The two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
You
CongHui
C
National Laboratory of Protein Engineering and Plant Genetic Engineering, The College of Life Sciences, Peking University, 100871, Beijing, PR China. cyou@pasteur.fr
Lu
HongYan
H
Sekowska
Agnieszka
A
Fang
Gang
G
Wang
YiPing
Y
Gilles
Anne-Marie
AM
Danchin
Antoine
A
D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016680:Genome, Bacterial; D063208:Methionyl Aminopeptidases; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000201:enzymology
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases