Selective cloning of Bacillus subtilis xylose isomerase and xylulokinase in Escherichia coli genes by IS5-mediated expression.

A fragment of Bacillus subtilis DNA coding for xylose isomerase and xylulokinase was isolated from a BamHI restriction pool by complementation of an isomerase-defective Escherichia coli strain. The spontaneous insertion of IS5, which occurred during the very slow growth of the E. coli xyl- cells on xylose, allowed the expression of the cloned Bacillus genes in E. coli. Without IS5 insertion, the xylose genes were inactive in E. coli. Deletion experiments indicated that the control of the expression resides within a 270-bp long region at the right end of IS5. Deletion of this region led to a loss of expression, which could be restored by insertion of the lacUV5 promoter fragment at the deletion site. Sequence analysis showed that the site of IS5 insertion is 195 bp upstream from the putative ATG initiation codon of the xylose isomerase structural gene. This ATG is preceded by a ribosome binding sequence and two hexamers also found in promoter regions of other Bacillus genes. Deletion and mutagenesis analysis led to a preliminary map of the Bacillus xylose operon.     

Regulatory mutants of the aroF-tyrA operon of Escherichia coli K-12.

The regulatory region of the aroF-tyrA operon was fused to the chloramphenicol acetyltransferase (cat) gene on a plasmid vector. Expression of the cat gene was subject to repression by tyrR+. This fusion was used to isolate regulatory mutants with increased expression of the cat gene in which repression by tyrR+ was affected. Nucleotide sequencing of these mutants has led to the identification of three sites involved in the repression of aroF by tyrR+. The existence of a functional promoter divergently transcribing from the aroF regulatory region was also demonstrated by using the cat fusion vector. The expression of this promoter is also regulated by tyrR+.     

IS50-mediated inverse transposition: specificity and precision

The IS50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn5, can move in unison carrying with them any interstitial DNA segment. In consequence, DNA molecules such as a lambda::Tn5 phage genome are composed of two overlapping transposons - the kan segment bracketed by IS50 elements (Tn5), and lambda bracketed by IS50 elements. During direct transposition, mediated by IS50 "O" (outside) ends, the kan gene is moved and the lambda vector is left behind. During inverse transposition, mediated by the "I" (inside) ends of the IS50 elements, the lambda vector segment is moved and the kan gene is left behind. Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982). We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a lambda::Tn5 phage into the amp and tet genes of plasmid pBR322. Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively. Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn5 (by direct transposition). The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS50 junctions, and in eleven others by sequencing one junction. The sequence of the "I" end of IS50 was preserved and 9-bp target sequence duplications were present in every case analyzed. GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites. Our data indicate that transposition mediated by pairs of "I" ends is similar in its specificity and precision to the more frequent transposition mediated by IS50 "O" ends.     

Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator

A41-bp AluI restriction fragment from the trp promoter-operator region has been cloned into the PvuII site of pBR322, regenerating PvuII sites. Transformants were selected on media that allowed the selection of trp-operator-bearing plasmids. The cloned 41-bp fragment can be released from the vector by PvuII digestion, and it possesses a functional promoter and operator as demonstrated by in vivo tests. The 41-bp fragment contains several restriction sites: HincII, TaqI, RsaI, and a HpaI site that is located at the center of the operator sequence. Two new operator derivatives, symmetrical about the HpaI site, were prepared from the 41-bp fragment by joining two right-side, or two left-side PvuII-HpaI pieces together at the HpaI site. These derivatives showed in vivo operator activity. Plasmids containing up to five copies of the 41-bp trp-promoter-operator fragment have been constructed. These plasmids should be useful in preparing large amounts of the 41-bp fragment.     

Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.     

ISPst9, an ISL3-like insertion sequence from Pseudomonas stutzeri AN10 involved in catabolic gene inactivation

A novel insertion sequence (IS), ISPst9, from Pseudomonas stutzeri AN10 was cloned and characterized. ISPst9 is a typical bacterial IS, consisting of a 2472-bp element flanked by 24-bp perfect inverted repeats that generates 8-bp AT-rich target duplications upon insertion. The sequence also contains a gene that encodes an active transposase (TnpA) with significant amino acid identity to members of the ISL3 family. Southern blot analysis of digested genomic DNA of strain AN10 and its 4-chlorosalicylate-degrading derivative strain AN142 demonstrated that native ISPst9 transposes in multiple copies, with one of them responsible for the nahH insertional inactivation observed in strain AN142. Precise excision of ISPst9 yielded NahH+ revertants of AN142 at high frequencies (up to 10-6). In vivo transposition, mainly in multiple copies, of an ISPst9 derivative containing a KmR cassette cloned into a suicide vector was also demonstrated. Hybridization experiments carried out with different strains of P. stutzeri and with 292 phylogenetically distinct environmental isolates suggested that the presence of an ISPst9-like IS occurs in diverse bacteria together with the presence of aromatic hydrocarbon-degrading determinants.     

Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria.

A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria. This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters. Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters. Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria. Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed. The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida.