BACKGROUND
ribosome-mediated protein synthesis is always initiated with either methionine  or n-formylmethionine   <cit> .
however, after removal of the n-formyl group from the polypeptide by peptide deformylase , the n-terminal methionine of a large number of proteins is cleaved by methionine aminopeptidase   <cit> .
the efficiency of removal of the initiator methionine is defined by a highly conserved local substrate specificity, which is determined by both methionine and its adjacent residue.
map hydrolytically removes the n-terminal methionine only when the penultimate residue bears a small and uncharged side chain   <cit> , and residues downstream of the penultimate residue have little impact on the reaction.
all maps studied to date were reported to be cobalt-dependent metalloproteases  <cit> .
however, some reports showed that maps exhibited activity in the presence of other divalent ions, such as zn, fe or mn  <cit> .
according to their sequence homology, maps are grouped into two subtypes, type i and type ii  <cit> .
an insertion of approximately  <dig> residues in the c-terminal domain of type ii methionine aminopeptidases is the only difference between the two types  <cit> .
eukaryotes possess at least two map genes, of both type i and type ii  <cit> , while there is only one map gene in most prokaryotic genomes, either type i  or type ii   <cit> .
map is distributed throughout living organisms, where it plays an important physiological role.
the deletion of the single map gene in prokaryotes such as escherichia coli  <cit>  or salmonella typhimurium  <cit>  is lethal.
in yeast, the deletion of any one of the two map genes  causes a slow growth phenotype, while deletion of both genes is lethal  <cit> .
why might map activity have an essential role in living cells?
previous studies showed that many proteins needed to have their n-terminal methionine removed to have normal biological activity, proper subcellular localization and eventual degradation .
as a result, it is possible that map is essential because of the essentiality of its downstream targets.
besides essentiality, map changes the dynamics of the sulfur containing metabolites, which may have an important role in the homeostasis of the cell.
in general, there is only one map gene in the genome of prokaryotes, with the exception of a cyanobacteria strain, which has three functional methionine aminopeptidases  <cit> .
surprisingly, in the genomes of bacilli, two or even more putative map genes can be detected by sequence alignment.
in b. subtilis, two putative genes responsible for map activity, map and yflg, can be identified  <cit> .
kobayashi et al.
<cit>  reported that map is an essential gene in b. subtilis while yflg is not, an apparent paradox if yflg codes for a methionine aminopeptidase.
in an attempt to better understand the functional role of map, we studied the evolutionary trend of maps in silico and demonstrated that both the map and yflg genes from b. subtilis code for methionine aminopeptidases in vitro and in vivo; furthermore, the finding of a high expression level for map in parallel with a low expression of yflg in b. subtilis, may account for their essentiality and dispensability, respectively.
RESULTS
sequence alignment and evolutionary pattern analyses
in the genome sequence of b. subtilis, two genes could code for methionine aminopeptidases, map and yflg, respectively  <cit>  .
the sequence similarity and identity between e. coli map  and b. subtilis map  is 65% and 46%, respectively.
similarity between map_ec and yflg from b. subtilis  is 55% and their identity is 34% and similarity between map_bs and yflg_bs is 58% and their identity is 36%.
however, the sequence of yflg_bs differs slightly from the prosite consensus for type i maps , which is -x-g-h-g- --x-h-x--x--.
in particular it displays one extra residue next to the conserved ghg metal-binding motif.
interestingly, map_bs is more similar to map_ec and yflg_bs is more similar to the only putative methionine aminopeptidase of staphylococcus aureus .
overall the proteins are very similar in their physico-chemical properties .
moreover, as shown in figure  <dig>  the metal  binding site  is conserved.
it was therefore important to investigate whether both proteins had methionine aminopeptidase activity.
this difference in evolution of proteins considered as essential in related organisms  is intriguing, and we explored the evolution pattern of map in a study meant to identify persistent genes in bacteria  <cit> .
when matched with the evolution of 16s rna, the map gene does not follow a linear course of evolution, as do most ribosomal proteins  <cit> , but shows an erratic pattern .
