Fumio Imamoto

Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli.

Insertion and deletion mutations of the hupB and hupA genes, which encode the HU-1 and HU-2 proteins, respectively, of Escherichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome. The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively. A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene. Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain. The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 degrees C. Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress. The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature.

Requirement of integration host factor (IHF) for growth of Escherichia coli deficient in HU protein

Escherichia coli mutants deficient in histone-like protein, HU, and integration host factor (IHF) were constructed and their growth characteristics were examined. Mutants deficient in both HU and IHF grew slowly and were filamentous at both 30 degrees C and 37 degrees C. These mutants scarcely grew in LB broth at 42 degrees C. They formed minute colonies on LB plates at 42 degrees C and no colonies at 46 degrees C, indicating temperature-sensitive (ts) growth. On the contrary, mutants deficient in either HU or IHF were not ts for growth. These results indicate that IHF compensates for the absence of HU and permits normal cell growth; this suggests functional similarity between HU and IHF.

ROLE OF HU PROTEINS IN FORMING AND CONSTRAINING SUPERCOILS OF CHROMOSOMAL DNA IN ESCHERICHIA COLI

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12-13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying both gyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.

Maintenance of plasmids in HU and 1HF mutants of Escherichia coli

SummaryComplementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the ΔhupA-ΔhupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the ΔhupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the ΔhupA-ΔhupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.

Preferential replication-dependent mutagenesis in the lagging DNA strand inEscherichia coli

The mutation frequencies attributable to −1 frameshift or one-base substitution in the structural genes coding for resistance to chloramphenicol (Cm) and tetracycline (Tc) were followed over several cycles of DNA replication, and found to differ several-fold, depending on the orientation of the gene on the plasmid with respect to the direction of (unidirectional ColE1-type) replication. The mutation frequency was higher when the reporter gene was present in the plasmid in the same orientation as the direction of the origin, i.e., when the transcription template is the lagging daughter strand, than when the gene was inserted in the opposite orientation. This significant difference in reversion frequencies of genes with different polarities was demonstrated only for a brief period of cell growth (several cycles of replication) after induction of thednaQ49 mutator, but was not observed when an increased number of replication cycles, was permitted, most probably due to fixation of the mutation into both strands. The mutated intermediate DNA which possesses a misaligned basepair in the Cm gene was demonstrated to be replicated into two progeny DNA molecules; one is the chloramphenicol-resistant (CmR) DNA synthesized from the template strand having the mutation and the other is the Cms DNA from the template strand without mutation. Our results suggest that replication-dependent mutagenesis may occur preferentially in the lagging strand.

HU-1 mutants of Escherichia coli deficient in DNA binding

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.

Genetic transformation of Lactobacillus casei by electroporation

Lactobacillus casei IAM1045 was transformed with a plasmid pAM beta 1-1, a tra deleted derivative of pAM beta 1, by electroporation. Effective transformation was achieved in electroporation buffers of a wide range of pH values, and in all phases of cell growth tested, with highest frequency in the early log phase. Polyethylene glycol increased the transformation frequency, whereas divalent cations such as Mg2+, Ca2+ and Mn2+ at 0.25 mM decreased the frequency by 2 to 3 orders. Highly efficient transformation of approximately 10(-4)/viable cell was achieved under optimal conditions. A plasmid harboring the trpD, C, F, B and A genes from L casei RNL7 was introduced by electroporation into tryptophan auxotrophic L casei JCM1053. The resulting transformant was found to express the trp genes introduced.

Three tandemly repeated structural genes encoding tRNAf1Met in the metZ operon of Escherichia coli K-12 ☆

The metZ operon of Escherichia coli K-12 consists of three tandemly repeated structural genes encoding tRNA(f1Met) separated by two well-conserved spacer sequences. A multicopy plasmid carrying an intact metZ operon is unstable, deleting tRNA(f1Met)-coding regions by RecA-dependent recombination during cell growth.

Construction and characterization of a rad51rad52 double mutant as a host for YAC libraries.

RAD52 or RAD51 recombination-deficient yeast strains stabilize otherwise unstable YACs containing ribosomal DNA or the human color vision locus (Kohno et al., 1994). Thus the RAD52RAD51 pathways(s) are apparently involved in the instability of YACs containing tandem repeat loci, presumably by promoting recombination-based deletion formation. Some other genomic loci are still unstable or unrecoverable in those strains, but we now find that greater stability is observed in a rad51rad52 double mutant strain that we have newly constructed. YACs containing a highly unstable region around DXS49 or centromeric regions throw off a variety of products in single mutants, but are much more stable in the rad51rad52 strain, which could therefore provide a better host for library construction and maintenance.

Characterization of HU-like protein from Bifidobacterium longum

A HU-like protein (HBl) of Bifidobacterium longum was purified and characterized. HBl is heat-stable and acid-resistant, and has a molecular weight of about 9.1 kDa as estimated by its mobility on electrophoresis. HBl is intermediate in basicity (pI 9.8) between the HU-1 and HU-2 proteins of Escherichia coli, and is dissociated from a calf thymus DNA-cellulose column at 300-400 mM NaCl. Its amino acid composition shows many similarities with that of E coli HU. The NH2-terminal amino acid sequence of HBl also shows significant similarities to the consensus sequence deduced from the sequences of eleven HU-like proteins from prokaryotic sources. Chemical crosslinking analysis indicated that the HBl protein predominantly forms a homotypic dimer.