- Research article
- Open Access
The two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis
- CongHui You†1, 2,
- HongYan Lu†3,
- Agnieszka Sekowska4Email author,
- Gang Fang2,
- YiPing Wang1,
- Anne-Marie Gilles2Email author and
- Antoine Danchin2
© You et al; licensee BioMed Central Ltd. 2005
- Received: 01 August 2005
- Accepted: 05 October 2005
- Published: 05 October 2005
Two putative methionine aminopeptidase genes, map (essential) and yflG (non-essential), 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 (MAP_Bs) and YflG (YflG_Bs) 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 50 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.
- Green Fluorescent Protein Fusion
- Primer Extension Analysis
- lacZ Fusion
- Methionine Aminopeptidase
- Synthetic Peptide Substrate
Ribosome-mediated protein synthesis is always initiated with either methionine (in eukaryotes) or N-formylmethionine (in prokaryotes and eukaryotic organelles) . However, after removal of the N-formyl group from the polypeptide by peptide def ormylase (DEF, EC 22.214.171.124), the N-terminal methionine of a large number of proteins is cleaved by m ethionine a minop eptidase (MAP, EC 126.96.36.199) [2, 3]. 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 (Gly, Ala, Ser, Thr, Pro, Val, or Cys) [4–9], 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(II), Fe(II) or Mn(II) [10–12]. According to their sequence homology, MAPs are grouped into two subtypes, type I and type II [13, 14]. An insertion of approximately 60 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 (Archaea) or type II (Bacteria) .
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 (type I or type II) 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 (reviewed in ). 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
Enzyme activity in vitro
It is often recognized that gene function identification solely based on sequence comparisons could be misleading [24, 25]. 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 27409.17 ± 0.92 Da and 27209.35 ± 2.14 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.
Reaction rate of MAP_Bs and YflG_Bs with different peptides as the substrates
Specific Activity (U/mg)
Peptide (4 mM)
2.6 ± 0.3
232.0 ± 8.2
2.0 ± 0.1
41.0 ± 5.5
1.2 ± 0.1
1.3 ± 0.1
MAP-defective mutants are rescued by MAP_Bs or YflG_Bs supplied in trans
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 pDG148 (30–50 copies using the pUB110 replicon from B. subtilis ), map::lacZ disruptants in the chromosome of B. subtilis were obtained (BSHP7046 and BSHP7037). 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 (data not shown).
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.
Promoter localization of the yflG gene
YvoA showed a slight repression effect on yflG expression
As shown in Figure 8B, the promoter of yflG showed a low but consistently higher activity in the yvoA gene disrupted strain BSIP8004 when compared with the wild type parent BFS4611 (two-fold higher activity). This suggested that YvoA could contribute to the regulation of yflG expression.
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 (Table 1 and Figure 3). 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 (Figure 5). 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 (strains BSHP7046 and BSHP7037). 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 (Figure 7). We further showed that this expression is modulated by YvoA (Figure 8), 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 (data not shown). We also explored expression of yflG::lacZ fusions during sporulation and germination. No enhancement was observed (data not shown). As a consequence, we surmise that the high expression level of map accounts for its essentiality in B. subtilis.
MAP activity in crude cell extracts of E. coli and B. subtilis
Specific Activity (U/mg)
Peptide (4 mM)
E. coli (BL5)
B. subtilis wild type (168)
B. subtilis yflG mutant (BFS4611)
1.5 ± 0.2
1.5 ± 0.1
1.5 ± 0.1
1.1 ± 0.2
1.1 ± 0.1
1.1 ± 0.2
0.6 ± 0.1
0.8 ± 0.2
0.4 ± 0.1
Distribution of def and map genes in selected genomes
Numbers in Each Genome
Nocardia farcinica IFM 10152
Shewanella oneidensis MR-1
Silicibacter pomeroyi DSS-3
Bacillus cereus ZK
Bacillus thuringiensis serovar konkukian
Bacillus anthracis 'Ames Ancestor'
Bacillus anthracis Sterne
Bacillus cereus ATCC 10987
Bacillus anthracis Ames
Bacillus cereus ATCC 14579
Leifsonia xyli subxyli CTCB07
Synechocystis PCC 6803
Bacillus clausii KSM-K16
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.
