Identification of Capsular Polysaccharide Synthesis Loci Determining Bacteriophage Susceptibility in Tetragenococcus halophilus.

Bacteriophages infecting Tetragenococcus halophilus, a halophilic lactic acid bacterium, have been a major industrial concern due to their detrimental effects on the quality of food products. Previously characterized tetragenococcal phages displayed narrow host ranges, but there is little information on these mechanisms. Here, we revealed the host's determinant factors for phage susceptibility using two virulent phages, phiYA5_2 and phiYG2_4, that infect T. halophilus YA5 and YG2, respectively. Phage-resistant derivatives were obtained from these host strains, and mutations were found at the capsular polysaccharide (CPS) synthesis (cps) loci. Quantification analysis verified that capsular polysaccharide production by the cps derivatives from YG2 was impaired. Transmission electron microscopy observation confirmed the presence of filamentous structures outside the cell walls of YG2 and their absence in the cps derivatives of YG2. Phage adsorption assays revealed that phiYG2_4 adsorbed to YG2 but not its cps derivatives, which suggests that the capsular polysaccharide of YG2 is the specific receptor for phiYG2_4. Interestingly, phiYA5_2 adsorbed and infected cps derivatives of YG2, although neither adsorption to nor infection of the parental strain YG2 by phiYA5_2 was observed. The plaque-surrounding halos formed by phiYA5_2 implied the presence of the virion-associated depolymerase that degrades the capsular polysaccharide of YA5. These results indicated that the capsular polysaccharide is a physical barrier rather than a binding receptor for phiYA5_2 and that phiYA5_2 specifically overcomes the capsular polysaccharide of YA5. Thus, it is suggested that tetragenococcal phages utilize CPSs as binding receptors and/or degrade CPSs to approach host cells. IMPORTANCE T. halophilus is a halophilic lactic acid bacterium that contributes to the fermentation processes for various salted foods. Bacteriophage infections of T. halophilus have been a major industrial problem causing fermentation failures. Here, we identified the cps loci in T. halophilus as genetic determinants of phage susceptibility. The structural diversity of the capsular polysaccharide is responsible for the narrow host ranges of tetragenococcal phages. The information provided here could facilitate future studies on tetragenococcal phages and the development of efficient methods to prevent bacteriophage infections.

Adjacent double branches exist in the branching structure of starch.

Starch is composed of two giant polymers of glucose-linked amylose and amylopectin but is not considered to have a branched structure with adjacent α-1,6-linkages (adjacent double branches). We previously discovered that the oligosaccharide DP6-1 contains adjacent double branches in sake, a Japanese rice wine. In this study, we analyzed the oligosaccharides generated from rice starch following porcine pancreatic α-amylase and glucoamylase digestion by hydrophilic interaction chromatography coupled with time-of-flight/mass spectrometry. We found that their elution times were identical to those of the oligosaccharides containing adjacent double branches observed in sake. Nuclear magnetic resonance (NMR) analysis confirmed that one of the oligosaccharides was identical to DP6-1. This indicates for the first time that adjacent double branches exist in the branching structure of starch. The adjacent double branches were estimated to be present at a ratio of approximately 1:100 of the total branching points in rice starch, suggesting that the contribution of the adjacent double branch to the entire structure of starch may be limited. Nonetheless, we found adjacent double branches in starch from rice, maize, wheat, and potato, which indicates that this novel branching pattern is universally present in starch.

Ribitol-Containing Wall Teichoic Acid of Tetragenococcus halophilus Is Targeted by Bacteriophage phiWJ7 as a Binding Receptor.

