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Accretionary prisms are composed mainly of ancient marine sediment scraped from the subducting oceanic plate at convergent plate boundaries. Anoxic groundwater is stored in deep aquifers associated with accretionary prisms and can be collected via deep wells. We investigated how such groundwater pumping affects the microbial community in a deep aquifer. Groundwater samples were collected from a deep well drilled down to 1500 m every six months (five times in total) after completion of deep well construction and the start of groundwater pumping. Next-generation sequencing and clone-library analyses of 16S rRNA genes were used to describe the subterranean microbial communities in the samples. The archaea: the prokaryote ratio in groundwater increased significantly from 1 to 7% (0 and 7 months after initiating groundwater pumping) to 59 to 72% (13, 19, and 26 months after initiating groundwater pumping), and dominant prokaryotes changed from fermentative bacteria to sulfate-reducing archaea. The optimal growth temperature of the sulfate-reducing archaea, estimated based on the guanine-plus-cytosine contents of their 16S rRNA genes, was 48–52 °C, which agreed well with the groundwater temperature at the deep-well outflow. Our results indicated that, in deep aquifers, groundwater pumping enhances groundwater flow, and the supply of sulfate-containing seawater activates the metabolism of thermophilic sulfate-reducing archaea.

<jats:p>Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of <jats:italic>Escherichia coli</jats:italic> and <jats:italic>Pseudomonas putida</jats:italic> in an artificial bacterial consortium comprising <jats:italic>E. coli</jats:italic>, <jats:italic>P. putida</jats:italic>, <jats:italic>Pseudomonas fluorescens</jats:italic>, and <jats:italic>Lactiplantibacillus plantarum</jats:italic>. Moreover, the growth inhibition of <jats:italic>P. putida</jats:italic> promoted the growth of <jats:italic>P. fluorescens</jats:italic> and inhibited the growth of <jats:italic>L. plantarum</jats:italic>. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.</jats:p>

BACKGROUND: There has been an increasing demand for optically pure d-lactic and l-lactic acid for the production of stereocomplex-type polylactic acid. The d-lactic acid production from lignocellulosic biomass is important owing to its great abundance in nature. Corn steep liquor (CSL) is a cheap nitrogen source used for industrial fermentation, though it contains a significant amount of l-lactic acid, which decreases the optical purity of d-lactic acid produced. METHOD AND RESULTS: To remove l-lactic acid derived from the CSL-based medium, l-lactate oxidase (LoxL) from Enterococcus sp. NBRC 3427 was expressed in an engineered Lactiplantibacillus plantarum (formally called Lactobacillus plantarum) strain KOLP7, which exclusively produces d-lactic acid from both hexose and pentose sugars. When the resulting strain was applied for d-lactic acid fermentation from the mixed sugars consisting of the major constituent sugars of lignocellulose (35 g L-1 glucose, 10 g L-1 xylose, and 5 g L-1 arabinose) using the medium containing 10 g L-1 CSL, it completely removed l-lactic acid derived from CSL (0.52 g L-1 ) and produced 41.7 g L-1 of d-lactic acid. The l-lactic acid concentration was below the detection limit, and improvement in the optical purity of d-lactic acid was observed (from 98.2% to > 99.99%) by the overexpression of LoxL. CONCLUSION AND IMPLICATIONS: The LoxL-mediated consumption of l-lactic acid would enable the production of optically pure d-lactic acid in any medium contaminated by l-lactic acid.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%-100%) than the CRISPR/SpCas9 system (50%-79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.

Linoleic acid (LA) has garnered much attention due to its potential applications in the oleochemical and nutraceutical industries. The oleaginous yeast Rhodotorula toruloides has outstanding lipogenecity, and is considered a potential alternative to the current plant-based platforms for LA production. Δ12-fatty acid desaturases (Δ12-Fads) are involved in LA synthesis in various fungi and yeasts, but their functions in R. toruloides remain poorly understood. To achieve the production of LA-rich lipids in R. toruloides, we investigated the function of the native Δ12-FAD (RtFAD2). First, the overexpression of RtFAD2 and its co-overexpression with RtFAD1 (encoding R. toruloides Δ9-Fad) and their effects on LA production in R. toruloides were investigated. The function of RtFad2 was confirmed by heterologous expression in Saccharomyces cerevisiae. Overexpression of RtFAD2 significantly elevated the LA contents and titers in the wild-type strain R. toruloides DMKU3-TK16 (TK16) and in a thermotolerant derivative of TK16 (L1-1). Additionally, overexpression of RtFAD2 in R. toruloides strains also increased the lipid titer and content. Overexpression of RtFAD1 was down-regulated in the RtFAD1 and RtFAD2 co-overexpressing strains, suggesting that the elevated LA content may function as a key regulator of RtFAD1 expression to control C18 fatty-acid synthesis in R. toruloides. We characterized the function of RtFAD2 and showed that its overexpression in R. toruloides increased the lipid and LA production. These findings may assist in the rational design of metabolic engineering related to LA or polyunsaturated fatty acid production in R. toruloides.