this suggests that map might be involved in different biological processes besides its role as methionine aminopeptidase according to the organisms.
this prompted us to explore the evolution of map with proteins that might be functionally related, using the criteria we used to identify persistent genes.
we defined persistent genes as the genes present as orthologs in more than 85% of the genomes of a clade , after removing obligatory endosymbionts from the list .
the protein divergence  of map in b. subtilis and its orthologs from other firmicutes were compared with bacterial evolutionary distance .
similar analyses were performed with a general protease, clpp, def, ribosomal protein s <dig>  rpsd and ykrb, a second deformylase present in b. subtilis .
interestingly, in contrast to the expected evolutionary pattern of rpsd, map exhibited the same erratic evolutionary way as def and ykrb, the two functional deformylases in b. subtilis, suggesting co-evolution of methionine aminopeptidase and deformylase.
enzyme activity in vitro
it is often recognized that gene function identification solely based on sequence comparisons could be misleading  <cit> .
therefore, we first determined whether map and yflg were authentic map genes by overproducing the proteins in e. coli and characterizing their enzymatic activity.
both products of map and yflg genes were purified to more than 90% homogeneity.
mass spectrometry showed that the molecular mass of map_bs and yflg_bs was  <dig>  ±  <dig>  da and  <dig>  ±  <dig>  da, respectively.
interestingly, both proteins purified from e. coli retained their initial methionine, consistent with the nature of the second residue, isoleucine in both cases.
as seen in table  <dig>  both enzymes exhibited a methionine aminopeptidase activity in vitro with the synthetic peptide substrates tested.
they differed widely, however, in the extent of their activity: the specific activity of yflg_bs was about 90- and 20-fold higher than that of map_bs with the tetrapeptide mgmm and tripeptide mas as the substrates, respectively.
both enzymes preferred the tetrapeptide mgmm as the substrate.
with mas and mg as the substrates, map_bs activity was  <dig> % and  <dig> % of that with mgmm as the substrate, respectively, while yflg_bs retained only  <dig> % and  <dig> % relative activity, respectively, with these substrates.
the effect of divalent ions  on enzyme activity was investigated .
map_bs had significant activity with both mn2+ and co2+.
map_bs retained appreciable activity  after replacement of co2+ with cd2+, zn2+, ca2+, mg2+, cu2+, sr2+ and ba2+, but not ni2+.
in contrast, yflg_bs showed a strong preference for co2+ with a decrease in activity to below 20% with any other divalent ions tested.
subcellular localization
to investigate whether map_bs and yflg_bs had any difference in their subcellular localization, we constructed n-terminal green fluorescent protein  fusions of both proteins.
as previously reported, the catalytic domain of the methionine aminopeptidase is located in the c-terminal part of the polypeptide chain  <cit> , therefore n-terminal gfp fusions of both proteins were not expected to interfere with their functions.
in addition, we also constructed c-terminal gfp fusions of both map_bs and yflg_bs, but because of the possible degradation of the fusion proteins in vivo or some unknown reasons, we failed to detect any fluorescence in either case .
cells from strains bsip <dig>  and bsip <dig>  in the mid-exponential growth phase were collected and visualized by fluorescence microscopy, respectively.
both gfp-map_bs  and gfp-yflg_bs  proteins distributed evenly all over the cells.
as was expected, the intensity of the fluorescence of both proteins increased with the increasing concentration of xylose used to induce expression of the gfp fusions ) .
map-defective mutants are rescued by map_bs or yflg_bs supplied in trans
the e. coli mutant em <dig> contains an engineered map gene, whose expression is under the control of isopropyl β-d-thiogalactoside , so that the strain cannot grow unless iptg is added to the growth medium  <cit> .
using the replicative vector pbad, which holds an arabinose-inducible promoter, the map and yflg genes from b. subtilis were individually introduced into strain em <dig>  as shown in figure  <dig>  the transformants could complement the map defect when iptg was omitted  if a high concentration of arabinose was supplied, in the case of plasmids carrying either b. subtilis map or yflg genes.
