The genetics of conidiophore pigmentation in Aspergillus nidulans

The grey-brown pigmentation of Aspergillus nidulans conidiophores depends on the functions of two 'ivory' loci. ivoB codes for a developmental specific phenol oxidase, and mutants accumulate its substrate N-acetyl-6-hydroxytryptophan. ivoA mutants are unable to make this substrate. ygA mutants are also poorly pigmented, and extracts require copper salts to activate both the phenol oxidase and conidial laccase. ivoA and ivoB mutants partially suppress the spore colour phenotype of ygA mutants. Comparisons of morphology, phenol oxidase and substrate accumulation in morphological mutants at the brlA locus suggest that the brlA protein regulates ivoA, ivoB and morphogenetic loci independently. The medA locus, which also affects morphology and pigmentation, may code for a modifier of brlA function. abaA mutants which are blocked at a later stage of development than brlA or medA mutants have low phenol oxidase levels, implying that by this stage of development the activity of the ivoB locus is declining.     

Identification of Developmental Regulatory Genes in Aspergillus Nidulans by Overexpression

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [ alcA (p) ] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA (p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA (p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA (p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA (p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA (p) :: brlA induction. Sequence analyses of the DNA fragments under alcA (p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA (p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.     

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.     

Isolation of Aspergillus nidulans Mutants That Overcome brlA-Induced Growth Arrest

The Aspergillus nidulans brlA gene is a primary regulator of development-specific gene expression during conidiation. Forced activation of brlA in vegetative cells leads to inappropriate induction of conidiophore formation and causes growth to stop. In fact, when conidia containing a nutritionally inducible brlA gene fusion are placed on inducing medium, they fail to germinate. We used this phenotype to select 174 mutants that continue growing following such forced brlA activation. Forty-six of these mutants also produced abnormal developmental structures during air-induced conidiation as expected if the mutations resulted in an altered response to BrlA (designated sbr mutants for suppressors of brlA response). The predominant mutant class identified was defective in a known developmental regulatory gene, abaA. We also identified mutants with defects in the previously characterized early acting developmental regulatory genes flbB and flbD and in four previously undescribed loci designated sbrA-D. sbrA mutants represent the second largest group and are characterized by production of conidiophore stalks that lack a normal vesicle and form branching sterigmata that rarely make spores. Because abaA expression could not be detected in sbrA mutants following brlA activation we propose that sbrA functions as a developmental modifier, participating in brlA-dependent activation of other developmental regulators.     

Aspergillus Fabm Encodes an Essential Product That Is Related to Poly(a)-Binding Proteins and Activates Development When Overexpressed

Overexpression of several different Aspergillus nidulans developmental regulatory genes has been shown to cause inappropriate developmental activation and growth inhibition. We previously exploited this observation that induced development caused growth inhibition in designing a screen to identify other genes that could activate development when overexpressed. We identified 16 mutants in which induced expression of different random genomic DNA sequences caused growth inhibition, accumulation of mRNA corresponding to the brlA developmental regulatory locus, and in several cases sporulation. This phenotype was designated FAB for Forced expression Activation of brlA and the genes were called fabA through fabP. Here we describe one of these genes, fabM, which is predicted to encode a poly(A)-binding protein (PABP) that is constitutively expressed and is essential for viability. While it is unclear why overexpression of the fabM caused sporulation, we showed that this activity required other known early developmental regulators including brlAβ, flbA, flbB, flbC, and fluG. We propose that fabM is an example of a gene that is not only required for growth, but also has specific functions early in development that assist developmental induction, presumably by allowing translation of specific mRNAs like brlA.     

TmpA, a member of a novel family of putative membrane flavoproteins, regulates asexual development in Aspergillus nidulans

Asexual reproduction (conidiation) in Aspergillus nidulans is induced by environmental signals like exposure to air or nutrient starvation, and depends on brlA gene activation. The study of 'fluffy' mutants showing delayed asexual development and reduced brlA expression has defined the fluG pathway, involved in regulation of this differentiation process. Genetic characterization of a 'fluffy' mutant identified tmpA as a new gene involved in regulation of conidiation. TmpA defines a new family of putative transmembrane proteins of unknown function, widespread in filamentous fungi and plants, with homologues showing similarity to non-ribosomal peptide synthetases. The deletion of tmpA resulted in decreased brlA expression and conidiation in air-exposed colonies. This defect was suppressed when DeltatmpA mutants were grown next to wild-type or DeltafluG mutant colonies, even without direct contact between hyphae. In liquid culture, tmpA was essential for conidiation induced by nitrogen but not by carbon starvation, whereas the overexpression of different tmpA tagged alleles resulted in conidiation. The overexpression of fluG-induced conidiation independently of tmpA and DeltatmpADeltafluG double mutants showed an additive 'fluffy' phenotype, indicating that tmpA and fluG regulate asexual sporulation through different pathways. TmpA and its homologues appear to have diverged from the ferric reductase family, retaining overall transmembrane architecture, NAD(P), flavin adenine dinucleotide (FAD) and possibly haem-binding domains. Based on our results, we propose that TmpA is a membrane oxidoreductase involved in the synthesis of a developmental signal.     

Enhancers of Conidiation Mutants in Aspergillus Nidulans

Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies.     

An intragenic map of the brlA locus of Aspergillus nidulans.

Summary We have constructed an intragenic map for the Aspergillus nidulans brlA gene, mutants in which are distinguishable by visual criteria only. Most of the leaky phenotype mutants map near the right (3) end. The gene shows distinct recombinational polarity consistent with recombination initiation at the promoter (centromereproximal) end of the gene. brlA12 and brlA20 mutants gave abnormal DNA restriction patterns consistent with the III; VIII and VI; VIII translocations, respectively, determined by haploidization.     

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.     

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions.

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.