Fumiyasu Fukumori

Roles of putative His-to-Asp signaling modules HPT-1 and RRG-2, on viability and sensitivity to osmotic and oxidative stresses in Neurospora crassa

Neurospora crassa has a putative histidine phosphotransfer protein (HPT-1) that transfers signals from 11 histidine kinases to two putative response regulators (RRG-1 and RRG-2) in its histidine-to-aspartate phosphorelay system. The hpt-1 gene was successfully disrupted in the os-2 (MAP kinase gene) mutant, but not in the wild-type strain in this study. Crossing the resultant hpt-1; os-2 mutants with the wild-type or os-1 (histidine kinase gene) mutant strains produced no progeny with hpt-1 or os-1;hpt-1 mutation, strongly suggesting that hpt-1 is essential for growth unless downstream OS-2 is inactivated. hpt-1 mutation partially recovered the osmotic sensitivity of os-2 mutants, implying the involvement of yeast Skn7-like RRG-2 in osmoregulation. However, the rrg-2 disruption did not change the osmotic sensitivity of the wild-type strain and the os-2 mutant, suggesting that rrg-2 did not participate in the osmoregulation. Both rrg-2 and os-2 single mutation slightly increased sensitivity to t-butyl hydroperoxide, and rrg-2 and hpt-1 mutations increased the os-2 mutant’s sensitivity. Although OS-1 is considered as a positive regulator of OS-2 MAP kinase, our results suggested that HPT-1 negatively regulated downstream MAP kinase cascade, and that OS-2 and RRG-2 probably participate independently in the oxidative stress response in N. crassa.

Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

The third cellulase of alkalophilic Bacillus sp. strain N-4: evolutionary relationships within the cel gene family

The third cellulase gene (celC) of Bacillus sp. strain N-4 was cloned in plasmid pBR322 and was located within a 5.5-kb HindIII fragment. The cellulase encoded by this fragment had an Mr of about 100,000 and showed optimum activity around pH 9. These properties were different from those of the enzymes encoded by the celA and celB genes of the same organism. The amino acid sequence deduced from the nucleotide sequence was found to be highly homologous to the CEL-F enzyme from Bacillus sp. strain No. 1139 [Fukumori et al., J. Gen. Microbiol. 132 (1986) 2329-2335]. An evolutionary relationship observed among the four cellulases of alkalophilic Bacillus strains and that of Bacillus subtilis endoglucanase suggested that ancestral genes for alkaline and neutral cellulases diverged early in the evolution of these enzymes.

ATF-1 transcription factor regulates the expression of ccg-1 and cat-1 genes in response to fludioxonil under OS-2 MAP kinase in Neurospora crassa.

The ATF/CREB family transcriptional factors are regulated by stress-activated MAP kinase in yeast. The disruptants of the atf-1 gene, which encodes an ATF/CREB family transcriptional factor, were isolated and characterized in Neurospora crassa. The characteristic phenotypes in the os-2 MAP kinase strain, such as osmotic sensitivity and fludioxonil resistance, were not observed in the Deltaatf-1 strain; however, like the os-2 strain, up-regulation of the catalase gene cat-1 and the clock-controlled gene ccg-1 by treatment with fludioxonil (1 microg/mL) or 4% NaCl was almost completely abolished in the Deltaatf-1 strain. A gel shift assay indicated that ATF-1 bound to the cat-1 and ccg-1 promoters probably through the CRE motifs. The enzyme activity of large-subunit catalase CAT-1, the major conidial catalase, was not detected in the Deltaatf-1 strain, suggesting that the production of CAT-1 during formation of conidia is largely dependent on ATF-1. Among 11 clock-controlled genes, the expression of ccg-1, ccg-9, ccg-13, and ccg-14 was induced by fludioxonil in an OS-2-dependent manner; however, induction of ccg-13 and ccg-14 was observed in the Deltaatf-1 strain, suggesting the existence of another transcription factor regulated by OS-2. The homozygous cross between the Deltaatf-1 strains produced perithecia and ascospores; however, their ascospores never germinated. These findings suggest that ATF-1 acts as one of the transcriptional factors downstream of the OS-2 MAP kinase and probably regulates some genes involved in conidiation, circadian rhythm, and ascospore maturation in N. crassa.

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION*

Background: The bacterial pathway of l-hydroxyproline metabolism has not been identified. Results: Different types of d-hydroxyproline dehydrogenases and unique Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase involved in the bacterial l-hydroxyproline pathway were identified and characterized for the first time. Conclusion: l-Hydroxyproline degradation by bacteria was elucidated at the molecular level. Significance: Our results suggest that d-hydroxyproline dehydrogenases evolved convergently, and we discovered a unique deaminase enzyme likely within the aldolase protein family. l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ1-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α4β4γ4), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism.

