Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes

Abstract

Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamHl fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.The secretion of proteins through the prokaryotic cell envelope has been a major subject of study over the past two decades. Early research concentrated mainly on secretion by gram-positive organisms, since it was presumed that gram-negative bacteria secrete few proteins. However, it is now clear that many gram-negative organisms are able to secrete proteins to the periplasm, the outer membrane, and the medium (14). Extracellular proteins appear to cross the cytoplasmic membrane by a similar mechanism in grampositive and in gram-negative bacteria. They are synthesized as precursers with N-terminal signal sequences which are required for translocation through the cytoplasmic membrane. The signal peptide is subsequently cleaved by a peptidase to release the protein (26). There appear to be different mechanisms for the secretion of extracellular proteins to the outer membrane and the extracellular milieu. The subject has been reviewed extensively (14,22,25,26). It is clear that more work is required to determine the factors involved in the correct targeting of extracellular proteins, especially in gram-negative bacteria.Members of the genus Lysobacter are gram-negative, gliding bacteria which produce a number of extracellular enzymes. For references, see reference 40. Epstein and Wensink (7) have cloned and sequenced the gene for the 19.8-kDa a-lytic protease from Lysobacter enzymogenes. It codes for a polypeptide chain that is twice the size of the mature protease. It is synthesized with a propeptide which inhibits its activity until the enzyme is released from the cell (32). Other microbial proteases also are synthesized with an N-terminal extension (25).The characterization and comparison of genes for extracellular enzymes from an organism such as L. enzymogenes might help to determine the mechanism of the targeting process. The organism produces two extracellular phosphatases. One, a 25-kDa enzyme, is secreted into the medium, and the other, a 69-kDa phosphatase, is associated with the outer membrane (39,41). This report describes the cl...

Identification of a Regulated Alkaline Phosphatase, a Cell Surface-Associated Lipoprotein, inMycobacterium smegmatis

“…coli PhoA has been studied extensively and used as a reporter for the identification of secreted proteins (9,31,32,46,48,50,57), because it is an exported enzyme which is activated only after its translocation into the bacterial periplasmic space. PhoA homologues have also been described in other bacteria (2,3,20,30,34). The PhoA precursor protein in E. coli has a classic amino-terminal signal peptide and is translocated via a Sec-dependent pathway to the periplasmic side of the cytoplasmic membrane.…”

-Lactamase of Lysobacter enzymogenes: cloning, characterization and expression of the gene and comparison of the enzyme to other lactamases

“…The sources of restriction endonucleases, T4 DNA ligase, polynucleotide kinase, the large fragment of E. coli DNA polymerase, T7 DNA polymerase, [ c +~~P ]~A T P and [Y-~~PIATP and other reagents and chemicals have been reported previously (Au et al, 1991 ;von Tigerstrom & Boras, 1990).…”

“…Genomic DNA was prepared from cells grown in 3 % (w/v) trypticase soy broth. Culture conditions for E. coli LE392 and MVI 193 have been described previously (Au et al, 1991). For the production ofp-lactamase by E. coli MVll93 transformed with pUCll8 or with pGB-2a, the cells were grown aerobically at 37 "C for 17 h in 2 x YT [1.6 YO (w/v) tryptone, 1.0 % (w/v) yeast extract, 0.5 % (w/v) NaCl] containing 100 pg ampicillin ml-' and 0.5 mM-IPTG.…”

“…Oligonucleotide probes based on the N-terminal amino acid sequence of the Lysobacter B-lactamase (von Tigerstrom & Boras, 1990) and 17mer oligonucleotide primers for the sequencing reactions were synthesized in our department (Au et al, 1991). EcoRI fragments, obtained by digestion of pGB-2a and isolation by agarose gel electrophoresis were labelled by the random primer method (Feinberg & Vogelstein, 1983, 1984 for use as hybridization probes of the restriction digests of L. enzymogenes DNA.…”

“…However, we thought it was important to describe the enzyme in detail to compare it to other lactamases and trace its origin. Furthermore, a comparison of the plactamase gene with the genes of the a-lytic protease (Epstein & Wensink, 1987;Silen et al, 1989) and the 0001-7847 0 1993 SGM G. J. Boras and others secreted phosphatase (Au et al, 1991) of L. enzymogenes should allow us to gain more insight into the secretion and the targeting of extracellular enzymes by this organism.…”