Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

Cloning and characterization of the Azotobacter vinelandii recA gene and construction of a recA deletion mutant

Summary The recA gene of Azotobacter vinelandii was isolated from a genomic library by heterologous complementation of an Escherichia coli recA mutation for resistance to UV radiation. The A. vinelandii recA gene was localized on adjacent PstI fragments of 1.3 and 1.7 kb. The cloned A. vinelandii recA gene was functionally analogous to the E. coli recA gene. It was also able to complement the E. coli recA mutation for homologous recombination. A recA deletion mutant of A. vinelandii was constructed. This mutant was sensitive to DNA-damaging agents like UV rays, methyl methane sulfonate (MMS) and nalidixic acid and was deficient in homologous recombination.     

Recombination protein A gene,recA, fromSpirulina platensis IAM-M135

We report the cloning and sequencing of therecA gene fromSpirulina platensis. A genomic library ofSpirulina was constructed in pUC19 and screened by PCR using oligonucleotides corresponding to the conserved amino acid sequences ofAnabaena variabilis andSynechococcus RecA proteins. TheSpirulina recA gene consists of an open reading frame (ORF) of 1095 nucleotides encoding a protein (365 residues) which shares an identity of 79%, 70% and 57% with the RecA proteins ofAnabaena variabilis, Synechococcus andEscherichia coli respectively. TherecA gene is located close to one end of the clonedBglII fragment and has only 53 bp of 5 nucleotides. The isolation of this gene has implications for the development of gene transfer system(s) forSpirulina.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

A novel ABA-hypersensitive mutant in Arabidopsis defines a genetic locus that confers tolerance to xerothermic stress

Hot and dry air (harmattan or xerothermic climate) greatly inhibits plant growth, particularly flowering and seed setting of crops. Little is known about the mechanism of plant response to this extreme environmental stress due to the lack of valuable genetic resource. Here, we report the isolation and characteristics of a unique Arabidopsis mutant, hat1 (h armattan t olerant 1), which shows high tolerance to hot and dry air. Under normal growth conditions, the mutant does not differ in morphology and soil drought tolerance compared to the wild type. When subjected to high temperature (42°C) and low humidity (10–15%), however, it could survive up to 6 days, while the wild type (Col-0) died after 24 h. The hat1 mutant also exhibits enhanced tolerance to soil drought, but only under xerothermic conditions. Mutant plants tightly close their stomata to retain water under xerothermic stress, and are more tolerant to high salinity at all developmental stages, accumulating less Na⁺ and more K⁺ than wild-type plants during NaCl treatment. Interestingly, hat1 plants are also ABA-hypersensitive. Genetic analysis revealed that the hat1 phenotype is caused by a dominant mutation at a single nuclear locus. Mapping studies indicate that Hat1 is located at an interval of 168 kb on chromosome 5 in which 21 genes are known to be regulated by diverse abiotic stresses. A mutant of this kind, to our knowledge, has not been previously reported. Thus, this report serves as a starting point in the genetic dissection of the plant response to xerothermic stress, and provides physiological and genetic evidence of the existence of a novel abiotic stress response pathway that is also ABA-dependent.     

A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters

A promoter-trap screen allowed us to identify an Arabidopsis line expressing GUS in the root vascular tissues. T-DNA border sequencing showed that the line was mutated in the caffeic acid O-methyltransferase 1 gene (AtOMT1) and therefore deficient in OMT1 activity. Atomt1 is a knockout mutant and the expression profile of the AtOMT1 gene has been determined as well as the consequences of the mutation on lignins, on soluble phenolics, on cell wall digestibility, and on the expression of the genes involved in monolignol biosynthesis. In this mutant and relative to the wild type, lignins lack syringyl (S) units and contain more 5-hydroxyguaiacyl units (5-OH-G), the precursors of S-units. The sinapoyl ester pool is modified with a two-fold reduction of sinapoyl-malate in the leaves and stems of mature plants as well as in seedlings. In addition, LC-MS analysis of the soluble phenolics extracted from the seedlings reveals the occurrence of unusual derivatives assigned to 5-OH-feruloyl malate and to 5-OH-feruloyl glucose. Therefore, AtOMT1 enzymatic activity appears to be involved not only in lignin formation but also in the biosynthesis of sinapate esters. In addition, a deregulation of other monolignol biosynthetic gene expression can be observed in the Atomt1 mutant. A poplar cDNA encoding a caffeic acid OMT (PtOMT1) was successfully used to complement the Atomt1 mutant and restored both the level of S units and of sinapate esters to the control level. However, the over-expression of PtOMT1 in wild-type Arabidopsis did not increase the S-lignin content, suggesting that OMT is not a limiting enzyme for S-unit biosynthesis.these authors contributed equally to this workthese authors contributed equally to this work     

Characterization of a sunflower (Helianthus annuus L.) mutant,deficient in carotenoid synthesis and abscisic-acid content,induced by in-vitro tissue culture

Genetic variation induced by tissue culture has been characterized in many species. The present study was conducted to genetically and phenotypically characterize an albino mutant in sunflower induced by in-vitro culture. A single recessive gene defective in carotenoid biosynthesis eventually leads to a chlorophyll loss due to photobleaching, absence of seed dormancy, and a low level of endogenous abscisic acid (ABA) in cotyledons and leaves. Further characterization has shown that the endogenous level of the hormone does not increase after drought stress and that the mutation prevents anthocyanin synthesis.     

Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant

An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.     

Expression of the recA gene of Escherichia coli in several species of gram-negative bacteria

Summary A broad host range plasmid containing an operon fusion between the recA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria — 30 species belonging to 20 different genera — to study the expression of the recA gene after DNA damage. These included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae and Azotobacteraceae. Results obtained show that all bacteria tested, except Xanthomonas campestris and those of the genus Rhodobacter, are able to repress and induce the recA gene of E. coli in the absence and in the presence of DNA damage, respectively. All these data indicate that the SOS system is present in bacterial species of several families and that the LexA-binding site must be very conserved in them.     

Protein deficient chloroplast ribosomes in a yellow mutant of Chlamydomonas reinhardii

Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analyzed and compared by two-dimensional gel electrophoresis. The mixothrophically grown yellow-76 mutant differs from wild-type cells in lowered chlorophyll content and photosynthetic activity per chlorophyll unit. The latter is connected with the decreased activity of the ribulose-I,5-diphosphate-carboxylase enzyme. Analytical ultracentrifugation of cell extracts shows a normal amount of free 70S ribosomes and 50S subunit in the mutant cells. Two-dimensional gel electrophoresis shows considerable alterations in the protein composition of the 70S ribosomes of the mutant. Two proteins are absent from the electrophoretograms of the yellow-76 mutant, and seven proteins are present in reduced amounts. The genetical analysis shows a Mendelian pattern of inheritance, indicating that protein alterations presumably are localized in nuclear DNA.Abbreviation MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Engineering, biophysical characterisation and binding properties of a soluble mutant form of annexin A2 domain IV that adopts a partially folded conformation

The four approximately 75-residue domains (repeats) that constitute the annexin core structure all possess an identical five-alpha-helix bundle topology, but the physico-chemical properties of the isolated domains are different. Domain IV of the annexins has previously been expressed only as inclusion bodies, resistant to solubilisation. Analysis of the conserved, exposed hydrophobic residues of the four annexin domains reveals that domain IV contains the largest number of hydrophobic residues involved in interfacial contacts with the other domains. We designed five constructs of domain IV of annexin A2 in which several interfacial hydrophobic residues were substituted by hydrophilic residues. The mutant domain, in which all fully exposed hydrophobic interfacial residues were substituted, was isolated as a soluble protein. Circular dichroism measurements indicate that it harbours a high content of alpha-helical secondary structure and some tertiary structure. The CD-monitored (lambda=222 nm) thermal melting profile suggests a weak cooperative transition. Nuclear magnetic resonance (1H-15N) correlation spectroscopy reveals heterogeneous line broadening and an intermediate spectral dispersion. These properties are indicative of a partially folded protein in which some residues are in a fairly structured conformation, whereas others are in an unfolded state. This conclusion is corroborated by 1-anilinonaphthalene-8-sulfonate fluorescence (ANS) analyses. Surface plasmon resonance measurements also indicate that this domain binds heparin, a known ligand of domain IV in the full-length annexin A2, although with lower affinity.     

Isolation and characterization of a nitrate reductase deficient mutant of <Emphasis Type="Italic">Chlorella ellipsoidea</Emphasis> (Chlorophyta)

Using X-ray mutagenesis and a chlorate-resistance selection method, a nitrate reductase (NR)-deficient mutant of Chlorella ellipsoidea nrm-4 was isolated, which is incapable of using NO₃⁻ as a nitrogen source. NR activity was not detected in nrm-4, suggesting this is a null mutation. Molecular analysis revealed that nrm-4 has a two base deletion at position 2348–49 of its DNA sequence, which produced a frame-shift mutation. The nrm-4 mutation is stable and nrm-4 algae could only use NH₄⁺ and NO₂⁻ as nitrogen sources. Expression of a wild-type NR gene could complement this NR-deficient mutant. Furthermore, this transgenic strain was able to grow in NO₃⁻ medium, when the growth rate of the nrm-4 strain was equal to that of the wide type alga. The nrm-4 strain could potentially be used as a bioreactor that uses nitrate as a selectable marker instead of an antibiotic or herbicide.     

Temperature sensitivity ofTumorous head,a homoeotic mutant ofDrosophila melanogaster

Summary A temperature-sensitive period during early embryogenesis for three stocks carrying thetuh-3 gene suggests that it is a homoeotic mutation involved in the initial determination of the eye-antennal disc rather in maintenance of the determination.     

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc⁺⁺, Nod ^−/+ phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.     

Characterization of expressed pigmented core light harvesting complex (LH 1) in a reaction center deficient mutant of Blastochloris viridis

The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Accumulation of an ABA analogue in the wilty tomato mutant,flacca

A new abscisic acid (ABA) analogue has been isolated from tomato plants. High levels of the compound are found in flacca mutants compared with normal isogenic controls. The analogue also accumulates in response to water stress. Three alternative structures, consistent with the mass spectrum, have been proposed. The possibility that the compound may be a biosynthetic precursor of ABA is considered.We are grateful for the financial support of the ARC.     

Photorespiratory N donors,aminotransferase specificity and photosynthesis in a mutant of barley deficient in serine: glyoxylate aminotransferase activity

A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO₂. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO₂ at twice the wild-type rate when it was fed [¹⁴C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO₂ concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE diethylaminoethyl - PFR photon fluence rate - SGAT serine:glyoxylate aminotransferase