Siderophore transport mutants ( sit ) in Neurospora crassa

Siderophore transport mutants (sit) in Neurospora crassa. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This research note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol30/iss1/2 Mycologia LXVIII: 388-401) of numerous isolates of the species N. crassa did not show any significant variability in non-repeated DNA sequences. Recent studies on restriction analysis of rDNAs of different cell types of N. crassa (Chambers and Dutta, unpublished) have also clearly shown no variability within the rDNAs of the same species. Hence, it is obvious that restriction analysis of rDNAs is a reliable means to study genetic characteristics, and should prove to be a very useful tool for identification of species of eukaryotic organisms. (Supported in part by a Department of Energy Grant.) Department of Genetics and Botany, and Cancer Research Center, Howard University, Washington, D.C. 20059. Charlang, G.W.* and N.P. Williams Siderophore transport mutants (sit) in Neurospora crassa. During the last several years we have isolated and worked with a number of sideraphore transport mutants (sit) in Neurospora crassa . The work is unfinished and nothing has been published about these mutants, except for their listing in the "Compendium" (Perkins, et al., 1982 Microbiol. Rev. 46: 426). Since one of us (GWC) is no longer working in this field, we have decided to make the mutants available to anyone interested. This note introduces the mutants, their background, isolation and general characteristics. They have been deposited in the Stock Center (FGSC 4211 through 4231) in the current stock list. Background: The parent strain is the triple mutant, arg-5,ota,aga (FGSC 2744), which was obtained from R.H. Davis. This mutant is blocked in all known pathways of ornithine synthesis and produces so little ornithine (Charlang and Ng, 1982 Neurospora Newsl. 29: 15) that it becomes siderophore-dependent when grown in medium with glycerol as carbon source. This observation formed the basis for use in "the hunt for" siderophore transport mutants. Isolation: To reduce siderophores pools in conidia, the triple mutant was grown without ornithine prior to the hunt. UV-treatment of conidia was followed by filtration enrichment in glycerol medium supplemented with ferricrocin (FC) (Vogel's N-free salts; glycerol and asparagine). All media contained putrescine and arginine (recrystallized) which were filter-sterilized and added to the autoclaved medium. All liquid media also contained Tween 80 [four drops (56 mg) per 100 ml]. FC was added to the autoclaved medium at 10 to 20 μg per 100 ml. Ungerminated conidia were plated on sorbose medium supplemented with ornithine. One thousand isolates from these plates were tested for growth in liquid glycerol medium (same medium as above, except Winkelmann's salts (Winkelmann and Zahner 1973 Arch. Mikrobiol. 88: 49) instead of Vogel's, with trace elements added (but not Fe or citrate) supplemented with either ornithine or FC. Those 120 isolates that grew on ornithine but not on FC were tested for 3HFC uptake. The medium used in the uptake was the liquid glycerol medium (Winkelmann's salts) used in the growth tests of the original isolates. Conidia were germinated for 3 hours at 30°C before measuring uptake. Eleven isolates were found to be uptake deficient. To check specificity of the uptake defect they were tested for 14C-phenylalanine transport. Six of the 11 isolates appeared to have a general transport defect as they were also deficient in phenylalanine uptake. They have been set aside and not analyzed any further. (They are not in the Stock Center but GWC still has silica gels, if anyone would like to have them.) The remaining five isolates were normal as far as phenylalanine transport is concerned, and they were designated as sit mutants. Crosses: The five sit mutants were crossed to 74a in order to remove the parental marker genes. Progeny were first analyzed in low water activity (aw) medium with or without ferricrocin. This test was based on our earlier finding that, in low a w media (a w ~0.93), conidia lose that part of their ferricrocin pool which is required for germination (Horowitz et al.,1976 J. Bacteriol. 127: 135). Germination will occur, however, if the medium is supplemented with the siderophore. Uptake mutants should show little or no response to added ferricrocin. The medium was the same as that used for uptake studies, except that sucrose was used as C-source and NaCl was added to lower the aw to .966. At this level wild type strain 74a still shows a response to added ferricrocin. Any aw lower is severely inhibitory to the triple mutant. Any progeny looking like uptake mutants were then tested for "HFC uptake and analyzed for presence of parental genes. Removal of the parental genes proved to be difficult in most cases because not only are arg-5,ota and aga in separate linkage groups, but two of them interfered with the progeny testing; arg-5 reduces ferricrocin uptake, and aga