Investigation of the Presence of Phenolic Compounds in Monocotyledonous Cell Walls, using UV Fluorescence Microscopy

Harris and Hartley (1976, 1980) demonstrated the presence offerulic acid in cell walls of certain monocotyledons using UVfluorescence microscopy (fluorescing green after treatment withammonium hydroxide solution). The presence or absence of thistype of fluorescence is apparently critical in higher levelsystematics of monocotyledons. In order to evaluate the significanceof this character, cell wall fluorescence was investigated ina range of monocotyledon species, particularly the AustralianXanthorrhoeaceae sensu lato (Bedford et al., 1986), which werenot investigated in earlier studies. This family is widely regardedas polyphyletic and was divided into several families by Dahlgren,Clifford and Yeo (1985). Some of its constituent genera, suchas Dasypogon, Kingia and Calectasia, have been linked with bothcommelinoid and non-commelinoid monocotyledons, and are of obscureaffinity. Some genera of Xanthorrhoeaceae sensu lato (Baxteria,Calectasia, Dasypogon and Kingia) show this type of green cellwall fluorescence and may therefore be more closely linked withthe commelinoid monocotyledons, rather than the Lilianae-Asparagales,as previously placed (Dahlgren et al., 1985).Copyright 1994,1999 Academic Press Asparagales, Dasypogonaceae, fluorescence, Hanguana, monocotyledons, systematics, Xanthorrhoeaceae     

Inhibition of tRNA-dependent ligase MurM from Streptococcus pneumoniae by phosphonate and sulfonamide inhibitors

Ligase MurM catalyses the addition of Ala from alanyl-tRNA^Ala, or Ser from seryl-tRNA^Ser, to lipid intermediate II in peptidoglycan biosynthesis in Streptococcus pneumoniae, and is a determinant of high-level penicillin resistance. Phosphorus-based transition state analogues were designed as inhibitors of the MurM-catalysed reaction. Phosphonamide analogues mimicking the attack of a lysine nucleophile upon Ala-tRNA^Ala showed no inhibition of MurM, but adenosine 3′-phosphonate analogues showed inhibition of MurM, the most active being a 2′-deoxyadenosine analogue (IC₅₀ 100 μM). Structure/function studies upon this analogue established that modification of the amino group of the aminoalkylphosphonate resulted in loss of potency, and modification of the adenosine 5′-hydroxyl group with either a t-butyl dimethyl silyl or a carbamate functional group resulted in loss of activity. A library of 48 aryl sulfonamides was also screened against MurM using a radiochemical assay, and two compounds showed sub-millimolar inhibition. These compounds are the first small molecule inhibitors of the Fem ligase family of peptidyltransferases found in Gram-positive bacteria.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Characterisation of AnBEST1, a functional anion channel in the plasma membrane of the filamentous fungus, Aspergillus nidulans

Two distant homologues of the bestrophin gene family have been identified in the filamentous fungus, Aspergillus nidulans (anbest1 and anbest2). AnBEST1 was functionally characterised using the patch clamp technique and was shown to be an anion selective channel permeable to citrate. Furthermore, AnBEST1 restored the growth of the pdr12Δ yeast mutant on inhibitory concentrations of extracellular propionate, benzoate and sorbate, also consistent with carboxylated organic anion permeation of AnBEST1. Similar to its animal counterparts, AnBEST1 currents were activated by elevated cytosolic Ca(2+) with a K(d) of 1.60μM. Single channel currents showed long (>10s) open and closed times with a unitary conductance of 16.3pS. Transformation of A. nidulans with GFP-tagged AnBEST1 revealed that AnBEST1 localised to the plasma membrane. An anbest1 null mutant was generated to investigate the possibility that AnBEST1 mediated organic anion efflux across the plasma membrane. Although organic anion efflux was reduced from anbest1 null mutants, this phenotype was linked to the restoration of uracil/uridine-requiring A. nidulans strains to uracil/uridine prototrophy. In conclusion, this study identifies a new family of organic anion-permeable channels in filamentous fungi. We also reveal that uracil/uridine-requiring Aspergillus strains exhibit altered organic anion metabolism which could have implications for the interpretation of physiological studies using auxotrophic Aspergillus strains.     

Genetic analysis of the TOR pathway in Aspergillus nidulans

We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.