Journal of Microbiology

Volume 51(6); December 2013

Reviews

MINIREVIEW] Overview: Replication of Porcine Reproductive and Respiratory Syndrome Virus: Sang-Im Yun , Young-Min Lee; J. Microbiol. 2013;51(6):711-723. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3431-z

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Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNAprocessing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.

MINIREVIEW] Shiga Toxins Expressed by Human Pathogenic Bacteria Induce Immune Responses in Host Cells: Moo-Seung Lee , Myung Hee Kim , Vernon L. Tesh; J. Microbiol. 2013;51(6):724-730. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3429-6

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Abstract: Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxinmediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), macrophage inflammatory protein-1α/β (MIP-1α/β), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groβ. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Research Support, Non-U.S. Gov'ts

Niabella terrae sp. nov. Isolated from Greenhouse Soil: Jae-Hyung Ahn , Eun-Hye Jo , Byung-Yong Kim , Jaekyeong Song , Soon-Wo Kwon , Hang-Yeon Weon; J. Microbiol. 2013;51(6):731-735. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3507-9

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Abstract: An orange-colored bacterial strain, ICM 1-15T, was isolated from greenhouse soil. The 16S rRNA gene sequence of this strain showed the highest sequence similarity with Niabella ginsengisoli GR10-1T (95.2%) and Niabella yanshanensis CCBAU 05354T (95.0%) among the type strains. The strain ICM 1-15T was a strictly aerobic, Gram-negative, non-sporeforming, non-motile, flexirubin pigment-producing, short rod-shaped bacterium. The strain grew at 15–35°C (optimum, 25°C), at a pH of 5.0–8.5 (optimum, pH 6.5), and in the presence of 0–3% NaCl (optimum, 1%). The DNA G+C content of strain ICM 1-15T was 43.6 mol%. It contained MK-7 as the major isoprenoid quinone and iso-C15:0 (38.9%), iso-C15:1 G (20.3%), and iso-C17:0 3-OH (12.9%) as the major fatty acids. On the basis of evidence from our polyphasic taxonomic study, we concluded that strain ICM 1-15T should be classified within a novel species of the genus Niabella, for which the name Niabella terrae sp. nov. is proposed. The type strain is ICM 1-15T (=KACC 17443T =JCM 19502T).

Aestuariibaculum scopimerae sp. nov., Isolated from the Globular Ghost Crab, Scopimera globosa: Jae-Bong Lee , Byung-Chun Kim , Hyangmi Kim , Kyung Sook Bae , Jae-Hyeong Yang , Young-Yull Chun , Seong-Joon Park , Doo-Sang Park; J. Microbiol. 2013;51(6):736-740. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3499-5

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Abstract: A Gram-staining-negative, non-motile, catalase- and oxidasepositive bacterium, designated strain I-15T, was isolated from a crab of the Yellow Sea, Korea. On the basis of a 16S rRNA gene sequence analysis, strain I-15T was shown to belong to Bacteroidetes, related to the genus Aestuariibaculum. Sequence similarity between strain I-15T and the only type strain of the genus Aestuariibaculum, Aestuariibaculum suncheonense SC17T, was 96.7%. Strain I-15T grew at 0.5–6.0% (w/v) NaCl, at 10–42°C and at pH 4.5–8.0. It could hydrolyze starch and Tweens 80. Menaquinone-6 was the only respiratory quinone, and summed features 3 (C16:1 ω7c/C16:1 ω6c) (16.4%), iso-C15:0 (15.6%), and iso-C15:1 G (12.6%) were the major cellular fatty acids. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids. The DNA G+C content was 39.0 mol%. Polyphasic data allowed genotypic and phenotypic distinction of strain I-15T from the only validly published Aestuariibaculum species. Therefore, the organism is considered a novel species of the genus Aestuariibaculum, for which the name Aestuariibaculum scopimerae sp. nov. is proposed. The type strain is I-15T (=KCTC 32459T =JCM 19486T).

Sunxiuqinia dokdonensis sp. nov., Isolated from Deep Sub-Seafloor Sediment: Dong-Ho Chang , Jae-Bong Lee , Geun-Hye Lee , Moon-Soo Rhee , Haewon Lee , Kyung Sook Bae , Doo-Sang Park , Byoung-Chan Kim; J. Microbiol. 2013;51(6):741-746. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3492-z

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Abstract: A novel facultatively anaerobic strain DH1T was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island) in the East Sea of the Republic of Korea. The new strain was characterized using polyphasic approaches. The isolate was Gram-stain-negative, motile by gliding, non-spore-forming rods, oxidase-negative, and catalase-positive; and formed colonies of orange-red color. The NaCl range for growth was 0.5–7.0% (w/v) and no growth was observed in the absence of NaCl. The isolate grew optimally at 30°C, with 2% (w/v) NaCl and at pH 7. The cell-wall hydrolysates contained ribose as a major sugar. The DNA G+C content was 40.8 mol%. The closest related strains are Sunxiuqinia faeciviva JAM-BA0302T and Sunxiuqinia elliptica DQHS-4T (97.9 and 96.3% sequence similarity, respectively). The level of DNADNA relatedness between strain DH1T and S. faeciviva JAMBA0302T was around 41% (but only 6% between DH1T and S. elliptica DQHS-4T). The major cellular fatty acids of the isolate were contained iso-C15:0 (25.9%), anteiso-C15:0 (16.7%), and summed feature 9 (comprising C16:0 3-OH and/or unknown fatty acid of dimethylacetal ECL 17.157; 13.2%). The predominant menaquinone was MK-7. On the basis of polyphasic evidence from this study, the isolate was considered to represent a novel species of the genus Sunxiuqinia, for which the name Sunxiuqinia dokdonensis sp. nov. is proposed; the type strain is DH1T (=KCTC 32503T =CGMCC 1.12676T =JCM 19380T).

Diversity of the Bacterial Community in the Rice Rhizosphere Managed Under Conventional and No-tillage Practices: Zubair Aslam , Muhammad Yasir , Hwan Sik Yoon , Che Ok Jeon , Young Ryun Chung; J. Microbiol. 2013;51(6):747-756. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-2528-8

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Abstract: Bacterial diversity in the rice rhizosphere at different rice growth stages, managed under conventional and no-tillage practices, was explored using a culture-based approach. Actinobacteria are among the bacterial phyla abundant in the rice rhizosphere. Their diversity was further examined by constructing metagenomic libraries based on the 16S rRNA gene, using actinobacterial- and streptomycete-specific polymerase chain reaction (PCR) primers. The study included 132 culturable strains and 125 clones from the 16S rRNA gene libraries. In conventional tillage, there were 38% Proteobacteria, 22% Actinobacteria, 33% Firmicutes, 5% Bacteroidetes, and 2% Acidobacteria, whereas with no-tillage management there were 63% Proteobacteria, 24% Actinobacteria, 6% Firmicutes, and 8% Bacteroidetes as estimated using the culturedependent
method
during the four stages of rice cultivation. Principal coordinates analysis was used to cluster the bacterial communities along axes of maximal variance. The different growth stages of rice appeared to influence the rhizosphere bacterial profile for both cultivation practices. Novel clones with low similarities (89–97%) to Actinobacteria and Streptomyces were retrieved from both rice fields by screening the 16S rRNA gene libraries using actinobacterial- and streptomycete-specific primers. By comparing the actinobacterial community retrieved by culture-dependent and molecular methods, it was clear that a more comprehensive assessment of microbial diversity in the rice rhizosphere can be obtained using a combination of both techniques than by using either method alone. We also succeeded in culturing a number of bacteria that were previously described as unculturable. These were in a phylogenetically deep lineage when compared with related cultivable genera.

Diversity of Cyanobacterial Species and Phylotypes in Biofilms from the Littoral Zone of Lake Baikal: Ekaterina G. Sorokovikova , Olga I. Belykh , Anna S. Gladkikh , Oleg V. Kotsar , Irina V. Tikhonova , Oleg A. Timoshkin , Valentina V. Parfenova; J. Microbiol. 2013;51(6):757-765. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3240-4

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Abstract: The majority of naturally occurring biofilms contain numerous microorganisms that have not yet been cultured. Additionally, there is little information available regarding the genetic structure and species diversity of these communities. Therefore, we characterised the species diversity, structure and metagenome of biofilms grown on stones and steel plates in the littoral zone of Lake Baikal (East Siberia, Russia) by applying three different approaches. First, light microscopy enabled identification of the species diversity of biofilm-forming cyanobacteria on different substrates with the dominance of Rivularia rufescens, Tolypothrix limbata, Chamaesiphon fuscus, Сh. subglobosus, and Heteroleibleinia pusilla. Additionally, scanning electron microscopy was used to show the spatial structure of biofilms. Finally, sequence analysis of 30,660 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to Cyanobacteria (8–46% sequences), Proteobacteria (14–43%), and Bacteroidetes (10– 41%). Rivularia sp., Pseudanabaena sp., and Chamaesiphon spp. were the dominant cyanobacterial phylotypes.

