Ralstonia pseudosolanacearum is a plant-pathogenic bacterium that causes bacterial wilt in economically important crops. Chemotaxis is required for full virulence in R. pseudosolanacearum. R. pseudosolanacearum Ps29 possesses 20 methyl-accepting chemotaxis proteins (MCPs) and 2 MCP-like chemoreceptors. To understand the role of chemotaxis in plant infection, we are characterizing the functions of these 20 MCPs. Out of 20 MCPs, 8 MCPs have been characterized. To characterize the remaining MCPs, we deleted the 8 genes encoding characterized MCPs in R. pseudosolanacearum Ps29 to construct R. pseudosolanacearum D8. R. pseudosolanacearum D8 was examined for chemotactic responses to several chemically undefined materials including vegetable juices and tryptic soy broth (TSB) to find attractants. R. pseudosolanacearum D8 showed strong responses to green pepper and carrot juices and TSB. We constructed a mutant library of R. pseudosolanacearum D8 by deleting each of the MCP genes. Chemotaxis assays to TSB revealed that an MCP which we named McpD was responsible for sensing an attractant(s) in TSB. Because amino acids are the major constituents of TSB, we measured chemotactic responses of R. pseudosolanacearum D8 to 20 proteinogenic amino acids and found Asp and Glu as the major attractants of McpD and Cys as the minor attractant. R. pseudosolanacearum Ps29 can utilize Asp and Glu as sole carbon and nitrogen sources, suggesting that the role of McpD-mediated chemotaxis is finding growth substrates.

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites. We found that a position between the -35 box and the -10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of better SAM biosensing systems.

Amplicon sequencing is a widely used method for surveying biological diversity. However, the technique is disturbed by PCR bias leading to errors in community composition analyses. In this study, microbial community composition was evaluated in twenty-eight locations of hot spring water with temperatures between 87-48°C at Nakabusa Hot Springs, Japan, using amplicon sequencing analysis with the V4 region of the 16S rRNA gene. In discrepancy with the greenish color and the absorption spectra of the microbial samples, the relative abundance of amplicon sequence variants (ASVs) in the major photosynthetic organisms, Chloroflexus spp., were scarce in any sample when using the annealing temperature of 50°C in amplicon PCR. Changing the annealing temperature to 68ºC significantly improved the detection efficiency of Chloroflexus ASVs, and the obtained numbers were consistent with the presence of the photosynthetic pigments. The abundance of many other microbial ASVs was also dependent on the annealing temperature. The log ratio in the abundance of major ASVs between two annealing temperatures was correlated with the GC content of the 16S rRNA gene, suggesting that even some other major ASVs in the community are seriously affected by PCR bias due to the GC content. Combined usage of results from two different annealing temperatures, rather than a result using a single annealing temperature, seems to be a better way to obtain community structure information with less PCR bias in thermophilic organisms of high 16S rRNA GC content.

Serratia nuclease Nuc A is a non-specific nucleotide hydrolase that has been widely used in large-scale protein purification or eliminating nucleic acid contamination from purified proteins. To enhance the enzyme production, the Serratia nuclease gene was synthesized and expressed in Bacillus licheniformis 2709, a robust strain capable of secreting native and heterologous proteins selectively or non-selectively. To further increase the secretory expression level of the enzyme, different strong promoters and signal peptides were fused with the mature Nuc A-encoding gene at various genetic loci. The highest expression level of Nuc A was observed under the control of regulatory elements PaprE, which occur naturally in B. licheniformis 2709 for the alkaline protease (AprE) expression. Through maximizing the number of copies of PaprE-nucA expression cassette at different integration sites, the yield of nuclease Nuc A reached approximately 31954 U/mL after 60 hours of cultivation in shake flasks. The specific activity of the recombinant nuclease reached 1.58×107 U/mg, which is about 9 times higher than that expressed in Escherichia coli strain. Additionally, the recombinant Nuc A exhibited high catalytic activities in the pH range of 7-10. Furthermore, it was resistant to 0.2% SDS, 1.0 mM PMSF, and 0.4% Triton X-100. After 8 M Urea treatment, residual activity is measured. The high expression levels and positive characteristics of recombinant Nuc A provide an effective solution for large-scale production and industrial application of the nuclease.