lower concentrations of arabinose only partially supported growth.
this demonstrated that both map and yflg from b. subtilis can supply the map function to e. coli in vivo.
in b. subtilis, map is an essential gene, while yflg is not  <cit> .
however, with a back-up map or yflg copy under the control of iptg provided in multi-copy plasmid pdg <dig> , map::lacz disruptants in the chromosome of b. subtilis were obtained .
nevertheless, as witnessed in both cases by the systematic formation of variegated blue white streaked colonies on x-gal plates, the clones were extremely unstable, leading to the loss of transformants after a few generations .
map expression is considerably higher than yflg expression
the fact that map is essential while yflg is not  <cit> , together with the ability of yflg gene to rescue map_bs function when supplied at a high level, points to substantial differences in the expression level of the two genes.
to test this possibility, the expression of map and yflg genes in b. subtilis in vivo was studied using lacz as the reporter gene.
strains bshp <dig> and bfs <dig> were grown in minimal medium and were used to monitor the expression of map and yflg, respectively.
the map gene showed the higher promoter activity .
its activity increased gradually during the log phase of growth and a nearly four-fold increase was detected  between early exponential and stationary growth phases.
in contrast, the yflg gene showed very low activity under all the conditions tested  .
in summary, the map gene promoter showed  <dig> to 100-fold higher activity than that of yflg using lacz as the reporter gene.
promoter localization of the yflg gene
in the b. subtilis genome, the yflg gene is located upstream of the yflh gene, followed by a putative rho-independent transcription terminator.
rt-pcr experiments demonstrated that yflg and yflh belonged to a common transcription unit, making an operon .
furthermore, lacz transcription fusion with yflh gene  showed the same activity changes as that of yflg gene during all the conditions tested , substantiating the rt-pcr results.
in order to identify the promoter of the yflg-yflh operon, we carried out primer extension analysis.
two primers complementary to two different regions inside the yflg gene were used yielding identification of the same transcription start point.
as shown in figure  <dig>  the start is located  <dig> nt upstream of the atg translation start codon and regions weakly similar to consensus - <dig>  and - <dig>  regions are found upstream of this start point, separated by  <dig> nt, in an at-rich region.
it is difficult at this point to correlate the structure of this promoter with the poor expression of yflg in all conditions tested.
yvoa showed a slight repression effect on yflg expression
as the expression of yflg in b. subtilis in vivo was very low, we endeavoured to uncover conditions to enhance its expression.
no significant difference was found when the carbon, nitrogen and sulfur sources were changed, and no difference was found in conditions of sporulation or germination .
as genes proximal in the chromosome often code for proteins with related functions, the neighbourhood of yflg was analysed.
gene nagp, which encodes a putative phosphoenolpyruvate-dependent transport system n-acetylglucosamine-specific enzyme iicb component, is located upstream of yflg and transcribed divergently .
it could share common control elements.
exploring the corresponding intergenic region with a sliding window  <dig> nt-long we uncovered sequence aattggtatagatcactag which has a very significant counterpart  upstream of the nagab operon, encoding two n-acetylglucosamine metabolic genes and a putative transcriptional regulator  gene, yvoa .
this prompted us to investigate whether yvoa was a possible regulator for yflg.
using a lacz fusion, we monitored the expression of yflg in a yvoa-disrupted strain, bsip <dig>
as shown in figure 8b, the promoter of yflg showed a low but consistently higher activity in the yvoa gene disrupted strain bsip <dig> when compared with the wild type parent bfs <dig> .
this suggested that yvoa could contribute to the regulation of yflg expression.
discussion
in prokaryotes, it is usually accepted that there is only one gene responsible for methionine aminopeptidase.
however, the b. subtilis genome program predicted the existence of two methionine aminopeptidase genes, map and yflg  <cit> .
we have shown that both map_bs and yflg_bs are functional methionine aminopeptidases in vitro .
furthermore, gene rescue experiments showed that both map and yflg genes expressed on replicative plasmids could supply the map function in e. coli and b. subtilis, demonstrating that both map_bs and yflg_bs are functional methionine aminopeptidases in vivo .
interestingly, kobayashi et al.
