An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-β-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species. Received: 5 February / 20 March 2000     

Regulatory region of the Aspergillus nidulans argB gene

Summary We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5 upstream sequence of the argB gene. The pro^– arg^– strain of A. nidulans was transformed with the above plasmids. Several arg⁺ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between –150 and –50 by upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.Abbreviations bp base pairs - kb 10³ base pairs - OTC ornithine carbamoyltransferase - ORF open reading frame     

Disruption of the Aspergillus niger argB gene: a tool for transformation

We disrupted the Aspergillus niger gene argB, encoding ornithine transcarbamylase. Full characterisation of the argB deletion was performed by Southern blot analysis, growth tests and by means of mitotic recombination, complementation and transformation. The argB locus was found to be physically removed, thus creating an auxotrophic mutation. The latter can be supplemented by addition of arginine into the culture medium. The argB gene and its disruption do not correlate to the argI13 (formerly argB13) allele described. The delta argB is on chromosome I whereas argI13 is on V. In addition, the argI13 mutation can only be complemented by the A. nidulans argB gene, whereas the new argB deletion can be complemented by both the A. niger and A. nidulans argB genes. The delta argB strain has been used to generate several strains in a breeding programme and to study the expression of important genes, such as areA and kexB.     

Complementation of Rickettsia rickettsii RelA/SpoT restores a nonlytic plaque phenotype

Spotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains of Rickettsia rickettsii, the virulent R strain and the avirulent Iowa strain. A nonlytic variant of R. rickettsii R, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated as relA/spoT-like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. In R. rickettsii R, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intact relA/spoT gene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncated relA/spoT gene in the Iowa lytic plaque variant restored the nonlytic phenotype. The relA/spoT mutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.     

The α-factor enhancement of hybrid formation by protoplast fusion in Saccharomyces cerevisiae can be mimicked by other procedures

Summary Slow growing mutants of Neurospora crassa were obtained by ethidium bromide treatment of the wild type strain. A particular mutant ER-3 showed stopper phenotype accompanied by deficient cytochrome spectra. The mutant showed an altered restriction pattern of the mtDNA which indicated a deletion of 25,000bp. The phenotype of the ethidium bromide induced mutant ER-3 seem to be related to the loss of several essential genes due to a deletion in its mtDNA.     

Deletion of the Escherichia coli uup gene encoding a protein of the ATP binding cassette superfamily affects bacterial competitiveness

Bacteria use a variety of mechanisms for intercellular communication. Here we show that deletion of the uup gene, which encodes a soluble ATP binding cassette (ABC) ATPase, renders the mutant strain sensitive to its parent when they are grown together in the same medium. Our data suggest that the decrease in viability of the mutant is dependent on direct cell-to-cell contact with the parent strain. Furthermore, we show that the presence of intact Walker B motifs in Uup is required for immunity or resistance to the parental strain, suggesting that ATP hydrolysis is an important determinant of this phenotype.     

HYP1, a hydrophobin gene from Aspergillus fumigatus, complements the rodletless phenotype in Aspergillus nidulans.

Aspergillus fumigatus produces conidia that are highly dispersable and resistant to degradation. We have sought to analyze these properties by studying the rodlets which form the outer spore coat protein. Degenerate primers based on hydrophobins in other fungi were applied to genomic DNA from A. fumigatus in PCR. A product of this reaction with similarity to an Aspergillus nidulans gene as judged by Southern hybridization was chosen for further study. Cloning and sequencing revealed a gene with two introns which encodes a protein of 159 amino acids. Structural characteristics consistent with those of other fungal hydrophobin genes, especially conserved cysteine residues, are present. The expression of the gene is limited to the developmental stages in which maturing conidiophores are present. This A. fumigatus gene, HYP1, was used to transform a mutant strain of A. nidulans that lacks rodlets. Transformants with a single copy of HYP1 expressed a rodlet layer on their conidia as observed by freeze-fracture electron microscopy.     

Construction of genetically defined aroA mutant of a native E. coli O78:K80 isolated from avian colibacillosis, in Iran

A precisely defined, single gene deletion of aroA was made systematically by Datsenko and Wanner based approach in native Escherichia coli O78: K80, and its nature was examined. The aroA gene encodes 5-enolpyrovylshikimate3-phosphate synthase and participates in the aromatic amino acids and folic acid, which are universal metabolic pathways of bacteria. For construction of mutant strain, the gene was replaced by recombination with a chloramphenicol cassette, flanked by FLP recognition target site. Primers designed to create in-frame deletion upon excision of the resistance cassette. The aroA deletion was confirmed by both polymerase chain reaction and failure of the mutant to replicate and grow on minimum medium lacking aromatic amino acids. On the other hand, the wild-type parent strain grows well in this medium. The sequence of the aroA gene in native strain was different, and knowing this subject was important to produce a mutant strain with deletion-based methods of mutation. Furthermore, use of antibiotic cassette replacement facilitates mutant construction in a more cost-effective manner in comparison with other techniques such as use of suicide vectors.     

Molecular characterization of the argJ mutation in Neisseria gonorrhoeae strains with requirements for arginine, hypoxanthine, and uracil.

Arginine auxotrophs are commonly encountered among clinical isolates of Neisseria gonorrhoeae. Arginine auxotrophs which also require hypoxanthine and uracil (AHU strains) compose a unique set of strains that are highly homogeneous and are believed to be clonally derived. The Arg- phenotype of these strains is due to a lesion in the argJ gene encoding ornithine acetyltransferase. We have cloned the mutant argJ gene from an AHU strain and compared the sequence of this gene to the wild-type argJ gene. The mutant gene contained a 3-bp deletion within a repetitive region of the argJ gene. This mutation was restored to the wild-type sequence in a naturally occurring Arg+ revertant of the AHU strain. This deletion was detected in a wide variety of other AHU strains but not in other ArgJ- strains or in ArgJ+ strains, supporting the theory that AHU strains are clonally derived.     

Genetic basis of the <Emphasis Type="Italic">ovc</Emphasis> phenotype of <Emphasis Type="Italic">Neurospora:</Emphasis> identification and analysis of a 77 kb deletion

The ovc mutant of Neurospora crassa accumulates more carotenoids than the wild type in the light, is sensitive to high osmotic pressure and exhibits an altered aerial development. The three traits are complemented by a single gene, cut-1, but only the two latter are exhibited by a mutant of this gene carrying a premature stop mutation. Targeted cut-1 deletion results in a normal carotenoid content, confirming the involvement of at least a second gene in the carotenoid-overproducing phenotype of the ovc strain. Molecular analysis of ovc genomic DNA indicates the absence of a large DNA segment affecting the gene cut-1. A PCR walking approach allowed the identification of a deletion extending along 77,078 bp on linkage group IV. The break-points are located in ApA/TpT sequences, suggesting the involvement of UV-induced thymine dimers in the origin of the deletion. The ovc mutant lacks 21 predicted ORFs, including cut-1 as the only known genetic marker, and four ORFs from a 22-member transmethylase gene family. Ten ORFs have no similarity with any predicted gene from other species. Three of them are closely related by sequence and linkage, evoking ancestral gene duplications.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene

Summary Conidial protoplasts of an A. nidulans amdS deletion strain (MH1277) have been transformed to the AmdS⁺ phenotype with a plasmid carrying the wild type gene (p3SR2). Optimalisation of transformation and plating conditions now has resulted in frequencies of 300–400 transformants per g of DNA.Analysis of DNA from AmdS⁺ transformants of MH1277 showed that transformation had occurred by integration of vector DNA sequences into the genome. In virtually all these transformants multiple copies of the vector were present in a tandemly repeated fashion, not preferentially at the resident, partially deleted amdS gene. It is suggested that the observed integration phenomena are dependent on the genetic background of the A. nidulans strain, used for transformation. A model to explain the tandem type of integration is proposed.Abbrevations bp base pairs - kb 1,000 bp - EtBr ethidiumbromide - PEG polyethyleneglycol - r-DNA ribosomal DNA - c.f.u. colony forming units     

Phenotype microarray analysis of the AdeRS two-component system in <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