S-Adenosylmethionine (SAM)-dependent methyltransferases are important tools for the biocatalytic methylation of diverse biomolecules. Methylation by a whole-cell biocatalyst allows the utilization of intrinsic SAM and its regeneration system, which consists of a cyclic and multi-step enzymatic cascade. However, low intracellular availability of 5-methyl-tetrahydrofolate (5-methyl-THF), which functions as a methyl group donor, limits SAM regeneration. Here, we integrated methanol metabolism with 5-methyl-THF formation into SAM-dependent methylation system in Escherichia coli, driven by heterologously expressed methanol dehydrogenase (MDH). The coupling of MDH-catalyzed methanol oxidation with the E. coli endogenous reactions enhances the formation of 5-methyl-THF using methanol as a source of methyl group, thereby promoting both the SAM regeneration and methylation reactions. Co-expression of the mutant MDH2 from Cupriavidus necator N-1 with the O-methyltransferase 5 from Streptomyces avermitilis MA-4680 enhanced O-methylation of esculetin 1.4-fold. Additional overexpression of the E. coli endogenous 5,10-methylene-THF reductase, which catalyzes the last step of 5-methyl-THF formation, further enhanced the methylation reaction by 1.9-fold. Together with deregulation of SAM biosynthesis, the titer of methylated compounds was increased about 20-fold (from 0.023 mM to 0.44 mM). The engineered E. coli strain with enhanced 5-methyl-THF formation is now available as a chassis strain for the production of a variety of methylated compounds.

Accretionary prisms are thick masses of sedimentary material scraped from the oceanic crust and piled up at convergent plate boundaries found across large regions of the world. Large amounts of anoxic groundwater and natural gas, mainly methane (CH4), are contained in deep aquifers associated with these accretionary prisms. To identify the subsurface environments and potential for CH4 production by the microbial communities in deep aquifers, we performed chemical and microbiological assays on groundwater and natural gas derived from deep aquifers associated with an accretionary prism and its overlying sedimentary layers. Physicochemical analyses of groundwater and natural gas suggested wide variations in the features of the six deep aquifers tested. On the other hand, a stable carbon isotope analysis of dissolved inorganic carbon in the groundwater and CH4 in the natural gas showed that the deep aquifers contained CH4 of biogenic or mixed biogenic and thermogenic origins. Live/dead staining of microbial cells contained in the groundwater revealed that the cell density of live microbial cells was in the order of 104 to 106‍ ‍cells‍ ‍mL-1, and cell viability ranged between 7.5 and 38.9%. A DNA analysis and anoxic culture of microorganisms in the groundwater suggested a high potential for CH4 production by a syntrophic consortium of hydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. These results suggest that the biodegradation of organic matter in ancient sediments contributes to CH4 production in the deep aquifers associated with this accretionary prism as well as its overlying sedimentary layers.

Growth temperature is one of the most representative biological parameters for characterizing living organisms. Prokaryotes have been isolated from various temperature environments and show wide diversity in their growth temperatures. We herein constructed a database of growth TEMPeratures of Usual and RAre prokaryotes (TEMPURA, http://togodb.org/db/tempura), which contains the minimum, optimum, and maximum growth temperatures of 8,639 prokaryotic strains. Growth temperature information is linked with taxonomy IDs, phylogenies, and genomic information. TEMPURA provides useful information to researchers working on biotechnological applications of extremophiles and their biomolecules as well as those performing fundamental studies on the physiological diversity of prokaryotes.

Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono- to diphosphates and di- to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono- (AMP) into triphosphate (ATP) not only through step-by-step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate-dependent pyrophosphorylation activity.

