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...
The gene for the periplasmic 19-lactamase of Lysobactev enzymogenes was isolated as part of a 1017 bp EcoRI fragment and the nucleotide sequence of the gene was determined. It has a G + C content of 71-5 % and encodes a 27 amino acid signal sequence and the mature /?-lactamase of 276 amino acids which has a mass of 29146 Da. The enzyme appears to be unique to L. enzymogenes but its amino acid sequence shows a high degree of homology with the amino acid sequences of the lactamase from Citvobactev diversus and other Class A 19-lactamases. The /?-lactamase gene of L. enzymogenes was expressed in Eschevichia coli using pUC118 as the vector. The production of active /?-lactamase was highest after the active growth phase of the expression host and reached levels which were about three times higher than those obtained with L. enzymogenes.
Four ADP-glucose pyrophosphorylase cDNA clones were isolated from mature leaves and pith of sago palm by the polymerase chain reaction (PCR) technique. Three of them (agpp10, agpp12 and agpl19) encoded the AGP large subunit, while the fourth clone (agpl1) encoded the small subunit. agpp10 and agpp12 were isolated from pith, agpl19 was isolated from mature leaves, while agpl1 from both tissues. In addition, a full-length cDNA of agpl1 was successfully isolated from a cDNA library of mature leaves by a PCR-based screening technique. Semi-quantitative analysis suggests that agpp10 and agpp12 were detectable only in pith, agpl19 only in leaves, while agpl1 was expressed in both leaves and pith tissues.
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