Acinetobacter baumannii is a nosocomial pathogen capable of resistance to multiple antimicrobials. The AdeRS two-component system (TCS) is associated with antimicrobial resistance by controlling the AdeABC efflux pump. To elucidate modulation by AdeRS, we made an A. baumannii mutant lacking the AdeRS TCS and characterized it using phenotype microarray (PM) analysis. After disrupting the adeRS operon, lower expression of AdeABC efflux pump was observed in the mutant strain. PM analysis showed that the AdeRS deletion strain and parental strain presented different tolerances to 91 compounds. Tolerance to 54 of the 91 compounds could be restored by complementing the AdeRS deleted strain with a plasmid carrying the adeRS gene. Compared to the parental strain, the AdeRS deletion strain was more sensitive to various inhibitors that target on-protein synthesis and function of cell membrane permeability. Tolerance to phleomycin of the AdeRS deletion strain reduced greatly and was further confirmed with minimum inhibitory concentration (MIC) determination and spot assay. The efflux pump inhibitor, NMP, could reduce phleomycin MIC four-fold at least for 29 (34.8%) of 81 tigecycline-resistant extensively drug-resistant A. baumannii (TGC-resistant XDRAB) clinical isolates. Our results suggested that the AdeRS TCS of A. baumannii was important for both elimination of antibiotics and tolerance to particular compounds.     

FvmnSOD is involved in oxidative stress defence,mitochondrial stability and apoptosis prevention in Fusarium verticillioides

Superoxide dismutases are key enzymes in elimination of the superoxide anion radical (O₂^•−) generated intracellularly or by exogenous oxidative stress eliciting agents, like menadione. In this study, we investigated the physiological role of the manganese superoxide dismutase-encoding gene in Fusarium verticillioides via the construction of a gene deletion mutant, ΔFvmnSOD and comparing its phenotype with that of the wild-type parental strain and a ΔFvmnSOD′ C strain, complemented with the functional manganese superoxide dismutase gene. Deletion of FvmnSOD had no effect on the relative intracellular superoxide ratio but increased the sensitivity of the fungus to menadione sodium bisulphite on Czapek-Dox stress agar plates. The lack of FvmnSOD caused changes in mitochondrial morphology and physiology: The volumetric ratio of these cell organelles in the second hyphal segment, as well as the total, the KCN-sensitive cytochrome c-dependent and the KCN+SHAM (salicylhidroxamic acid)-resistant residual respiration rates, were higher in the mutant as compared to the wild-type and the complemented strains. Nevertheless, changes in the respiration rates were attributable to the higher volumetric ratio of mitochondria found in the gene deletion mutant. Changes in the mitochondrial functions also brought about higher sensitivity to apoptotic cell death elicited by the Penicillium chrysogenum antifungal protein. The gene deletion mutant developed significantly thinner hyphae in comparison to the wild-type strain. Deletion of FvmnSOD had no effect on fumonisin B₁ and B₂ production of the fungus grown in Myro medium as a static culture.     

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus

Thymidine-auxotrophic small colony variants (SCVs) of Staphylococcus aureus are frequently isolated from the chronically infected airways of patients suffering from cystic fibrosis. To date, little is known regarding the molecular mechanisms leading to the formation of this special phenotype, but the auxotrophism for thymidine suggests that impaired thymidine metabolism might play a major role. Sequence analysis of the thymidylate synthase-encoding thyA gene of six clinical thymidine-auxotrophic S. aureus SCVs revealed that all isolates had mutations within thyA. In five isolates the function of the thymidylate synthase was definitely impaired: three of them showed a truncation of the thyA coding sequence by nonsense or frame-shift mutations, in one further isolate the active site of the enzyme was affected by an internal 12-bp deletion, and another isolate had a 173-bp deletion spanning the 5'-terminal region of thyA and the preceding DNA sequence. The sixth isolate showed two amino acid substitutions within the thyA gene product. To confirm the importance of impaired thymidylate synthase synthesis or activity for the formation of the thymidine-auxotrophic SCV phenotype, we constructed a thyA knock-out mutant of a wild-type S. aureus strain. This mutant showed all characteristics of clinical SCVs, such as slow growth, decreased pigment production, reduced hemolytic activity, auxotrophism for thymidine, resistance to trimethoprim/sulfamethoxazol, and reduced plasma coagulase activity. Complementation of the thyA knock-out mutant with intact thyA in trans nearly restored the normal phenotype. In conclusion, these data confirm at the molecular level that impaired thymidylate synthase function is causative for the formation of the thymidine-auxotrophic SCV phenotype in S. aureus.     

The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance

Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.