- Volume 141, Issue 5, 1995

Zoospores of the plant pathogen Phytophthora palmivora use a number of tactic responses to target specific infectible regions of host roots. Although the dominant one is believed to be chemotaxis, it has been shown that zoospores of oomycetes may also use the exogenous proton/electrical currents generated by plant roots for guidance. Since these proton currents also generate significant pH gradients in the rhizosphere, the tactic response of zoospores to changes in pH was examined. Using ‘swim-in’ capillary tests, zoospores of P. palmivora were found to be repelled by solutions of high pH and attracted to solutions of low pH, relative to a control at neutrality. This in vitro tactic response was generally consistent with the measured pH at sites of zoospore accumulation around intact and wounded roots. However, the endogenous pH gradient around host roots could be abolished with buffers and this treatment did not affect the extent or pattern of zoospore accumulation. Therefore, detection of root-generated pH gradients is unlikely to have a major role in the homing response of zoospores towards plant roots.

Mycobacterium avium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. avium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. avium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. avium complex and M. paratuberculosis clinical isolates with TM4-derived luciferase reporter phages demonstrated that the majority of these isolates were susceptible to TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied to isolate kanamycin-resistant transformants of M. avium complex and M. paratuberculosis with Escherichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformants without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species.

The insect pathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (aw) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0.05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951-0.935 aw (. 95.1-93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 aw (. 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930-0.920 aw). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.

The uptake of nitrate or ammonium (at a concentration of 10 mol I-1) by marine phytoplankton was studied in relation to cell size. Initial specific nitrate uptake rates by small (35000 m3) and large (130000 m3) cells of the diatom Ditylum brightwellii did not differ significantly. However, the larger cells maintained a high uptake rate for a longer time. Therefore, they accumulated nitrate in a higher biomass-specific pool than the smaller cells. In the dark, this effect was even more pronounced. Two smaller diatom species, Lauderia borealis (7474 m3) and Thalassiosira pseudonana (98 (m3), had lower initial specific nitrate uptake rates and lower intracellular pools. Transient-state ammonium uptake did not result in accumulation of large intracellular pools of ammonium. Theoretically, and on the basis of the presented results, we stress the dualistic functional role of the vacuole. A large vacuole is an effective way for larger algal species to possess a minimum cell nutrient quota/cell surface ratio which is in the range of smaller species. Furthermore, by functioning as a storage reservoir it reduces inhibition of the uptake rate by cytoplasmic accumulated nutrients. The effect of the latter mechanism is that larger algal species are better at nitrate uptake under fluctuating conditions. These results imply that, in nitrogen-controlled marine systems, resource competition under fluctuating nutrient concentrations can only lead to a shift towards larger phytoplankton species if nitrate rather than ammonium is the main nitrogen source. From theoretical considerations it is argued that the maximum growth rate of algae is determined by nutrient assimilation properties rather than by photosynthetic capacity.

The enzyme glutamine synthetase (GS) is described for the first time in Dictyostelium discoideum. The appearance of this enzyme is developmentally regulated. The level of activity is low in vegetative cells and increases more than threefold during differentiation. Furthermore this enzyme is shown to be differentially localized in prespore cells, the specific activity being approximately fourfold higher than in prestalk cells. The enzyme has a pH optimum of 7.8 and 8.2 in the γ-glutamyltransferase and γ-glutamylsynthetase assays, respectively, and a temperature optimum of 45°C. Kinetic studies of GS revealed apparent Km values of 5.9 mM, 0.009 mM and 8.6 mM for glutamine, ADP and NH2OH, respectively, in the γ-glutamyltransferase assay, and of 2.2 mM, 0.12 mM and 0.64 mM for glutamate, ATP and NH2OH, respectively, in the γ-glutamylsynthetase assay.

Nucleotide sequencing of a region downstream from the Pseudomonas aeruginosa 1244 pilin structural gene, pilA, revealed an ORF potentially able to code for a protein of M r 50862. This ORF, called pilO, was flanked by a tRNAthr gene, which was followed by a transcriptional termination sequence. The tRNAthr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P. aeruginosa strains. A 2200 base mRNA strand, which contained both the pilO and pilA transcripts, was produced from this region, while a 650 base transcript containing only pilA was present in a 100-fold excess over the longer transcript. Hyperexpression of the pilA gene in a PilO- strain resulted in normal pilus-specific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparent M r and a more neutral pI compared to that produced by a strain containing a functional pilO gene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functional pilO gene.

Chemical mutagenesis of the AmpC -lactamase-hyperinducible Escherichia coli strain SN0301/pNu305 carrying the cloned ampC and ampR genes from Citrobacter freundii OS60 gave four independent mutants in which -lactamase was no longer inducible, or was inducible only to a low level, by -lactam antibiotics. The genes ampC, ampR, ampD and ampE, which were essential for -lactamase induction, were functional in these mutants. In all four mutants, the sites of mutation were mapped to 9.9 min on the E. coli chromosome. Complementation with wild-type ampG restored inducibility of -lactamase to wild-type levels. The nucleotide sequence of all four mutant ampG alleles (ampG1, ampG3, ampG4 and ampG5) was determined. In three of the mutants, a single base exchange led to an amino acid change from glycine to aspartate at different sites in the deduced amino acid sequence. In the fourth mutant (ampG4), with low-level inducibility, the nucleotide sequence was identical to wild-type ampG. Spontaneous back-mutation of the chromosomal ampG1 mutant resulted in restoration of wild-type inducibility and a return to the wild-type ampG sequence. Unspecific induction by components of the growth medium was also dependent on intact ampG function.