increases sensitivy to low aw. We did succeed in removing the parental genes from the mutants in all but one case; sit-5 still carries ota. Before these crosses were completed others were set up between sit mutants to test for allelism. The five mutations appear to be nonallelic. (Some of the double mutants obtained at that time have been deposited in the Stock Center. Many still carry the arg-5 genotype and may be of limited usefulness in siderophore transport studies.) The mutations have not been mapped. General Description: The sit mutants grow well on minimal medium. sit-1 germinates slowly. sit-2 has a deep orange color, and its conidia tend to remain connected in twos or threes. sit and sit-4 are very poor protoperithecial parents. None of the mutants is totally unable to transport FC or coprogen. There is some passive FC and coprogen uptake in Neurospora, and this may be what we are observing in these mutants. sit-1 and sit appear to be binding mutants. Their ability to bind FC to the cell surface (in presence of NaN3 which inhibits active transport) is much lower than that of wild type strain 74A. The other sit mutants have normal binding. Current Work: Shortly before the demise of our group at Caltech, we had begun a biochemical search for the siderophore receptor using sit-l, sit-2 and a double mutant (sit-l , sit-2). This work was initiated by Lennart Adler who is continuing the studies at Gothenburg University, Sweden. One of us (NPW) plans to continue the genetic analysis of the mutants to establish the location of the five sit loci in the Neurospora linkage groups. We thank N.H. Horowitz for support, guidance and encouragement. (Supported by Public Health Service Grant AI-15739.) Division of Biology, California Institute of Technology, Pasadena, California 91125 and Department of Botany, Howard University, Washington, DC 20059. *Present address: 255 S. Sierra Bonita Avenue, Pasadena, California 91106. Grindle M. and W. Temple We are using N. crassa as a model organism to investigate the mode of action of agricultural fungicides and the genetic and bioFungicide resistance of smco chemical bases for fungicide resistance. Certain aromatic hydrocarbon and dicarboximide fungicides ("AHD" fungicides) presumably mutants of Neurospora crassa have a similar mode of action because mutants are usually crossresistant to them. For example, Vin and os mutants of N.crassa are resistant to the aromatic hydrocarbons dicloran, chlaroneb and quintozene and the dicarboximides iprodiane, procymidone and vinclozolin (Grindle and Temple, 1982 Neurospora Newsl. 29: 16-17). The fungicides are used on a wide range of crops, especially grapes, soft fruits and glasshouse plants, to combat pathogens such as species of Botrytis, Rhizoctonia and Sclerotinia. There is increasing concern that dicarboximide-resistant mutants of Botrytis cinerea might become a practical problem in agriculture and horticulture. The sensitivity of Vin and os mutants to media of high osmolarity suggests that there are defects in the cell wall-plasma membrane complex of AHD-resistant mutants. Many morphological mutants of N. crassa are believed to have abnormal cell walls or membranes, and the biochemical lesions and enzyme defects of some mutants have been determined (Scott 1976, Ann. Rev. Microbiol. 30: 85-104; Mishra 1977, Adv. Genet.. 19: 341.405). Representative morphological mutants were analysed for resistance to AHD fungicides. Each mutant was grown on dishes of Vogel's minimal medium (MM) and on MM containing 2-10 μg/ml vinclozolin to detect isolates that were more resistant than the wild type 74-OR8-la. Colony diameters (mean of 2 measurements per colony) were noted after 18 hr and 24 hr growth, and the growth rate (mm/24 hr increase in diameter) was calculated from the growth during 6 hr. Growth rates on MM and on fungicide-supplemented MM were used to determine the amounts of fungicide that reduced colony diameters by 50% (ED50) and by 95% (ED95). The ED50

During the last several years we have isolated and worked with a number of sideraphore transport mutants (sit) in Neurospora crassa .The work is unfinished and nothing has been published about these mutants, except for their listing in the "Compendium" (Perkins, et al., 1982 Microbiol. Rev. 46: 426).Since one of us (GWC) is no longer working in this field, we have decided to make the mutants available to anyone interested.This note introduces the mutants, their background, isolation and general characteristics.They have been deposited in the Stock Center (FGSC 4211 through 4231) in the current stock list.Background: The parent strain is the triple mutant, arg-5,ota,aga (FGSC 2744), which was obtained from R.H. Davis.This mutant is blocked in all known pathways of ornithine synthesis and produces so little ornithine (Charlang and Ng, 1982 Neurospora Newsl.29: 15) that it becomes siderophore-dependent when grown in medium with glycerol as carbon source.This observation formed the basis for use in "the hunt for" siderophore transport mutants.
