Mutations affecting replication and copy number control in plasmid mini-F both reside in the gene for the 29-kDa protein

We have isolated and characterized cop, copts, and repam mutants of plasmid mini-F after in vitro mutagenesis with hydroxylamine. cop mutants exhibit a copy number of about 10 per cell. The copts mutants are cold-sensitive and have, at 25 degrees C, a copy number of about 30-40 copies per cell, which drops to 4 copies at 42 degrees C. The cop and repam mutations affect the 29-kDa E protein. The Copts phenotype results from the simultaneous occurrence of two mutations, a cop mutation in the E protein and a temperature-dependent mutation (termed ecp) enhancing the Cop phenotype at low temperature. The latter new type of mutation is located within the DNA region 44.1-44.85F. Complementation experiments with plasmid cointegrates show that the wild-type gene is dominant over the cop allele. The nucleotide sequences of the cop and the repam mutations have been determined.     

Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants.

Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.     

Copy mutant of mini-Rts1: lowered binding affinity of mutated RepA protein to direct repeats.

Nucleotide sequence analysis of mini-Rts1 and its copy mutant disclosed the presence of two clusters of direct-repeat sequences flanking the coding region for the 33,000-dalton RepA protein and two base substitutions on the mini-Rts1cop1 genome (Kamio et al., J. Bacteriol. 158:307-312, 1984). On subcloning of the left cluster (incI) that is located downstream from repA, the five 24-base-pair repeats expressed a stronger incompatibility toward mini-Rts1 than did the four repeats. The right cluster (incII) that contains three 21-base-pair repeats also exhibited strong incompatibility toward mini-Rts1. By separating the two base substitutions of mini-Rts1cop1, the mutation that is responsible for the copy increase was determined to be a single base change in the RepA coding region. Both clusters of the repeats, cloned separately into the vector plasmid, showed a weaker incompatibility toward mini-Rts1cop1 than to the wild-type mini-Rts1. These findings suggest a lowered binding affinity of the mutated RepA protein to the direct repeats.     

Regulatory hierarchy of photomorphogenic loci: allele-specific and light-dependent interaction between the HY5 and COP1 loci.

Previous studies suggested that the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) gene product represses photomorphogenic development in darkness and that light signals reverse this action. In this report, we used genetic analysis to investigate the regulatory hierarchical relationship of COP1 and the loci encoding the photoreceptors and other signaling components. Our results showed that cop1 mutations are epistatic to the long hypocotyl mutations hy1, hy2, hy3, and hy4, suggesting that COP1 acts downstream of the phytochromes and a blue light receptor. Although epistasis of a putative null cop1-5 mutation over a hy5 mutation implied that COP1 acts downstream of HY5, the same hy5 mutation can suppress the dark photomorphogenic phenotypes (including hypocotyl elongation and cotyledon cellular differentiation) of the weak cop1-6 mutation. This, and other allele-specific interactions between COP1 and HY5, may suggest direct physical contact of their gene products. In addition, the synthetic lethality of the weak deetiolated1 (det1) and cop1 mutations and the fact that the cop1-6 mutation is epistatic to the det1-1 mutation with respect to light control of seed germination and dark-adaptative gene expression suggested that DET1 and COP1 may act in the same pathway, with COP1 being downstream. These results, together with previous epistasis studies, support models in which light signals, once perceived by different photoreceptors, converge downstream and act through a common cascade(s) of regulatory steps, as defined by DET1, HY5, COP1, and likely others, to derepress photomorphogenic development.     

A single amino acid difference between Rep proteins of P1 and P7 affects plasmid copy number

P1 and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A P1 replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the P1 RepA protein (RepA1) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of P1 cop mutants drops to that of P1 cop+. However, when RepA7 is supplied, the copy number of P1 cop and P1 cop+ is higher than that when RepA1 is supplied. This indicates that the single amino acid difference between the two versions of the RepA protein plays an important role in determining the plasmid copy number.     

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.     

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.     

An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control

Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.     

COP9: a new genetic locus involved in light-regulated development and gene expression in arabidopsis.

We report here the identification and characterization of a new Arabidopsis light-regulatory locus, COP9, mutation that leads to a constitutive photomorphogenic phenotype. Dark-grown cop9 seedlings exhibit many morphological characteristics of light-grown seedlings, including short hypocotyls and open and enlarged cotyledons with cell-type and chloroplast differentiation. Furthermore, the cop9 mutation leads to high-level expression of light-inducible genes in the absence of light, probably by altering the promoter activities of these genes. These properties imply that the mutation in the COP9 locus uncouples the light/dark signals from morphogenesis and light-regulated gene expression. In addition, light-grown cop9 mutants are severely dwarfed and are unable to reach maturation and flowering. This adult-lethal phenotype indicates that the COP9 locus also plays a critical role for normal development of the light-grown plant. Similar to cop1 mutants, but not det1, the cop9 mutants show (1) no effect on the phytochrome control of seed germination and (2) deficiency in the dark-adaptive change of expression of light-regulated genes. Our results suggest that the cop9 and cop1 mutations result in the same range of phenotypes and therefore COP9 and COP1 loci may encode closely related components in the same regulatory pathway.     

Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop.

The replication control functions of a mutant of plasmid R1 that replicates without control at temperatures above 35 degrees C have been analyzed. Although the mutations have not been mapped precisely, the data indicate that the gene (cop) previously identified on the wild-type plasmid (S. Molin and K. Nordström, J. Bacteriol. 141:111-120, 1980) as being responsible for expressing a trans-acting replication inhibitor, as well as for incompatibility of plasmid R1, is not affected in this mutant. Thus, the conditional lack of replication control observed in this plasmid mutant presumably is not caused by the loss of inhibitor activity of the cop gene.     

Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis.

pE194, a 3.5-kilobase multicopy plasmid, confers resistance to the macrolide-lincosamide-streptogramin B antibiotics in Bacillus subtilis. By molecular cloning and deletion analysis we have identified a replication segment on the physical map of this plasmid, which consists of about 900 to 1,000 base pairs. This segment contains the replication origin. It also specifies a trans-acting function (rep) required for the stable replication of pE194 and a negatively acting copy control function which is the product of the cop gene. The target sites for the rep and cop gene products are also within this region. Two incompatibility determinants have been mapped on the pE194 genome and their properties are described. One (incA) resides within the replication region and may be identical to cop. incB, not located in the replication region, expresses incompatibility toward a copy control mutant (cop-6) but not toward the wild-type replicon.     

Stability and expression of a plasmid-containing killer toxin cDNA in batch and chemostat cultures of saccharomyces cerevisiae

A chimeric plasmid (pYT760-ADH1) containing the yeast killer toxin-immunity cDNA was transformed into a leucine-histidine mutant (AH22) and into four industrial toxin-sensitive yeasts. The chimeric plasmid was very stable and expressed toxin production (89.5 +/- 4.8% killer cells) in two of the transformed yeasts that contained the 2mu plasmid, but was lost within 10 generations from two other transformed pickle yeasts that did not contain the 2mu plasmid. It suggested that plasmid stability was dependent on the presence of the 2mu plasmid which is naturally present in some yeasts. The plasmid was extremely stable (100% killer cells) and expressed more toxin in the mutant strain AH22. The effects of dilution rate, D(h(-1)) on plasmid stability and toxin expression were studied in transformed AH22 (AH22/T3) and Montrachet 522 (522/T1) wine yeast grown in glucose-limited chemostat cultures. The results show that killer toxin production by AH22/T3 cells increased as a function of D(h(-1)) and that plasmid stability reached 100% at D >/= 0.09 +/- 0.01 h(-1). However, with Montrachet 522/T1 transformed cells, 100% plasmid stability was seen at D >/= 0.18 +/- 0.02. h(-1). We also challenged the AH22/T3 in chemostat culture (D = 0.25 h(-1)) with an equal number of untransformed cells (AH22). Transformed cells dominated the population (100%) within 8-10 h of growth, a time equivalent to two mean residence time.     

Flowering of Arabidopsis cop1 mutants in darkness

To elucidate the role of the COP1 gene in flowering, we analyzed flowering of cop1 mutant lines in darkness. When grown in the presence of 1% (w/v) sucrose, the cop1-6 mutant flowered in darkness, but cop1-1 and cop1-4 did not. However, cop1-1 and cop1-4 flowered in darkness when grown in the presence of 5% (w/v) sucrose. Therefore, the COP1 gene represses not only photomorphogenesis in seedlings but also flowering in darkness. Comparison of mRNAs levels of floral identity genes in cop1-6 and wild-type plants grown in darkness revealed increased mRNA levels of genes that act downstream of CO and reduced FLC mRNA level in cop1-6. Double mutants of cop1-6 and each of the late-flowering mutations cry2-1, gi-2, co-1, and ld-1 flowered in darkness. All of the double mutants except cry2-1 cop1-6 flowered later than cop1-6, demonstrating that cop1-6 is epistatic to cry2-1 for early flowering. The ld-1 cop1-6 double mutant flowered much earlier than the ld-1 mutant. The delay in flowering in the double mutants was not strongly influenced by the light conditions, whereas that of the gi-2 cop1-6 double mutant was reduced in darkness.     

Characterization of exon skipping mutants of the COP1 gene from Arabidopsis

The removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1–1 was a G→A change 4 nt upstream from the 3′ splice site of intron 5, while the mutation in cop1–2 was a G→A at the first nucleotide of intron 6, abolishing the conserved G within the 5′ splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1–8 was G→A in the final nucleotide of intron 10 abolishing the conserved G within the 3′ splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.     

Identification and expression of a copy number control gene in the IncFIII hemolytic plasmid pSU316.

A DNA fragment carrying both the IncFIII determinant and a copy number control gene of the hemolytic plasmid pSU316 has been cloned in pBR322. Deletion derivatives of the hybrid plasmid generated by Bal 31 digestion, which no longer exhibit the IncFIII phenotype, fall into two complementation groups when tested against a pSU316 miniplasmid derivative. Type 1 mutants exhibit the copy number control (Cop+) phenotype whereas type 2 mutants do not. Restriction analysis of type 1 and type 2 mutants allowed us to locate the cop gene of pSU316 in a 700-base-pair fragment adjacent to the IncFIII determinant. Plasmid expression in a minicell system suggests that the product of the cop gene of pSU316 could be a 13,000-dalton protein.     

Identification of the number of mutant copies of the genetic transfer factor AP42

The copy number mutant of genetic transfer factor pAP42 (incFIX) was induced by the treatment of E. coli AP115 cells carrying the plasmid by N-methyl-N-nitro-N-nitrosoguanidine. The copy number mutant pAP42::Tn1cop1 is characterized by the increased copy number. The cells carrying the copy number mutant have a higher ampicillin resistance and higher beta-lactamase activity. Mutant plasmid pAP42: :Tn1cop1 is incompatible with plasmid pAP42 and compatible with plasmids of the other inc-groups of F-like plasmids.