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is used in the fermentation process of soy sauce manufacturing. For many years, bacteriophage infections of T. halophilus have been a major industrial problem that causes fermentation failure. However, studies focusing on the mechanisms of tetragenococcal host-phage interactions are not sufficient. In this study, we generated two phage-insensitive derivatives from the parental strain T. halophilus WJ7, which is susceptible to the virulent phage phiWJ7. Whole-genome sequencing of the derivatives revealed that insertion sequences were transposed into a gene encoding poly(ribitol phosphate) polymerase (TarL) in both derivatives. TarL is responsible for the biosynthesis of ribitol-containing wall teichoic acid, and WJ7 was confirmed to contain ribitol in extracted wall teichoic acid, but the derivative was not. Cell walls of WJ7 irreversibly adsorbed phiWJ7, but those of the phage-insensitive derivatives did not. Additionally, 25 phiWJ7-insensitive derivatives were obtained, and they showed mutations not only in tarL but also in tarI and tarJ, which are responsible for the synthesis of CDP-ribitol. These results indicate that phiWJ7 targets the ribitol-containing wall teichoic acid of host cells as a binding receptor. IMPORTANCE Information about the mechanisms of host-phage interactions is required for the development of efficient strategies against bacteriophage infections. Here, we identified the ribitol-containing wall teichoic acid as a host receptor indispensable for bacteriophage infection. The complete genome sequence of tetragenococcal phage phiWJ7 belonging to the family Rountreeviridae is also provided here. This study could become the foundation for a better understanding of host-phage interactions of tetragenococci.

New role of a histone chaperone, HirA: Involvement in kojic acid production associated with culture conditions in Aspergillusoryzae.

Kojic acid (KA) is a representative secondary metabolite of Aspergillus oryzae, but the underlying molecular mechanisms that regulate KA production are unknown. This study tried to find a genetic factor of KA production in A. oryzae, with a special focus on liquid cultures. We screened a gene predicted to encode HirA, a subunit of the histone chaperon, the HIR complex. A gene disruption strain of hirA showed decreased KA production in liquid culture, whereas it showed increased KA production in plate culture. We confirmed that a decrease/increase of KA production observed by hirA disruption was caused by altered expression of kojA and kojR. These observations suggested the regulatory role of histone chaperon in secondary metabolism in filamentous fungi. So far as we know, this report is the first showing that disruption of a gene resulted in the opposite effect on KA production in liquid and plate cultures in A. oryzae.

Comparative Genomics of Closely Related Tetragenococcus halophilus Strains Elucidate the Diversity and Microevolution of CRISPR Elements.

Tetragenococcus halophilus - a halophilic lactic acid bacterium - is frequently used as a starter culture for manufacturing fermented foods. Tetragenococcus is sometimes infected with bacteriophages during fermentation for soy sauce production; however, bacteriophage infection in starter bacteria is one of the major causes of fermentation failure. Here, we obtained whole-genome sequences of the four T. halophilus strains YA5, YA163, YG2, and WJ7 and compared them with 18 previously reported genomes. We elucidated five types of clustered regularly interspaced short palindromic repeat (CRISPR) loci in seven genomes using comparative genomics with a particular focus on CRISPR elements. CRISPR1 was conserved in the four closely related strains 11, YA5, YA163, and YG2, and the spacer sequences were partially retained in each strain, suggesting that partial deletions and accumulation of spacer sequences had occurred independently after divergence of each strain. The host range for typical bacteriophages is narrow and strain-specific thus these accumulation/deletion events may be responsible for differences in resistance to bacteriophages between bacterial strains. Three CRISPR elements, CRISPR1 in strains 11, YA5, YA163, and YG2, CRISPR2 in strain WJ7, and CRISPR2 in strain MJ4, were inserted in almost the same genomic regions, indicating that several independent insertions had occurred in this region. As these elements belong to class 1 type I-C CRISPR group, the results suggested that this site is a hotspot for class 1, type I-C CRISPR loci insertion. Thus, T. halophilus genomes may have acquired strain-specific bacteriophage-resistance through repeated insertion of CRISPR loci and accumulation/deletion events of their spacer sequences.

Novel glucoamylase-resistant gluco-oligosaccharides with adjacent α-1, 6 branches at the non-reducing end discovered in Japanese rice wine, sake.

Sake, a traditional Japanese rice wine, contains various oligosaccharides (Sake oligosaccharides; SAOs) derived from rice starch. We previously found that SAOs reach a high degree of polymerization (DP). In this study, we developed a hydrophilic interaction liquid chromatography-time-of-flight/mass spectrometry (HILIC-TOF/MS) based analytical method to separate isomeric SAOs. Isomers of SAOs with DP = 6, 7, and 8, which were named DP6-1, DP7-1, DP8-1 and DP8-2, respectively, were purified from sake and their structures were determined by two-dimensional NMR spectroscopy. These were novel oligosaccharides containing two α-1, 6 bonded branches on an α-1, 4-linked glucose main chain. Interestingly, adjacent double α-1, 6 branches that have not been identified in starch, were found in DP6-1, DP7-1, and DP8-1, suggesting the presence of the branching pattern in starch. DP6-1 was poorly digested by fungal glucoamylase, and this may be attributed to its adjacent double branches at the non-reducing end.