<cit>  reported that disruption of the map gene in b. subtilis is lethal, while the deletion of yflg is not.
in the present study, we obtained b. subtilis map mutants when map or yflg genes were expressed in multi-copy replicative plasmids under the control of an iptg induced promoter .
this observation suggests that the single copy of the yflg gene expressed in the chromosome could not supply the map function in b. subtilis, substantiating that under our conditions its expression is extremely low, making map the only significant methionine aminopeptidase available.
this is corroborated by the lacz fusion studies showing that the map gene is expressed some 50–100-fold higher than yflg under all conditions tested.
despite the weak expression of yflg, however, we detected a sigma a promoter using primer extension .
we further showed that this expression is modulated by yvoa , suggesting a possible connection between n-acetylglucosamine metabolism and methionine aminopeptidase.
in this respect it is worth noting that in e. coli glucosamine-6-phosphate deaminase activity is modulated by the amino-terminal methionine of the enzyme  <cit> .
we tried several growth conditions involving n-acetylglucosamine as a carbon or nitrogen source, or both, but did not find any modulation of yflg activity .
we also explored expression of yflg::lacz fusions during sporulation and germination.
no enhancement was observed .
as a consequence, we surmise that the high expression level of map accounts for its essentiality in b. subtilis.
the published map_ec activity  <cit>  is about 5– <dig> times higher than that of map_bs in our assay conditions.
this may be related to the observation that the map gene in e. coli does not belong to a highly expressed ribosome protein operon, in contrast to the situation in b. subtilis  <cit> .
as a result, the relative expression level of map in e. coli might be lower than that of the map gene in b. subtilis.
for still unknown reasons it might be impossible for the yflg gene to function as the main methionine aminopeptidase in b. subtilis in vivo in spite of the high activity of yflg_bs in vitro.
as shown in table  <dig>  the enzyme activity of maps in crude cell extracts of e. coli , b. subtilis  and yflg disrupted b. subtilis strain  showed not much difference in vitro under all the condition tested.
different expression levels of map genes in a genome carrying more than one methionine aminopeptidase genes have been previously suggested to be the main reason for their different physiological roles in vivo, but no direct evidence was provided  <cit> .
methionine aminopeptidases in vivo are active on proteins, i.e.
on complex polypeptides  <cit>  and not on the short peptides in our in vitro assays.
it could well be that specific targets require a specific aminopeptidase.
a similar conclusion holds for other apparently paralogous genes.
for example, there are also two functional peptide deformylases in b. subtilis, but only one is probably the predominant deformylase with concomitant high gene expression  <cit> .
the finding of the co-evolutionary parallel trend of methionine aminopeptidase and deformylase in firmicutes  is consistent with functional relationship in vivo, which correlates well with the fact that deformylase removes all n-formyl groups as a prerequisite for the subsequent function of map  <cit> .
this co-evolution may be the signature of protein-protein interactions  <cit> , an intriguing conjecture, which seems to be supported by the consistent duplication of deformylases in genomes with more than one methionine aminopeptidase .
CONCLUSIONS
we proved that both map_bs and yflg_bs are functional methionine aminopeptidases in b. subtilis and we suggested that map gene is essential because of its high expression level, while yflg is nonessential possibly because of its low expression level making that it can not take over or compensate the function of map when map is not expressed, or because of specific targets dedicated to only one of the two maps.
conservation of several map-like genes in bacilli suggests involvement in a process specific to this group of organisms, which may involve also peptide deformylase.

