Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Isolation of Saccharomyces cerevisiae Mutants Defective in Acyl Coenzyme A Synthetase

Mutants of Saccharomyces cerevisiae defective in acyl-CoA synthetase (EC 6.2.1.3) were isolated. The mutants were concentrated by the radiation-suicide technique with the use of tritiated palmitic acid. Selection of the mutants was based on the premise that acyl-CoA synthetase activity would become indispensable when yeast cells in which fatty acid synthesis de novo is blocked are grown in a medium supplemented with fatty acid. The mutant strains isolated exhibited low acyl-CoA synthetase activity in vitro. Furthermore, they accumulated markedly more of the incorporated palmitic acid in the nonesterified form than did the wild- type strain. Some of the mutants showed thermosensitive acyl-CoA synthetase activity, indicating a mutation of the structural gene of the enzyme. Genetic studies on these mutants indicated that their phenotype resulted from a single, recessive mutation of a nuclear gene, designated faa 1 (fatty acid activation).     

Pathogenicity of Pseudomonas syringae subsp. savastanoi Mutants Defective in Phytohormone Production

The present study compares the pathogenicity on olive and oleander plants of three wild-type strains of Pseudomonas syringae subsp. savastanoi (ITM317 and PBa230 from olive and ITM519 from oleander) and three phytohormone-deficient mutants of ITM519: ITM519-41 (Iaa⁺/cytokinins-), ITM519-7 (Iaa⁻/cytokinins⁺), ITM519-6 (Iaa⁻/cytokinins⁻), Mutants not producing IAA (ITM519-7and ITM519-6) only induced necrosis of the inoculated tissues (ITM519-,6) or swellings on the stems attributed to cytokinin production accompanied by necrosis (ITM519-7). By contrast, the Iaa⁺/cytokinins⁻ mutant (ITM519-41) induced attenuated symptoms on stems and knots similar to those obtained with the parent strain on oleander leaves. Olive strains induced necrosis of oleander leaves and were virulent and avirulent, respectively, on olive and oleander stems.
Strain ITM519 and its three mutants were all able to multiply in oleander leaves at similar rates, reaching the same final populations. By contrast, the two olive strains multiply poorly, reaching populations c. 10²-fold lower.
These results confirm that expression of IAA genes alone is sufficient to initiate the development of knots on oleander while cytokinins are necessary for the full expression of the disease symptoms (determining knot size). The findings also indicate that the plant tissues (stems and leaves) react differently to the various strains of the bacterium and, furthermore, suggest that, besides phytohormones, other pathogenetic factors could be involved in this host-pathogen interaction. The necrotic reaction of oleander leaves heavily inoculated with olive strains was interpreted as a possible form of hypersensitivity reaction.     

Sugar transport in the light leaf spot pathogen Pyrenopeziza brassicae

Abstract In the light leaf spot fungus Pyrenopeziza brassicae , the kinetics of uptake of sucrose, glucose and fructose are all biphasic. At low and high concentrations, glucose and fructose share a high-affinity and a low-affinity hexose carrier respectively, with K _m s of 3.5 and 4.6 μM for uptake of glucose and fructose respectively by the high-affinity system, and K _s s of 690 and 750 uM for uptake of glucose and fructose by the low-affinity system. The data also suggest the existence of separate high-affinity and low-affinity uptake systems for sucrose. P. brassicae possesses both soluble and paniculate invertase activity, with pH optima of 5.0 and 4.0 respectively. Activity of the particulate invertase is considerably in excess of the highest rates of sucrose uptake.     

Isolation and Characterization of Pichia heedii Mutants Defective in Xylose Uptake

To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.     

Isolation and Characterization of Dictyostelium Mutants Defective in Sexual Cell Fusion

Sexual cell fusion in the cellular slime mold Dictyostelium discoideum occurs between cells of opposite (heterothallic system) or same (homothallic system) mating types. It also requires certain environmental conditions such as darkness and abundance of water, and thus offers an interesting model system for analyzing mechanisms of cell recognition and of cellular response to environmental factors. We have been studying the mechanism of sexual cell fusion, using two heterothallic strains, NC4 and HM1 of D. discoideum. Two cell-surface glycoproteins, gp70 and gp138, have been identified as relevant molecules in the cell fusion of these strains. The former is specific to mat a cells (HM1) and the latter, common to both mat a and mat A (NC4). Involvement of cell-surface carbohydrates has also been suggested. However, the fuctions of the above fusion-related molecules are still elusive. In the present study, we isolated fusion-deficient mutants from a mutagenized mat A strain of D. discoideum to set up combined genetic and biochemical analyses. Among the three nonconditional mutants obtained, two were normal in the fruiting-body formation, asexual development, but one was aggregateless ( agg ⁻). Further analysis of these mutants would provide detailed information on the mechanism of sexual cell fusion.     

