Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

Expression of the yeast galactokinase gene in Escherichia coli.

In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964). However, the molecular mechanisms which control the expression of these genes are not well understood. A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK. The presence of the yeast gene was demonstrated by (i) complementation of the E. coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts. The yeast DNA fragment is 4700 base pairs long, and enables the host E. coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h. The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E. coli fully induced with fucose.     

Molecular cloning of the structural gene for Acinetobacter citrate synthase

The structural gene for citrate synthase of Acinetobacter anitratum has been cloned in Escherichia coli in a form which expresses the enzyme. A library of EcoRI fragments of Acinetobacter genomic DNA was prepared in the vector lambda gt10, and clones were screened by hybridization with an E. coli citrate synthase clone under conditions of reduced stringency. A 6.5 kbp clone was obtained which was subcloned into pBR322, and shown to direct the formation of Acinetobacter citrate synthase in E. coli hosts. The promoter was located within a BglII fragment, and from this information the orientation of the gene was deduced.     

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.     

Identification of the galE gene and a galE homolog and characterization of their roles in the biosynthesis of lipopolysaccharide in a serotype O:8 strain of Yersinia enterocolitica.

A clone that complements mutations in Yersinia enterocolitica lipopolysaccharide (LPS) core biosynthesis was isolated, and the DNA sequence of the clone was determined. Three complete open reading frames and one partial open reading frame were located on the cloned DNA fragment. The first, partial, open reading frame had homology to the rfbK gene. The remaining reading frames had homology to galE, rol, and gsk. Analysis of the galE homolog indicates that although it can complement an Escherichia coli galE mutant, its primary function in Y. enterocolitica is not in the production of UDP galactose but, instead, some other nucleotide sugar required for LPS biosynthesis. This gene has been renamed lse, for LPS sugar epimerase. The rol homolog has been demonstrated to have a role in Y. enterocolitica serotype 0:8 O-polysaccharide antigen chain length determination. An additional galE homolog has been identified in Y. enterocolitica by homology to the E. coli gene. The product of this gene has UDP galactose 4-epimerase activity in both E. coli and Y. enterocolitica. This gene is linked to the other genes of the galactose utilization pathway, similar to what is seen in other members of the family Enterobacteriaceae. Although Y. enterocolitica 0:8 strains are reported to have galactose as a constituent of LPS, a strain containing a mutation in this galE gene does not exhibit any LPS defects.     

Characterization of a second lysine decarboxylase isolated from Escherichia coli.

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

Cloning of the galactokinase gene (galK) from Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) and Streptomyces lividans 66 strains were shown to be sensitive to the galactose analogue 2-deoxy-D-galactose. Spontaneous resistant mutants were isolated that were Gal- and lacked the enzyme galactokinase. The galK gene (structural gene for galactokinase) from S. coelicolor was cloned into S. lividans using the low copy number vector pIJ922. The resulting plasmid (pMT650), which contained a 14 kb insert, complemented gal mutations in both species. The presence of the galK gene on a 2.8 kb EcoRI fragment was confirmed by expressing it in Escherichia coli where it complemented a well characterized galK mutation.     

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.     

苏云金芽孢杆菌以色列亚种130kd杀蚊蛋白基因的...

The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe. The crystal protein containing the component of 130kd toxic protein was purified. The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay. The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E. coli TG1. From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization. The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3. For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation. It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis. The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.     

Galactose-specific messenger ribonucleic acid contents in Escherichia coli

A method is described for measuring the proportion of galactose-specific mRNA (gal-mRNA) in the total RNA extracted from pulse-labelled cells of Escherichia coli K12, by DNA-RNA hybridization with DNA prepared from bacteriophage lambdadg. RNA from wild-type E. coli was compared with RNA from a homogenote carrying the gal operon both in the chromosome and in a substituted sex-factor, and with RNA from a deletion strain that carried the galactose operon only in the exogenote. In each case the cultures were induced with fucose. Under these conditions the amount of gal-mRNA was found to be proportional to the content of galactokinase in the different cultures, and to the gene frequency. The amounts of gal-mRNA in an O(c) mutant and an R(-) mutant were also proportional to the observed contents of galactokinase. In cultures repressed for the enzymes of the galactose operon with thiomethylgalactoside, the content of gal-mRNA was higher than expected from the content of galactokinase. Possible explanations of this finding are discussed.     

Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici")

The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.     

I-CeuI fragment analysis of the Shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri

I-CeuI fragments of four Shigella species were analyzed to investigate their taxonomic distance from Escherichia coli and to collect substantiated evidence of their genetic relatedness because their ribosomal RNA sequences and similarity values of their chromosomal DNA/DNA hybridization had proved their taxonomic identity. I-CeuI digestion of genomic DNAs yielded seven fragments in every species, indicating that all the Shigella species contained seven sets of ribosome RNA operons. To determine the fragment identities, seven genes were selected from each I-CeuI fragment of E. coli strain K-12 and used as hybridization probes. Among the four Shigella species, S. boydii and S. sonnei showed hybridization patterns similar to those observed for E. coli strains; each gene probe hybridized to the I-CeuI fragments with sizes similar to that of the corresponding E. coli fragment. In contrast, S. dysenteriae and S. flexneri showed distinct patterns; rcsF and rbsR genes that located on different I-CeuI fragments in E. coli, fragments D and E, were found to co-locate on a fragment. Further analysis using an additional three genes that located on fragment D in K-12 revealed that some chromosome rearrangements involving the fragments corresponding to fragments D and E of K-12 took place in S. dysenteriae and S. flexneri.     

The gal Genes for the Leloir Pathway of Lactobacillus casei 64H

The gal genes from the chromosome of Lactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of the L. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking the HindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKp promoter is inducible by galactose in the medium, while galEp constitutes a semiconstitutive promoter located in galK.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.     

Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli.

The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in lambda gtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by lambda Tn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [14C]fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [14C]fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis.     

Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype.

Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated. The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene. This was done through complementation with a mini-mu plasmid E. coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C. The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations. It was mapped to 68 min on the E. coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E. coli chromosome. The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment. DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr. Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria. By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment. This ORF defines a previously undiscovered gene in E. coli, called sohA (suppressor of htrA).