genes are femX null mutants are viable in the presence of vancomycin but die in its absence. of the type and and mutant of on in the presence of vancomycin for carrying a gene in the and a resistance gene in the S. coelicolor for resistance the and no on that femX vancomycin to the and as femX mutants and the femX mutants on vancomycin, to that femX is in the of vancomycin but in its presence a in S. to vanK and is a gene elsewhere on the S. coelicolor chromosome, which we no the presence of and of the gene no in of the of the chromosome, that and that a of genes S. S. S. S. S. S. whereas vanK and femX in the of the to VanK recognize d-Ala-d-Ala-containing precursors as The van genes are expressed in the of vancomycin VanK is exposed to d-Ala-d-Ala-containing precursors because vancomycin expression of VanHAX the same as to stem as VanK vanK in the of we a in which vanK the of the and the femX null expression of vanK the femX null mutant to in the of vancomycin that VanK recognize precursors as this we the femX null mutant carrying the in the of vancomycin, peptidoglycan and a with a of to in and and that VanK add the branch to d-Ala-d-Ala-containing expression of vanK the of a femX null The femX null mutant carrying the in the of vancomycin but the same carrying the with is viable in the of the of peptidoglycan precursors the femX null mutant carrying the in the of vancomycin. The is the for and to and in that the of the are than of the because of the of of the in this is as FemX the branch to the stem pentapeptide of S. coelicolor cell wall precursors of this branch is for cell wall FemX is of the genes cell wall precursors such that the stem pentapeptide of d-Ala-d-Ala, and FemX cannot recognize d-Lac-containing precursors. the FemX d-Lac-containing precursors The femX gene is the van genes are a femX null mutants are viable in the presence of vancomycin but die in its absence. only VanK recognize d-Lac-containing expression of VanHAX in a vanK mutant is and vanK is required for vancomycin of VanK in vancomycin resistance in S. coelicolor. VanK the Gly branch to cell wall precursors FemX the of the Gly branch to precursors but cannot recognize precursors as a vanK is required for vancomycin resistance in S. is that a is for resistance in S. coelicolor but in the of S. coelicolor vanK is present in the resistance gene with the cluster for the in Streptomyces S. evidence that FemX of recognize d-Lac-containing van of the cell of the that the of FemX in the the and the in the of vancomycin, the branch of the same in the presence of vancomycin a branch that d-Lac-containing precursors are for and the enzymes that the branch in S. but as to the of a branch and the of the and to present a of FemX in which FemX recognize d-Lac-containing precursors FemX cannot the presence of vanK VanRS with the in to to the of a viable cell but that is in to the wall in the of vancomycin. The of the S. and of that the of a to yet to is evidence that the to with d-Lac-containing present for of in to Streptomyces and actinomycete of S. the The of of with the resistance genes and a encoded elsewhere on the S. S. S. of the with to the with in S. for the presence of in the resistance gene cluster is that cannot recognize as a is and cannot to is required for resistance S. S. S. The of vancomycin resistance is of and in the the of vancomycin resistance to a in has a with the of of S. high S. high in the S. S. S. and cell wall in but in because cannot the the of peptidoglycan precursors on the of the and this the of to its substrate, thus of the that the cell wall its of the of of vancomycin, in cannot to this that resistance to vancomycin because to the peptidoglycan the the of and to reprogram cell wall such that the pentapeptide of peptidoglycan precursors in rather than in S. The of vancomycin for precursors in is than for precursors S. the of cell wall precursors which a and a that that peptidoglycan precursors terminate in previously that the Streptomyces coelicolor carries a gene cluster conferring inducible, resistance to vancomycin S. coelicolor is the of a of for the of of the S. coelicolor a but all of the are the family to which the in the that S. coelicolor and that the van gene cluster confers a Further, is that all resistance genes are actinomycete which genes to Consistent with this the S. coelicolor resistance genes are with a The S. coelicolor cluster of seven genes, are to the genes in and a two-component system that of the seven van genes in to vancomycin. and vanK are because are genes with no counterpart in previously characterized which a is required for vancomycin but vanK to for vancomycin resistance VanK is a of the Fem family of enzymes, which are that add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. S. has a branch and Fem and are in its S. S. S. that femX is gene in S. and that of its expression in the of peptidoglycan precursors. and mutants are of to of the branch to whereas of the branch to a that FemX the the and and the and S. S. S. has in a in and FemX has to as for the in Streptomyces the branch is a yet S. coelicolor has and The of the peptidoglycan enzymes VanHAX and the VanRS two-component system the that VanK for resistance in S. coelicolor the of vanK in the of VanK is a of the Fem family of we to vanK is required for vancomycin resistance that S. coelicolor to the of the peptidoglycan branch to in the of the branch to in of resistance in The that the FemX of S. coelicolor recognize only precursors that terminate as a substrate, and VanK required for vancomycin resistance because is the only that add the Gly branch to precursors of precursors a branch is in because of the