Emeritus Professor David Catcheside

Abstracts

- Influence of culture parameters on extracellular peroxidase activity and transformation of low-rank coal by Phanerochaete chrysosporium

J.P. Ralph and D.E.A. Catcheside, School of Biological Sciences, Flinders University, South Australia. Appl. Microbiol. Biotechnol., (1998) 49:438-444

Abstract: The white-rot fungus Phanerochaete chrysosporium can degrade macromolecules in low rank coal, offering the potential for converting coal to specific products. We investigated the influence of temperature, veratryl alcohol and oxygen on transformation of a solubilised fraction of Morwell brown coal (SWC6 coal) and on the activity of lignin peroxidase (LiP) and manganese peroxidase (MnP) in N-limited cultures of P. chrysosporium. After 20 days, the mass and OD₄₀₀ of SWC6 coal recovered from cultures containing 0.03% SWC6 coal, incubated at 28C under hyperbaric oxygen, were reduced by over 95%. The modal apparent molecular mass of the residuum was reduced by 50%. Addition of 2mM veratryl alcohol had little effect on the transformation of SWC6 coal. The extent of transformation was reduced in cultures incubated at 37C or under air. In cultures under air, coal molecules were transiently polymerised. Decolourisation of SWC6 coal reflects conversion to products that cannot be recovered from the medium, not to destruction of chromophores within recoverable material. The activity of LiP, measured in cultures free of SWC6 coal to avoid interference with the assay, correlates directly with the degradation of SWC6 coal as measured by the decline in OD₄₀₀. The data suggests that LiP is more important than MnP in conversion of SWC6 coal to products that are assimilated by cells.


- Involvement of Manganese Peroxidase in the transformation of macromolecules from low-rank coal by Phanerochaete chrysosporium

James P. Ralph and David E.A. Catcheside, School of Biological Sciences, Flinders University, South Australia. Appl. Microbiol. Biotechnol., (1998) 50:778-784

Abstract: Manganese peroxidase (MnP) catalyses the oxidation of Mn(II) to Mn(III), a diffusible non-specific oxidant likely to be involved in the transformation of polyphenolic macromolecules from brown coal by the white-rot fungus Phanerochaete chrysosporium. We report here that solubilised macromolecules from Morwell brown coal were depolymerised by Mn(III) ions when incubated under hyperbaric O₂. However, under N₂ or air they were polymerised, suggesting that net depolymerisation by Mn(III) requires molecular oxygen to inhibit coupling of coal radicals. Coal macromolecules were also polymerised when separated by a semipermeable membrane from a culture of P. chrysosporium or from a solution of MnP, Mn(II) and H₂O₂, probably by Mn(III) crossing the membrane. In oxygenated cultures in which MnP was up-regulated by Mn(II), the extent of depolymerisation correlated with cumulative MnP activity suggesting that MnP-generated Mn(III) has a central role in initial depolymerisation of coal molecules in vivo. However, mutant ME446-B17-1 which produces MnP but not lignin peroxidase (LiP), polymerised coal macromolecules in oxygenated cultures. In sum, it appears MnP can both polymerise and depolymerise brown coal macromolecules and that in vivo, both hyperbaric O₂ and LiP are also required to force net depolymerisation to products assimilable by cells.


- Analysis of conversion tracts associated with recombination events at the am locus of Neurospora crassa

Frederick J. Bowring and David E.A. Catcheside, School of Biological Sciences, Flinders University, South Australia. Current Genetics, (1998) 294:43-49

Abstract: In cross B163, heteroallelic am¹ am⁶ and heterozygous for both conventional genetic flanking markers and closer molecular markers, we found that the majority of flanker exchanges were remote from events that generated prototrophic recombinants (Bowring and Catcheside, 1996). We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am. Segregation of these markers in B163 prototrophs confirms that the majority of meiotic recombination events at am resulted from gene conversion. Conversion of am⁶, the distal allele, is more frequent than conversion of am¹. 12% of tracts in am⁶ convertants were discontinuous while 30% of continuous tracts converting am⁶ extend less than 741 bp.


