Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae

The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance to high levels of zinc. The two genes are not linked and have been mapped; COT1 to chromosome XV and ZRC1 to chromosome XIII. Phenotypes related to metal homeostasis have been examined in strains with varied COT1 and ZRC1 gene doses. Overexpression of COT1 confers tolerance to moderately toxic levels of zinc and ZRC1 confers tolerance to moderately toxic levels of cobalt. Strains that carry null alleles at both loci are viable. The metal-hypersensitive phenotypes of mutations in either gene are largely unaffected by changes in dosage of the other. COT1 and ZRCI function independently in conferring tolerance to their respective metals, yet the uptake of cobalt ions by yeast cells is dependent on the gene dosage of ZRC1 as well as of COT1 Strains that overexpress ZRC1 have increased uptake of cobalt ions, while ZRCI null mutants exhibit decreased cobalt uptake. The defects in cobalt uptake due to mutations at COT1 and ZRC1 are additive, suggesting that the two genes are responsible for the majority of cobalt and zinc uptake in yeast cells. The function of either gene product seems to be more important in metal homeostasis than is the GRR1 gene product, which is also involved in metal metabolism. Mutations in the GRR1 gene have no effect on the cobalt-related phenotypes of strains that have altered gene dosage of either COT1 or ZRC1.     

Interactions between gene products involved in divalent cation transport in Saccharomyces cerevisiae

The COT1 and ZRC1 genes of Saccharomyces cerevisiae are structurally related dosage-dependent suppressors of metal toxicity. COT1 confers increased tolerance to high levels of cobalt; ZRC1 confers increased tolerance to high levels of zinc. The two genes are not linked and have been mapped; COT1 to chromosome XV and ZRC1 to chromosome XIII. Phenotypes related to metal homeostasis have been examined in strains with varied COT1 and ZRC1 gene doses. Overexpression of COT1 confers tolerance to moderately toxic levels of zinc and ZRC1 confers tolerance to moderately toxic levels of cobalt. Strains that carry null alleles at both loci are viable. The metal-hypersensitive phenotypes of mutations in either gene are largely unaffected by changes in dosage of the other. COT1 and ZRCI function independently in conferring tolerance to their respective metals, yet the uptake of cobalt ions by yeast cells is dependent on the gene dosage of ZRC1 as well as of COT1 Strains that overexpress ZRC1 have increased uptake of cobalt ions, while ZRCI null mutants exhibit decreased cobalt uptake. The defects in cobalt uptake due to mutations at COT1 and ZRC1 are additive, suggesting that the two genes are responsible for the majority of cobalt and zinc uptake in yeast cells. The function of either gene product seems to be more important in metal homeostasis than is the GRR1 gene product, which is also involved in metal metabolism. Mutations in the GRR1 gene have no effect on the cobalt-related phenotypes of strains that have altered gene dosage of either COT1 or ZRC1.     

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.     

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH₂-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH₂-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

Use of oligonucleotide probes to identify members of two-component regulatory systems in Xanthomonas campestris pathovar campestris

Summary Two-component regulatory systems comprising a sensor and a regulator protein, both with highly conserved amino acid domains, and commonly genetically linked, have been described in a range of bacterial species and are involved in sensing environmental stimuli. We used two oligonucleotide probes matching the postulated coding regions for domains of sensor and regulator proteins respectively in Xanthomonas campestris pathovar campestris (Xcc) to identify possible two-component regulatory systems in Xcc. Two different fragments of Xcc DNA with homology to both of these probes were cloned. The DNA sequence of part of one of these fragments encompassed a potential open reading frame (ORF), the predicted amino acid sequence of which had extensive homology with regulator proteins of two-component regulatory systems. Analysis of the predicted amino acid sequence for the 3 end of an adjacent ORF revealed a very high level of homology with the C-terminal end of sensor proteins. Strains of Xcc with Tn5-induced mutations in the regulator gene were affected in extracellular polysaccharide production, and also in resistance to salt and chloramphenicol. No effects of mutation in the second clone were observed.     

