Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000.

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the HindIII site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of Tn1000 into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of Tn1000 insertions in the "plus" or gamma delta orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the Tn1000 insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter. The hutIG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of Tn1000 insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.     

Cloning of mycobacterial histidine synthesis genes by complementation of a Mycobacterium smegmatis auxotroph

Histidine-requiring auxotrophs of Mycobacterium smegmatis were isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. One of these mutants, his5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial-specific biosynthetic pathways involved in the virulence of pathogenic species to be studied.     

Construction of a correlated physical and genetic map of the Klebsiella pneumoniae hisDGO region using transposon Tn5 mutagenesis.

Multicopy plasmids containing the hisDG region of Klebsiella pneumoniae were mutagenized with transposon Tn5. The resulting plasmids were examined for their ability to complement hisD and hisG mutations in Escherichia coli. The physical location of Tn5 on each of the hisD::Tn5 and hisG::Tn5 plasmids was determined by restriction endonuclease analysis. By combining the two types of data, a precise correlated physical and genetic map of the K. pneumoniae hisDG region was constructed. Based on this analysis, the minimum sizes of the hisD and the hisG genes were calculated to be 1100 bp and 900 bp, respectively. The hisO(P) region was also identified. The insertional specificity of transposon Tn5 was shown to be very low. One unanticipated result was obtained: Tn5 insertions in the plasmid-borne hisG gene were not polar on hisD.     

Cloning and expression of the histidine operon of Salmonella typhimurium in cells of Escherichia coli

The histidine operon of Salmonella typhimurium and its fragments were cloned in Escherichia coli cells on a multicopy plasmid. Expression of the cloned genes and histidine production by the variants possessing the hisG mutation which desensibilizes the ATP phosphoribosyl transferase for histidine were studied. Amplification of the complete operon including the hisG gene enables histidine accumulation of 2-3 g/l after 72 hours of fermentation.     

Conditions affecting the mutagenicity of sodium bisulfite in Salmonella typhimurium

Sodium bisulfite is a weak mutagen at pH 5 and 6 in S. typhimurium strains carrying the hisG46 and hisD6610 mutations, but is not mutagenic in strains with the hisC3076 or hisD3052 mutations. The bisulfite-induced base-pair substitution mutations were slightly enhanced by the presence of the plasmid, pKM101, but inhibited by the presence of the uvrB and rfa mutations. The hisO1242 mutation which causes constitutive expression of the histidine operon, produced a slight enhancement of frameshift (hisD6610), but not base-pair substitution (hisG46) mutations. Bisulfite-induced mutations appear to be the result of two different mechanisms which may be a function of the repair capacity of the strains. The data suggest that the deamination of cytosine may not be responsible for frameshift mutations, but may be responsible for base-pair substitution mutagenesis. Because the rate of bisulfite autooxidation appears to play a role in the mutagenic process, we are suggesting that the deamination of cytosine may be the result of oxidative damage rather than through the direct formation of a cytosine-bisulfite adduct. This is further supported by the much lower concentrations of bisulfite needed to cause mutagenicity than the 1 M concentrations cited to produce cytosine-bisulfite adducts.     

Isolation and study of hybrid plasmids carrying Escherichia coli threonine operon genes

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.     

Genetic and physical maps of Klebsiella aerogenes genes for histidine utilization (hut)

Summary Deletion derivatives of the hut-containing plasmid pCB101 were tested against point mutants defective in individual genes of the histidine utilization (hut) operons using a complementation/recombination assay. Location of the genes of the right operon, hutU and hutH, was confirmed by direct assay of the gene products, urocanase and histidase; location of the repressor gene was identified by measuring the ability of the plasmid-carried genes to repress the formation of histidase from a chromosomal location. The analysis of eight deletion plasmids unambiguously confirms the map order of the hut genes as hutI-G-C-U-H, and demonstrates that, in Klebsiella aerogenes, the hutU and hutH genes are transcribed from their own promoter. In addition, the genetic map of hut can be aligned with the restriction map of the hut DNA in plasmid pCB101. One of the deletion plasmids studied apparently encodes a defective histidase subunit that is trans-dominant to active histidase. Another deletion, which completely removes the left operon, hutIG, allows high level expression of the hutUH operon and thus overproduction of a toxic intermediate.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.     

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.     

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids.

Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.