Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus <Emphasis Type="Italic">Sordaria macrospora</Emphasis>

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.     

A <Emphasis Type="Italic">Sordaria macrospora</Emphasis> mutant lacking the <Emphasis Type="Italic">leu1</Emphasis> gene shows a developmental arrest during fruiting body formation

Developmental mutants with defects in fruiting body formation are excellent resources for the identification of genetic components that control cellular differentiation processes in filamentous fungi. The mutant pro4 of the ascomycete Sordaria macrospora is characterized by a developmental arrest during the sexual life cycle. This mutant generates only pre-fruiting bodies (protoperithecia), and is unable to form ascospores. Besides being sterile, pro4 is auxotrophic for leucine. Ascospore analysis revealed that the two phenotypes are genetically linked. After isolation of the wild-type leu1 gene from S. macrospora, complementation experiments demonstrated that the gene was able to restore both prototrophy and fertility in pro4. To investigate the control of leu1 expression, other genes involved in leucine biosynthesis specifically and in the general control of amino acid biosynthesis (“cross-pathway control”) have been analysed using Northern hybridization and quantitative RT-PCR. These analyses demonstrated that genes of leucine biosynthesis are transcribed at higher levels under conditions of amino acid starvation. In addition, the expression data for the cpc1 and cpc2 genes indicate that cross-pathway control is superimposed on leucine-specific regulation of fruiting body development in the leu1 mutant. This was further substantiated by growth experiments in which the wild-type strain was found to show a sterile phenotype when grown on a medium containing the amino acid analogue 5-methyl-tryptophan. Taken together, these data show that pro4 represents a novel mutant type in S. macrospora, in which amino acid starvation acts as a signal that interrupts the development of the fruiting body. Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00438-005-0021-8     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Genetic analysis ofCochliobolus heterostrophus polyoxin-resistant mutants

Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.     

Genetic evaluation of peroxisomal and cytosolic acetoacetyl-CoA thiolase isozymes in n-alkane-assimilating diploid yeast, Candida tropicalis

The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic. Thiolase Is, the site-directed mutation leading Thiolase I ΔC6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1bΔ-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1aΔ/t1bΔ-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrated that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.     

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD⁺-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Δ and gpd2Δ were studied under alcoholic fermentation conditions. gpd1Δ mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Δ mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Δ) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Δ) but a slight decrease in the strain ANG2 (gpd2Δ). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Δ) and ANG2 (gpd2Δ) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction

Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-component system-defective mutants, ΔgacA and ΔgacS, and a double mutant, ΔgacAΔgacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/GenBank/EMBL databank with the accession numbers AB266103, AB266104, AB266105, AB266106, AB266107, AB266108.     

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and β-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

丝状真菌Podospora anserina中光敏色素基因的鉴定及功能分析

光敏色素在细菌和植物发育中起着关键作用,但它们在真菌中的生物学功能尚不完全清楚。【目的】探究光敏色素基因PaPhy1和PaPhy2在Podospora anserina有性生殖和无性发育中的作用及其调控机制。【方法】利用同源重组方法对P.anserina中2个光敏色素基因PaPhy1和PaPhy2进行定点敲除,获得光敏色素基因缺失菌株ΔPaPhy1和ΔPaPhy2,并通过遗传杂交构建双重突变体ΔPaPhy1ΔPaPhy2;分析突变型菌株和野生型菌株在不同光照下有性生殖、无性发育、生长速率和活性氧代谢等方面的差异,明确光敏色素基因在P.anserina中的主要功能。【结果】白光和蓝光诱导P.anserina子实体的形成,ΔPaPhy在光照下产生子实体的数量减少,ΔPaPhy的生命周期延长。【结论】光敏色素基因与P.anserina有性生殖密切相关;ΔPaPhy的衰老延迟和活性氧代谢有关。本研究的结果为进一步探索光照对丝状真菌繁殖调控机制以及抗衰老研究提供了新的思路。     

Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the β-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus–E. coli plasmid shuttle vectors were constructed, which genetically complemented ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.     

转录组分析罗伯茨绿僵菌精胺合成酶MrSPS介导真菌血腔定殖功能

【背景】精胺在植物应对逆境胁迫、动物抵抗疲劳和衰老、真菌生长代谢等过程中发挥重要作用,但目前在昆虫病原真菌中的研究未见报道。【目的】在分子水平上探究罗伯茨绿僵菌精胺合成关键酶——精胺合成酶在昆虫血腔定殖中的作用机制。【方法】显微注射法测定Mrsps敲除株ΔMrsps的致病力变化,并观察血腔中ΔMrsps生长状态;收集ΔMrsps和野生型WT注射侵染30 h后的大蜡螟血淋巴进行转录组测序,分别与罗伯茨绿僵菌和大蜡螟参考基因组进行比对分析,并结合定量PCR进行验证。【结果】与WT和回补株ΔMrsps-cp相比较,ΔMrsps致病力显著下降,而且随着注射浓度的降低,ΔMrsps致病力下降越显著。侵染36 h后WT和ΔMrsps孢子都能正常萌发且开始以类酵母状态生长,60 h后,相较于WT,ΔMrsps的生长繁殖数量较少。转录组共检测到3 202个罗伯茨绿僵菌基因,其中1 769个基因在ΔMrsps中表达上调,922个基因表达下调;差异表达基因涉及碳水化合物代谢、运输、分解代谢、翻译和氨基酸代谢等多条途径;筛选出28个血腔致病相关基因全部在ΔMrsps中表达下调;定量PCR检测发现在整个血腔定殖阶段免疫逃避蛋白Mcl1基因和血腔定殖Colonization of hemocoel 1基因在WT和ΔMrsps-cp中的表达量高于ΔMrsps。共检测到13 249个大蜡螟基因,其中4 026个差异表达基因;KEGG注释分析显示大量差异表达基因富集到内分泌系统和免疫系统等途径;深入分析发现22个差异表达基因归属于Toll和Imd信号通路,其中18个基因在ΔMrsps侵染的大蜡螟中表达上调,表明ΔMrsps侵染大蜡螟过程中更易引起免疫系统的激活。【结论】揭示了Mrsps在罗伯茨绿僵菌血腔定殖阶段作用的分子机制,为进一步揭示精胺在真菌中的作用机理提供了理论基础。     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

Deletion analysis of the C-terminal region of a molecular chaperone DnaK from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain. Wan-Chi Liang and Min-Guan Lin contributed equally to this work.     

Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.     

Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris

Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA.