argB Gene Research Articles

Overproduction of L-piperazic acid by overexpression of ArgB gene in Aureobasidium melanogenum DFAK1

L-piperazic acid (L-Piz) is one of components in many bioactive compounds synthesized by Streptomyces spp. Therefore, it is very important to develop a sustainable and eco-friendly bioprocess to produce L-Piz from renewable glucose. In this study, it was found that the engineered Aureobasidium melanogenum DFAK-ArgB6 strain overexpressing the ArgB gene encoding N-acetylglutamate kinase could produce 3.0 g/L L-Piz from glucose during the fed-batch fermentation. The yield was 55.6 mg L-Piz/g of glucose and the productivity was 24.8 mg/L/h. This was the highest amount of L-Piz produced by microorganisms so far. Finally, the partially purified L-Piz had purity of 86.4%.

CiaR impacts biofilm formation by regulating an arginine biosynthesis pathway in Streptococcus sanguinis SK36

Streptococcus sanguinis is an early colonizer of the tooth surface and competes with oral pathogens such as Streptococcus mutans to maintain oral health. However, little is known about its mechanism of biofilm formation. Here, we show that mutation of the ciaR gene, encoding the response regulator of the CiaRH two-component system in S. sanguinis SK36, produced a fragile biofilm. Cell aggregation, gtfP gene expression and water-insoluble glucan production were all reduced, which suggested polysaccharide production was decreased in ΔciaR. RNA sequencing and qRT-PCR revealed that arginine biosynthesis genes (argR, argB, argC, argG, argH and argJ) and two arginine/histidine permease genes (SSA_1568 and SSA_1569) were upregulated in ΔciaR. In contrast to ΔciaR, most of strains constructed to contain deletions in each of these genes produced more biofilm and water-insoluble glucan than SK36. A ΔciaRΔargB double mutant was completely restored for the gtfP gene expression, glucan production and biofilm formation ability that was lost in ΔciaR, indicating that argB was essential for ciaR to regulate biofilm formation. We conclude that by promoting the expression of arginine biosynthetic genes, especially argB gene, the ciaR mutation reduced polysaccharide production, resulting in the formation of a fragile biofilm in Streptococcus sanguinis.

Molecular characterization of arginine deiminase pathway in Laribacter hongkongensis and unique regulation of arginine catabolism and anabolism by multiple environmental stresses.

The betaproteobacterium Laribacter hongkongensis is associated with invasive bacteremic infections and gastroenteritis. Its genome contains two adjacent arc gene cassettes (arc1 and arc2) under independent transcriptional control, which are essential for acid resistance. Laribacter hongkongensis also encodes duplicate copies of the argA and argB genes from the arginine biosynthesis pathway. We show that arginine enhances the transcription of arcA2 but suppresses arcA1 expression. We demonstrate that ArgR acts as a transcriptional regulator of the two arc operons through binding to ARG operator sites (ARG boxes). Upon temperature shift from 20°C to 37°C, arcA1 transcription is upregulated while arcA2, argA2, argB2 and argG are downregulated. The transcription of arcA1 and arcA2 are augmented under anaerobic and acidic conditions. The transcription levels of argA1, argA2, argB1, argB2 and argG are significantly increased under anaerobic and acidic conditions but are repressed by the addition of arginine. Deletion of argR significantly decreases bacterial survival in macrophages, while expression of both arc operons, argR and all five of the anabolic arg genes increases 8 h post-infection. Our results show that arginine catabolism in L. hongkongensis is finely regulated by controlling the transcription of two arc operons, whereas arginine anabolism is controlled by two copies of argA and argB.

Analysis of the arginine biosynthetic gene cluster argCJBDFR of Corynebacterium crenatum

Objective: Corynebacterium crenatum AS1.542, a Gram-positive bacterium and indigenous nonpatho-genic corynebacteria, is widely exploited for the in-dustrial production of amino acids. The objective of this paper is to clarify the genetic information of the arginine biosynthetic pathway, and further more contribute to the improvement of arginine produc-tion. Methods: Polymerase chain reaction (PCR) technology was employed for obtaining the arginine biosynthetic gene sequence, and softwares eg. Laser-gene, BPROM, RNAshapes were used for the analysis of obtained sequences. Results: Arginine biosynethetic gene cluster of C. crenatum, comprising argJ, argB, argD, argF, argR and part of argC, has been ampli-fied and sequenced. The gene order has been estab-lished as argCJBDFR, with a entire length of 6.08kb. Conclusion: An internal promoter was found in the upstream of argB gene, four argBDFR ORFs are lo-cated in a same transcription unit, and the tran-scripiton termination of argC gene is irrelevant with the rho-factor. Comparison with ornithine acetyl-transferase (coded by argJ gene) from C. glutamate, ornithine acetyltransferase from C. crenatum also belongs to the monofunctional enzymes.