Bacterial strains, plasmids, and culture conditions
Bacterial strains and plasmids used or created in this study
Source or Reference
K12 supE 44 hsdR 17 recA 1 endA 1 gyrA 46 thi relA 1 lac- F'[proAB+ lacIq lacZΔM 15 Tn10(tetR)]
K12 supE hsdΔ 5 thi Δ (lac-proAB) F'[traD 36 proA+ proB+ lacIq lacZ ΔM 15]
PT7-map (on chromosome)
Chiron Culture Collection Centre
trp C2 yflG::lacZ
Functional analysis projecta
trp C2 yvoA::lacZ
Functional analysis projecta
trp C2 map-lacZ
trp C2 pDG148-map
trp C2 map::lacZ pDG148-map
trp C2 map::lacZ pDG148-yflG
trp C2 pDG148-yflG
trp C2 yflH::lacZ
trp C2 amyE::gfp-map
trp C2 amyE::gfp-yflG
trp C2 yvoA::[(lacZ-ery)::pEC23(Kan)]
trp C2 yflG::lacZ yvoA::[(lacZ-ery)::pEC23(Kan)]
Source or Reference
expression vector, KanR
genetic rescue vector, AmpR
expression vector, AmpR KanR
cloning vector, CmR
N-terminal GFP fusion vector, AmpR SpecR
integrative plasmid, KanR
M. Simon and P. Stragier, unpublished
DNA purification, restriction enzyme digestion, ligation and transformation of E. coli were performed according to standard protocols . For cloning purpose, YieldAce or Pfu DNA polymerase (Stratagene) was used. All the DNA sequences in the plasmids constructed in this work were determined using the dideoxy-chain termination method and Thermo Sequenase Kit (Amersham Pharmacia Biotech). B. subtilis cells were transformed with plasmid DNA following the two-steps protocol described previously . Transformants were selected on LB plates containing corresponding antibiotics and IPTG when needed.
Plasmid and strain constructions
In order to clone the genes from B. subtilis for protein producing in E. coli, both map and yflG genes were amplified by PCR from genomic DNA of B. subtilis 168 and cloned into expression vector pET24b(+) (Novagen) using Nde I and Xho I restriction sites. The amplified fragments included nucleotides +1 to +744 and nucleotides +1 to +747 relative to the translational start point of map and yflG, respectively. The resulting constructs, plasmids pIPP8003 and pIPP8004, were transformed into E. coli BL5 giving strains ECIP8003 and ECIP8004 for overproducing MAP and YflG, respectively.
To construct map and yflG GFP fusion plasmids and strains, both map and yflG genes were amplified by PCR from genomic DNA of B. subtilis 168 and cloned into N-terminal GFP fusion vector pSG1729  using Xho I and Eco RI restriction sites. The amplified fragments included nucleotides +1 to +744 and nucleotides +1 to +747 relative to the translational start point of map and yflG, respectively. The resulting plasmids, pIPP8001 and pIPP8002, were linearized by the Sca I restriction enzyme and were transformed into B. subtilis 168. Clones were selected for the amylase deficient phenotype after double cross-over recombination at amyE site , giving strains BSIP8001 and BSIP8002, respectively.
To obtain map and yflG expression plasmids for further map gene rescue analysis in a conditional map gene deletion E. coli strain, EM9 , both map (nucleotides -30 to +744 relative to the map translation start point) and yflG (nucleotides -21 to +754 relative to the yflG translation start point) genes were amplified by PCR from genomic DNA of B. subtilis 168 with the creation of Bam HI and Bgl II sites. Both purified PCR fragments were cleaved by Bam HI and Bgl II restriction enzymes and inserted between the same two sites in plasmid pBAD containing arabinose inducible promoter (a modified pBAD-αβγ plasmid containing an additional Bam HI cloning site was used after removing a Bam HI and Bgl II insert ), producing plasmids pHPP1017 and pHPP1018, respectively. The proper orientation was checked by the ability of cloned fragment to be recovered by double digestion using Bam HI and Bgl II restriction enzymes. Plasmids pHPP1017, pHPP1018 and the pBAD vector were transformed into E. coli strain EM9 individually and selected on LB plates containing ampicillin, chloramphenicol and IPTG, giving strains ECHP1005, ECHP1006 and ECHP1007, respectively.
To obtain a back-up map and yflG copies for further conditional map disruptant construction in B. subtilis, the map gene (nucleotides -43 to +744 relative to the map translation start point) and yflG gene (nucleotides -54 to 733 relative to the yflG translation start point) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing a Sal I cloning site at the 5' end and a Sph I cloning site at the 3' end, respectively. Both fragments were then inserted into the Sal I and Sph I sites of the IPTG-inducible replicative vector pDG148 , producing plasmid pHPP7021 and pHPP7022, respectively. The wild type strain B. subtilis 168 was transformed with these plasmids, giving strains BSHP7045 and BSHP7038, respectively.