The Proportion of Non-Aflatoxigenic Strains of the Aspergillus flavus/oryzae Complex from Meju by Analyses of the Aflatoxin Biosynthetic Genes: Seung-Beom Hong , Mina Lee , Dae-Ho Kim , Soo-Hyun Chung , Hyeon-Dong Shin , Robert A. Samson; J. Microbiol. 2013;51(6):766-772. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3128-3

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Abstract: Strains of the Aspergillus flavus/oryzae complex are frequently isolated from meju, a fermented soybean product, that is used as the starting material for ganjang (soy sauce) and doenjang (soybean paste) production. In this study, we examined the aflatoxin producing capacity of A. flavus/oryzae strains isolated from meju. 192 strains of A. flavus/oryzae were isolated from more than 100 meju samples collected from diverse regions of Korea from 2008 to 2011, and the norB-cypA, omtA, and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 178 strains (92.7%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and 14 strains (7.3%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). Only 7 strains (3.6%) in the aflatoxin-producible group produced aflatoxins on Czapek yeast-extract medium. The aflatoxin-producing capability of A. flavus/ oryzae strains from other sources in Korea were also investigated, and 92.9% (52/56) strains from air, 93.9% (31/33) strains from rice straw, 91.7% (11/12) strains from soybean, 81.3% (13/16) strains from corn, 82% (41/50) strains from peanut, and 73.2% (41/56) strains from arable soil were included in the non-aflatoxigenic group. The proportion of non-aflatoxigenicity of meju strains was similar to that of strains from soybean, air and rice straw, all of which have an effect on the fermentation of meju. The data suggest that meju does not have a preference for non-aflatoxigenic or aflatoxin-producible strains of A. flavus/oryzae from the environment of meju. The non-aflatoxigenic meju strains are proposed to be named A. oryzae, while the meju strains that can produce aflatoxins should be referred to A. flavus in this study.

Enhanced Production of Biomass and Lipids by Supplying CO2 in Marine Microalga Dunaliella sp.: Hancheol Jeon , Yew Lee , Kwang Suk Chang , Choul-Gyun Lee , EonSeon Jin; J. Microbiol. 2013;51(6):773-776. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3256-9

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Abstract: Non-food-based biofuel feedstocks are in high demand worldwide. Among the various feedstocks, microalgae are the most promising feedstock for mitigating atmospheric CO2 and producing biodiesel. In this study, various concentrations of CO2, from 0.03 to 12%, were used to investigate their effect on the cell growth, biomass and lipid production and fatty acid composition of Dunaliella sp. in a closed photobioreactor. The results showed that the highest biomass and total lipids, 521 mg/L/d and 40 mg/L/d, respectively, were produced with 5% CO2 aeration during the logarithmic growth phase. The oleic acid (18:1n9c) and elaidic acid (18:1n9t) contents were increased approximately two fold. The physiological responses of Dunaliella sp. at 10% CO2 were similar to those at 5% CO2. Therefore, the present results suggest that 5–10% is a suitable CO2 concentration range for Dunaliella sp. growth to mitigate atmospheric CO2 and increase biofuel production.

