Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4.
In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH approximately 32 aa approximately E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has < 50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.
Xie
H X
HX
State Key Laboratory of Freshwater Ecology and Biotechnology, and Laboratory of Fish Diseases, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, P.R. China.
Nie
P
P
Sun
B J
BJ
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers; D004926:Escherichia coli; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D015723:Gene Library; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D000085822:Prolyl Oligopeptidases; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012697:Serine Endopeptidases / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D008666:Metalloendopeptidases