Molecular cloning and sequence analysis of the lysR gene from the extremely thermophilic eubacterium, Thermus thermophilus HB27.

We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27. This mutant showed the lysA- or lysR- genotype since it could not grow on the minimal plate which contained diaminopimelic acid. Sequence analysis of the clones which could rescue the Lys- mutant indicated the lysR gene. The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6. In the Lys- mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys- and kanamycin-resistant phenotype. The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli. The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4%. We also determined that the argC gene encoding N-acetyl-gamma-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.

Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae

We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host ( niaD − sC −). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain ( niaD − sC − adeA −) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains ( niaD − sC −Δ argB adeA − or niaD − sC −Δ argB adeB −) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain ( niaD − sC −Δ argB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.

Establishment of mRFP1 as a fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in vitro (GATEWAY).

The advent of fluorescent proteins as vital dyes had a major impact in many research fields. Different green fluorescent protein (GFP) variants were established in prokaryotic and eukaryotic organisms within the past 10 years, and other fluorescent proteins were discovered and applied. We expressed the Discosoma red fluorescent protein, DsRed (T4), the improved monomeric red fluorescent protein (mRFP1) and the blue fluorescent protein (BFP) in the filamentous fungus Aspergillus nidulans. Whereas DsRed requires tetramer formation for fluorescence, mRFP1 functions as monomer. We used sGFP, DsRed (T4), mRFP1 and BFP for nuclear and/or mitochondrial labelling. To facilitate gene tagging, we established a number of cloning vectors for the efficient, simultaneous fusion of any protein with mRFP1, BFP and sGFP or the haemagglutinin epitope, 3xHA. A PCR-amplified gene of interest can be inserted into the expression vectors without cloning but using homologous recombination in vitro (GATEWAY). The vectors contain the argB gene as a selection marker for A. nidulans and the inducible alcA promoter for control of expression. The system allows labelling of a protein with several tags in one recombination reaction. Both the nutritional marker gene and the promoter are frequently used in other fungi, suggesting that this set of expression vectors will be very useful tools for gene analysis on a genome-wide scale.

Disruption of the Aspergillus fumigatus argB gene using a novel in vitro transposon-based mutagenesis approach.

We disrupted the Aspergillus fumigatus argB gene, encoding ornithine transcarbamylase, using a novel in vitro transposon-based mutagenesis approach. This approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Using this approach, we obtained arginine auxotrophs of A. fumigatus. Full characterization of the argB insertion was performed by Southern blot analysis. These strains can be supplemented by addition of arginine into the culture medium and can be fully rescued to arginine prototrophy by transformation with the intact A. fumigatus argB gene.

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.

Repression of chsB expression reveals the functional importance of class IV chitin synthase gene chsD in hyphal growth and conidiation of Aspergillus nidulans.

The functions of two previously identified chitin synthase genes in Aspergillus nidulans, chsB and chsD, were analysed. First, a conditional chsB mutant was constructed in which the expression of chsB is under the control of a repressible promoter, the alcA promoter, of A. nidulans. Under repressing conditions, the mutant grew slowly and produced highly branched hyphae, supporting the idea that chsB is involved in normal hyphal growth. The involvement of chsB in conidiation was also demonstrated. Next, double mutants of chsB and chsD were constructed, in which chsB was placed under the control of the alcA promoter and chsD was replaced with the argB gene of A. nidulans. These double mutants grew more slowly than the chsB single mutant under high-osmolarity conditions. The hyphae of the double mutant appeared to be more disorganized than those of the chsB single mutant. It was also found that ChsD was functionally implicated in conidiation when the expression of chsB was limited. These results indicate the importance of the ChsD function in the absence of chsB expression. The roles of ChsB and ChsD in hyphal growth and in conidiation were supported by the analysis of the spatial expression patterns of chsB and chsD, using lacZ of Escherichia coli as a reporter gene.