peptidoglycan to cell Here we that the and and are in cell wall S. coelicolor in of for and with of and with for Streptomyces The with Streptomyces The with and the to of the of peptidoglycan to in and exposed to vancomycin for and for as previously and in this S. coelicolor Streptomyces The S. the resistance gene and the of S. S. of and a carrying a vanK with and with in for the of to Streptomyces femX femX femX femX in a of and vanK and null in which the with a carrying the resistance gene and of of and S. The S. carrying S. and the gene of the with the the femX and femX for and and for The and carrying and S. coelicolor for the of and the and and a mutant the of no on that of on vancomycin in to and the of femX and femX mutant The vanK mutant the the of S. of vanK in the femX expression of the gene the of the carrying a and a and with to vanK and and of the gene and to vanK as a and with to the femX mutant and with of peptidoglycan precursors a of the of and in The with for and the on a The and in a of on a S. S. a in in and the of precursors and in a in but the of a in precursors with and the of and and the a of we in which to precursors to with that the of a the of its a the of that that this which is is for the of the precursors. and and are in cell wall S. coelicolor in of for and with of and with for Streptomyces The with Streptomyces The with and the to of the of peptidoglycan to in and exposed to vancomycin for and for as previously of and vanK and null in which the with a carrying the resistance gene and of of and S. The S. carrying S. and the gene of the with the the femX and femX for and and for The and carrying and S. coelicolor for the of and the and and a mutant the of no on that of on vancomycin in to and the of femX and femX mutant The vanK mutant the the of S. of vanK in the femX expression of the gene the of the carrying a and a and with to vanK and and of the gene and to vanK as a and with to the femX mutant and with of peptidoglycan precursors a of the of and in The with for and the on a The and in a of on a S. S. a in in and the of precursors and in a in but the of a in precursors with and the of and and the a of we in which to precursors to with that the of a the of its a the of that that this which is is for the of the precursors. in to the of vancomycin on the stem peptidoglycan S. coelicolor in the presence of vancomycin, and the of and characterized The branch with a in all and exposure to vancomycin. and VanK the stem that VanK to the Gly to the mutant is to vancomycin, to the of vancomycin on its cell wall because cell wall in to vancomycin to the of vancomycin on in the S. coelicolor and the mutant to in Streptomyces The the of peptidoglycan exposed to vancomycin for precursors and The of the are in in the of vancomycin, the van genes no the and the mutant the precursors with and the branch with a of a that the of and a of that the of and The of its of cell wall precursors. pentapeptide and are of the than of the because of the of of and in a with of and to The with a of and the same to The of is as are of that of the in is but that of the in is The of the are than of the because of the of to vancomycin the of the and the in and a with a of the and the a to and the wild a of to and the d-Lac-containing with the Gly in the mutant The of its femX that present in the wild type exposed to vancomycin but undetectable in the mutant treated in the same we that the of VanK to add the Gly branch to d-Lac-containing precursors and that the constitutively expressed FemX cannot recognize precursors as a the Gly branch is to for peptidoglycan and cell VanK required for vancomycin this is that gene in S. S. S. in S. coelicolor. VanK for FemX the cell FemX expression of the van genes is vancomycin this we to femX in the presence and of vancomycin, the of S. the of this S. coelicolor genes on in are with a this the that the of genes because the gene is in rather than in S. for the of femX is constitutively but vanK expression is vancomycin. FemX the branch to the stem pentapeptide of S. coelicolor cell wall precursors of this branch is for cell wall femX is gene the van cluster a FemX which recognize precursors as a a femX is that the van genes are femX null mutants are viable in the presence of vancomycin but die in its absence. of the type and and mutant of on in the presence of vancomycin for carrying a gene in the and a resistance gene in the S. coelicolor for resistance the and no on that femX vancomycin to the and as femX mutants and the femX mutants on vancomycin, to that femX is in the of vancomycin but in its presence a in S. to vanK and is a gene elsewhere on the S. coelicolor chromosome, which we no the presence of and of the gene no in of the of the chromosome, that and that a of genes S. S. S. S. S. S. whereas vanK and femX in the of the to VanK recognize d-Ala-d-Ala-containing precursors as The van genes are expressed in the of vancomycin VanK is exposed to d-Ala-d-Ala-containing precursors because vancomycin expression of VanHAX the same as to stem as VanK vanK in the of we a in which vanK the of the and the femX null expression of vanK the femX null mutant to in the of vancomycin that VanK recognize precursors as this we the femX null mutant carrying the in the of vancomycin, peptidoglycan and a with a of to in and and that VanK add the branch to d-Ala-d-Ala-containing expression of vanK the of a femX null The femX null mutant carrying the in the of vancomycin but the same carrying the with is viable in the of the of peptidoglycan precursors the femX null mutant carrying the in the of vancomycin. The is the for and to and in that the of the are than of the because of the of The