To disrupt the map gene, a 246 bp-long fragment within the map gene (nucleotides +142 to +388 relative to the map translation start point) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing an Eco RI cloning site at the 5' end and a Bam HI cloning site at the 3' end of the fragment. This fragment was then inserted into the Eco RI and Bam HI sites of plasmid pJM783 , which contains a promoterless lacZ reporter gene, producing plasmid pHPP7025. The plasmid was introduced into the chromosome of BSHP7045 and BSHP7038 strains by a single cross-over event, giving strains BSHP7046 and BSHP7037, respectively. To ascertain that they were correctly inserted, clones were checked by PCR.
To construct a map transcriptional fusion with the lacZ gene, a DNA fragment downstream from the map gene (nucleotides from + 423 relative to the translation start point to the gene's stop codon) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing an Eco RI cloning site at the 5' end and a Bam HI cloning site at the 3' end of the fragment, then inserted into the Eco RI and Bam HI sites of plasmid pJM783 , producing plasmid pHPP7017. The plasmid was introduced into the chromosome of B. subtilis 168 by a single crossover event, giving strain BSHP7042.
The yflG transcriptional fusion with the lacZ gene (yflG::lacZ disruptant, strain BFS4611) was constructed within the framework of the European Union and Japanese project for the functional analysis of the genome of B. subtilis, where more than 2000 genes have been disrupted by fusion with lacZ reporter gene [22, 46, 47].
To construct a yflH transcriptional fusion with the lacZ gene, a DNA fragment downstream from the yflH gene (nucleotides from + 32 to +310 relative to the translation start point) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing an Eco RI cloning site at the 5' end and a Bam HI cloning site at the 3' end of the fragment, then inserted into the Eco RI and Bam HI sites of plasmid pJM783 , producing plasmid pHPP7018. The plasmid was introduced into the chromosome of B. subtilis 168 by a single cross-over event, giving strain BSHP7043.
To obtain the disruptant yvoA strain compatible with BFS4611, the BFS817 strain (yvoA::lacZ disruptant, from the European Union and Japanese consortium, see above) was transformed with the Sca I linearized pEC23 plasmid (which carries a kanamycin resistance gene, M. Simon and P. Stragier, unpublished) for replacement with the kanamycin resistance gene of the lacZ and Erm genes belonging to the pMutin plasmid (erythromycin resistant) integrated into the genome, by homologous recombination. The resulting clones were checked for their inability to grow on erythromycin and chloramphenicol. The resulting strain was named BSIP8003. The chromosomal DNA of BSIP8003 was prepared and used to transform the BFS4611 strain, producing the double yvoA and yflG disrupted strain BSIP8004.
Expression and purification of MAP_BS and YflG_Bs proteins
The strains ECIP8003 and ECIP8004 were grown at 37°C in LB medium containing kanamycin (25 μg/ml) and induced with 0.5 mM IPTG for four hours. Cell pellets were harvested by centrifugation and stored at -20°C. Cells were suspended in 20 mM Tris-HCl (pH 8.0) and sonically disrupted. The cellular debris was removed by centrifugation at 12,000 g for 40 min. The supernatants were applied to a DEAE-sephacel column chromatography (Amersham). The protein (MAP_Bs or YflG_Bs) was eluted with 0.1 M NaCl in the same buffer. Protein concentration was determined by the Bradford's method  using protein assay kit (Bio-Rad Laboratories). The purity of isolated proteins was determined by SDS-PAGE . Ion mass spectrometry was used to measure the molecular mass of each purified protein as well as checking the existence of the first methionine in each of the proteins.
The methionine aminopeptidase assay was performed as described by Arie Ben-Bassat et al.  with small modifications. 10 μl protein solution was added to 90 μl of the substrate solution (4 mM peptide in 0.1 M K2HPO4 (pH7.5), and 0.2 mM CoCl2), then incubated at 37°C for 10 min. The reaction was stopped by incubation at 100°C for 2 min. After addition of 900 μl of the colour development mixture (containing 0.2 mg of L-amino acid oxidase, 0.03 mg of horseradish peroxidase, and 0.2 mg of o-dianisidine dihydrochloride in 0.1 M Tris-HCl (pH 7.4)), the tube was incubated at 37°C for 10 min, then the absorbance at 440 nm was recorded. Standard curve with known concentration of L-methionine in the colour development buffer was plotted for quantitative analysis. The absorbance of 1 μmol of methionine per ml at 440 nm is equivalent to 8.6 . One Unit of activity was defined as 1 μmol of methionine produced per min under the assay conditions used. Preference of metal ions was tested by changing the cobalt in the substrate solution into other divalent metal ions at a final concentration of 0.2 mM.
