New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Identification of two new genes,mukE andmukF,involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Escherichia coli cell cycle control genes affect chromosome superhelicity

We have used ethidium bromide titration for direct measurement of the changes in the negative supercoiling of Escherichia coli chromosome caused by mutations inactivating the cell cycle functions mukB and seqA. The amounts of the intercalative agent required to relax the supercoiled chromosome in mukB and seqA mutants were lower and higher, respectively, than for the wild-type parent, confirming that these cell cycle genes modulate the topology of the E. coli chromosome. Plasmid superhelicity measured in these mutant strains showed similar effects albeit of reduced magnitude. As the effects of mukB and seqA mutations were not restricted to the chromosome alone, MukB and SeqA proteins possibly interact with factors involved in the maintenance of intracellular DNA topology. To our knowledge, this is the first direct demonstration of the influence of mukB and seqA genes on the superhelicity of the E. coli chromosome.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB⁺ gene of the F plasmid, rifampin, added at 42°C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257–258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784–789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolidigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (M_r 12 000) newly synthesized at 42°C in the presence of rifampin appeared to be the product of the srnB⁺ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30°C, or the addition of Mg²⁺ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB⁺ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB⁺ gene.     

High level production of human proapo A-I by fed-batch culture of recombinant Escherichia coli

Three Escherichia coli strains, two recA⁻ strains (DH1 and YK537) and one recA⁺ strain (KS476) harboring human proapo A-I expression plasmid pUS(pAI), were cultivated in fed-batch mode using a synthetic medium and the amounts of human proapo A-I accumulation were compared under various cultivation conditions. In the expression plasmid, nine proapo A-I genes were tandemly ligated downstream of the tac promoter. Experimental results indicated that selection of the host strain and cultivation temperature was important. Among the three E. coli strains checked, strain DH1 yielded the most effective production of human proapo A-I at 30°C.     

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele,encoding TonBQ160K,which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3′ end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

On the genetic control of cell cycles during morphogenesis in Ulva mutabilis

Development of the wild type and two temperature-sensitive mutants of the multicellular green alga Ulva mutabilis is compared. The mutants develop normal phenotypes at 22°C and abnormal phenotypes at 15°C. Normal development starts by formation of a filament consisting of a row of cells. The growth rate, the generation times, and the cell length at division change in a coordinated manner according to the positions of the cells within the filament. In the mutant cs₂ transfer to 15°C inhibits all cytoplasmic divisions during early development. In the mutant cs₆ the first three divisions proceed normally. Then cytoplasmic division is blocked in the most distal cells, while the proximal cells continue to divide according to a branched pattern. In the cs₂ mutant cell determination seems to occur at 15°C, while the differentiation of the determined cells can only occur at 22°C. In the mutant cs₆ the cells are not determined at 15°C. The cs₆⁺ gene, as well as the previously described Slender-like genes, presumably has a short period of activity and is concerned with more fundamental epigenetic processes than the cs₂⁺-gene and the previously described precocious-like genes, which seem to have more prolonged periods of activity.     

Identification of two new genes,mukE andmukF, involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase α and is required for cell cycle progression in G2 / M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cΔ) shows slow growth at low temperature (15°?C), while at 30°?C and 37°?C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Poll, the catalytic subunit of DNA polymerase α. The pol1-1 /yjr043cΔ double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cΔ. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cΔ mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication.     

Specific mutation of transglutaminase gene from <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> H197 and characterization of microbial transglutaminase

Microbial transglutaminase (MTG) gene (mtg) from Streptomyces hygroscopicus H197 strain was cloned by PCR and mutated by deleting a specific 84 bp fragment using overlapping extension PCR. The mutant MTG and the wild MTG genes expressed by recombinant plasmid pET32a⁺-mutant mtg and pET32a⁺-mtg, respectively, and were harvested by alternating freeze–thaw steps and purified by Ni column. The purified mutant MTG and the wild MTG exhibited 0.22 U/mg and 0.16 U/mg activity, respectively, and 0.69 U/mg and 0.54 U/mg activity, respectively, after activated by trypsin. The molecular weight of mutant MTG was estimated as 67 kDa by SDS-PAGE. Both MTGs showed optimum activity at pH 6–8 for hydroxamate formation from N-CBZ-Gln-Gly and hydroxylamine, and exhibited higher stability at 40°C and 1–3% salinity. The two types of MTG were not stable in the presence of Zn(II), Cu(II), Hg(II), Pb(II), Fe(III), and Ag(I), suggesting that they could possess a thiol group. In addition, the mutant MTG and the wild MTG were strongly affected by ethanol. Furthermore, the mutant MTG was obviously (P < 0.05 or P < 0.01) more stable than the wild MTG at 50°C and 60°C, at pH 4, 5, and 9, at 7% and 9% salinity, 30% and 35% ethanol concentration, and in the presence of Li(I) and Ag(I). The polyhydroxy compounds as protein stabilizers could elevate MTG stability.     