Haloarcula strains, which are halophilic archaea, harbour two to three copies of 16S rRNA genes (rrsA, rrsB and rrsC) in their genomes. While rrsB and rrsC (rrsBC) show almost identical sequences, rrsA shows 4-6% sequence difference and 1-3% guanine-plus-cytosine content (PGC) difference compared to rrsBC. Based on the strong correlation between the PGC of 16S rRNA genes and the growth temperatures of the prokaryotes, we hypothesised that high-PGCrrsA and low-PGCrrsBC are expressed at high and low temperatures, respectively. To verify the hypothesis, we performed sequence analyses and expression surveys of each 16S rRNA gene in eight Haloarcula strains. The secondary structure prediction of the 16S rRNA via computer simulation showed that the structural stability of 16S rRNAs transcribed from rrsA was higher than that of 16S rRNAs transcribed from rrsBC. We measured expression levels of rrsA and rrsBC under various temperature conditions by reverse-transcriptase quantitative PCR. The expression ratio of high-PGCrrsA to low-PGCrrsBC increased with cultivation temperatures in seven of eight Haloarcula strains. Our results suggest that the transcription of high-PGCrrsA and low-PGCrrsBC may be regulated in response to environmental temperature, and that 16S rRNAs transcribed from high-PGCrrsA function under high temperature conditions close to the maximum growth temperature.

Accretionary prisms are thick layers of sedimentary material piled up at convergent plate boundaries. Large amounts of anaerobic groundwater and methane (CH4) are contained in the deep aquifers associated with accretionary prisms. In order to identify microbial activity and CH4 production processes in the deep aquifers associated with the Cretaceous accretionary prism in Okinawa Island, Japan, we performed geochemical and microbiological studies using anaerobic groundwater and natural gas (mainly CH4) samples collected through four deep wells. Chemical and stable hydrogen and oxygen isotope analyses of groundwater samples indicated that the groundwater samples obtained from each site originated from ancient seawater and a mixture of rainwater and seawater, respectively. Additionally, the chemical and stable carbon isotopic signatures of groundwater and natural gas samples suggested that CH4 in the natural gas samples was of a biogenic origin or a mixture of biogenic and thermogenic origins. Microscopic observations and a 16S rRNA gene analysis targeting microbial communities in groundwater samples revealed the predominance of dihydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. Moreover, anaerobic cultures using groundwater samples suggested a high potential for CH4 production by a syntrophic consortium of H2-producing fermentative bacteria and H2-utilizing methanogenic archaea through the biodegradation of organic substrates. Collectively, our geochemical and microbiological data support the conclusion that the ongoing biodegradation of organic matter widely contributes to CH4 production in the deep aquifers associated with the Cretaceous accretionary prism.

The halophilic archaeon Haloarcula hispanica harbors three ribosomal RNA (rRNA) operons (rrnA, rrnB, and rrnC) that contain the 16S rRNA genes rrsA, rrsB, and rrsC, respectively. Although rrsB and rrsC (rrsBC) have almost identical sequences, the rrsA and rrsBC sequences differ by 5.4%, and they differ by 2.5% with respect to guanine-plus-cytosine content (PGC). The strong correlation between the typical growth temperatures of archaea and PGC of their 16S rRNA genes suggests that H. hispanica may harbor different 16S rRNA genes having different PGC to maintain rapid growth in a wide range of temperatures. We therefore performed reverse transcription-coupled quantitative PCR to assess expression levels of rrsA (P-GC, 58.9%) and rrsBC (PGC, 56.4-56.5%) at various temperatures. The expression ratio of rrsA to rrsBC increased with culture temperature. Mutants with complete deletions of one or two of the three rRNA operons were constructed and their growth rates at different temperatures compared to that of the wild-type. The growth characteristics of the rRNA operon single-mutant strains were indistinguishable from the wild-type. The rRNA operon double-mutant strains maintained the same temperature range as wild-type but displayed reduced growth rates. In particular, the double-mutant strains grew much slower than wild-type at low temperature related to minimum growth temperature of the wild-type. On the other hand, at physiologically high temperatures the wild-type and the double-mutant strain which harbors only rmA with high-P-GC rrsA grew significantly faster than the double-mutant strain which harbors only rrmC with low-P-GC rrsC. These findings suggest the importance of 16S rRNAs transcribed from rrsA with high-P-GC in maintaining rapid growth of this halophilic archaeon at raised growth temperatures.

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂ -producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂ -producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂ -producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH4 and H2 production utilizing anaerobic groundwater containing an active subterranean microbial community.