Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli.

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

Expression of cloned monkey metallothionein in Escherichia coli.

Expression vectors were constructed in which a cDNA specifying the monkey kidney metallothionein-II (MT-II) was linked directly to the lambda PR promoter. Enhanced expression of MT-II in Escherichia coli was observed when two initiation signals were tandemly linked to the MT-II gene and the lambda cI+ host cells were induced by nalidixic acid.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Expression of cloned monkey metallothionein in Escherichia coli

Expression vectors were constructed in which a cDNA specifying the monkey kidney metallothionein-II (MT-II) was linked directly to the lambda PR promoter. Enhanced expression of MT-II in Escherichia coli was observed when two initiation signals were tandemly linked to the MT-II gene and the lambda cI+ host cells were induced by nalidixic acid.     

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.     

Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.     

Expression of cloned calf prochymosin gene sequence in Escherichia coli

An expression plasmid for calf prochymosin (prorennin) cDNA was constructed. The plasmid (pCR301) contains the lacUV5 promoter in front of the fused gene in which the codons for the N-terminal four amino acids of prochymosin cDNA were replaced with those for the N-terminal ten amino acids of beta-galactosidase. Synthesis of the fused protein with the expected Mr was detected immunologically in Escherichia coli harboring pCR301. The product seemed to be localized in the cell membrane of the bacterial host.     

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region

The nucleotide sequence of the Vibrio alginolyticus sucrose uptake-encoding region was determined, and contained two genes, scrA and scrK. The scrA gene encodes an enzyme IISucrose (EIIScr) protein of the phosphoenolpyruvate dependent phosphotransferase system and the scrK gene encodes a fructokinase. The deduced amino acid (aa) sequence for the V. alginolyticus EIIScr protein was homologous with the EIIScr proteins from Streptococcus mutans, Salmonella typhimurium (pUR400 system) and Bacillus subtilis. The deduced aa sequence for the V. alginolyticus fructokinase was homologous with the Escherichia coli enzymes, 6-phosphofructokinase (isoenzyme 2) and ribokinase. Transposon phoA mutagenesis experiments indicated that the EIIScr protein was a membrane-bound protein with a region that extended into the periplasm.     

A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains

Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy.     

Deletion analysis of sucrose metabolic genes from a Salmonella plasmid cloned in Escherichia coli K12

The sucrose operon from pUR400, a 78-kbp conjugative Salmonella plasmid, was cloned in Escherichia coli K12. The operon was located in a 5.7-kbp SalI restriction fragment and was subcloned, in each of two possible orientations, using the expression vector pUC18. The insert DNA was restriction mapped and duplicate restriction sites in the insert and in the polylinker of the vector were used to create various deletions promoter distal in the operon sequence. Additional deletions were made with the restriction exonuclease Bal31. Cells containing hybrid plasmids with specified deletions lacked the ability to transport sucrose or were constitutive for hydrolase and/or uptake activities. The scrA (enzyme IIScr) and scrR (regulatory) genes resided within 2900-bp SmaI-SalI DNA fragment and were assigned the order scrB, scrA, scrR. An amplified sucrose-inducible gene product, Mr 68,000, was detected only in the membrane fraction from recombinant cells that contained plasmid with the intact operon sequence. This protein represented 11% of the total membrane protein and was resistant to extraction with 0.5 M sodium chloride, 2% Triton X-100, and 0.5% sodium deoxycholate. The protein did not appear to be the product of either the scrA, scrB, or scrR gene and may therefore represent a previously unidentified membrane-bound sucrose protein. A new gene, scrC, is proposed. In addition, the cloned 5.7-kbp SalI and 2.5-kbp SmaI-SalI DNA fragments failed to hybridize to chromosomal DNA from Bacillus subtilis, Streptococcus lactis, Streptococcus mutans, and Lactobacillus acidophilus as well as to DNA from a sucrose plasmid from Salmonella tennessee. However, the probes showed weak homology with a 20-kbp EcoRI restriction fragment from Klebsiella pneumoniae.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.     

Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.     

Existence of an additional structural component in the basal bodies of Escherichia coli and Vibrio alginolyticus flagellae

The structure of Escherichia coli and Vibrio alginolyticus flagella was studied using electron microscopy. An additional protein structure was shown to exist in the basal bodies of intact flagella in these organisms. It is possible that this structure involves three proteins important for the assembly of flagella, energy transduction, and a change-over in the direction of flagellar rotation.     

Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.