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

Summary A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.Abbreviations IGP imidazole glycerolphosphate - HP histidinolphosphate     

Reduced but accurate translation from a mutant AUA initiation codon in the mitochondrial COX2 mRNA of Saccharomyces cerevisiae

The gene encoding the XorII methyltransferase (M · XorII) was cloned from Xanthomonas oryzae pv. oryzae and characterized in Escherichia coli. The M · XorII activity was localized to a 3.1 kb BamHI-BstXI fragment, which contained two open reading frames (ORFs) of 1272 nucleotides (424 amino acids) and 408 nucleotides (136 amino acids). Ten polypeptide domains conserved in other M⁵ cytosine methyltransferases (MTases) were identified in the deduced amino acid sequence of the 1272 ORF. E. coli Mrr⁺ strains were transformed poorly by plasmids containing the XorII MTase gene, indicating the presence of at least one ^MCG in the recognition sequence for M · XorII (CGATCG). The 408 nucleotide ORF was 36% identical at the amino acid level to sequences of the E. coli dcm-vsr gene, which is required for very short patch repair. X. oryzae pv. oryzae genomic DNA that is resistant to digestion by Pvul and XorII hybridizes with a 7.0 kb fragment containing the XorII MTase gene and vsr homolog, whereas DNA from strains that lack M · XorII activity do not hybridize with the fragment.The sequence presented in this paper has been submitted to NCBI; the accession number is U06424     

The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Mutants of Corynebacterium glutamicum that were unable to grow under mild alkaline pH conditions were isolated by mutagenesis. Strain AL-43 exhibiting the highest sensitivity to alkaline pH among the mutants was selected and used to clone a DNA fragment that could complement the phenotype. Sequencing and subcloning of the cloned 4.0-kb EcoRI DNA fragment showed that the Cgl1281 gene was responsible for the complementation. The deduced amino acid sequence of Cgl1281 was found to show significant sequence similarity with CzcD, a Me²⁺/H⁺(K⁺) antiporter, from Bacillus subtilis and also possess the features of the cation diffusion facilitator (CDF) family: the presence of 6 putative transmembrane segments and a signature sequence, indicating that the gene product is a member of the CDF family. Chromosomal disruption of the Cgl1281 rendered C. glutamicum cells sensitive to alkaline pH as well as cobalt, while expression of the gene from a plasmid restored alkali-tolerance to the wild-type level and also led to increased cobalt resistance. These results demonstrated that the putative transporter of the CDF family mediates resistance to cobalt and also plays a physiological role in alkaline pH tolerance in C. glutamicum.     

Cloning, characterization and regulation of a protein disulfide isomerase from the fission yeast Schizosaccharomyces pombe

To elucidate the physiological roles and regulation of a protein disulfide isomerase (PDI) from the fission yeast Schizosaccharomyces pombe, the full-length PDI gene was ligated into the shuttle vector pRS316, resulting in pPDI10. The determined DNA sequence carries 1,636 bp and encodes the putative 359 amino acid sequence of PDI with a molecular mass of 39,490 Da. In the amino acid sequence, the S. pombe PDI appears to be very homologous to A. thaliana PDI. The S. pombe cells harboring pPDI10 showed increased PDI activity and accelerated growth, suggesting that the cloned PDI gene is functioning and involved in the yeast growth. The 460 bp upstream region of the PDI gene was fused into promoterless β-galactosidase gene of the shuttle vector YEp367R to generate pYUPDI10. The synthesis of β-galactosidase from the PDI–lacZ fusion gene was enhanced by oxidative stress, such as superoxide anion and hydrogen peroxide. It was also induced by some non-fermentable and fermentable carbon sources. Nitrogen starvation was able to enhance the synthesis of β-galactosidase from the PDI–lacZ fusion gene. The enhancement by oxidative stress and fermentable carbon sources did not depend on the presence of Pap1. The PDI mRNA levels were increased in both Pap1-positive and Pap1-negative cells treated with glycerol. Taken together, the S. pombe PDI gene is involved in cellular growth and response to nutritional and oxidative stress.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Cloning and analysis of the inulinase gene from Kluyveromyces cicerisporus CBS4857

A highly expressed inulinase gene, KcINU1 was cloned and sequenced from Kluyveromyces cicerisporus CBS4857 a strain which secrets high levels of inulinase into the growth medium. The result of DNA sequencing showed that KcINU1 contained a 1665 bp ORF, coding for a 555 amino acid protein, in which a 23 amino acid signal peptide was included. The sequence has the GenBank Accession no. AF 178979. The analysis of conserved domain in the ORF indicated there was a consensus sequence about 470 amino acids long. The 0.7 Kb promoter and 0.9 Kb terminator were also cloned and sequenced.     

Isolation and characterization of a new gene, sre , which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ⁻ strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ⁻ mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells. Received: 20 January 1998 / Accepted: 23 April 1998     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079