p-Anisaldehyde, a fragrance and flavour with important roles in food, cosmetics, and drug industries, is currently synthesized through chemical methods. Production of p-anisaldehyde by chemical oxidation of trans-anethole in industry gives rise to excessive by-products and adverse environmental impacts, whereas biological process would address such problems. Here, we presented a process of biotransformation of trans-anethole for production of p-anisaldehyde. The tao gene encoding for trans-anethole oxygenase (TAO) from Paraburkholderia sp. MR185 was fused with a solubilization tag GST and ProS2, respectively. GST did not exhibit solubility enhancement effect, whereas fusion with ProS2 significantly improved TAO's soluble expression in E. coli and the fusion protein ProS2-TAO-Sil3K accounted for more than 40% of total soluble proteins. ProS2-TAO-Sil3K was purified by simple silica affinity and its activity did not require addition of NADH, NADPH, and FAD. Metal ions Co2+, Zn2+, Ni2+, and Cu2+ displayed significant inhibition effect on TAO activity, and addition of Fe2+ improved enzyme activity by 32.6%. After induction, engineered E. coli cells were used as whole-cell biocatalyst for transformation of trans-anethole, and the final concentration of p-anisaldehyde reached 10.18 mM (1.38 g/L), with the volumetric productivity of 0.11 g/L/h and conversion rate of 67.9%. These results reveal that the biosynthesis of p-anisaldehyde has a great potential in practice.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The Km value and kcat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s-1 respectively. The Km value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

Polyamide 4 (PA4) is expected to solve the issue of marine plastic pollution due to its excellent mechanical properties and biodegradability. In this study, to reveal the mechanism of PA4 biodegradation in the marine environment, we isolated 5 strains of PA4-degrading bacteria belonging to Aliiglaciecola, Dasania, and Pseudophaeobacter from a marine environment. The isolated 5 strains are novel PA4-degrading bacteria that are phylogenetically distinct from those isolated in previous studies. In addition, we compared the PA4-degrading activities and structures of the PA4-degrading enzymes secreted by the 5 strains and PA4-degrading strains isolated in our previous study. The PA4-degrading activity in the supernatant of the cultivation solutions differed among the strains. Native-PAGE and zymography using a polyacrylamide gel containing a PA4 emulsion demonstrated that PA4-degrading enzymes are classified into no less than three types of structures. These results suggested that marine PA4-degrading bacteria have multiple PA4-degrading enzymes. Our findings will contribute to a better understanding of the microbial degradation of PA4 in the marine environment.

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Most cyanobacterial genomes possess more than two copies of genes encoding cyAbrBs (cyanobacterial AbrB-like proteins) having an AbrB-like DNA-binding domain at their C-terminal region. Accumulating data suggest that a wide variety of metabolic and physiologic processes are regulated by cyAbrBs. In this study, we investigated the function of the essential gene cyabrB1 (sll0359) in Synechocystis sp. PCC 6803 by using CRISPR interference technology. The conditional knockdown of cyabrB1 caused increases of cyAbrB2 transcript and protein levels. However, the effect of cyabrB1 knockdown on global gene expression profile was quite limited compared to the previously reported profound effect of knockout of cyabrB2. Among 24 up-regulated genes, 16 genes were members of the divergently transcribed icfG and sll1783 operons related to carbon metabolism. The results of this and previous studies indicate the different contributions of two cyAbrBs to transcriptional regulation of genes related to carbon, hydrogen and nitrogen metabolism. Possession of a pair of cyAbrBs has been highly conserved during the course of evolution of the cyanobacterial phylum, suggesting physiological significance of transcriptional regulation attained by their interaction.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 β-1,3-glucanase.