Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as PT5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

Zn-deficiency, a global health challenge affects one-third of the world population. Zn-biofertilizer offer an efficient and cost-effective remedy. As Zn-biofertilizer can improve plant growth and grain's Zn-content ensuring improved dietary Zn-supply. This study sought to understand how silver and TiO2 nanoparticles in the rhizosphere affect the activity of Zn-solubilization bacteria (ZSB) and plant growth. Two ZSB strains Bacillus sp. D-7 and Pseudomonas sp. D-117 with excellent Zn-solubilization efficiency of 254 and 260%, respectively were isolated and characterized using polyphasic characterization including 16S rRNA gene sequencing to formulate an effective Zn-biofertilizer. The plant growth promoting activity of this biofertilizer in Mung bean was checked in the presence and absence of various doses of TiO2 and Ag-NPs and was compared with plant grown without biofertilizer. The change in rate of seed germination, vegetative growth (shoot and root length, fresh and dry weight), photosynthetic pigment and Zn-content was checked. Lower doses of nanomaterials (50 and 100 mg kg⁻¹ soil) slightly promoted the plant growth compared to control. While, higher doses (200 and 400 mg kg⁻¹ soil) inhibited the growth. A maximum decrease of shoot length, root length, fresh-weight, and dry-weight of 57.1, 53.9, 53.1, and 10.4% respectively was observed with 400 mg kg⁻¹ of Ag-NPs. However, in the presence of ZSB, the decrease at the same Ag-NP concentration was 41.6, 31.5, 27.4, and 6.6, respectively. These results strongly suggest that Zn-solubilizing bacteria improve resilience to nanoparticles toxicity and helps in Zn fortification in Mung bean even under nanomaterial stress.

High salt wastewater is produced in industries, including seafood and pickling processing. The salinity in such wastewaters has been shown to negatively impact biological treatment efficacy. Little is known about the changes in the microbial community structure in the mature biological 2 treatment systems, the impacts of salinity on community composition, and the shifts over time during operation. This study aimed to identify the changes in the microbial community due to both salt and days of operation through 16s rRNA sequencing and KEGG functional predictions. Intermittent sand bioreactors (ISBs) with a focus on ammonia treatment were utilized. Results showed that the overall community structure and diversity were distinct as wastewater salinity varied from 0%-1.3%. At 1.3% salinity Zoogloea, a common genus in wastewater treatment plants, was not present and Aequorovita, Thauera and Dokdonella became the dominant genera. Nitrosomonas, an important ammonia oxidizing bacteria, increased in abundance with days of operation but was not significantly impacted by an increase in salinity. This finding was further supported by an increase in predicted nitrification potential with time of operation within all intermittent sand bioreactors tested. These results provide a deeper understanding of the impacts of salinity on microbial community development in biological treatment systems and elucidate the shifts in community structure occurring during early operations and into system maturity.

The culture filtrates of the predominant bacterial strains isolated from soil samples have been shown to increase the microbial colony counts on agar plates used for the isolation of uncultured bacteria. One of the factors in the culture filtrates responsible for this increase was identified to be superoxide dismutase (SOD). The generation of reactive oxygen species (O2-, H2O2, and ・OH) was detected from conventional laboratory agar media. The use of agar media supplemented with radical scavengers (SOD, catalase, ascorbic acid, or rutin) effectively increased the colony counts and kinds of microbial strains that grew from soil samples. Taxonomical studies on these isolates revealed new taxa for phylum Actinomycetota; one family, three genera, and nine species were newly described. One of the strains, Patulibacter minatonensis KV-614T belonging to the new family Patulibacteraceae, was isolated on agar medium supplemented with SOD. P. minatonensis KV-614T represents a novel lineage within the phylum Actinomycetota. A polymerase chain reaction (PCR) study using specific primers for the detection of strains related to the genus Patulibacter, order Solirubrobacterales, showed a high distribution frequency, with detection in over 70% of the soil samples tested. These data suggest that the use of radical scavengers may facilitate the isolation of some hitherto-uncultivated microorganisms widely distributed in soil.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Mycosporine-like amino acids (MAAs) are low-molecular-weight UV-protective compounds, and porphyra-334 and shinorine are common MAAs. Porphyra-334 is synthesized via the conjugation of mycosporine-glycine with threonine, whereas substitution with serine yields shinorine. The terrestrial cyanobacterium Nostoc commune KU002 (NIES-2538) produces 7-O-(β-arabinopyranosyl)-porphyra-334, and the mysABCD gene cluster responsible for MAA biosynthesis has been isolated. The heterologous expression of the mysABC genes from N. commune KU002 in Escherichia coli cells led to mycosporine-glycine production regardless of the culture medium supplemented with serine, threonine, or xylose. When the mysABCD genes from N. commune KU002 were expressed in E. coli cells, porphyra-334 production occurred, and shinorine production was observed upon serine supplementation in the culture medium. Notably, threonine and xylose supplementation in the culture medium increased the amounts of porphyra-334 in both cellular extracts and culture medium extracts. When the mysD gene was replaced with that from the shinorine producer Actinosynnema mirum JCM 3225, shinorine was primarily synthesized instead of porphyra-334. Interestingly, the transformant expressing the chimeric KU002-mysABC-JCM3225-mysD produced a novel MAA derivative with an absorption maximum at 334 nm and a molecular mass of 346 when cultured in the medium supplemented with threonine and xylose. These results suggest that the substrate specificity of MysD, which catalyzes the conjugation of mycosporine-glycine and serine or threonine, alters the production of porphyra-334 or shinorine and that the supplements added to the culture medium affect the amount and composition of MAAs produced in the E. coli transformant.