Lactobacillus paracasei subsp. paracasei LC01 Positively Modulates Intestinal Microflora in Healthy Young Adults: Hao Zhang , Jing Sun , Xianting Liu , Chuan Hong , Yuanbo Zhu , Aiping Liu , Siqi Li , Huiyuan Guo , Fazheng Ren; J. Microbiol. 2013;51(6):777-782. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3279-2

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Abstract: Lactobacillus paracasei subsp. paracasei LC01 (LC01) can tolerate intestinal stresses and has antioxidant activity. To evaluate the effect of the bacterium on human intestinal microflora, a randomized, double-blind, placebo-controlled human trial was carried out. Fifty-two healthy adult volunteers were randomized equally to two groups. One group consumed 12% (wt/vol) skimmed milk supplemented with 1010 CFU of LC01 each day for the 4-week treatment period, and then consumed placebo in the next treatment period, separated by a 2-week washout. The other group followed the reverse order. Group-specific real-time PCR and biochemical analyses was used to determine the intestinal bacterial composition of fecal samples collected at the end of every period, and the concentration of short-chain fatty acids and ammonia. A significant inhibition in fecal Escherichia coli and increase in Lactobacillus, Bifidobacterium, and Roseburia intestinalis were observed after consumption of LC01. Acetic acid and butyric acid were significantly higher in the probiotic stage and fecal ammonia was significantly lower. The results indicated a modulation effect of LC01 on the intestinal microflora of young adults, suggesting a beneficial effect on bowel health. LC01 may have potential value as a probiotic.

Influence of Acetobacter pasteurianus SKU1108 aspS Gene Expression on Escherichia coli Morphology: Kannipa Tasanapak , Uraiwan Masud-Tippayasak , Kazunobu Matsushita , Wichien Yongmanitchai , Gunjana Theeragool; J. Microbiol. 2013;51(6):783-790. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-2619-6

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Abstract: The aspS gene encoding Aspartyl-tRNA synthetase (AspRS) from a thermotolerant acetic acid bacterium, Acetobacter pasteurianus SKU1108, has been cloned and characterized. The open reading frame (ORF) of the aspS gene consists of 1,788 bp, encoding 595 amino acid residues. The highly conserved Gly-Val-Asp-Arg ATP binding motif (motif 3) is located at the position 537-540 in the C-terminus. Deletion analysis of the aspS gene upstream region suggested that the promoter is around 173 bp upstream from the ATG initiation codon. Interestingly, transformation with the plasmids pGEM-T138, pUC138, and pCM138 synthesizing 138 amino acid C-terminal fragments of AspRS, that carry the ATP binding domain, caused E. coli cell lengthening at 37 and 42°C. Moreover, E. coli harboring pUC595 (synthesizing all 595 amino acids) and a disordered aspS gene in pGEM-T138 had normal rod shapes. The normal rod shape was observed in E. coli harboring pD539V following site-directed mutagenesis of the ATP binding domain. We propose that overproduction of truncated C-terminal peptides of AspRS may cause sequestration of intracellular ATP in E. coli, leaving less ATP for cell division or shaping cell morphology.

Trans-Membrane Transport of n-Octadecane by Pseudomonas sp. DG17: Fei Hua , Hong Qi Wang , Yi Li , Yi Cun Zhao; J. Microbiol. 2013;51(6):791-799. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3259-6

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Abstract: The trans-membrane transport of hydrocarbons is an important and complex aspect of the process of biodegradation of hydrocarbons by microorganisms. The mechanism of transport of 14C n-octadecane by Pseudomonas sp. DG17, an alkane-degrading bacterium, was studied by the addition of ATP inhibitors and different substrate concentrations. When the concentration of n-octadecane was higher than 4.54 μmol/L, the transport of 14C n-octadecane was driven by a facilitated passive mechanism following the intra/extra substrate concentration gradient. However, when the cells were grown with a low concentration of the substrate, the cellular accumulation of n-octadecane, an energy-dependent process, was dramatically decreased by the presence of ATP inhibitors, and n-octadecane accumulation continually increased against its concentration gradient. Furthermore, the presence of non-labeled alkanes blocked 14C n-octadecane transport only in the induced cells, and the trans-membrane transport of n-octadecane was specific with an apparent dissociation constant Kt of 11.27 μmol/L and Vmax of 0.96 μmol/min/mg protein. The results indicated that the transmembrane transport of n-octadecane by Pseudomonas sp. DG17 was related to the substrate concentration and ATP.