 ribosome-mediated protein synthesis is always initiated with either methionine or n-formylmethionine. however, after removal of the n-formyl group from the polypeptide by peptide deformylase, the n-terminal methionine of a large number of proteins is cleaved by methionine aminopeptidase. the efficiency of removal of the initiator methionine is defined by a highly conserved local substrate specificity, which is determined by both methionine and its adjacent residue. map hydrolytically removes the n-terminal methionine only when the penultimate residue bears a small and uncharged side chain, and residues downstream of the penultimate residue have little impact on the reaction. all maps studied to date were reported to be cobalt-dependent metalloproteases. however, some reports showed that maps exhibited activity in the presence of other divalent ions, such as zn, fe or mn. according to their sequence homology, maps are grouped into two subtypes, type i and type ii. an insertion of approximately residues in the c-terminal domain of type ii methionine aminopeptidases is the only difference between the two types. eukaryotes possess at least two map genes, of both type i and type ii, while there is only one map gene in most prokaryotic genomes, either type i or type ii. map is distributed throughout living organisms, where it plays an important physiological role. the deletion of the single map gene in prokaryotes such as escherichia coli or salmonella typhimurium is lethal. in yeast, the deletion of any one of the two map genes causes a slow growth phenotype, while deletion of both genes is lethal. why might map activity have an essential role in living cells? previous studies showed that many proteins needed to have their n-terminal methionine removed to have normal biological activity, proper subcellular localization and eventual degradation. as a result, it is possible that map is essential because of the essentiality of its downstream targets. besides essentiality, map changes the dynamics of the sulfur containing metabolites, which may have an important role in the homeostasis of the cell. in general, there is only one map gene in the genome of prokaryotes, with the exception of a cyanobacteria strain, which has three functional methionine aminopeptidases. surprisingly, in the genomes of bacilli, two or even more putative map genes can be detected by sequence alignment. in b. subtilis, two putative genes responsible for map activity, map and yflg, can be identified. kobayashi et al. reported that map is an essential gene in b. subtilis while yflg is not, an apparent paradox if yflg codes for a methionine aminopeptidase. in an attempt to better understand the functional role of map, we studied the evolutionary trend of maps in silico and demonstrated that both the map and yflg genes from b. subtilis code for methionine aminopeptidases in vitro and in vivo; furthermore, the finding of a high expression level for map in parallel with a low expression of yflg in b. subtilis, may account for their essentiality and dispensability, respectively.  sequence alignment and evolutionary pattern analyses in the genome sequence of b. subtilis, two genes could code for methionine aminopeptidases, map and yflg, respectively. the sequence similarity and identity between e. coli map and b. subtilis map is 65% and 46%, respectively. similarity between map_ec and yflg from b. subtilis is 55% and their identity is 34% and similarity between map_bs and yflg_bs is 58% and their identity is 36%. however, the sequence of yflg_bs differs slightly from the prosite consensus for type i maps, which is -x-g-h-g- --x-h-x--x--. in particular it displays one extra residue next to the conserved ghg metal-binding motif. interestingly, map_bs is more similar to map_ec and yflg_bs is more similar to the only putative methionine aminopeptidase of staphylococcus aureus. overall the proteins are very similar in their physico-chemical properties. moreover, as shown in figure the metal binding site is conserved. it was therefore important to investigate whether both proteins had methionine aminopeptidase activity. this difference in evolution of proteins considered as essential in related organisms is intriguing, and we explored the evolution pattern of map in a study meant to identify persistent genes in bacteria. when matched with the evolution of 16s rna, the map gene does not follow a linear course of evolution, as do most ribosomal proteins, but shows an erratic pattern. this suggests that map might be involved in different biological processes besides its role as methionine aminopeptidase according to the organisms. this prompted us to explore the evolution of map with proteins that might be functionally related, using the criteria we used to identify persistent genes. we defined persistent genes as the genes present as orthologs in more than 85% of the genomes of a clade, after removing obligatory endosymbionts from the list. the protein divergence of map in b. subtilis and its orthologs