Transglycosylation Forms Novel Glycoside Ethyl α-Maltoside and Ethyl α-Isomaltoside in Sake during the Brewing Process by α-Glucosidase A of Aspergillus oryzae.

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.

Investigation of relationship between sake-making parameters and sake metabolites using a newly developed sake metabolome analysis method.

We developed a sake metabolome analysis method using liquid chromatography-quadrupole/time-of-flight mass spectrometry to investigate the metabolome of various types of sakes and other alcohol beverages. Our method identified 198 compounds by comparison with standard metabolites. Using this method, we investigated the relationship between several sake-making parameters and sake metabolites by conducting combination experiments of these parameters using small-scale fermentation. The results indicated that all parameters significantly affected sake metabolites (P < 0.005) and most peaks were affected by multiple sake-making parameters. Interestingly, the effect of the rice cultivar on sake metabolites was higher for koji rice than for kake-rice. This result suggests that the rice cultivar used has a greater effect on the characteristics of Aspergillus oryzae compared to sake yeast and affects sake metabolites. In this study, we also evaluated the combined effect of several parameters. We demonstrated the different effects of each parameter on several amino acids. The results showed a new aspect of the science of sake making. For example, the amount of α-ethylglucoside, which can affect the taste of sake, was negatively correlated with α-glucosidase activity in koji (r = -0.84). In this study, various unidentified peaks were observed; detectable peaks can be increased by analyzing additional standard reagents. Investigating these unidentified peaks and accumulating datasets for sake-making parameters will give us insight into how to improve sake taste and quality.

A unique Zn(II)2-Cys6-type protein, KpeA, is involved in secondary metabolism and conidiation in Aspergillus oryzae.

Aspergillus oryzae is an important microorganism in the bio- and food industries; therefore, understanding the mechanism underlying its secondary metabolism regulation is important for ensuring its safe use. Here, we screened a novel Zn(II)2-Cys6-type protein-encoding gene, AO090003001186, designated as kpeA (kojic acid production enhancement A), from an A. oryzae disruption mutant library of transcriptional regulators. kpeA is highly conserved among filamentous fungi and encodes a protein with Zn(II)2-Cys6 motif located in the middle of the sequence. Phylogenetic analysis revealed that KpeA was classified into a distal group compared to other fungal Zn(II)2-Cys6-type transcriptional regulators. A Cys to Ala substitution mutant of KpeA showed identical phenotype to the kpeA disruption strain, confirming that KpeA is novel type Zn(II)2-Cys6 binding protein. Colonies of the kpeA disruption strain (ΔkpeA) had longer aerial hyphae and showed decreased conidia production. Microscopic analysis suggested that the reduced vesicle size and conidial head formation in ΔkpeA strain account for the decreased conidia production. Transcriptional levels of brlA and downstream abaA and wetA were decreased in ΔkpeA strain. Moreover, ΔkpeA strain produced 6-fold more kojic acid than the control strains, and the expression of kojR and kojA was increased in ΔkpeA strain. Therefore, KpeA is a novel Zn(II)2-Cys6-type protein likely involved in conidiation and kojic acid production at the transcriptional level.

Analysis of the oligosaccharides in Japanese rice wine, sake, by hydrophilic interaction liquid chromatography-time-of-flight/mass spectrometry.

A traditional Japanese alcoholic beverage, sake, contains several oligosaccharides, which are associated with the taste of sake; however, little is known about the specific molecular species and concentrations of oligosaccharides in sake. Here, we developed an analytical method using hydrophilic interaction liquid chromatography-time-of-flight/mass spectrometry (HILIC-TOF/MS) which successfully detects the oligosaccharides in sake. A series of oligosaccharides with successive degree of polymerization (DP) values up to 18 were identified in sake for the first time, which we have named sake oligosaccharides (SAOs). The concentrations of the SAOs with DP = 3-8 were estimated to be in the range of 200-2000 ppm. Quantitative analysis of 6 different sake samples for SAOs with DP=2-8 and the other saccharides showed that the amount of each SAO differs significantly among the sake samples. Enzymatic digestion analysis suggested that the SAOs are probably branched maltooligosaccharides in structure, which are resistant to ß-amylase.

Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis.

Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.

Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi.

Filamentous fungi, particularly Aspergillus species, have recently attracted attention as host organisms for recombinant protein production. Because the secretory yields of heterologous proteins are generally low compared with those of homologous proteins or proteins from closely related fungal species, several strategies to produce substantial amounts of recombinant proteins have been conducted. Codon optimization is a powerful tool for improving the production levels of heterologous proteins. Although codon optimization is generally believed to improve the translation efficiency of heterologous genes without affecting their mRNA levels, several studies have indicated that codon optimization causes an increase in the steady-state mRNA levels of heterologous genes in filamentous fungi. However, the mechanism that determines the low mRNA levels when native heterologous genes are expressed was poorly understood. We recently showed that the transcripts of heterologous genes are polyadenylated prematurely within the coding region and that the heterologous gene transcripts can be stabilized significantly by codon optimization, which is probably attributable to the prevention of premature polyadenylation in Aspergillus oryzae. In this review, we describe the detailed mechanism of premature polyadenylation and the rapid degradation of mRNA transcripts derived from heterologous genes in filamentous fungi.

The transfer of stable ¹³³Cs from rice to Japanese sake.

Possible contamination by radioactive cesium (Cs), released by the Fukushima Daiichi Nuclear Plant Accident in Japan on March 2011, has been a matter of concern with respect to Japanese sake made from rice grains cultivated in affected fields. In this study, the behavior of stable (133)Cs, which is a useful analogue for predicting the behavior of radioactive Cs, was investigated in the production of sake using rice grains harvested in Japan in 2010. The concentration of stable (133)Cs in the polished rice grains decreased gradually with decreasing milling ratios until a ratio of 70% was reached, and below that point, it did not change significantly. The (133)Cs concentration in the 70% polished rice was approximately 20% of that found in brown rice. The sake was brewed on a small scale using 70% polished rice, and the transfer of (133)Cs from rice to sake was examined. Approximately 30-40% of (133)Cs in the 70% polished rice was removed during the washing and the steeping of the rice grains, and approximately 40% of the (133)Cs in the 70% polished rice was transferred to the sake. If the radioactive Cs species behaves similarly, these results suggest that brown rice containing 100 Bq/kg radioactivity of Cs would generate 70% polished rice grains containing 20 Bq/kg and that the sake brewed from these grains would contain 3-5 Bq/kg.

Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry.

Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 µM in sake. The analysis was validated by dipeptide recovery of 64.0-107.2% (2.5 µM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

Transcripts of a heterologous gene encoding mite allergen Der f 7 are stabilized by codon optimization in Aspergillus oryzae.

We have previously demonstrated that transcripts of an AT-biased heterologous gene encoding mite allergen Der f 7 from Dermatophagoides farinae were polyadenylated prematurely within the coding region when native cDNA was expressed in Aspergillus oryzae, and that this premature polyadenylation was prevented by the codon optimization of the Der f 7 gene, resulting in increased steady-state mRNA levels. In this study, we tested the stability of transcription products derived from expression constructs of the native and codon-optimized Der f 7 gene in A. oryzae using 1,10-phenanthroline as a transcription inhibitor. Transcription products of native Der f 7 cDNA fused to the A. oryzae glucoamylase gene (glaA) were rapidly degraded; the half-life of the mRNA was approximately 13 min. However, the half-life of codon-optimized Der f 7 mRNA fused to glaA was approximately 43 min, which was highly similar to that of endogenous glaA mRNA. These results indicate that Der f 7 mRNA is significantly stabilized by codon optimization. In addition, Der f 7 mRNA was stabilized by the codon optimization of only the 3'-half region, where premature polyadenylation sites were exclusively situated; the half-life of the chimeric Der f 7 mRNA was approximately 39 min. This suggested that destabilization of native Der f 7 mRNA is mainly triggered by premature polyadenylation within the coding region. To the best of our knowledge, this is the first report to provide experimental evidence that heterologous mRNA is significantly stabilized by codon optimization in eukaryotic cells.

Functional analysis of the cyclopiazonic acid biosynthesis gene cluster in Aspergillus oryzae RIB 40.

