The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide

Abstract

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtD EC , prtE EC , and prtF EC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtF EC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF EC system.The 62-kDa extracellular lipase of Serratia marcescens has no typical N-terminal signal sequence, but a sequence consisting of multiple repeats of nine amino acid residues (GGXGXD XXX), which is characterized as a glycine-and aspartic acidrich region, is situated in the C-terminal moiety. This sequence was found in the following extracellular proteins of gram-negative bacteria: metalloprotease from Erwinia chrysanthemi (5, 6); hemolysin, encoded by the hlyA gene in Escherichia coli (9); leukotoxin, encoded by the lktA gene in Pasteurella haemolytica (26); cyclolysin, a multifunctional protein carrying an adenylate cyclase activity and a hemolytic activity, encoded by the cyaA gene in Bordetella pertussis (11); and Ca 2ϩ -binding protein, encoded by the nodO gene in Rhizobium leguminosarum (7). The colicin V protein, the cvaC gene product of E. coli (10), possesses a repeated glycine-rich sequence which is not homologous to the GGXGXDXXX sequence but shares some characteristics with it. Since the E. coli cells carrying the S. marcescens lipA gene encoding the lipase did not secrete the lipase protein into the medium, the lipase is expected to be secreted extracellularly via a signal peptide-independent s...

Quorum-sensing-directed protein expression in Serratia proteamaculans B5a

“…4b) presented one band with lipolytic activity, which had an approximate mass of 58 kDa, presumably corresponding to the 62 kDa lipase LipA from S. marcescens (Akatsuka et al, 1995). The quantitative enzymic assay for lipolytic activity (Fig.…”

“…The structural organization and sequence conservation of the genes encoding the ABC protein translocation systems is highly conserved among the Gram-negative bacteria . The lipB operon of S. marcescens consists of three genes, lipBCD, encoding an inner-membrane ATPase (the ABC protein), a membrane fusion protein and an outer-membrane polypeptide, respectively (Akatsuka et al, 1995(Akatsuka et al, , 1997. S. liquefaciens MG1 has been demonstrated to be under the transcriptional control of N-butyryl-L-homoserine lactone (C4-HSL), which renders the production of extracellular protease activity indirectly under the control of quorum sensing .…”

Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins

“…chrysanthemi prtDE genes (Letoff6 et al, 1990), we also found significant homology to the secretion guard for the Serratia marcescens lipase lipBC genes (Akatsuka et al, 1995) and metalloprotease hasDE genes (Letoff6 et al, 1993).…”

“…Although the genes involved in the synthesis and secretion of the extracellular proteins are usually clustered, the lipase and metalloprotease genes are not linked to lipBCD in S. marcescens but are linked to genes encoding products homologous to GDP-mannose pyrophosphorylase, an enzyme involved in the biosynthesis of lipopolysaccharide in Salmonella typhimurium and E. coli (Akatsuka et al, 1995). The structure of LPS plays an important role in the localization of secretory components such as TolC, PrtF and even PrtE in the outer membrane (Wandersman & Letoffk, 1993).…”

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus

“…In each case the protease is secreted via an ABC transporter, often linked to the protease structural gene in the same operon. However, the genetic organization of the protease (prt) operon varies, as does the number of proteases secreted (Ahn et al, 1999;Akatsuka et al, 1995Akatsuka et al, , 1997Duong et al, 1992;Ghigo & Wandersman, 1992a, b;Guzzo et al, 1991b;Kawai et al, 1999).…”