Genetic and phenotypic characterization of cop1 mutants of Arabidopsis thaliana

Ten Arabidopsis lines that carry recessive mutations in the cop1 (constitutively photomorphogenic) locus have been isolated. These lines define at least four different alleles. All of the mutant lines produce dark-grown seedlings that mimic wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings includes: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in cotyledons. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown siblings. The four mutant alleles represent two types of mutations: three alleles (cop 1-1, cop 1-2, and cop 1-3) have severely affected phenotypes whereas one allele (cop 1-4) has a less severe phenotype. Compared to the severe alleles, the cop 1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. The cop1–1/cop1-4 hybrid seedlings are intermediate in many physiological properties under both dark- and light-growth conditions, relative to the two parents. These results may suggest that the extent of residual cop1 gene activity in the mutants dictates the degree to which the aberrant plant phenotype is expressed. Analysis of plants carrying both cop1 and hy, a mutation that results in a deficiency of active phyto-chrome, suggests that the cop1 gene product acts downstream of phytochrome. The differentiation of chloroplasts in the roots of light-grown cop1 plants but not in wild-type plants suggests that the wild-type cop1 gene product also normally plays a role in suppressing chloroplast development in the roots of light-grown plants. To aid the eventual molecular cloning of the cop1 locus, its chromosomal location has been mapped and a molecular marker that is located about 1 centimorgan away from the cop1 locus obtained.     

Copper Resistance Gene Homologs in Pathogenic and Saprophytic Bacterial Species from Tomato

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detected in chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato, suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some function other than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv. vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium by immunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of the homologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning and transferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility and implications of copper resistance gene exchange between different species and genera of pathogenic and saprophytic bacteria on tomato plants are discussed.     

Identification and characterization of a second copy number control gene in mini-F plasmids

Summary We previously reported the existence of a series of chemically induced trans recessive copy-number mutations (cop) for mini-F plasmids and the existence of a similar series of cop mutations induced by insertion of the ampicillin resistance transposon Tn3. In this paper we describe the experiments showing that these two series of mutations are in different genes. Briefly, the experiments show that the one mutant series can complement the other, that the mutations map in distinct but adjacent regions, that the copy numbers of double mutants are the products of the copy numbers determined by the single mutations, and that Tn3 does not elevate copy number by a polar effect on the adjacent cop gene defined by chemical mutagenesis. We term the latter gene copA and the gene mutated by Tn3, copB. We also demonstrate here that copB mutations are recessive to the wild type allele. Further, we have characterized copB by deletion and recombinational analysis as the series of five 19- to 22-base-pair directly repeated sequences that had previously been designated incC-that is, one of the incompatibility genes. The evidence for this conclusion is that plasmids lacking two, three or five direct repeats have their copy number elevated proportionately. Possible mechanisms for copB control of replication are discussed.     

Comparison of the CopB systems of plasmids R1 and Co1V2-K94: A single base alteration in CopB gene is responsible for the increased copy number of the low copy number plasmid CoIV2-K94

Summary We have isolated a deletion mutation and a point mutation in the copB gene of the replication region Repl of the IncFI plasmid Co1V2-K94. Subsequently, this copB gene with and without point mutation was cloned and sequenced, and the point mutation was mapped in the coding region of copB with a change of one amino acid from arginine to serine. Furthermore, this copB mutant had an approximately 10-fold increase in copy number. The CopB-phenotype of Co1V2-K94 could be complemented in trans by the copB gene of coresident IncFII plasmids such as R1 and R538, but not R100, suggesting that ColV2-K94 and R1 or R538 contain the same copB allele.     