- Long, interrupted conversion tracts initiated by cog in Neurospora crassa

PJ, Yeadon and DEA Catcheside. School of Biological Sciences, Flinders University of South Australia, Bedford Park, SA 5042. Genetics (1998)148:113-122

Abstract: Multiple polymorphisms distinguish Emerson and Lindegren strains of Neurospora crassa within the histidine-3 gene and in its distal flank. Restriction site and sequence length polymorphism in a set of 14 PCR products covering this 6.9 kb region were used to identify the parental origin of DNA sequence information in prototrophic progeny of crosses heterozygous for auxotrophic mutations in his-3 and the silent sequence differences. 41% of conversion tracts are interrupted. Where the absence of rec-2⁺ permits activity of the recombination hotspot cog, conversion appears to originate at cog and conversion tracts are up to 5.9 kb long. The chromosome bearing cog^L, the dominant allele which confers a high frequency of recombination, is almost invariably the recipient of information. In progeny from crosses heterozygous rec-2/rec-2⁺, conversion tracts are much shorter, most are not initiated at cog and either chromosome seems equally likely to be converted. Although 32% of his-3 prototrophs have a crossover which may be associated with conversion, it is suggested that the apparent association between conversion and crossing over at this locus may be due to confounding of coincidental events rather than to a mechanistic relationship.


- Transformations of low rank coal by Phanerochaete chrysosporium and other wood rot fungi

J.P. Ralph and D.E.A. Catcheside. School of Biological Sciences, Flinders University of South Australia, Bedford Park, SA 5042. Fuel Processing Technology (1997) 52: 79-93.

Abstract: There is a substantial body of evidence that the organic fraction of low rank coal (LRC) is chemically transformed by wood-rot fungi. These fungi and the oxidases they secrete have variously been shown to solubilise, polymerise, depolymerise and decolourise macromolecules derived from LRC. The white-rot fungus, Phanerochaete chrysosporium, is able to depolymerise and decolourise alkali-soluble acid-precipitable LRC macromolecules (AS-coal), converting them to a form not recoverable by alkali washing. Transformation of AS-coal is enhanced in N-limiting media under hyperbaric oxygen and is believed to be due, at least in part, to oxidation by manganese peroxidase (MnP) and lignin peroxidase (LiP). The precise role these enzymes play is not yet clear but enzyme and mutant studies show AS-coal can be both polymerised and depolymerised by MnP and its mimetic Mn(III), without change to its absorbance at 400nm and LiP decolourises AS-coal without apparently altering its molecular mass. Culture filtrates containing MnP and LiP acting on methylated AS-coal yield an array of low molecular mass moieties. Coal-derived monomers have not been recovered from cultures of P. chrysosporium, consistent with them being taken-up by the fungal cells. This suggests that cellular transformations may permit the diverse catabolic products derived from LRC to be converted to specific low molecular mass compounds in usable yield.


- Size exclusion chromatography of solubilised low rank coal

J.P. Ralph and D.E.A. Catcheside. School of Biological Sciences, Flinders University of South Australia, Bedford Park, SA 5042. Journal of Chromatography A (1996) 724: 97-105.

Abstract: Solubilised brown coal is a polydisperse and chemically heterogeneous polyanion. The parameters appropriate for estimation of its molecular mass (Mr) by Size exclusion chromatography using a stationary phase comprising glyceryl-propyl silica were investigated. Ion exclusion occurred when the ionic strength (I) was zero, being most pronounced for coal soluble at low pH. Reverse phase partitioning appeared at low ionic strength. Non size-exclusion interactions were minimised when I lay between 0 and 0.033. The estimates for modal MW of solubilised coal ranged between 355 kD and 33 kD. These were consistent with ultrafiltration data, suggesting SEC is a practical method for determining coal MW.


- Gene conversion alone accounts for more than 90% of recombination events at the am locus of Neurospora crassa

F.J. Bowring and D.E.A. Catcheside. School of Biological Sciences, Flinders University of South Australia, Bedford Park, SA 5042. Genetics (1996) 142: 129-136.

Abstract: We have used closely flanking molecular markers located approximately 4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am¹ am⁶. 93% of prototrophs were generated by events which did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (~0.07) than estimated previously using more distant flanking markers. This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favour of retaining the parental association of flanking sequences. Since estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (~0.35), our observation may have general significance.