Amplification of synthesis and secretion of haemolysin using a run-away plasmid in Escherichia coli

Molin and co-workers have described the construction of a ‘run-away’ plasmid, pOU71 which could be useful for the amplification of cloned genes at high temperature when the plasmid replicates to high copy number.In this paper we describe the kinetics of synthesis of a plasmid-coded gene product, β-lactamase, concomitant with pOU71 amplification at 42°C. Maximum amplification was obtained by shifting a culture growing at 30–42°C for 60 min resulting in a 70- to 80-fold amplification for the β-lactamase gene product when the culture was returned to 30°C.The haemolytic determinant LE2001 from an Escherichia coli strain of human origin was cloned into plasmid pOU71 giving rise to plasmid pLG570. Using an identical amplification procedure a 20-fold amplification of the synthesis and secretion of haemolysin was achieved.     

Cloning of the sacS gene encoding a positive regulator of the sucrose regulon in Bacillus subtilis

The sacS gene controls the expression of 2 saccharolytic enzymes in Bacillus subtilis (sucrase and levansucrase).This paper describes a recombinant plasmid containing a mutant allele, sacS^c. The plasmid was isolated from a B. subtilis DNA bank established in Escherichia coli. Moreover, it was shown that the sacS^c allele, placed on a high-copy plasmid, is dominant over the wild-type chromosomal sacS⁺ allele. This result strongly suggests that the sacS gene encodes a positive regulatory protein.     

Hyperproduction of phenylalanine by Escherichia coli: application of a temperature-controllable expression vector carrying the repressor-promoter system of bacteriophage lambda

For the high production of phenylalanine by Escherichia coli, we cloned the pheA^FR and aroF^FR genes (FR = feedback resistant), which encoded chorismate mutase P-prephenate dehydratase and 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase that are feedback inhibition-free as to the endproducts, into a temperature-controllable expression vector composed of the P_R and P_L promoter and a temperature sensitive repressor, cI₈₅₇, of bacteriophage lambda. The plasmid obtained was designated as pSY130-14, and the temperature dependency of expression of the cloned genes and of phenylalanine production was investigated at different temperatures between 30 and 42°C using the strain AT2471 harbouring the plasmid. Above 35°C, the pheA^FR gene and aroF^FR gene expressions, and activities of both enzymes continued to increase up to 42°C. The cell concentration remained constant up to 38.5°C, but started to decrease sharply above 40°C, while the cell concentration of the host strain, AT2471, remained constant at all temperatures tested. The concentration of phenylalanine also depended on the temperature, and the highest production of phenylalanine, 18.6 g l⁻¹, was obtained from glucose at 38.5°C in a 2.5 1 reactor.     

Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli

A pBR322-T.leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the β-IPM dehy drogenase (product of leuB) activity, the optimal temperature of which was 80°C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37°C decreased the temperature optimum from 80°C to 75°C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37°C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.     

Host mutant (tabD)-induced inhibition of bacteriophage T4 late transcription: II. Genetic characterization of mutants

In this paper we show that the tabD mutants, selected with ts553 or tsCB53, and described in the accompanying paper (Coppo et al., 1975): (a) are recessive to tab⁺; (b) fail to complement each other, and thus map in the same cistron; (c) by their linkage to rif and their dominance relationships with well characterized amber mutations in the Escherichia coli RNA polymerase operon, probably all map in the gene controlling the synthesis of the β′ subunit of the enzyme. We also describe the isolation of a ts⁺, k^D mutant in phage T4 gene 55, used in the selection of a new tabD mutant (tabD^k292). This tab mutant: (a) generates a defective phenotype which differs somewhat from that of the other tabD mutants; (b) complements the other tabD mutants; (c) by its dominance relationship to amber mutants in the RNA polymerase operon, can be assigned to the structural gene coding for the β subunit of the enzyme.A new type of interaction between T4 genes 55 and 45 is also described. The k^D properties of ts553 (gene 55) are suppressed at 30 °C, by a temperature-sensitive mutation in gene 45. This type of interaction between missense mutations in genes 45 and 55 apparently occurs even in tab⁺ strains, since temperature-sensitive mutations in gene 45 accumulate in lysates of two gene 55 mutants (ts553 and tsA81).