Aureispira marina is a marine bacterium with gliding motility isolated from the southern coastline of Thailand. It contained ceramide as a major cellular lipid composed of saturated or unsaturated branched chain 2-hydroxy-fatty acid and sphingosine. The structure of unsaturated 2-hydroxy-fatty acid was investigated in our previous study, but the geometric configuration of the double bond remained unclear. In the present study, 14-methyl-∆2-pentadecenol (∆2-iso-C16:1-ol) was prepared from D-2-hydroxy-15-methyl-∆3-hexadecenoic acid (D-2-OH-∆3-iso-C17:1) of the ceramide component, and analyzed by 1H and 13C NMR in comparison with ∆2-trans-hexadecenol (∆2-trans-n-C16:1-ol) derived from commercially available D-sphingosine. From the coupling constants of protons in the double bond and the chemical shift value of allylic carbon, the configuration of the double bond was determined as trans. Since the structure of 2-hydroxy-fatty acids was clarified, cellular fatty acids of A. marina and A. maritima, another species of the genus Aureispira, were reexamined, and the description on the cellular fatty acid composition of the genus Aureispira in the previous papers (Hosoya et al., 2006, Int. J. System. Evol. Microbiol., 56, 2931-2935; Hosoya et al., 2007, Int. J. System. Evol. Microbiol., 57, 1948-1951) lacking the description of 2-hydroxy-fatty acids was emended.

Naphthalene is a persistent environmental pollutant for its potential teratogenic, carcinogenic and mutagenic effects. In this study, 10 strains of bacteria capable of degrading naphthalene were isolated from crude-oil contaminated soil. Among them, Pseudomonas plecoglossicida 2P exhibited prominent growth with 1000 mg/L naphthalene as the sole carbon source and degraded 94.15% of naphthalene in 36 h. Whole genome sequencing analysis showed that P. plecoglossicida 2P had a total of 22 genes related to naphthalene degradation, of which 8 genes were related to the salicylic acid pathway only, 5 genes were related to the phthalic acid pathway only, 8 genes were common in both the salicylic acid and phthalic acid pathways, and 1 gene was related to the gentisic acid pathway. P. plecoglossicida 2P was applied in a two-phase partition bioreactor (TPPB) to degrade naphthalene in wastewater. The optimal operating conditions of the reactor were obtained through response surface optimization: initial naphthalene concentration (C0) =1600 mg/L, bacterial liquid concentration (OD600) = 1.3, and polymer-to-wastewater mass ratio (PWR) = 2%. Under these conditions, the naphthalene degradation rate was 98.36% at 24 h. The degradation kinetics were fitted using the Haldane equation with a high coefficient of determination (R2=0.94). The present study laid foundations for naphthalene degradation mechanism of genus Pseudomonas and its potential application in TPPB.

To enhance the value of surimi, efforts have been made to develop a fermentation method with lactic acid bacteria (LAB) to proteolyze fish protein. However, fermenting unheated surimi poses a spoilage risk due to its high bacterial content. Surimi heat treatment can prevent spoilage, but gel formation induced by heating introduces another technical issue: it hinders uniform fermentation. Thus, this study aims to observe the proteolysis and enhance the functionality of seafood product through lactic acid fermentation of kamaboko, a heated surimi. Upon analyzing the kamaboko fermented with Lactobacillus helveticus JCM1004, we observed that LAB produced protease, resulting in the degradation of myosin heavy chain and actin during fermentation. Lactic acid fermentation significantly augmented the peptide content of kamaboko, subsequently elevating the angiotensin Ⅰ-converting enzyme (ACE) inhibitory activity in 200-fold diluted extract of fermented kamaboko to approximately 70% and higher. Notably, our investigation revealed that proteolysis was confined to the surface of kamaboko, as evidenced by SDS-PAGE analysis. This observation implies that the surface area of kamaboko influences the ACE inhibitory activity. Through a comparative analysis of various bacterial strains, we demonstrated that the increase in ACE inhibitory activity is contingent on the protease generated by LAB. These results suggest that LAB-mediated proteolysis of fish proteins liberates bioactive peptides, thereby manifesting in the ACE inhibitory activity. In summary, this study underscores that the fermentation of kamaboko employing proteolytic LAB holds promise in the development of novel functional seafood products.