in to the of vancomycin on the stem peptidoglycan S. coelicolor in the presence of vancomycin, and the of and characterized The branch with a in all and exposure to vancomycin. and VanK the stem that VanK to the Gly to the mutant is to vancomycin, to the of vancomycin on its cell wall because cell wall in to vancomycin to the of vancomycin on in the S. coelicolor and the mutant to in Streptomyces The the of peptidoglycan exposed to vancomycin for precursors and The of the are in in the of vancomycin, the van genes no the and the mutant the precursors with and the branch with a of a that the of and a of that the of and The of its to vancomycin the of the and the in and a with a of the and the a to and the wild a of to and the d-Lac-containing with the Gly in the mutant The of its femX that present in the wild type exposed to vancomycin but undetectable in the mutant treated in the same we that the of VanK to add the Gly branch to d-Lac-containing precursors and that the constitutively expressed FemX cannot recognize precursors as a the Gly branch is to for peptidoglycan and cell VanK required for vancomycin this is that gene in S. S. S. in S. coelicolor. VanK for FemX the cell FemX expression of the van genes is vancomycin this we to femX in the presence and of vancomycin, the of S. the of this S. coelicolor genes on in are with a this the that the of genes because the gene is in rather than in S. coelicolor. carrying a gene in the and a resistance gene in the S. coelicolor for resistance the and no on that femX vancomycin to the and as femX mutants and the femX mutants on vancomycin, to that femX is in the of vancomycin but in its presence a in S. to vanK and is a gene elsewhere on the S. coelicolor chromosome, which we no the presence of and of the gene no in of the of the chromosome, that and that a of genes S. S. S. S. S. S. whereas vanK and femX in the of the VanK to VanK recognize d-Ala-d-Ala-containing precursors as The van genes are expressed in the of vancomycin VanK is exposed to d-Ala-d-Ala-containing precursors because vancomycin expression of VanHAX the same as to stem as VanK vanK in the of we a in which vanK the of the and the femX null expression of vanK the femX null mutant to in the of vancomycin that VanK recognize precursors as this we the femX null mutant carrying the in the of vancomycin, peptidoglycan and a with a of to in and and that VanK add the branch to d-Ala-d-Ala-containing precursors. of the in this is as FemX the branch to the stem pentapeptide of S. coelicolor cell wall precursors of this branch is for cell wall FemX is of the genes cell wall precursors such that the stem pentapeptide of d-Ala-d-Ala, and FemX cannot recognize d-Lac-containing precursors. the FemX d-Lac-containing precursors The femX gene is the van genes are a femX null mutants are viable in the presence of vancomycin but die in its absence. only VanK recognize d-Lac-containing expression of VanHAX in a vanK mutant is and vanK is required for vancomycin is that a is for resistance in S. coelicolor but in the of S. coelicolor vanK is present in the resistance gene with the cluster for the in Streptomyces S. evidence that FemX of recognize d-Lac-containing van of the cell of the that the of FemX in the the and the in the of vancomycin, the branch of the same in the presence of vancomycin a branch that d-Lac-containing precursors are for and the enzymes that the branch in S. but as to the of a branch and the of the and to present a of FemX in which FemX recognize d-Lac-containing precursors FemX cannot the presence of vanK VanRS with the in to to the of a viable cell but that is in to the wall in the of vancomycin. The of the S. and of that the of a to yet to is evidence that the to with d-Lac-containing present for of in to Streptomyces and actinomycete of S. the The of of with the resistance genes and a encoded elsewhere on the S. S. S. of the with to the with in S. for the presence of in the resistance gene cluster is that cannot recognize as a is and cannot to is required for resistance S. S. S. of the in this is as FemX the branch to the stem pentapeptide of S. coelicolor cell wall precursors of this branch is for cell wall FemX is of the genes cell wall precursors such that the stem pentapeptide of d-Ala-d-Ala, and FemX cannot recognize d-Lac-containing precursors. the FemX d-Lac-containing precursors The femX gene is the van genes are a femX null mutants are viable in the presence of vancomycin but die in its absence. only VanK recognize d-Lac-containing expression of VanHAX in a vanK mutant is and vanK is required for vancomycin is that a is for resistance in S. coelicolor but in the of S. coelicolor vanK is present in the resistance gene with the cluster for the in Streptomyces S. evidence that FemX of recognize d-Lac-containing van of the cell of the that the of FemX in the the and the in the of vancomycin, the branch of the same in the presence of vancomycin a branch that d-Lac-containing precursors are for and the enzymes that the branch in S. but as to the of a branch and the of the and to present a of FemX in which FemX recognize d-Lac-containing precursors FemX cannot the presence of vanK VanRS with the in to to the of a viable cell but that is in to the wall in the of vancomycin. The of the S. and of that the of a to yet to vancomycin. is evidence that the to with d-Lac-containing present for of in to Streptomyces and actinomycete of S. the The of of with the resistance genes and a encoded elsewhere on the S. S. S. of the with to the with in S. for the presence of in the resistance gene cluster is that cannot recognize as a is and cannot to is required for resistance S. S. S. and for on the