The Aspergillus nidulans homoaconitase gene lysF is negatively regulated by the multimeric CCAAT-binding complex AnCF and positively regulated by GATA sites.

In beta-lactam-antibiotic-producing fungi, such as Aspergillus (Emericella) nidulans, L-alpha-aminoadipic acid is the branching point of the lysine and penicillin biosynthesis pathways. To obtain a deeper insight into the regulation of lysine biosynthesis genes, the regulation of the A. nidulans lysF gene, which encodes homoaconitase, was studied. Band-shift assays indicated that the A. nidulans multimeric CCAAT-binding complex AnCF binds to two of four CCAAT motifs present in the lysF promoter region. AnCF consists at least of three different subunits, designated HapB, HapC, and HapE. In both a delta hapB and a delta hapC strain, the expression of a translational lysF-lacZ gene fusion integrated in single copy at the chromosomal argB gene locus was two to three-fold higher than in a wild-type strain. These data show that AnCF negatively regulates lysF expression. The results of Northern blot analysis and lysF-lacZ expression analysis did not indicate a lysine-dependent repression of lysF expression. Furthermore, mutational analysis of the lysF promoter region revealed that two GATA sites matching the GATA consensus sequence HGATAR positively affected lysF-lacZ expression. Results of Northern blot analysis also excluded that the global nitrogen regulator AreA is the responsible trans-acting GATA-binding factor.

An Aspergillus nidulans uvsC null mutant is deficient in homologous DNA integration.

The Aspergillus nidulans uvsC gene was identified as a homolog of RAD51 and recA of Saccharomyces cerevisiae and Escherichia coli, respectively, whose role in genetic recombination and recombinational repair has been extensively studied. Like many other filamentous fungi, A. nidulans shows no bias towards either homologous or ectopic integration of exogenous DNA. Therefore it is a unique and useful organism for the study of the mechanisms of DNA integration. Homologous integration of a 1.7-kb argB gene was not detected in 50 transformants obtained from a uvsC null mutant. In contrast, the frequency of homologous integration in uvsC+ control strains varied from 41 to 86%. Another feature observed with the uvsC null mutant was that an increased number of transformants had undergone ectopic integrations at multiple sites in the genome. These results are consistent with the established function of Rad51/RecA, and further indicate the involvement of redundant pathways in integration of exogenous DNA. This study provides direct evidence for the involvement of uvsC in exogenous DNA integration and should contribute to the improvement of genetic manipulations in general, but particularly in fungi.

The chsA gene from Aspergillus nidulans is necessary for maximal conidiation.

A fragment from the open reading frame of the cloned chsA gene from Aspergillus nidulans was deleted and replaced with the argB gene. The resulting construct was used to replace the wild-type chsA gene in an argB deletion strain. The growth and morphology of the vegetative hyphae from the resulting chsA disruptant strain were indistinguishable from those of a wild-type strain but the chitin content of the hyphae from the disruptant was reduced to approximately 90% of that of wild-type. The disruptant showed reduced ability to produce the asexual spores (conidia) that are formed by differentiated aerial hyphae called conidiophores. The ability to form undifferentiated aerial hyphae was not impaired in the disruptant. The conidiophores and conidia produced by the disruptant were indistinguishable from those of wild-type. Conidium formation by the disruptant grown on a variety of media was reduced to about 30% of the wild-type. A chsE null strain did not show a defect in conidiation but a strain in which both chsA and chsE were inactivated produced about 3% of the conidia of wild-type. That finding supports the hypothesis that chsA and chsE encode a partially redundant function necessary for conidiophore development.

The chsA gene from Aspergillus nidulans is necessary for maximal conidiation

A fragment from the open reading frame of the cloned chsA gene from Aspergillus nidulans was deleted and replaced with the argB gene. The resulting construct was used to replace the wild-type chsA gene in an argB deletion strain. The growth and morphology of the vegetative hyphae from the resulting chsA disruptant strain were indistinguishable from those of a wild-type strain but the chitin content of the hyphae from the disruptant was reduced to approximately 90% of that of wild-type. The disruptant showed reduced ability to produce the asexual spores (conidia) that are formed by differentiated aerial hyphae called conidiophores. The ability to form undifferentiated aerial hyphae was not impaired in the disruptant. The conidiophores and conidia produced by the disruptant were indistinguishable from those of wild-type. Conidium formation by the disruptant grown on a variety of media was reduced to about 30% of the wild-type. A chsE null strain did not show a defect in conidiation but a strain in which both chsA and chsE were inactivated produced about 3% of the conidia of wild-type. That finding supports the hypothesis that chsA and chsE encode a partially redundant function necessary for conidiophore development.