- Extracellular oxidases and the transformation of solubilised fractions of low-rank coal by wood-rot fungi

Ralph, JP, LA Graham and DEA Catcheside (1996) Applied Microbiology and Biotechnology 46:226-232.

Abstract: The involvement of extracellular oxidases in biotransformation of low rank coal was assessed by correlating the ability of nine white-rot and brown-rot fungi to alter macromolecular material in alkali solubilised brown coal with the spectrum of oxidases they produce when grown on low nitrogen medium. The coal fraction used was that soluble at 3.0<pH<6.0 (SWC6 coal). In 15 ml cultures, Gloeophyllum trabeum, Lentinus lepideus and Trametes versicolor produced little or no lignin peroxidase (LiP), manganese peroxidase (MnP) or laccase activity and caused no change to SWC6 coal. Ganoderma applanatum and Pycnoporus cinnabarinus also produced no detectable LiP, MnP or laccase yet increased the absorbance at 400nm of SWC6 coal. G. applanatum which produced veratryl alcohol oxidase also increased the modal apparent molecular mass (MAMr). SWC6 coal exposed to Merulius tremellosus and Perenniporia tephropora which secreted MnP and laccase and Phanerochaete chrysosporium which produced MnP and LiP was polymerised but had unchanged or decreased absorbance. In the case of both P. chrysosporium and M. tremellosus polymerisation of SWC6 coal was most extensive, leading to the formation of a complex insoluble in 100 mM NaOH. Rigidoporus ulmarius which produced only laccase both polymerised and reduced the A₄₀₀ of SWC6 coal. P. chrysosporium, M. tremellosus and P. tephropora grown in 10 ml cultures produced a spectrum of oxidases similar to that in 15 ml cultures but in each case caused more extensive loss of A₄₀₀ and P. chrysosporium depolymerised SWC6 coal. It is concluded that the extracellular oxidases of white-rot fungi can transform low rank coal macromolecules and that increased oxygen availability in the shallower 10 ml cultures favours catabolism over polymerisation.


- Recovery and analysis of solubilised brown coal from cultures of wood-rot fungi

Ralph JP and DEA Catcheside (1996) J. Microbiological Methods 27: 1-11

Abstract: The effectiveness of alkali-washing as a method for recovering fractions of brown coal soluble in alkali (AS-coal) from cultures of brown and white-rot fungi was investigated. AS- coal was found to precipitate in fungal growth medium, causing an increase in modal apparent molecular mass (MAMr), and to bind to medium components and fungal mycelia. In consequence, fungal-mediated degradation of AS-coal cannot be determined by direct analysis of AS-coal present in culture fluid. Total recovery of AS-coal was achieved by (i) acidifying cultures and pelleting fungal mycelia and AS-coal by centrifugation (ii) incubating AS-coal and mycelia in NaOH to resolubilise the AS-coal (iii) separating AS-coal from mycelia by filtration, (iv) re-precipitation of AS-coal with acid and (iv) resuspending the AS-coal in phosphate buffer for analysis. Fourier transform infrared spectroscopy showed this process to remove all contaminants derived from growth medium except oleic acid and Tween-80. These compounds do not change the absorbance or MAMr of AS-coal as determined by size exclusion chromatography (SEC). The presence of 100 mM ortho phosphate, pH 6.9, had a differential effect on the MAMr of AS-coal depending upon the pH used for its initial solubilisation. Coal fractions soluble below pH 4.5 had a lower MAMr in phosphate than in water. In contrast, a coal fraction solubilised between pH 4.5 and 6, had an increased MAMr in phosphate buffer. These effects are probably due to changes in the hydrodynamic volume of coal macromolecules, making SEC appropriate for determining relative but not absolute changes in the Mr of solubilised coal.


