Keith Johnstone

Pulling the trigger: the mechanism of bacterial spore germination

In spite of displaying the most extreme dormancy and resistance properties known among living systems, bacterial endospores retain an alert environment‐sensing mechanism that can respond within seconds to the presence of specific germinants. This germination response is triggered in the absence of both germinant and germinant‐stimulated metabolism. Genes coding for components of the sensing mechanism in spores of Bacillus subtilis have been cloned and sequenced. However, the molecular mechanism whereby these receptors interact with germinants to initiate the germination response is unknown. Recent evidence has suggested that in spores of Bacillus megaterium KM, proteolytic activation of an autolytic enzyme constitutes part of the germination trigger reaction.

Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii

Abstract The biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii, were investigated. An erythrocyte lysis bioassay was developed and used to assay tolaasin activity. Tolaasin-induced haemolysis was dose dependent, maximal between pH 6·0 and 7·0, increased with temperature and was inhibited by the addition of divalent metal ions. Tolaasin production commenced during exponential growth of P. tolaasii and continued into stationary phase. Culture filtrate from wild type P. tolaasii was able to pit mushroom tissue and lyse erythrocytes, whereas culture filtrate from a tolaasin defective mutant had no lytic activity. Ultrastructural studies showed disruption of the Agaricus bisporus plasma membrane and vacuole membranes by both P. tolaasii and tolaasin, but not by a tolaasin defective mutant. Pre-incubation of tolaasin with multilamellar liposomes resulted in loss of lytic activity and osmotic protectants prevented tolaasin-induced haemolysis, demonstrating that tolaasin partitions into membranes, forms pores in erythrocyte membranes and causes lysis by a colloid osmotic mechanism. Tolaasin was phytotoxic when infiltrated into leaves of Nicotiana tabacum and was shown to be active against a range of basidiomycetes and Gram-positive bacteria. Gram-negative bacteria were resistant to tolaasin, but became susceptible when treated concomitantly with sub-minimal inhibitory concentrations of polymyxin B. P. tolaasii was resistant to tolaasin even in the presence of polymyxin B.

Evidence for the involvement of the surface active properties of the extracellular toxin tolaasin in the manifestation of brown blotch disease symptoms by Pseudomonas tolaasii on Agaricus bisporus

Pseudomonas tolaasii produces an ion channel forming haemolytic lipodepsipeptide toxin tolaasin which has been shown to be a potent biosurfactant. Following purification by high performance liquid chromatography, the critical micellar concentration and the surface tension at the critical micellar concentration of purified toxin were determined in distilled water as 0·46 mg ml −1 and 41 mN m −1 respectively by the drop weight method and as 0·42 mg ml −1 and 38 mN m −1 respectively by the contact angle method. The critical micellar concentration of tolaasin varied as a function of pH, with a minimum critical micellar concentration of 1·2 mg ml −1 in 0·1 m Na borate buffer at pH 6·3. However the surface tension at the critical micellar concentration of tolaasin remained almost constant at 40–42 mN m −1 over the pH range 3·0–10·0. The surfactant properties of a mutant truncated form of the toxin (tolaasin 144), which is unable to lyse erythrocytes at low concentrations but which reproduces disease symptoms, were also studied. Tolaasin 144 was also shown to be a biosurfactant with a critical micellar concentration in water of 1·2 mg ml −1 , a surface tension at the critical micellar concentration of 34 mN m −1 and was capable of reducing the surface tension of distilled water to 29 mN m −1 . Both tolaasin and tolaasin 144 (which is unable to form ion channels in artificial lipid bilayers) were able to induce rapid erythrocyte lysis in the presence or absence of the ion channel blocking agent Zn 2+ at concentrations above their critical micellar concentration. Since tolaasin 144 is only able to cause lysis of intact mushroom caps, whereas tolaasin can lyse both mushroom caps and cut mushroom tissue, these results suggest that the ion channel forming activity of tolaasin plays a role in lysis of cut mushroom tissue, whereas the surface active properties of tolaasin and tolaasin 144 are required for lysis of mushroom caps. A second role for tolaasin in brown blotch disease is suggested by the observation that the ability to synthesize tolaasin or tolaasin 144 increased the rate of spread of P. tolaasii colonies in vitro . These results are discussed in relation to the biology of brown blotch disease.

Determination of the structure of an extracellular peptide produced by the mushroom saprotroph pseudomonas reactans

Abstract The primaiy structure of WLIP, a lipodepsipeptide produced by the mushroom saprotroph P. reactans, is determined by a combination of chemical and spectroscopic techniques. The conformation of WLIP in DMSO-d6 is investigated by molecular modelling calculations using constraints obtained from two-dimensional NMR spectroscopy.

Evidence for the requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape

Using a detached cotyledon test for pathogenicity, a UV-induced, non-pathogenic mutant of Pyrenopeziza brassicae was isolated which was also deficient in extracellular protease production in vitro . The proteolytic activity in the wild type was apparently due to a single cysteine protease with a mol. wt of 34 k, a temperature optimum of 40°C and a pH optimum of 8. When the mutant was crossed with a wild-type isolate of P. brassicae , the non-proteolytic and non-pathogenic traits co-segregated in the resulting progeny. The protease − mutant was transformed with clones from a genomic library of P. brassicae and a transformant obtained which had a single cosmid insert and showed concomitant restoration of pathogenicity and proteolytic activity in vitro . These results suggest that extracellular protease is a pathogenicity determinant of P. brassicae and possible functions for this protease in the disease process are discussed.