A Putative APSES Transcription Factor Is Necessary for Normal Growth and Development of Aspergillus nidulans: Ji-Yeon Lee , Lee-Han Kim , Ha-Eun Kim , Jae-Sin Park , Kap-Hoon Han , Dong-Min Han; J. Microbiol. 2013;51(6):800-806. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3100-2

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Abstract: The nsdD gene encoding a GATA type transcription factor positively controls sexual development in Aspergillus nidulans. According to microarray data, 20 genes that were upregulated by deleting nsdD during various life cycle stages were randomly selected and deleted for functional analysis. None of the mutants showed apparent changes in growth or development compared with those of the wild-type except the AN3154 gene that encodes a putative APSES transcription factor and is an ortholog of Saccharomyces cerevisiae swi4. Deleting AN3154 resulted in retarded growth and development, and the gene was named rgdA (retared growth and development). The rgdA deletion mutant developed a reduced number of conidia even under favorable conditions for asexual development. The retarded growth and development was partially suppressed by the veA1 mutation. The conidial heads of the mutant aborted, showing reduced and irregular shaped phialides. Fruiting body development was delayed compared with that in the wild-type. The mutant did not respond to various nutritional or environmental factors that affected the development patterns. The rgdA gene was expressed at low levels throughout the life cycle and was not significantly affected by several regulators of sexual and asexual development such as nsdD, veA, stuA, or brlA. However, the rgdA gene affected brlA and abaA expression, which function as key regulators of asexual sporulation, suggesting that rgdA functions upstream of those genes.

Isolation and Functional Characterization of a Delta 6-Desaturase Gene from the Pike Eel (Muraenesox cinereus): Sun Hee Kim , Kyung Hee Roh , Jung-Bong Kim , Kwang-Soo Kim , Nam Shin Kim , Hyun Uk Kim , Kyeong-Ryeol Lee , Jong-Sug Park , Jong-Bum Kim; J. Microbiol. 2013;51(6):807-813. Published online October 5, 2013; DOI: https://doi.org/10.1007/s12275-013-3144-3

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Abstract: Stearidonic acid (STA; 18:4n-3) and γ-linolenic acid (GLA; 18:3n-6) are significant intermediates in the biosynthetic pathway for the very-long-chain polyunsaturated fatty acids of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6), respectively. To develop a sustainable system for the production of dietary polyunsaturated fatty acids, we focused on the action of the enzyme delta 6-desaturase (D6DES) on the essential acids, linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3). A 1,335-bp full-length cDNA encoding D6DES (McD6DES) was cloned from Muraenesox cinereus using degenerate PCR and RACE-PCR
methods
. To investigate the enzymatic activity of McD6DES in the production of n-6 and n-3 fatty acids, a recombinant plasmid expressing McD6DES (pYES-McD6DES) was transformed into and expressed in Saccharomyces cerevisiae. The exogenously expressed McD6DES produced GLA and STA at conversion rates of 14.2% and 45.9%, respectively, from the exogenous LA and ALA substrates. These results indicate that McD6DES is essentially a delta 6-desaturase involved in very-long-chain polyunsaturated fatty acid synthesis.

Research Support, U.S. Gov't, Non-P.H.S.

Structure and Function of the Mating-type Locus in the Homothallic Ascomycete, Didymella zeae-maydis: Sung-Hwan Yun , Olen C. Yoder , B. Gillian Turgeon; J. Microbiol. 2013;51(6):814-820. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3465-2

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Abstract: Homothallic Didymella zeae-maydis undergoes sexual reproduction by selfing. Sequence analysis of the mating type (MAT) locus from this fungus revealed that MAT carries both MAT1-1-1 and MAT1-2-1 genes found in heterothallic Dothideomycetes, separated by ~1.0 kb of noncoding DNA. To understand the mechanistic basis of homothallism in D. zeae-maydis, each of the MAT genes was deleted and the effects on selfing and on ability to cross in a heterothallic manner were determined. The strain carrying an intact MAT1-1-1 but defective MAT1-2-1 gene (MAT1-1-1;ΔMAT1-2-1) was self-sterile, however strains carrying an intact MAT1-2-1 but defective MAT1-1-1 gene (ΔMAT1-1-1;MAT1-2-1), when selfed, showed delayed production of a few ascospores. Attempts to cross the two MAT deletion strains yielded fewer ΔMAT1-1-1;MAT1-2-1 than MAT1-1-1;ΔMAT1-2-1 progeny and very few ascospores overall compared to WT selfs. This study demonstrates that, as in the other homothallic Dothideomycetes, both MAT genes are required for full fertility, but that, in contrast to other cases, the presence of a single MAT1-2-1 gene can induce homothallism, albeit inefficiently, in D. zeae-maydis.

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