- Guest: a 98 bp inverted repeat transposable element in Neurospora crassa

by P. Jane Yeadon and David E. A. Catcheside, School of Biological Sciences, Flinders University, PO Box 2100, Adelaide SA 5001, Australia. Molecular and General Genetics (1995) 247:105-109

Abstract: The region immediately 3' of histidine-3 has been cloned and sequenced from two laboratory strains of the Ascomycete fungus Neurospora crassa; St Lawrence 74A and Lindegren, which have different derivation from wild collections. Amongst the differences distinguishing these sequences are insertions ranging in size from 20 to 101 bp present only in St Lawrence. The largest of these is flanked by a 3 bp direct repeat, has terminal inverted repeats (TIR) and shares features with several known transposable elements. At 98 bp, it may be the smallest transposable element yet found in eukaryotes. There are multiple copies of the TIR in the Neurospora genome, similar but not identical to the one sequenced. PCR amplification of Neurospora genomic DNA using 26 bp of the TIR as a single primer, gave products of discrete sizes ranging from 100 bp to about 1.3 kb, suggesting that the element isolated (Guest) may be a deletion derivative of a family of larger transposable elements. Guest appears to be the first DNA intermediate transposable element reported for fungi.


- The chromosomal region which includes the recombinator cog in Neurospora crassa is highly polymorphic

by P. Jane Yeadon and David E. A. Catcheside School of Biological Sciences, Flinders University, PO Box 2100, Adelaide SA 5001, Australia. Current Genetics (1995) 28: 155-163

Abstract: The St Lawrence ST74-OR23-IVA and Lindegren Y8743 strains of Neurospora crassa have different provenance from wild collections and dissimilar cog alleles; that in Lindegren, cog^La (previously designated cog⁺), is a more efficient recombinator than cog^S74A and cog^Ea (previously cog), the alleles in St Lawrence and Emerson a respectively. Restriction fragment length polymorphisms (RFLPs) and sequence polymorphisms (SPs) were used to map the difference between cog^La and cog^S74A to a region that extends from 2.3 to 3.2 kb 3' of the his-3 coding sequence. The DNA sequences from 400 bp 3' of his-3 to 120 bp 3' of the cog-region in these strains were found to be homologous but diverge by 3.5%. The differences include single base pair changes, short insertion/deletions, differences in the length of poly-T tracts and three longer sequences present only in St Lawrence: a 98 bp inverted repeat transposable element we have called Guest (Yeadon and Catcheside 1995) that has generated a 3 bp direct repeat of the target site present in Lindegren and 15 bp and 20 bp sequences that have no obvious structural features nor similarity to Guest. Southern analysis of other laboratory strains revealed four major and several minor variants of this region. All strains assayed are descendants of Lindegren A, Lindegren a, Abbott 4A and Abbott 12a and it is clear that each of these progenitors collected from the wild population had a different variant of the cog region. Sequence divergence of this degree seems remarkable, even for an intergenic region, for fully interfertile strains of a single species.


- The orientation of gene maps by recombination of flanking markers for the am locus of Neurospora crassa

by Frederick J. Bowring and David E.A.Catcheside. School of Biological Sciences, Flinders University. Bedford Park, South Australia, 5042. Current Genetics (1995) 29: 27-33

Abstract: Fincham (1967), Smyth (1973b) and Rambosek and Kinsey (1983) have each generated fine-structure maps of the am gene of Neurospora crassa. Each map had a consistent linear order of alleles but the assignment of an orientation with respect to other linkage group V loci differed. Fincham found the end marked by the am⁶ allele to be at the distal end of the locus, Smyth found am⁶ to be at the proximal end while the data of Rambosek and Kinsey did not suggest an orientation. Smyth's orientation has been adopted as the standard but not unreservedly. We have aligned the genetic and physical maps of the am gene, showing that am⁶ is at the distal end, supporting Fincham's orientation. However, we suggest that an assumption used to orient fine structure genetic maps is flawed and that the conflicting orientation between these three studies follows from the different choice of flanking markers. - Involvement of Lignin and Manganese Peroxidases and other agents in the Degradation of Brown Coal by the White-Rot Fungus Phanerochaete chrysosporium

by J.P. Ralph and D.E.A. Catcheside. Flinders University of South Australia, Bedford Park, SA 5042, 6th International Conference on Biotechnology in the Pulp and Paper Industry, Vienna, 11-15 June 1995.

Abstract: Brown coal, like lignin, is a complex aromatic polymer. Degradation of brown coal by the fungus Phanerochaete chrysosporium appears to involve, in addition to lignin peroxidase and manganese peroxidase, other undefined agents. If such agents exist, they are anticipated to contribute to the catabolism of lignin and other aromatic compounds and may have diverse applications in biotechnology.

Papers for which abstracts are held at other sites

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