Isolation and Characterization of Paramecium Mutants Defective in Their Response to Magnesium

Four mutant strains of Paramecium tetraurelia with a reduced ability to respond behaviorally to Mg(2+) have been isolated. Voltage-clamp analyses showed that their Mg(2+) insensitivity is associated with a reduced Ca(2+) -dependent Mg(2+) current. The four mutants, which have been dubbed ``eccentric,' result from recessive mutations in two unlinked loci, xntA and xntB. Further analysis of xntA(1) showed it to be unlinked to any of the behavioral mutants of P. tetraurelia described previously, but it is allelic to d4-521, a ``K(+)-resistant' strain, and d4-596, a ``Ba(2+)-shy' mutant. The varied pleiotropic effects of xntA(1), which include increased resistance to Ni(2+) and Zn(2+) poisoning, suggest that the locus encodes a central regulator of cell function in Paramecium.     

Transport of Glutamate and Glutamine in the Light Leaf Spot Pathogen Pyrenopeziza brassicae

The kinetics of glutamic acid and glutamine uptake in the light leaf spot fungus, Pyrenopeziza brassicae , are biphasic. At low and high concentrations, glutamic acid and glutamine share a high-affinity and a low-affinity carrier, respectively, with K_ms of 4.0 and 4.4 μ m for uptake of glutamic acid and glutamine, respectively, by the high-affinity system, and K_m s of 580 and 560 μ m for uptake of glutamic acid and glutamine by the low-affinity system. The data suggest that glutamic acid and glutamine are taken up by a different system to that responsible for the uptake of ornithine, arginine, lysine and asparagine, and may represent system IV described in Neurospora crassa for the uptake of acidic amino acids.     

The Isolation and Characterization of Mutants Defective in Nitrate Assimilation in NEUROSPORA CRASSA

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth tests that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing co-factor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-7, nit-8 and nit-9), all of which are genetically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar to the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one locus (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.     

Isolation of Mutants Defective in Early Steps of Meiotic Recombination in the Yeast Saccharomyces Cerevisiae

Using a selection based upon the ability of early Rec- mutations (e.g., rad50) to rescue the meiotic lethality of a rad52 spo13 strain, we have isolated 177 mutants. Analysis of 56 of these has generated alleles of the known Rec genes SPO11, ME14 and MER1, as well as defining five new genes: REC102, REC104, REC107, REC113 and REC114. Mutations in all of the new genes appear to specifically affect meiosis; they do not have any detectable mitotic phenotype. Mutations in REC102, REC104 and REC107 reduce meiotic recombination several hundred fold. No alleles of RED1 or HOP1 were isolated, consistent with the proposal that these genes may be primarily involved with chromosome pairing and not exchange.     

Isolation and Phenotypes of Mutants from Salmonella typhimurium Defective in Formate Hydrogenlyase Activity

A new method is described for the isolation of different mutants defective in formic dehydrogenase activity measured with benzyl viologen as electron acceptor.     

Isolation of Rhodospirillum centenum Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis

The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.Behavioral change in response to alterations in the quality and quantity of light in the environment is a ubiquitous trait among motile photosynthetic bacteria. Three distinct types of responses to light have been described in the literature (14, 19, 36, 37). The scotophobic response (fear of darkness) is characterized by a tumbling, stop, or reversal that occurs when a swimming bacterium experiences a temporal, or spatial, step down in light intensity. Photokinesis describes an alteration in the rate of motility caused by differences in light intensity. A phototactic response, which has been studied most extensively in algae and cyanobacteria, involves an oriented movement of a cell toward or away from a light source (19). An important distinction is that the direction of irradiation is not relevant to scotophobic or photokinetic responses, whereas it is a critical determinant in phototaxis. Thus, phototactic organisms are uniquely capable of migrating towards a light source, irrespective of whether they are going up or down a gradient of light intensity (37).The various photosensory behaviors exhibited by anoxygenic photosynthetic bacteria have been studied mainly by physiological and biochemical tests, with little supporting genetic data (3, 4, 8, 9, 13, 16, 27, 38). The few genetic tests that have been undertaken have demonstrated that mutations which functionally impair the photosystem also disrupt the ability of cells to respond to light (3, 20). This indicates that a product of photosynthesis, such as the generation of proton motive force or photosynthesis-driven electron transfer, is most likely the signal that controls photosensory behavior, rather than direct absorption of light by a chromophore-containing receptor. This conclusion is supported by recent physiological studies which have shown that specific inhibitors of cyclic photosynthesis-driven electron transport inhibit photosensory behavior in Rhodobacter sphaeroides (13, 16) and Rhodospirillum centenum (38). By using a site-directed mutational approach, we have shown that the scotophobic and phototactic responses of the purple nonsulfur photosynthetic bacterium R. centenum involve components of the chemotaxis phosphorylation cascade (25, 26). This suggests that a sensor of photosynthetic activity may have features similar to that of chemoreceptors. However, which component of the photosynthesis electron transfer chain is being sensed and what is actually sensing alterations in electron transfer are unknown.To identify components responsible for prokaryotic behavioral responses to light, it is essential that techniques be developed for the isolation of mutants that are specifically defective in photosensory behavior. One of the reasons why screens for photosensory mutants have not been developed is the inherent difficulty of assaying for photosensory behavior. Until recently, screening for such mutants involved the onerous task of microscopically assaying individual cells from liquid-grown cultures for a response to a step up or down in light intensity. Since statistically meaningful results require that multiple cells be assayed, this “brute force” approach is infeasible. A significant advance in the isolation of prokaryotic photosensory mutants was recently provided by our observation that colonies of the purple photosynthetic bacterium R. centenum are capable of macroscopic phototactic motility (36, 37). Cells of R. centenum are dimorphic, existing in liquid medium as swim cells bearing a single polar flagellum or as hyperflagellated swarm cells on solid surfaces (36, 37). A unique feature of R. centenum swarming colonies is that they are capable of migrating rapidly (up to 75 mm/h) toward an infrared light source or away from a visible light source (36, 37). This behavior allows us to rapidly screen for mutants that are deficient in photosensory responses by simply assaying colonies for aberrant light-directed migration. In this study, we have utilized mini-Tn5-mediated mutagenesis to isolate numerous mutants that exhibit defects in light-directed motility. The phenotypes of specific classes of mutants provide some unique observations on photosensory behavior, as well as on the mechanism of swim cell to swarm cell differentiation.     

Isolation of Temperature-Sensitive Mutants of Arabidopsis thaliana That Are Defective in the Redifferentiation of Shoots

Three temperature-sensitive mutants of Arabidopsis thaliana that were defective in the redifferentiation of shoots were isolated as tools for the study of organogenesis. M3 lines were constructed by harvesting M3 seeds separately from each M2 plant. Comparative examination of shoot redifferentiation in root explants of 2700 M3 lines at 22[deg]C (permissive temperature) and at 27[deg]C (restrictive temperature) led to the identification of seven temperature-sensitive mutant lines. Genetic tests of three of the seven mutant lines indicated that temperature-sensitive redifferentiation of shoots in these three lines resulted from single, nuclear, recessive mutations in three different genes, designated SRD1, SRD2, and SRD3. The morphology of root explants of srd mutants cultured at the restrictive temperature suggests that the products of these SRD genes function at different stages of the redifferentiation of shoots.     

Isolation of Bacteriophage T4 Mutants Defective in the Ability to Degrade Host Deoxyribonucleic Acid

A method was devised for identifying nonlethal mutants of T4 bacteriophage which lack the capacity to induce degradation of the deoxyribonucleic acid (DNA) of their host, Escherichia coli. If a culture is infected in a medium containing hydroxyurea (HU), a compound that blocks de novo deoxyribonucleotide biosynthesis by interacting with ribonucleotide reductase, mutant phage that cannot establish the alternate pathway of deoxyribonucleotide production from bacterial DNA will fail to produce progeny. The progeny of 100 phages that survived heavy mutagenesis with hydroxylamine were tested for their ability to multiply in the presence of HU. Four of the cultures lacked this capacity. Cells infected with one of these mutants, designated T4nd28, accumulated double-stranded fragments of host DNA with a molecular weight of approximately 2 x 10(8) daltons. This mutant failed to induce T4 endonuclease II, an enzyme known to produce single-strand breaks in double-stranded cytosine-containing DNA. The properties of nd28 give strong support to an earlier suggestion that T4 endonuclease II participates in host DNA degradation. The nd28 mutation mapped between T4 genes 32 and 63 and was very close to the latter gene. It is, thus, in the region of the T4 map that is occupied by genes for a number of other enzymes, including deoxycytidylate deaminase, thymidylate synthetase, dihydrofolate reductase, and ribonucleotide reductase, that are nonessential to phage production in rich media.     

Isolation and Properties of Enterococcus hirae Mutants Defective in the Potassium/Proton Antiport System

A K+/H+ antiporter regulates cytoplasmic pH in Enterococcus hirae growing at alkaline pH. Mutants defective in this antiport activity were alkaline pH sensitive. One mutant, Pop1, lacked both K+/methylamine exchange at pH 9.5 and concomitant acidification of cytoplasmic pH. Pop1 grew well at pHs below 8 but did not at pHs above 9, conditions under which cytoplasmic pH was not fully acidified.     

Isolation of Degradation-Deficient Mutants Defective in the Targeting of Fructose-1,6-Bisphosphatase into the Vacuole for Degradation in Saccharomyces Cerevisiae

The key regulatory enzyme in the gluconeogenesis pathway, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are grown in medium containing a poor carbon source. FBPase is targeted to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. To identify genes involved in the FBPase degradation pathway, mutants that failed to degrade FBPase in response to glucose were isolated using a colony-blotting procedure. These vacuolar import and degradation-deficient (vid) mutants were placed into 20 complementation groups. They are distinct from the known sec, vps or pep mutants affecting protein secretion, vacuolar sorting and vacuolar proteolysis in that they sort CpY correctly and regulate osmotic pressure normally. Despite the presence of FBPase antigen in these mutants, FBPase is completely inactivated in all vid mutants, indicating that the c-AMP-dependent signal transduction pathway and inactivation must function properly in vid mutants. vid mutants block FBPase degradation by accumulating FBPase in the cytosol and also in small vesicles in the cytoplasm. FBPase may be targeted to small vesicles before uptake by the vacuole.     

Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis

In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1–5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion.     

Isolation and Genetic Analysis of Caulobacter Mutants Defective in Cell Shape and Membrane Lipid Synthesis

In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). Expression of the latter two phenotypes required complex media and both were repressed by glucose. However, the lesions were mapped to loci that are separated by a substantial distance. The hclA and the fatA genes mapped close together, possibly implying that comutation had occurred in AE6002. Data are presented that allow the unambiguous identification of a second Fat gene (fatB) in C. crescentus. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO₄ auxotroph gpsA505, was also determined. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location.     

Molecular evidence that the extracellular cutinase Pbc1 is required for pathogenicity of Pyrenopeziza brassicae on oilseed rape

Recent evidence has suggested that cutinase is required for cuticular penetration and, therefore, is essential for pathogenicity of Pyrenopeziza brassicae, the causal organism of light leaf spot disease of oilseed rape and other brassicas. In order to acquire molecular evidence for the role of cutinase in pathogenesis, the single-copy P. brassicae cutinase gene Pbc1 was disrupted by a transformation-mediated approach. Southern hybridization analysis revealed that in one mutant, NH10-1224, the disruption was due to a tandem insertion of two copies of the disruption vector into the 5' coding region of Pbc1. In contrast to the wild type, no expression of Pbc1 was detected during in planta growth or in cutin-induced mycelium of NH10-1224 and no cutinase activity was detected in culture supernatants from NH10-1224 using p-nitrophenyl butyrate as substrate. Scanning electron microscopy of Brassica napus cotyledons infected with wild-type P. brassicae confirmed that entry into the host is by direct penetration of the cuticle. In contrast, the cutinase-deficient mutant NH10-1224 failed to penetrate the cuticular layer and was unable to develop disease symptoms. This evidence is consistent with the hypothesis that Pbc1 is required for P. brassicae to penetrate the plant cuticle. Demonstration that complementation of NH10-1224 with the Pbc1 wild-type gene restores both cutinase activity and pathogenicity will be required to definitively establish that cutinase is required for successful pathogenesis of brassicas by P. brassicae.