Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol. wt. 16.4 Md), a fragment cut by Bam HI endonuclease from lambda gal phage DNA (lambda D-J-gal-att-int) was joined to pMB9 and cloned in the gal-strain of E. coli, which was grown on selective media with galactose as a sole source of carbon. Plasmid pgal2 was derived from pgal 1 by elimination of the 1.1 Md fragment located between the two EcoRI sites and carrying the lambda att-int region and part of pMB9. To obtain pgal3, the 10.7 Md fragment of lambda DNA located between the two SmaI sites (lambda D-J and part of pMB9) in pgal2 was cut out and the resulting flush-end fragments were sealed by the T4DNA ligase. The mol. wt. of pgal3 containing one SmaI site amounted to 4.6 Md, while several pgal3 variants that had lost their SmaI site were still smaller. Plasmid pgal1 inhibited the growth of the gal- host cells, which effect could be overcome by the accompanying helper pMB9. The presence of pgal2 and pgal3 supported the growth and multiplication of gal- cells on selective media even without the helper plasmid. The total amount of pgal plasmid DNA per cell was constant and equalled 60--70 Md (4 copies of pgal1 or 15--16 copies of pgal3, ColE1 or pMB9). This might explain why the co-presence of pMB9 helper does alleviate the "harmful" effects of the plasmid pgal1 (which carries att-int genes), by reducing the copy number of the latter from four to one.     

Cloning and characterization of 4.5S and 5S RNA genes in tobacco chloroplasts

Tobacco chloroplast 4.5S and 5S RNAs were shown to hybridize with a 0.9 . 10(6) dalton EcoRI fragment of tobacco chloroplast DNA. Recombinant plasmids were constructed from fragments produced by partial digestion of the chloroplast DNA with EcoRI and the pMB9 plasmid as a vector. Five recombinants containing the 4.5S and 5S genes were selected by the colony hybridization technique. One of these plasmids contained also the 16S and 23S RNA genes and was mapped using several restriction endonucleases as well as DNA-RNA hybridization. The order of rRNA genes is 16S-23S-4.5S-5S and the four rRNA genes are coded for by the same DNA strand.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

Deletions within E. coli plasmids carrying yeast rDNA.

Deletions occur in recombinant DNA plasmids that contain yeast ribosomal DNA (rDNA) inserted into the E. coli plasmids pSC101 and pMB9. Deletions within a pMB9 plasmid containing an insert longer than one tandem rDNA repeat apparently are due to homologous recombination because (1) all of the independently derived deletion products of this plasmid lost one complete rDNA repeat (8.6 kb) and retained only a single copy of the segment repeated at the ends of the original insert and (2) deletions were detected only when the insert had terminal redundancy. Deletions also occur within a pSC101 plasmid containing a tandem duplication of a segment (4.7 kb) including both pSC101 DNA and rDNA. Once again these deletions appear to be due to the presence of a duplicated region because all deletion products have lost one complete repeat. Deletions within both of these plasmids took place in both rec+ and recA- host cells, but occurred more frequently in rec+ cells. Oligomerization of the deletion products also occurred in both hosts and was more frequent in rec+ cells.     

Cloning of the lacI gene into A ColE1 plasmid.

The binding of lac repressor to lac operator was utilized to isolate an EcoRI fragment of lambda h80dlac (i+ as well as iq) that contains the lacI gene and lac promoter-operator regions. Ligation of this fragment into EcoRI cleaved pMB9 yielded chimeric DNA molecules of mol. w.t. 9.6 . 10(-6) and 1.5 . 10(7) daltons. Transformed strains containing the plasmids were analyzed for repressor production in vivo and in vitro. Repressor production in one plasmid strain is 7-fold greater than that in heat-inducible lambda h80dlac(iq)lysogens.     

The nucleotide sequence of the araC regulatory gene in Salmonella typhimurium LT2

The nucleotide sequence of the araC regulatory gene of Salmonella typhimurium LT2 has been determined. This sequence and the predicted araC translational product are compared to their counterparts in Escherichia coli. The two genes code for similar products although the S. typhimurium protein is eleven amino acids shorter than the E. coli protein. The predicted amino acid sequences are 92% conserved and the DNA sequences are 82% conserved for the common regions of the two genes.     

The hisD-hisC gene border of the Salmonella typhimurium histidine operon

Summary We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner . The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.     

Isolation of F' plasmids carrying a portion of the Salmonella typhimurium histidine transport operon.

The transposable drug resistance element Tn10 was employed as a region of homology to direct the insertion of Tn10-containing derivatives of F'ts114 lac into the chromosome of a Salmonella typhimurium strain that carries a Tn10 insertion in the histidine transport operon. Based on the direction of transfer of the resulting Hfr strains, the chromosomal Tn10 insertion was determined to be in orientation "A." New F' plasmids were selectively generated from one of the Hfr strains. The F' factors carry an intact dhuA hisJ portion of the histidine transport operon. A Southern hybridization revealed that one of the F' plasmids was formed by a type II excision event.     

Mutation leading to gene fusion in the histidine operon of Salmonella typhimurium

A spontaneous polar mutation located in the region of an intercistronic border in the hiatidine operon of Salmonella was isolated in our laboratory. The mutant, R81, tests as a frameshift in reversion experiments but is prototrophic, capable of growth without histidine supplements despite lowered levels of certain histidine enzymes. The mutation affects the operator distal end of the D gene, causing production of an active histidinol dehydrogenase enzyme with an altered C-terminus. The mutation severely affects expression of the immediately succeeding gene in the translation sequence, hisC, suggesting either that the D–C border and possibly hisC are physically altered or that their normal function in translation is seriously impaired. We have previously described the fortuitous production from R81 of a non-polar derivative with fused D and C genes. This strain produces a bifunctional enzyme with normally separate dehydrogenase and aminotransferase activities present on dimers or multimers of a single fused polypeptide chain. We have now investigated in greater detail the R81 mutation by amino acid sequencing of the C-terminus of altered histidinol dehydrogenase. We find that the R81 mutation causes the addition of a “tail” of four amino acid residues to an otherwise normal dehydrogenase polypeptide chain. The results support our previous suggestion that the R81 mutation profoundly effects the D–C gene border and that this effect is prerequisite to gene fusion.     

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.     

Isolation and analysis of recombinant DNA molecules containing yeast DNA.

2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Escherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.     

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.