Relaxation of stable RNA synthesis by a plasmid-borne locus

Summary The presence of Clo DF13 copy mutants in Escherichia coli (Flac) cells results, in contrast to the presence of Clo DF13 wt plasmids, in a decreased transfer of Flac and a decrease in the efficiency of plating (EOP) of male specific RNA phages.The degree of reduction of these processes is correlated to the number of Clo DF13 copies per cell as was found by the use of copy mutants and a thermosensitive copy control mutant of Clo DF13. For instance, the presence of the Clo DF13 cop3 plasmid results in a hundredfold decrease in EOP of RNA phages and a tenfold decrease in transfer of the F plasmid. No interaction with the efficiency of plating of male specific RNA phages was measured when the wild type Clo DF13, ColE1, ColE2, ColE3 or ColK plasmid is present in the cell. Studies with both, insertion and deletion mutants of CLo DF13 cop3 showed that these effects are not due to a high number of plasmid DNA molecules itself but due to a high amount of plasmid gene products in the cell. Furthermore these studies enabled us to locate the genes involved in these interactions on the Clo DF13 physical map. It turned out that two Clo DF13 genes are involved in the observed phenomena: one gene, coding for polypeptide B (molecular weight 61,000 daltons) which is also involved in the mobilisation of Clo DF13, and one gene coding for polypeptide D (molecular weight 21,000 daltons). The possible role of these Clo DF13 gene products, involved in the decrease in transfer of Flac as well as the decrease in efficiency of plating of male specific RNA phages, is discussed.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance

Summary Clo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA^- and/or parB^- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.     

Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates.

Summary The role of E. coli dnaB and dnaC protein in the replication of plasmid ColE1 and RSF1030 DNA was investigated in a soluble in vitro system (Staudenbauer, 1976a). Extracts from dnaB and dnaC mutants which are phenotypically DNA initiationor DNA elongation-defective were examined for their replicative capacity. It was found that all mutants tested are deficient in the synthesis of supercoiled plasmid DNA. Deficient extracts of dnaB mutants could be partially complemented by purified dnaB wild type protein but required for full complementation dnaC wild type protein as well. The dnaB wild type protein could be replaced by a P1dnaB analog (ban) protein complexed with a dnaB ts protein. Deficient extracts of dnaC mutants were complemented by purified dnaC wild type protein alone.The in vitro plasmid replication cycle had been separated into an early and late stage (Staudenbauer, 1977). Analysis by CsCl velocity centrifugation of the plasmid DNA synthesized in mutant extracts indicates that the early stage, namely the synthesis of early replicative intermediates, proceeds in all dnaB and dnaC mutants tested. However, replication of the early intermediates during the late stage depends on both the dnaB and dnaC protein. These conclusions were confirmed using inhibitors of DNA synthesis.     

Genetic evidence that control of F replication is negative

Summary We have taken advantage of two situations in which the incompatibility barrier between F plasmids is overcome to show that wild-type genes controlling F copy number (cop ⁺) are dominant in trans over mutant genes. The simplest interpretation of our findings is that the cop mutations have inactivated a repressor gene that controls F replication. Since the cop. mutations all map in a region that others have shown by sequence analysis to theoretically encode four proteins, a strong possibility exists that one of these proteins is the repressor.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19

Summary The region of plasmid R1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wildtype R1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.     

Genotypic analysis of degenerative Cordyceps militaris cultured in the pupa of Bombyx mori

The chemical composition and pharmacological effects of Cordyceps militaris are similar to those of Cordyceps sinensis, with the former undergoing greater development and utilization. Strain degeneration is a common phenomenon that occurs with high frequency during the subculturing of C. militaris, however, and the mechanism underlying strain degeneration remains unclear. In this study, we used touch‐down PCR to compare the ITS1 + 5.8S + ITS2, 18S, 28S and mating‐type (MAT) regions sequence of wild‐type and degenerated strains of C. militaris. We also used quantitative real‐time PCR to analyze expression levels of the CmMAT gene. Sequence analysis showed that the ITS1 + 5.8S + ITS2 and 28S regions of degenerated and wild‐type strains were completely identical, the 18S region of the degenerated strain contained seven single‐base mutations, including six base substitutions and one single‐base insertion. Compared with the wild‐type strain, the degenerated strain contained a deletion of the MAT1–2‐1 region, three base substitutions in the MAT1–1‐1 region, and a base substitution in the MAT1–1‐2 region that causes a glycine‐to‐valine amino acid substitution. Quantitative real‐time PCR analysis detected no CmMAT1–2‐1 gene expression in the degenerated strain, confirming the deletion of the CmMAT1–2‐1 gene. Expression levels of the CmMAT1–1‐1 and CmMAT1–1‐2 genes were significantly down‐regulated to only 7.5 % and 4.4 %, respectively, that of the wild‐type strain. These results indicate that 18S and MAT region mutations, as well as down‐regulated of CmMAT gene expression levels, may play important roles in C. militaris degeneration. This study provides a theoretical basis for further elucidation of the molecular mechanisms of C. militaris degeneration.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia

Summary Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.     

