Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene

Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed.     

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype.

We cloned and sequenced the sohB gene of Escherichia coli. The temperature-sensitive phenotype of bacteria that carry a Tn10 insertion in the htrA (degP) gene is relieved when the sohB gene is present in the cell on a multicopy plasmid (30 to 50 copies per cell). The htrA gene encodes a periplasmic protease required for bacterial viability only at high temperature, i.e., above 39 degrees C. The sohB gene maps to 28 min on the E. coli chromosome, precisely between the topA and btuR genes. The gene encodes a 39,000-Mr precursor protein which is processed to a 37,000-Mr mature form. Sequencing of a DNA fragment containing the gene revealed an open reading frame which could encode a protein of Mr 39,474 with a predicted signal sequence cleavage site between amino acids 22 and 23. Cleavage at this site would reduce the size of the processed protein to 37,474 Mr. The predicted protein encoded by the open reading frame has homology with the inner membrane enzyme protease IV of E. coli, which digests cleaved signal peptides. Therefore, it is possible that the sohB gene encodes a previously undiscovered periplasmic protease in E. coli that, when overexpressed, can partially compensate for the missing HtrA protein function.     

A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens.

Divergicin A is a strongly hydrophobic, narrow-spectrum, nonlantibiotic bacteriocin produced by Carnobacterium divergens LV13. This strain of C. divergens contains a 3.4-kb plasmid that mediates production of, and immunity to, the bacteriocin. N-terminal amino acid sequencing of the purified divergicin A was used to locate the structural gene (dvnA). The structural gene encodes a prepeptide of 75 amino acids consisting of a 29-amino-acid N-terminal extension and a mature peptide of 46 amino acids. Directly downstream of dvnA there is a second open reading frame that encodes the immunity protein for divergicin A. Divergicin A has a calculated molecular mass of 4,223.89 Da. The molecular mass determined by mass spectrometry is 4,223.9 Da, indicating that there is no posttranslational modification of the peptide. The N-terminal extension of divergicin A has an Ala-Ser-Ala (positions -3 to -1) cleavage site and acts as a signal peptide that accesses the general export system of the cell (such as the sec pathway in Escherichia coli). This is the first bacteriocin of lactic acid bacteria to be reported that does not have dedicated maturation and secretion genes. Production of divergicin A was observed in heterologous hosts containing only the two genes associated with divergicin A production and immunity. Fusing alkaline phosphatase behind the signal peptide for divergicin resulted in the secretion of this enzyme in the periplasmic space and supernatant of E. coli.     

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA.

In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.     

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.     

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low-molecular-weight, heat-stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellular protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escherichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 condons. The N-terminal amino acid sequence of the purified inhibitor was also determined. It indicated the existence of an amino-terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.     

A lipopeptide-encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, Ile-Ala-Ala-Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host-range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2-[3H]-glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases.

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Molecular characterization of nosA, a Pseudomonas stutzeri gene encoding an outer membrane protein required to make copper-containing N2O reductase.

A Pseudomonas stutzeri gene (nosA) encoding an outer membrane protein was cloned into the broad-host-range vector pRK290 and expressed in a mutant lacking the protein. Deletion analysis identified the approximate extent of the nosA region which was sequenced, and it was found to contain an open reading frame encoding 683 amino acids including a presumed signal sequence of 44 amino acids. The putative processed form had a molecular weight of 70,218, characteristics typical of outer membrane proteins, and considerable amino acid sequence homology with Escherichia coli BtuB. A short stretch of amino acids was homologous with the E. coli TonB-dependent outer membrane proteins, BtuB, IutA, FepA, and FhuA, suggesting a homologous function: interaction with a periplasmic protein or uptake of a specific substrate.     

Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus

A periplasmic binding protein essential for high-affinity transport of the C4-dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand-binding properties characterized. The protein was not produced in a Tn5 insertion mutant unable to transport C4-dicarboxylates under aerobic conditions in the dark. Wild-type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5 kb section sequenced. A complete open reading frame of 999 bp was identified that encoded a 333-residue protein (DctP) with a molecular weight of 36,128 with a 26-residue amino-terminal signal peptide. The identify of this protein with the purified dicarboxylate-binding protein and the position of the predicted signal peptide cleavage site was confirmed by N-terminal sequencing. No significant homology with other proteins was detected in database searches. A GC-rich region of dyad symmetry was located 7 bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high-affinity dicarboxylate transport system in this bacterium.     

Molecular cloning and expression of an isomalto-dextranase gene from Arthrobacter globiformis T6.

The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli. A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found. The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases. Transformed E. coli cells carrying the 2.3-kb fragment overproduced isomalto-dextranase into the periplasmic space under control of the promoter of the imd gene itself.     

Sequence of an osmotically inducible lipoprotein gene.

The osmB gene of Escherichia coli, whose expression is induced by elevated osmolarity, was cloned and physically mapped to a 0.65-kilobase-pair NsiI-HincII DNA fragment at 28 min on E. coli chromosome. The OsmB protein was identified in minicells expressing the cloned gene. The nucleotide sequence of a 652-base-pair chromosomal DNA fragment containing the osmB gene was determined. The open reading frame encodes a 72-residue polypeptide with an Mr of 6,949. This reading frame was confirmed by sequencing the fusion joint of an osmB::TnphoA gene fusion. The amino-terminal amino acid sequence of the open reading frame is consistent with reported signal sequences of exported proteins. The sequence around the putative signal sequence cleavage site, Leu-Ser-Ala-Cys-Ser-Asn, is highly homologous to the consensus sequence surrounding the processing site of bacterial lipoproteins. The presence of a lipid moiety on the protein was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin. Preliminary localization of the authentic OsmB protein was determined in minicells harboring a plasmid that carries the NsiI-HincII fragment; it was primarily in the outer membrane. Surprisingly, an osmB mutant carrying the osmB::TnphoA insertion mutation was more resistant to the inhibition of metabolism by high osmolarity than the parent strain was.     

Expression and Export of Pseudomonas putida NTU-8 Creatinase by Escherichia coli Using the Chitinase Signal Sequence of Aeromonas hydrophila

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.