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

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 prtDEC, prtEEC, and prtFEC 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 prtFEC 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.     

Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin.

A 5.5 kb DNA fragment carrying the functions necessary for the specific secretion of the extracellular metalloproteases B and C produced by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi has been sequenced. The fragment contains four transcribed and translated genes: inh, which codes for a protease inhibitor and is not required for protease secretion, and prtD, prtE and prtF, which share significant homology with the hlyB, hlyD and tolC genes required for alpha-haemolysin secretion in Escherichia coli. Mutations in any of the three prt genes abolish protease secretion. The prtD and prtE products (60 and 50 kd) contain at least one hydrophobic segment and the prtF gene product contains a signal sequence.     

Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli.

Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the isolation, from a PAO1 DNA genomic bank, of a cosmid carrying the structural gene coding for alkaline protease. By in vivo mutagenesis using transposon Tn1735, which functions as a transposable promoter, the expression of an 8.8-kilobase DNA fragment under control the tac promoter was obtained. When expressed in Escherichia coli, active alkaline protease was synthesized and secreted to the extracellular medium in the absence of cell lysis.     

Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways.

A genetic locus implicated in the synthesis and secretion of alkaline protease (APR) in Pseudomonas aeruginosa has been previously described [Guzzo et al., J. Bacteriol. 172 (1990) 942-948]. The nucleotide sequence of the DNA fragment encoding these functions was determined and revealed the existence of five open reading frames: aprA, the structural gene encoding APR; aprI, which encodes a protease inhibitor; and aprD, aprE, aprF whose products are involved in protease secretion. The AprD, AprE and AprF proteins share significant homology with proteins implicated in secretion of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin. These results provide further evidence for the existence of a specialized secretory system widespread among Gram- bacteria.     

The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coliα-haemolysin

The extracellular alkaline protease produced by Pseudomonas aeruginosa is secreted by a specific pathway, independent of the pathway used by most of the other extracellular proteins of this organism. Secretion of this protease is dependent on the presence of several genes located adjacent to the apr gene. Complementation studies have shown that PrtD, E, and F, the three secretion functions for Erwinia chrysanthemi proteases B and C (Létoffé et al., 1990), can mediate the secretion of the alkaline protease by Escherichia coli. The secretion functions involved in alpha-haemolysin secretion in E. coli (hlyB, hlyD, tolC) can also be used to complement alkaline protease secretion by E. coli, although less efficiently. These data indicate that protease secretion mechanisms in Pseudomonas and Erwinia are very similar and are homologous to that of E. coli alpha-haemolysin.     

Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product

A clone expressing a Porphyromonas gingivalis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between BamHI and HindIII sites. When a 3.2-kb HindIII fragment of pYS307 was used as a probe in Southern hybridization, HindIII-digested chromosomal DNA of P. gingivalis W83, as well as those of W50 and W12, showed a single 3.2-kb hybridizing band, while that of P. gingivalis 33277 showed a 5.0-kb band. Colonies of E. coli containing pYS307 showed pronounced proteolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coli containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-dependent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coli JM83 containing pYS307 was prepared for electrophoresis in the absence of beta-mercaptoethanol. When samples were solubilized in the presence of beta-mercaptoethanol prior to electrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The activity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCl2.     

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.     

Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO.

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.     

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Molecular cloning and physical mapping of the nitrogenase structural genes from the filamentous, non-heterocystous cyanobacterium Pseudanabaena sp. PCC7409

Abstract Sequences homologous to the structural genes for dinitrogenase ( nifD and nifK ) and nitrogenase reductase ( nifH ) have been cloned from the filamentous, non-heterocystous cyanobacterium Pseudanabaena PCC7409. The nifHDK ⁻ homologous sequences were shown to reside on a 6.5-kb Eco RI restriction fragment by using a restriction fragment encoding the Klebsiella pneumoniae nifHDK genes as a heterologous hybridization probe. This 6.5-kb restriction fragment was cloned from a λ gt.wes Eco RI library of the Paseudanabaena sp. PCC7409 genome. This fragment was subcloned into the plasmid vector pUC9 to generate plasmid pPSU20. A detailed physical map of the insert in plasmid pPSU20 was determined, and relative positions of the nifH, nifD , and nifK homologous sequences on this fragment were determined by hybridization analysis with gene-specific fragments derived from the corresponding Anabaena sp. PCC7120 genes. The results indicate that these genes are contiguous in Pseudanabaena sp. PCC 7409 and are arranged in the order nifH, nifD , and nifK . This arrangement resembles that observed for other non-heterocystous cyanobacteria but differs from that observed for Anabaena, Calothrix , and Nostoc species.     

Cloning the BstVI restriction-modification system in Escherichia coli

A standard DNA modification methyltransferase (MTase) selection protocol was followed to clone the BstVI restriction and modification system from Bacillus stearothermophilus in Escherichia coli. Both genes were contained in a 4.4-kb EcoRI fragment from B. stearothermophilus V chromosomal DNA. The heterologous expression of these genes did not depend on their orientation in the vector, suggesting that the genes are expressed in E. coli under the control of promoters located on the cloned fragment. Subcloning experiments demonstrated that the bstVIR gene was expressed in the absence of its cognate MTase.     

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.     

Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.     

Molecular cloning of a new endoglucanase gene from Clostridium cellulolyticum and its expression in Escherichia coli

Summary Two genes coding for endoglucanase activity in Clostridium cellulolyticum were cloned and expressed in Escherichia coli by using plasmid pUC18. The sizes of two fragments harbouring endoglucanase genes are 4.4 kb and 2.0 kb, respectively. The 2.0-kb fragment was identical with a reported DNA fragment encoding an endoglucanase of C. cellulolyticum. The 4.4-kb fragment was obtained first in this study. Deletion analysis showed that a 1.3-kb portion of the 4.4-kb fragment is necessary for the endoglucanase expression by its own promoter. The 4.4-kb fragment hybridized with several different fragments of the genomic DNA in C. cellulolyticum.Offprint requests to: T. Kodama     

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.     

Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO.

xcp mutations have pleiotropic effects on the secretion of proteins in Pseudomonas aeruginosa PAO. The nucleotide sequence of a 1.2-kb DNA fragment that complements the xcp-1 mutation has been determined. Sequence analysis shows the xcpA gene product to be a 31.8-kDa polypeptide, with a highly hydrophobic character. This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. A very strong homology was found between XcpA and PulO, a membrane protein required for pullulanase secretion in Klebsiella pneumoniae. This suggests the existence of a signal sequence-dependent secretion process common to these two unrelated gram-negative bacteria.     

Insertion sequence-based cassette PCR: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.