from other firmicutes were compared with bacterial evolutionary distance. similar analyses were performed with a general protease, clpp, def, ribosomal protein s rpsd and ykrb, a second deformylase present in b. subtilis. interestingly, in contrast to the expected evolutionary pattern of rpsd, map exhibited the same erratic evolutionary way as def and ykrb, the two functional deformylases in b. subtilis, suggesting co-evolution of methionine aminopeptidase and deformylase. enzyme activity in vitro it is often recognized that gene function identification solely based on sequence comparisons could be misleading. therefore, we first determined whether map and yflg were authentic map genes by overproducing the proteins in e. coli and characterizing their enzymatic activity. both products of map and yflg genes were purified to more than 90% homogeneity. mass spectrometry showed that the molecular mass of map_bs and yflg_bs was ± da and ± da, respectively. interestingly, both proteins purified from e. coli retained their initial methionine, consistent with the nature of the second residue, isoleucine in both cases. as seen in table both enzymes exhibited a methionine aminopeptidase activity in vitro with the synthetic peptide substrates tested. they differed widely, however, in the extent of their activity: the specific activity of yflg_bs was about 90- and 20-fold higher than that of map_bs with the tetrapeptide mgmm and tripeptide mas as the substrates, respectively. both enzymes preferred the tetrapeptide mgmm as the substrate. with mas and mg as the substrates, map_bs activity was % and % of that with mgmm as the substrate, respectively, while yflg_bs retained only % and % relative activity, respectively, with these substrates. the effect of divalent ions on enzyme activity was investigated. map_bs had significant activity with both mn2+ and co2+. map_bs retained appreciable activity after replacement of co2+ with cd2+, zn2+, ca2+, mg2+, cu2+, sr2+ and ba2+, but not ni2+. in contrast, yflg_bs showed a strong preference for co2+ with a decrease in activity to below 20% with any other divalent ions tested. subcellular localization to investigate whether map_bs and yflg_bs had any difference in their subcellular localization, we constructed n-terminal green fluorescent protein fusions of both proteins. as previously reported, the catalytic domain of the methionine aminopeptidase is located in the c-terminal part of the polypeptide chain, therefore n-terminal gfp fusions of both proteins were not expected to interfere with their functions. in addition, we also constructed c-terminal gfp fusions of both map_bs and yflg_bs, but because of the possible degradation of the fusion proteins in vivo or some unknown reasons, we failed to detect any fluorescence in either case. cells from strains bsip and bsip in the mid-exponential growth phase were collected and visualized by fluorescence microscopy, respectively. both gfp-map_bs and gfp-yflg_bs proteins distributed evenly all over the cells. as was expected, the intensity of the fluorescence of both proteins increased with the increasing concentration of xylose used to induce expression of the gfp fusions ). map-defective mutants are rescued by map_bs or yflg_bs supplied in trans the e. coli mutant em contains an engineered map gene, whose expression is under the control of isopropyl β-d-thiogalactoside, so that the strain cannot grow unless iptg is added to the growth medium. using the replicative vector pbad, which holds an arabinose-inducible promoter, the map and yflg genes from b. subtilis were individually introduced into strain em as shown in figure the transformants could complement the map defect when iptg was omitted if a high concentration of arabinose was supplied, in the case of plasmids carrying either b. subtilis map or yflg genes. lower concentrations of arabinose only partially supported growth. this demonstrated that both map and yflg from b. subtilis can supply the map function to e. coli in vivo. in b. subtilis, map is an essential gene, while yflg is not. however, with a back-up map or yflg copy under the control of iptg provided in multi-copy plasmid pdg, map::lacz disruptants in the chromosome of b. subtilis were obtained. nevertheless, as witnessed in both cases by the systematic formation of variegated blue white streaked colonies on x-gal plates, the clones were extremely unstable, leading to the loss of transformants after a few generations. map expression is considerably higher than yflg expression the fact that map is essential while yflg is not, together with the ability of yflg gene to rescue map_bs function when supplied at a high level, points to substantial differences in the expression level of the two genes. to test this possibility, the expression of map and yflg genes in b. subtilis in vivo was studied using lacz as the reporter gene. strains bshp and bfs were grown in minimal medium and were used to monitor the expression of map and yflg, respectively. the map gene showed