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 Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli.

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secret sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra- and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVS100) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. coli(pVS100). Sucrose uptake was inhibited by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 microM and 6 nmol/mg of protein per min and 275 microM, respectively.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant.

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.     

Cloning of Pseudomonas putida genes responsible for the primary stages of oxidation of naphthalene in Escherichia coli cells

Data on cloning Pseudomonas putida D-plasmid pBS286 (IncP-9) genes which are responsible for primary stages of naphthalene oxidation as well as data on the expression of these genes in Escherichia coli cells are presented. Recombinant plasmid pBS959 containing the whole constitutive nahA locus encoding naphthalene dioxygenase, a key enzyme of the naphthalene oxidation pathway, has been constructed on the basis of the pUC19 vector. An evidence has been obtained that at least a portion of the sequence of structural nahB gene is cloned in the recombinant pBS959 plasmid. Constitutive expression of the nahA gene is controlled by its own promoter and leads to conversion of indole to indigo in E. coli cells, strain HB101. Plasmid pBS959 is characterized by high structural stability; some instability observed is due to segregation.     

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.     

Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells.     

Cloning, sequencing, and disruption of a levanase gene of Bacillus polymyxa CF43.

The Bacillus polymyxa CF43 lelA gene, expressing both sucrose and fructan hydrolase activities, was isolated from a genomic library of B. polymyxa screened in Bacillus subtilis. The gene was detected as expressing sucrose hydrolase activity; B. subtilis transformants did not secrete the lelA gene product (LelA) into the extracellular medium. A 1.7-kb DNA fragment sufficient for lelA expression in Escherichia coli was sequenced. It contains a 548-codon open reading frame. The deduced amino acid sequence shows 54% identity with mature B. subtilis levanase and is similar to other fructanases and sucrases (beta-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LelA appears to be a 512-amino-acid polypeptide containing no canonical signal peptide. The hydrolytic activities of LelA on sucrose, levan, and inulin were compared with those of B. subtilis levanase and sucrase, confirming that LelA is indeed a fructanase. The lelA gene in the chromosome of B. polymyxa was disrupted with a chloramphenicol resistance gene (cat) by "inter-gramic" conjugation: the lelA::cat insertion on a mobilizable plasmid was transferred from an E. coli transformant to B. polymyxa CF43, and B. polymyxa transconjugants containing the lelA::cat construct replacing the wild-type lelA gene in their chromosomes were selected directly. The growth of the mutant strain on levan, inulin, and sucrose was not affected.     

Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

Cloning and localization of the replication and mobilization regions in the D-plasmid of Pseudomonas putida pBS286 (IncP-9) with a broad host range

Rep-mob loci of naphthalene degradative plasmid pBS286 (IncP-9) have been cloned on the Escherichia coli vectors pUC19 and pUBR322. These loci confer to recombinant plasmids pBS952 and pBS953 the ability for effective mobilization by RP4 (IncP-1) and F plasmid, as well as constant maintenance in various gram-negative bacteria. Localization of cloned sequences in the restriction fragments of conservative part of the pBS286 genome was established. The data obtained correlate with the analysis of plasmids pBS950 and pBS951 which are spontaneous mini-derivatives of pBS286 and pBS292 (delta NPL1::Tn1/Tra+ Nah-) plasmids formed during transformation of E. coli HB101 cells. Plasmids pBS952 and pBS953 retain the incompatibility properties of parental IncP-9 replicon. These recombinant derivatives can be used for construction of bhr vectors with required properties and compatible with bhr vectors constructed on the basis of plasmids from the IncP-1 and IncP-4 groups.     

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.     

Toxins of Bacteroides fragilis and Bacteroides thetaiotaomicron rods as stimulators of adhesion molecule expression on the surface of vascular endothelial cells

Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF), and from three B. thetaiotaomicron strains were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the procedure of van Tassell et al. (1992). The influence of the examined toxins on the expression of adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human dermal microvascular endothelial cells) was assayed in ELISA test with monoclonal antibodies. Four concentrations of toxins were applied: 0.01, 0.1, 1.0 and 10.0 (micrograms/ml). Endothelial cells were activated for 24 hours (ICAM-1 and VCAM-1 expression) and for 4 hours (E-selectin expression). The coloured product of immunoenzymatic reaction was measured by reading the absorbance at wavelength 492 nm. Two controls were performed in each experiment: with resting HMEC-1 and E. coli O55:B5 LPS (Sigma, USA). Bacteroides fragilis and B. thetaiotaomicron lipopolysaccharides stimulated three adhesion molecules under investigation. Their activity was comparable, but weaker than the activity of E. coli O55:B5 LPS. ICAM-1 was the most stimulated molecule. B. fragilis enterotoxin induced two adhesion molecules: VCAM-1 and E-selectin demonstrating weaker stimulatory activity than E. coli LPS. Stimulation of adhesion molecules on vascular endothelial cells should be considered to be a biological activity of B. fragilis and B. thetaiotaomicron endotoxins and B. fragilis enterotoxin.     

Oxidation of n-alkanes by Pseudomonas aeruginosa strain carrying the plasmid pBS251

Pseudomonas aeruginosa PAO8 cannot use n-alkanes or their respective alcohols as a sole carbon source. However, it can grow on n-alkanes when plasmid pBS251 is transferred into its cells. The hybrid plasmid pBS251 is a plasmid RP4 containing genes which control the capability to grow on n-alkanes of the C6-C12 series. Studies of n-alkane oxidation by P. aeruginosa PAO8 carrying pBS251 have shown that this plasmid controls the inducible alkane and alcohol oxidizing activities; the subsequent steps of n-alkane oxidation controlled by chromosomal genes are constitutive.     

Cloning and expression of the Bacteroides fragilis YCH46 neuraminidase gene in Eschirichia coli and Bacteroides uniformis

Abstract A neuraminidase-encoding gene nanH of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The nanH gene was subcloned from the cosmid and was located within a 2.2-kb Xho I- Kpn I fragment. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial chromosome. Neuraminidase activity expressed in the initial Escherichia coli clone was approximately 3600-fold lower than that expressed in B. fragilis YCH46. However, when nanH was transferred from E. coli to B. uniformis by mobilization of a shuttle plasmid, the transconjugant expressed 1100-fold higher activity than the E. coli donor did. These results suggest that modes of nanH expression in E. coli and Bacteroides are heterologous.     

Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli.

We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type of NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda pR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and Tn1000 mutagenesis, the NANase structural gene and control region that functions in B. fragilis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tn1000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.