Inactivation of type I polyhydroxyalkanoate synthase in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC _mcl) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC _mcl gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.     

Copper Chaperone Cycling and Degradation in the Regulation of the<Emphasis Type="BoldItalic">Cop</Emphasis> Operon of <Emphasis Type="BoldItalic">Enterococcus Hirae</Emphasis>

Extensive insight into copper homeostasis has recently emerged. The Gram-positive bacterium Enterococcus hirae has been a paradigm for many aspects of the process. The cop operon of E. hirae consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. CopA and CopB accomplish copper uptake and export, respectively, and the expression of the cop operon is regulated by copper via the CopY repressor and the CopZ chaperone. The functions of the four Cop proteins have been extensively studied in vivo as well as in vitro and a detailed understanding of the regulation of the cop operon by copper has emerged.     

Abolition of the cyclic variations in radiosensitivity during meiosis in a sporulation mutant blocked in premeiotic DNA synthesis

Summary The response to ultraviolet light (254 nm) of two sporulation mutants during the meiotic process was compared to that of a wild type diploid strain of Saccharomyces cerevisiae. The cyclic pattern for cell killing and rho ^- induction characteristic of diploid wild type cells persists in a strain able to perform the premeiotic DNA synthesis but which is blocked in the further steps of meiosis (spo8 DMS1). On the contrary, these fluctations are abolished in a derived mutant (spo8 dsm1) which is blocked in the premeiotic DNA synthesis. Under these conditions, the response to cell killing can be dissociated from that observed for rho ^- induction.     

Chromosome-encoded inducible copper resistance inPseudomonas strains

NinePseudomonas strains were selected by their high copper tolerance from a population of bacteria isolated from heavy-metal polluted zones. Copper resistance (Cu ^r ) was inducible by previous exposure of cultures to subinhibitory amounts of copper sulfate. All nine strains possessed large plasmids, but transformation and curing results suggest that Cu ^r is conferred by chromosomal genes. Plasmid-lessPseudomonas aeruginosa PAO-derived strains showed the same level of Cu ^r as environmental isolates and their resistance to copper was also inducible. Total DNA from the environmentalPseudomonas, as well as fromP. aeruginosa PAO strains, showed homology to a Cu ^r P. syringae cop probe at low-stringency conditions but failed to hybridize at high-stringency conditions.     

Accumulation of Copper and Other Metals by Copper-Resistant Plant-Pathogenic and Saprophytic Pseudomonads

Copper-resistant strains of Pseudomonas syringae carrying the cop operon produce periplasmic copper-binding proteins, and this sequestration outside the cytoplasm has been proposed as a resistance mechanism. In this study, strain PS61 of P. syringae carrying the cloned cop operon accumulated more total cellular copper than without the operon. Several other copper-resistant pseudomonads with homology to cop were isolated from plants, and these bacteria also accumulated copper. Two highly resistant species accumulated up to 115 to 120 mg of copper per g (dry weight) of cells. P. putida 08891 was more resistant to several metals than P. syringae pv. tomato PT23, but this increased resistance was not correlated with an increased accumulation of metals other than copper. Several metals were accumulated by both PT23 and P. putida, but when copper was added to induce the cop operon, there was generally no increase of accumulation of the other metals, suggesting that the cop operon does not contribute to accumulation of these other metals. The exceptions were aluminum for PT23 and iron for P. putida, which accumulated to higher levels when copper was added to the cultures. The results of this study support the role of copper sequestration in the copper resistance mechanism of P. syringae and suggest that this mechanism is common to several copper-resistant Pseudomonas species found on plants to which antimicrobial copper compounds are applied for plant disease control.