the higher promoter activity. its activity increased gradually during the log phase of growth and a nearly four-fold increase was detected between early exponential and stationary growth phases. in contrast, the yflg gene showed very low activity under all the conditions tested. in summary, the map gene promoter showed to 100-fold higher activity than that of yflg using lacz as the reporter gene. promoter localization of the yflg gene in the b. subtilis genome, the yflg gene is located upstream of the yflh gene, followed by a putative rho-independent transcription terminator. rt-pcr experiments demonstrated that yflg and yflh belonged to a common transcription unit, making an operon. furthermore, lacz transcription fusion with yflh gene showed the same activity changes as that of yflg gene during all the conditions tested, substantiating the rt-pcr results. in order to identify the promoter of the yflg-yflh operon, we carried out primer extension analysis. two primers complementary to two different regions inside the yflg gene were used yielding identification of the same transcription start point. as shown in figure the start is located nt upstream of the atg translation start codon and regions weakly similar to consensus - and - regions are found upstream of this start point, separated by nt, in an at-rich region. it is difficult at this point to correlate the structure of this promoter with the poor expression of yflg in all conditions tested. yvoa showed a slight repression effect on yflg expression as the expression of yflg in b. subtilis in vivo was very low, we endeavoured to uncover conditions to enhance its expression. no significant difference was found when the carbon, nitrogen and sulfur sources were changed, and no difference was found in conditions of sporulation or germination. as genes proximal in the chromosome often code for proteins with related functions, the neighbourhood of yflg was analysed. gene nagp, which encodes a putative phosphoenolpyruvate-dependent transport system n-acetylglucosamine-specific enzyme iicb component, is located upstream of yflg and transcribed divergently. it could share common control elements. exploring the corresponding intergenic region with a sliding window nt-long we uncovered sequence aattggtatagatcactag which has a very significant counterpart upstream of the nagab operon, encoding two n-acetylglucosamine metabolic genes and a putative transcriptional regulator gene, yvoa. this prompted us to investigate whether yvoa was a possible regulator for yflg. using a lacz fusion, we monitored the expression of yflg in a yvoa-disrupted strain, bsip as shown in figure 8b, the promoter of yflg showed a low but consistently higher activity in the yvoa gene disrupted strain bsip when compared with the wild type parent bfs. this suggested that yvoa could contribute to the regulation of yflg expression. discussion in prokaryotes, it is usually accepted that there is only one gene responsible for methionine aminopeptidase. however, the b. subtilis genome program predicted the existence of two methionine aminopeptidase genes, map and yflg. we have shown that both map_bs and yflg_bs are functional methionine aminopeptidases in vitro. furthermore, gene rescue experiments showed that both map and yflg genes expressed on replicative plasmids could supply the map function in e. coli and b. subtilis, demonstrating that both map_bs and yflg_bs are functional methionine aminopeptidases in vivo. interestingly, kobayashi et al. reported that disruption of the map gene in b. subtilis is lethal, while the deletion of yflg is not. in the present study, we obtained b. subtilis map mutants when map or yflg genes were expressed in multi-copy replicative plasmids under the control of an iptg induced promoter. this observation suggests that the single copy of the yflg gene expressed in the chromosome could not supply the map function in b. subtilis, substantiating that under our conditions its expression is extremely low, making map the only significant methionine aminopeptidase available. this is corroborated by the lacz fusion studies showing that the map gene is expressed some 50–100-fold higher than yflg under all conditions tested. despite the weak expression of yflg, however, we detected a sigma a promoter using primer extension. we further showed that this expression is modulated by yvoa, suggesting a possible connection between n-acetylglucosamine metabolism and methionine aminopeptidase. in this respect it is worth noting that in e. coli glucosamine-6-phosphate deaminase activity is modulated by the amino-terminal methionine of the enzyme. we tried several growth conditions involving n-acetylglucosamine as a carbon or nitrogen source, or both, but did not find any modulation of yflg activity. we also explored expression of yflg::lacz fusions during sporulation and germination. no enhancement was observed. as a consequence, we surmise that the high expression level of map accounts for its essentiality in b. subtilis. the published