Pectinase production by a diploid construct from two Aspergillus niger overproducing mutants

Arginine auxotrophy (Arg −) was induced in two Aspergillus niger strains, previously reported as 2-deoxy- d-glucose (2DG) resistant and pectinase overproducing mutants in either solid state fermentation (SSF) or submerged fermentation (SmF). Only one of these two arginine auxotrophs (strain Aw99ARG) was transformed with a plasmid containing the A. nidulands argB gene, indicating that this strain was affected in this particular gene. A diploid (D4) was generated via parasexual recombination between these two Arg − strains and was characterized with regard to 2DG sensitivity and pectinase production. This diploid showed wild type sensitivity to 2DG, suggesting that recessive mutations at two different loci were involved in 2DG resistance in each parent strain. The diploid had also increased pectinase production in SmF (127% higher) when compared to the wild type strain, from which all pectinase overproducing mutants were previously isolated, and 10% over Aw99-iii2 strain, formerly characterized as an overproducing strain in SmF.

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI).

We have used a plasmid containing the argB gene to transform an Aspergillus nidulans argB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and approximately 3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.

Molecular cloning and sequence analysis of the lysR gene from the extremely thermophilic eubacterium, Thermus thermophilus HB27

We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27. This mutant showed the lysA − or lysR − genotype since it could not grow on the minimal plate which contained diaminopimelic acid. Sequence analysis of the clones which could rescue the Lys − mutant indicated the lysR gene. The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6. In the Lys − mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys − and kanamycin-resistant phenotype. The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli. The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4%. We also determined that the argC gene encoding N-acetyl-γ-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.

Genetic and molecular analysis of Aspergillus nidulans transformants obtained by the biolistic process

Twenty transformants obtained by transforming the argB strain of Aspergillus nidulans by the biolistic process were analysed in detail. Eight lost the Arg+ phenotype very frequently and the other 12 were mitotically stable, even after 15 successive subculturing. To analyse the integration events, the mitotically stable transformants were submited to genetic and molecular analyses; 16.7% resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus, 8.3% showed replacement of the argB mutation by wild type allele, 41.7% showed transforming DNA integrated into nonhomologous chromosomal regions at chromosome III, and 33.3% showed transforming DNA integrated into nonhomologous chromosomal regions at chromosomes others than III. Among the mitotically stable transformants, the frequency of integration into nonhomologous sites was higher by the biolistic process than that reported by the literature for protoplast-mediated transformation.Key words: Aspergillus nidulans, fungal transformation, biolistic gene transfer, gene integration, argB gene.

Transformation of a mycoparasitic Trichoderma harzianum strain with the argB gene of Aspergillus nidulans

A transformation system was developed for the mycoparasitic filamentous fungus Trichoderma harzianum, based upon complementation of auxotrophic mutants. Prototrophic transformants were obtained using plasmids pSal23 and pAN5-41B, carrying the Aspergillus nidulans argB gene, and both the Aspergillus nidulans argB and Escherichia coli lacZ genes, respectively.

Gene expression from replicating plasmids in Aspergillus nidulans.

Plasmids bearing the AMA1 replicator from Aspergillus nidulans are capable of extrachromosomal replication in this fungus as well as in other species. Synthetic plasmids bearing the moderately expressed argB gene and the highly expressed, inducible beta-galactosidase gene (bgaS) were introduced into fungal cells. Expression of both genes was monitored by Northern hybridization. It was demonstrated that transcription of bgaS is induced and repressed normally, irrespective of whether the gene is integrated into the chromosome or maintained on an extrachromosomal supercoiled plasmid. Transcription of the strongly expressed bgaS gene stimulates transcription of the argB gene located on the same replicating plasmid irrespective of orientation. This effect also occurs with chromosomally integrated vectors, but to a lesser extent. Episomal vectors are present in 10 copies per nucleus, and the expression level of the argB gene is approximately proportional to copy number. However, the amount of mRNA transcribed from the highly expressed bgaS gene on the multi-copy replicating plasmid does not exceed that from single-copy integrants. High levels of expression of the plasmid-borne gene do not affect plasmid mitotic stability or copy number.