Influence of growth temperature on lipid and soluble carbohydrate synthesis by fungi isolated from fellfield soil in the maritime Antarctic

The effects of growth temperature on soluble carbohydrate and lipid content of Humicola marvinii, Geomyces pannorum and Mortierella elongata isolated from the Antarctic (Signy Island; 60° 43′S, 45°38′W) were investigated. Each of these fungi responded differently to suboptimal growth temperatures. At low temperatures Humicola marvinii accumulated cryoprotective carbohydrates (trehalose intracellularly and glycerol extracellularly), whereas Geomyces pannorum responded by altering its lipid composition with increases in unsaturated lipid content and overall unsaturation index. In the case of Mortierella elongata, features that may influence its ability to grow at low temperatures included the absence of detectable ergosterol, the presence of stearidonic acid and increased amounts of intracellular trehalose when grown at lower temperatures. The relative importance of lipid and carbohydrate metabolism in adaptation to temperature stress in these fungi is discussed.

Identification of a gene cluster encoding three high‐molecular‐weight proteins, which is required for synthesis of tolaasin by the mushroom pathogen Pseudomonas tolaasii

The extracellular lipodepsipeptide toxin tolaasin is the primary disease determinant of pathogenicity of Pseudomonas tolaasii on the cultivated mushroom, Agaricus bisporus. Transposon mutagenesis of P. tolaasii NCPPB 1116 with Tn5‐generated 5000 chromosomal insertions of which 35 (0.7%) were tolaasin negative and 12 (0.25%) produced a reduced amount of tolaasin. In addition, TnphoA mutagenesis yielded a single tolaasin‐negative mutant which was phoA active. Restriction enzyme mapping of mutant DNAs by Southern hybridization analysis revealed that the majority of Tn5 insertions were confined to a single genetic locus of approximately 65kbp. Pulsed‐field gel electrophoresis of representative Tn5 mutant DNAs showed that this region is at one end of a 640kbp Pacl chromosomal fragment and that the P. tolaasii genome is 6.7 Mbp. SDS‐PAGE analysis of protein extracts from wild‐type P. tolaasii demonstrated the presence of three high‐molecular‐Weight proteins (designated TL1, TL2 and TL3). Alterations in the presence of these proteins, as well as apparently truncated forms of the 465 kDa (TL1), 440 kDa (TL2) and 435 kDa (TL3) proteins were observed in some mutants, enabling the direction and order of the transcriptional units to be determined. Two other Tn5 mutations were also identified which resulted in a tolaasin‐negative phenotype, but which did not affect the expression of TL1, TL2, or TL3. One of these mutants is linked to the TL‐cluster, but the other is located outside this region. It is concluded that at least five genetic loci, including those encoding TL1, TL2 and TL3, are required for tolaasin synthesis.

THE CONTROL OF SEXUAL MORPHOGENESIS IN THE ASCOMYCOTINA

(1) A series of factors controls sexual morphogenesis in the Ascomycotina, a process involving the formation of novel structures such as ascocarps (fruit bodies) and asci (sacs containing spores) during sexual reproduction.

Spontaneous duplication of a 661 bp element within a two‐component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms

Spontaneous sectoring of Pseudomonas tolaasii colonies results in a phenotypic switch from the smooth, pathogenic form (designated 1116S) to the rough non‐pathogenic form (designated 1116R). This phenotypic switch can also be induced by mutation of the pheN master regulatory locus, which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins. Southern blot analysis of genomic DNA from 1116S and 1116R probed with a 3.4 kb XhoI–BamHI fragment containing the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R. In order to characterize the genetic basis of this variation, the pheN locus (designated pheN′ ) was cloned from 1116R and its nucleotide sequence determined. A 661 bp duplication was identified within pheN′ introducing a frameshift mutation in the predicted pheN open reading frame (ORF). A resulting predicted ORF of pheN′ designated ORF2 encodes a polypeptide of 706 amino acid residues, with a predicted molecular weight of 77 kDa, and which lacks part of the PheN sensor domain. Southern blot analysis of genomic DNA using a probe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form. Polymerase chain reaction (PCR) analysis of 25 independently isolated 1116R sectors using primers flanking the duplication site in pheN confirmed the presence of the duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants from 1116R to 1116S. Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form. The presence of a truncated PheN protein in 1116R was verified by Western blot analysis of total cell protein using a LemA antiserum, which revealed the presence of 99 kDa and 77 kDa cross‐reactive bands in 1116S and 1116R respectively. It is concluded that the spontaneous colony‐sectoring event that results in the 1116R phenotypic variant form of P. tolaasii arises owing to a 661 bp DNA duplication within the 5′ end of the pheN gene, which results in loss of the periplasmic sensor domain of PheN and elimination of normal PheN function.

An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy

Changes in membrane and macromolecular fluidity which may accompany the differentiation processes of sporulation and germination in Bacillus megaterium K.M. are examined by electron spin and nuclear magnetic resonance spectroscopy. No change in membrane lipid fluidity is observed in isolated forespores up to stage VI. Between stage VI and release of mature spores, the ESR spectrum of doxylstearic acid spin labels becomes polycrystalline. This change in spectral fluidity is completely reversed during germination and is paralleled by the rapid release of Ca2+ from the spore. NMR studies also show that the mature spore has reduced macromolecular mobility and an increased nonexchangeable water pool compared with vegetative cells.