A mutation in dnaK causes stabilization of the heat shock sigma factor σ32, accumulation of heat shock proteins and increase in toluene‐resistance in Pseudomonas putida

Heat shock gene expression is regulated by the cellular level and activity of the stress sigma factor σ(32) in Gram-negative bacteria. A toluene-resistant, temperature-sensitive derivative strain of Pseudomonas putida KT2442, designated KT2442-R2 (R2), accumulated several heat shock proteins (HSPs) under non-stress conditions. Genome sequencing of strain R2 revealed that its genome contains a number of point mutations, including a CGT to CCT change in dnaK resulting in an Arg445 to Pro substitution in DnaK. DNA microarray and real-time reverse transcription polymerase chain reaction analyses revealed that the mRNA levels of representative hsp genes (e.g. dnaK, htpG and groEL) were upregulated in R2 cells in the stationary phase. Wild-type and R2 cells showed similar heat shock responses at hsp mRNA and HSP levels; however, the σ(32) level in the mutant was not downregulated in the shut-off stage. Strain R2 harbouring plasmid-borne dnaK grew at 37°C, did not accumulate HSPs, and was more sensitive to toluene than strain R2. It is worth to note that that revertant of R2 able to grow at 37°C were isolated and exhibited a replacement of Pro445 by Ser or Leu in DnaK. Thus, the mutation in dnaK causes the temperature-sensitive phenotype, improper stabilization of σ(32) leading to HSP accumulation and increased toluene resistance in strain R2.

An AP-1-Like Transcription Factor, NAP-1, Regulates Expression of the Glutathione S-Transferase and NADH:Flavin Oxidoreductase Genes in Neurospora crassa

AP-1-like transcription factors play crucial roles in oxidative stress responses in yeast and filamentous fungi. The deletion of an AP-1-like transcription factor gene, nap-1, in Neurospora crassa slightly increased its sensitivity to oxidative stressors, including menadione. Microarray and quantitative real-time reverse transcriptase-PCR analyses were employed to identify menadione-inducible genes (migs) and the roles of NAP-1 in their regulation. N. crassa migs include three putative glutathione S-transferase genes and two NADH:flavin oxidoreductase genes, orthologs of OYE2 and OYE3, both of which play roles in menadione tolerance in Saccharomyces cerevisiae. Menadione induced nuclear localization of NAP-1, and oxidative upregulation of many of migs were NAP-1 dependent. Genes for a thioredoxin, a glutathione reductase, and a glutathione peroxidase were slightly upregulated by the chemical only in the wild-type strain, suggesting that NAP-1 is involved in their oxidative induction and probably dose not contribute to high-level constitutive expressions of such genes.

OxyR is involved in the expression of thioredoxin reductase TrxB in Pseudomonas putida

OxyR regulates the expression of a peroxiredoxin (AhpC) and two catalases (KatA and KatB), which play roles in peroxide protection, at the transcription level in Pseudomonas putida KT2442. Proteome analysis indicated significantly increased amounts of the enzymes AhpC, KatA, KatB, and a peroxiredoxin reductase (AhpF) in the oxyR1 mutant cells; these increases reflected the upregulation of the expression of the genes encoding these enzymes. Additionally, although the effect of oxyR1 mutation on the trxB transcript level was not clearly evident, it increased the amount of thioredoxin reductase (TrxB) by fivefold. Primer extension analysis revealed that trxB was constitutively transcribed from the P1 site; however, hydrogen peroxide treatment lowered the transcription of trxB from P1 but induced its transcription from P2. Adjacent to the -35 base of the P2 initiation site, sequences similar to those involved in the proposed OxyR binding in Escherichia coli were found in a region to which OxyR was shown to bind. These observations suggest that in P. putida, OxyR regulates TrxB expression by promoting trxB transcription from the P2 site when oxidative stresses lowered the transcription from the constitutive P1 site.

OS-2 Mitogen Activated Protein Kinase Regulates the Clock-Controlled Gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.

Deletion and expression analysis of beta-(1,3)-glucanosyltransferase genes in Neurospora crassa.

GPI(glycosylphosphatidylinositol)-anchored beta-(1,3)-glucanosyltransferases play an active role in cell wall biosynthesis in fungi. Neurospora crassa has 5 putative beta-(1,3)-glucanosyltransferase genes, namely, gel-1, gel-2, gel-3, gel-4, and gel-5, in its genome. Among them, the gel-3 gene is constitutively expressed at the highest level in growing hyphae, whereas gel-1 is expressed at the lowest level. The gel-3 deletion mutant displayed slow growth, while other gel gene disruptants exhibited normal growth. Although no gel gene disruption affected pH sensitivity and fertility, all Δgel mutants were resistant to cell wall degradation enzymes. Micafungin, a beta-(1,3)-glucan synthase inhibitor, induced gel-4 expression in the wild-type and 2 MAP kinase mutants mak-1 and mak-2 strains. In contrast, fludioxonil, an activator of OS-2 MAP kinase, strongly induced the gel-1 gene in the wild-type strain. Its induction was nearly abolished in the os-2 and in the atf-1/asl-1 mutant. These suggested that GEL-3 is a major factor in mycelial growth, while GEL-1 and GEL-4 may play important roles in cell wall remodeling in response to stress conditions or cell wall damage, respectively.