Amylase activity was detected after growth of B. subtilis strains on Tryptose Blood Agar Base (TBAB, Difco) supplemented with 10 g/l hydrolyzed starch (Sigma). Starch degradation was detected by sublimating iodine onto the plates .
β-galactosidase specific activity was measured as described by Miller . One Unit of β-galactosidase activity was defined as the amount of enzyme that produced 1 nmol of o-nitrophenol per min at 28°C. Specific activity was expressed in Units per mg protein. The experiments were performed in triplicates.
Subcellular localization analysis
To visualize GFP-MAP_Bs and GFP-YflG_Bs fusions locations in living cells, BSIP8001 and BSIP8002 strains were grown in minimal medium with 0.5% fructose as the sole carbon source supplemented with xylose to different final concentrations (0.1% to 1% (w/v)). Cells from the mid-exponential growth phase were collected and visualized by fluorescence microscopy. Microscope Axiovert 135 TV (ZEISS, Germany) was used in the experiment and fluorescence filter sets used to visualize GFP were obtained from Chroma Technology Corp (USA). Cells were visualized on 1% agarose (w/v) slides  using Axiovision 4 system (Allied High Tech Products, Inc.) with exposure time of 3–10 s.
Genetic rescue experiment
The growth of EM9, ECHP1005 (containing B. subtilis map gene), ECHP1006 (containing B. subtilis yflG gene) and ECHP1007 (containing pBAD vector alone), was compared on M63 minimal medium plates (37°C, overnight) in the presence or absence of IPTG (1 mM) with or without arabinose (0.02 %, 0.1 % and 0.5 %).
RNA isolation and manipulation
Total cellular RNA was extracted from B. subtilis 168 cells growing in minimal medium to an OD600 of 0.5 using High Pure RNA Isolation Kit from Roche. The RNA concentration was determined by light absorption at 260 nm and 280 nm. 2 μg of RNA were loaded onto 1.2% agarose gel to check the RNA purity and integrity. RT-PCR experiments were performed using RT-PCR System (Promega) as specified by the manufacturer.
Primer extension analysis was carried out using reverse transcriptase AMV (Roche) as described . Two oligonucleotides (+33 to +62 and +97 to +126 relative to translation start point of yflG) were used for the identification of yflG promoter. The same primers were used for the generation of sequence ladders. Reaction products were separated on 7% denaturing polyacrylamide gel containing 8 M urea. DNA sequences were determined using Sanger's dideoxy chain-termination method with "Thermo Sequenase radiolabeled terminator cycle sequencing kit" from Amersham Pharmacia Biotech.
DNA sequence and in silico analysis
DNA sequences were analyzed using the DNA Strider software . The program BLAST  was used to search for homologous sequences in the database. The program CLUSTALW was used for multiple alignment . B. subtilis sequences were analyzed using the SubtiList database [56, 57]. Accepted consensus sequences were extracted from PROSITE [58, 59].
Evolutionary pattern analyses
First, MAP orthologs (orthologs were defined using the BBH (Bi-directional Best Hit) strategy, i.e. if gene a of genome A is the most similar hit of gene b in genome B and vice versa, gene a and b are regarded as a pair of orthologs ) were retrieved between each pair of bacterial genomes. Secondly, the BBHs, with protein sequence similarity >50% and protein length difference <1.3, of the gene map in B. subtilis were collected. Thirdly, the orthologs of each of the BBHs were collected in all the genomes (a total of 206 bacterial genomes in EMBL as of July 1st, 2005), after recursive identification until the members in this cluster is no longer extended. Last, the protein divergence (sequence similarity divided by sequence length difference) of MAP in B. subtilis and its orthologs from other Firmicutes were compared with bacterial evolutionary distance (measured by 16S rRNA evolution). Same analyses were carried out on ClpP, Def, RpsD and YkrB.
The initial stage of the work was supported by the HKU-Pasteur Research Centre (SAR Hong Kong, China) and we would like to thank HF Kung and KY Yuen for their interest at that early moment. CH You was supported by EGIDE (France). We would like to thank JD Huang for his kind gift of pBAD-αβγ vector, Chiron Culture Collection Center for conditional map gene deletion E. coli mutant, EM9, U Mechold for kind suggestions to both the experiment and manuscript drafting, and YL Yap for contribution in silico analyses and discussion.
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