map_ec activity is about 5– times higher than that of map_bs in our assay conditions. this may be related to the observation that the map gene in e. coli does not belong to a highly expressed ribosome protein operon, in contrast to the situation in b. subtilis. as a result, the relative expression level of map in e. coli might be lower than that of the map gene in b. subtilis. for still unknown reasons it might be impossible for the yflg gene to function as the main methionine aminopeptidase in b. subtilis in vivo in spite of the high activity of yflg_bs in vitro. as shown in table the enzyme activity of maps in crude cell extracts of e. coli, b. subtilis and yflg disrupted b. subtilis strain showed not much difference in vitro under all the condition tested. different expression levels of map genes in a genome carrying more than one methionine aminopeptidase genes have been previously suggested to be the main reason for their different physiological roles in vivo, but no direct evidence was provided. methionine aminopeptidases in vivo are active on proteins, i.e. on complex polypeptides and not on the short peptides in our in vitro assays. it could well be that specific targets require a specific aminopeptidase. a similar conclusion holds for other apparently paralogous genes. for example, there are also two functional peptide deformylases in b. subtilis, but only one is probably the predominant deformylase with concomitant high gene expression. the finding of the co-evolutionary parallel trend of methionine aminopeptidase and deformylase in firmicutes is consistent with functional relationship in vivo, which correlates well with the fact that deformylase removes all n-formyl groups as a prerequisite for the subsequent function of map. this co-evolution may be the signature of protein-protein interactions, an intriguing conjecture, which seems to be supported by the consistent duplication of deformylases in genomes with more than one methionine aminopeptidase.  we proved that both map_bs and yflg_bs are functional methionine aminopeptidases in b. subtilis and we suggested that map gene is essential because of its high expression level, while yflg is nonessential possibly because of its low expression level making that it can not take over or compensate the function of map when map is not expressed, or because of specific targets dedicated to only one of the two maps. conservation of several map-like genes in bacilli suggests involvement in a process specific to this group of organisms, which may involve also peptide deformylase. 
both map_bs and yflg_bs are functional methionine aminopeptidases in vitro and in vivo. biochemical assays showed that both map and yflg from b. subtilis overproduced in escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. the high expression level of map and low expression level of yflg may account for their essentiality and dispensality in b. subtilis, respectively, when cells are grown under laboratory conditions. further work identified that yvoa acted as a possible weak repressor of yflg expression in b. subtilis in vivo. two putative methionine aminopeptidase genes, map and yflg, were identified in the genome sequence of bacillus subtilis. both map and yflg genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both e. coli and b. subtilis. in contrast, lacz gene transcriptional fusions showed that the promoter activity of map was to 100-fold higher than that of yflg. we investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in b. subtilis. compared with map_bs, yflg_bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates.
 two putative methionine aminopeptidase genes, map and yflg, were identified in the genome sequence of bacillus subtilis. we investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in b. subtilis.  in silico analysis of map evolution uncovered a coordinated pattern of map and deformylase that did not correlate with the pattern of 16s rna evolution. biochemical assays showed that both map and yflg from b. subtilis overproduced in escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. compared with map_bs, yflg_bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. both map and yflg genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both e. coli and b. subtilis. in contrast, lacz gene transcriptional fusions showed that the promoter activity of map was to 100-fold higher than that of yflg. primer extension analysis detected the transcription start site of the yflg promoter. further work identified that yvoa acted as a possible weak repressor of yflg expression in b. subtilis in vivo.  both map_bs and yflg_bs are functional methionine aminopeptidases in vitro and in vivo. the high expression level of map and low expression level of yflg may account for their essentiality and dispensality in b. subtilis, respectively, when cells are grown under laboratory conditions. their difference in activity on synthetic substrates suggests that they have different protein targets in vivo. 