Isolation: To reduce siderophores pools in conidia, the triple mutant was grown without ornithine prior to the hunt.UV-treatment of conidia was followed by filtration enrichment in glycerol medium supplemented with ferricrocin (FC) (Vogel's N-free salts; glycerol and asparagine).All media contained putrescine and arginine (recrystallized) which were filter-sterilized and added to the autoclaved medium.All liquid media also contained Tween 80 [four drops (56 mg) per 100 ml].
FC was added to the autoclaved medium at 10 to 20 µg per 100 ml.Ungerminated conidia were plated on sorbose medium supplemented with ornithine.One thousand isolates from these plates were tested for growth in liquid glycerol medium (same medium as above, except Winkelmann's salts (Winkelmann and Zahner 1973 Arch.Mikrobiol.88: 49) instead of Vogel's, with trace elements added (but not Fe or citrate) supplemented with either ornithine or FC.Those 120 isolates that grew on ornithine but not on FC were tested for 3 HFC uptake.
The medium used in the uptake was the liquid glycerol medium (Winkelmann's salts) used in the growth tests of the original isolates.Conidia were germinated for 3 hours at 30°C before measuring uptake.
Eleven isolates were found to be uptake deficient.To check specificity of the uptake defect they were tested for 14 C-phenylalanine transport.
Six of the 11 isolates appeared to have a general transport defect as they were also deficient in phenylalanine uptake.They have been set aside and not analyzed any further.(They are not in the Stock Center but GWC still has silica gels, if anyone would like to have them.)The remaining five isolates were normal as far as phenylalanine transport is concerned, and they were designated as sit mutants.
Crosses: The five sit mutants were crossed to 74a in order to remove the parental marker genes.Progeny were first analyzed in low water activity (a w ) medium with or without ferricrocin.This test was based on our earlier finding that, in low a w media (a w ~0.93), conidia lose that part of their ferricrocin pool which is required for germination (Horowitz et al.,1976 J. Bacteriol.127: 135).Germination will occur, however, if the medium is supplemented with the siderophore.Uptake mutants should show little or no response to added ferricrocin.The medium was the same as that used for uptake studies, except that sucrose was used as C-source and NaCl was added to lower the a w to .966.
At this level wild type strain 74a still shows a response to added ferricrocin.Any a w lower is severely inhibitory to the triple mutant.Any progeny looking like uptake mutants were then tested for "HFC uptake and analyzed for presence of parental genes.
Removal of the parental genes proved to be difficult in most cases because not only are arg-5,ota and aga in separate linkage groups, but two of them interfered with the progeny testing; arg-5 reduces ferricrocin uptake, and aga increases sensitivy to low a w .We did succeed in removing the parental genes from the mutants in all but one case; sit-5 still carries ota.Before these crosses were completed others were set up between sit mutants to test for allelism.The five mutations appear to be nonallelic.(Some of the double mutants obtained at that time have been deposited in the Stock Center.Many still carry the arg-5 genotype and may be of limited usefulness in siderophore transport studies.) The mutations have not been mapped.

General Description:
The sit mutants grow well on minimal medium.sit-1 germinates slowly.sit-2 has a deep orange color, and its conidia tend to remain connected in twos or threes.sit and sit-4 are very poor protoperithecial parents.
None of the mutants is totally unable to transport FC or coprogen.There is some passive FC and coprogen uptake in Neurospora, and this may be what we are observing in these mutants.sit-1 and sit appear to be binding mutants.Their ability to bind FC to the cell surface (in presence of NaN 3 which inhibits active transport) is much lower than that of wild type strain 74A.The other sit mutants have normal binding.
Current Work: Shortly before the demise of our group at Caltech, we had begun a biochemical search for the siderophore receptor using sit-l, sit-2 and a double mutant (sit-l , sit-2).This work was initiated by Lennart Adler who is continuing the studies at Gothenburg University, Sweden.
One of us (NPW) plans to continue the genetic analysis of the mutants to establish the location of the five sit loci in the Neurospora linkage groups.