The cyclopiazonic acid (CPA) nonproducing strain, Aspergillus oryzae RIB 40, does not biosynthesize cyclo-acetoacetyl-L-tryptophan (cAATrp) due to a truncation in the responsible PKS-NRPS gene. We found that RIB 40 converted cAATrp to 2-oxocyclopiazonic acid, the final product of CPA biosynthesis in A. oryzae. This indicates that the CPA biosynthesis gene cluster, except for the PKS-NRPS gene, is functional in RIB 40.

Genetic safeguard against mycotoxin cyclopiazonic acid production in Aspergillus oryzae.

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Its inability to produce mycotoxins, due to mutation or transcriptional repression of the genes responsible for their biosynthesis, is consistent with the hypothesis that A. oryzae is a domesticated species derived from A. flavus, a wild species that is a well-known producer of aflatoxin. In contrast, the cyclopiazonic acid (CPA) biosynthetic gene (cpa) cluster in A. oryzae contains genes that have been lost in A. flavus. Through targeted gene inactivation, isolation of the corresponding metabolite, and evaluation of biological activity of the metabolite, we demonstrated that an A. oryzae-specific gene-cpaH-mediates the conversion of CPA into the less toxic 2-oxocyclopiazonic acid, a new analogue of CPA. The detoxifying properties of cpaH, which have been lost in the A. flavus pathway, reflect the relationship of the two species.

Simple metabolite extraction method for metabolic profiling of the solid-state fermentation of Aspergillus oryzae.

Solid-state culture of microorganisms is an important style of culture both in the traditional food industry and in the modern fermentation industry. We propose here a simple method for metabolite extraction from the solid-state fermentation of a filamentous fungus, Aspergillus oryzae, which is known as rice-koji. To evaluate the efficiency of metabolite extraction, liquid chromatography-mass spectrometry (LC/MS) was used for simultaneous detection of a wide range of metabolites including amino acids, organic acids and vitamins, which are of interest in rice-koji making. Among eleven different metabolite extraction methods tested, we found that extraction using acetonitrile/water (50:50), coupled with boiling at 70 °C for 5 min, was advantageous in terms of both extraction yield and metabolism quenching. We consider that our method provides a technical basis for metabolome analysis of a solid-state fermentation.

Genetic analysis of conidiation regulatory pathways in koji-mold Aspergillus oryzae.

Conidia of koji-mold Aspergillus oryzae are often used as starters in the fermented food industry. However, little is known about conidiation regulation in A. oryzae. To improve the productivity of conidia in A. oryzae, it is necessary to understand conidiation regulation in the strain. Therefore, we analyzed the conidiation regulatory system in A. oryzae using 10 kinds of conidiation regulatory gene disruptants. The phenotypes of AorfluG, AorflbA, AorflbB, AorflbC, AorflbD, AorflbE, AorbrlA, AorabaA, AorwetA, and AorfadA mutants are almost identical to those of the corresponding mutants in Aspergillus nidulans. The results indicated that the functions of conidiation regulatory genes are almost conserved between A. oryzae and A. nidulans. However, the severely reduced conidiation phenotype of the AorfluG disruptant in A. oryzae differs from the phenotype of the corresponding mutant in Aspergillus fumigatus in air-exposed culture conditions. These results suggest that A. oryzae, A. nidulans, and A. fumigatus have a G-protein signaling pathway and brlA orthologs in common, and only A. fumigatus has particular brlA activation pathways that are independent of the fluG ortholog. Furthermore, the analyses of AorflbA disruptant and AorfadA dominant-active mutants implicated that AorFadA-mediated G-protein signaling suppresses vegetative growth of A. oryzae.

Functional expression of the Aspergillus flavus PKS-NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid.

alpha-Cyclopiazonic acid (CPA) is an indole tetramic acid mycotoxin. Based on our identification of the polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) hybrid gene cpaA involved in cyclopiazonic acid biosynthesis in Aspergillus fungi, we carried out heterologous expression of Aspergillus flavuscpaA under alpha-amylase promoter in Aspergillus oryzae and identified its sole product to be the CPA biosynthetic intermediate cyclo-acetoacetyl-l-tryptophan (cAATrp). This result rationalized that the PKS-NRPS hybrid enzyme CpaA catalyzes condensation of the diketide acetoacetyl-ACP formed by the PKS module and l-Trp activated by the NRPS module. This CpaA expression system provides us an ideal platform for PKS-NRPS functional analysis, such as adenylation domain selectivity and product releasing mechanism.