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Purple nonsulfur bacteria (PNSB) reportedly have probiotic effects in fish, but whether they are indigenous in the digestive tract of fish is a question that requires answering. We attempted to isolate PNSB from the digestive tract of ayu (Plecoglossus altivelis) from the Kuma River (Kumamoto, Japan) and successfully isolated 12 PNSB strains. All the isolated PNSB belonged to the genus Rhodopseudomonas. Five Rhodopseudomonas strains were also isolated from the soil samples collected along the Kuma River. The phylogenetic tree based on the partial sequence of pufLM gene indicated that the PNSB from ayu and soil were similar. The effects of NaCl concentration in growth medium on growth were also compared between the PNSB from ayu and soil. The PNSB from ayu showed a better growth performance at a higher NaCl concentration, suggesting that the intestinal tract of ayu, a euryhaline fish, might provide suitable environment for halophilic microorganisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The garden plant portulaca (Portulaca oleracea cv.) efficiently removes bisphenol A (BPA), an endocrine-disrupting chemical, from a hydroponic solution, but the molecular mechanisms underlying BPA metabolism by portulaca remain unclear. In this study, BPA metabolites converted by portulaca were analyzed by liquid chromatography coupled with tandem mass spectrometry. We observed the hydroxylation of BPA and the oxidization of it to quinone. Polyphenol oxidases are likely to contribute to BPA degradation by portulaca.

Portulaca (Portulaca oleracea cv.) efficiently removes phenolic pollutants from hydroponic solution. In plant roots, peroxidase (PRX) is thought to be involved in the removal of phenolic pollutants by the cross-linking them to cell wall polysaccharides or proteins at the expense of reduction of hydrogen peroxide (H(2)O(2)). In this study, we found that portulaca roots secreted an acidic PRX isozyme that had relatively high H(2)O(2) affinity. We isolated five PRX genes, and the recombinant PRX proteins produced in cultured tobacco cells were partially characterized. Among these genes, PoPRX2 probably encoded the acidic PRX isozyme. PoPRX2 had an extra N-terminal region which has not been reported for other PRX proteins. We found that PoPRX2 oxidized phenolic pollutants, including bisphenol A, octylphenol, nonylphenol, and 17β-estradiol. In addition, we found that the Cys261 residue of PoPRX2 played an important role in the determination of affinity for H(2)O(2) and stability toward H(2)O(2).

RNA interferences in the unicellular green alga, Chlamydomonas reinhardtii, can be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAi-transformed strain with successive cell divisions. Northern blot analyses revealed an inverse correlation between the copy number of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest the existence of an intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.

A novel gene with antistress activities against both salt (NaCl) and cadmium stresses was isolated from the cDNA library of halotolerant green alga Chlamydomonas sp. strain W80 by a functional expression screening with Escherichia coli. The C-terminal region of this protein is responsible for the antistress activity, because N-terminal truncated clone of this gene retains the antistress activity, and the C-terminal truncated clone loses the activity. In the C-terminal region, there is a histidine and aspartic acid-rich domain (HD-rich domain).

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.

By a functional expression screening with cyanobacterial cells, a new member of group 3 late embryogenesis abundant protein genes (designated cw80lea3) was isolated from the cDNA library of halotolerant green alga Chlamydomonas sp. strain W80. The principle of the screening method was based on the acquisition of NaCl salt-tolerance of the fresh water cyanobacterial cells carrying the halotolerant algal gene. The expression of cw80lea3 gene in the C. W80 cells was induced by salt- and cold-stresses, but not by abscisic acid treatment.

We analyzed the promoter of the genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO(2)-regulatory element. Cyanobacterial transformants were constructed with several DNA segments of the rbc promoter fused to the chloramphenicol acetyltransferase (CAT) gene, and their acetyltransferase activities were analyzed under 0.03% and 1% CO(2) conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO(2)-dependent repression. Fluorescent-labeled oligonucleotide probes of identical sequence to the AT-rich element were reacted with protein extracts from cells cultured under conditions of low and high CO(2) atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO(2), but only a weak signal from cells cultured under 1% CO(2). Moreover, a DNA affinity precipitation assay identified a 16-kDa protein that bound to nucleotide sequences within the AT-rich element. The partial amino acid sequence of the protein was similar to the deduced protein sequences of ORF129 and ORF155 from Synechocystis 6803. Our findings suggest that the AT-rich element plays a role as a negative CO(2)-regulatory element and its trans-acting factor possibly regulates the rbc transcription in response to CO(2) levels.

The preference for the optimal nucleotide of the mammalian translation initiation AUG context [GCCGCC(A/G)CCaugG] is generally more pronounced in the highly expressed genes than in the transcription factor genes at the -9 through -1 positions in humans. The influence of amino acid preference on the nucleotide choice at the +4 position was also examined.

We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The specific recombination sites on the smallest plasmid pAQ1 were localized by studying the properties of pAQ1-derived shuttle-vectors. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination. Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements. Interestingly, this element is over-represented not only in pAQ1 and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp. PCC6301, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these cyanobacteria.

Sequences of 118 mRNAs of the green alga Chlamydomonas reinhardtii in the GenBank data base were compiled to examine the consensus sequence surrounding the AUG translation initiation codon. The consensus sequence for C. reinhardtii was found to be gc(A/C)A(A/C)(A/C) AUGGC. The AUG context of chloroplast proteins (nuclear coded) and non-chloroplast proteins were compared by a separate compilation, and some distinctive features in AUG context of chloroplast proteins were found.

We have determined the complete nucleotide sequence of pAQ1, the smallest plasmid of the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The plasmid consists of 4,809 bp and has at least four open reading frames that potentially encode polypeptides of 50 or more amino acids. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, is over-represented not only in plasmid pAQ1 but also in the accumulated cyanobacterial genomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. It suggests that this sequence might mediate gene rearrangement, thus increasing genetic diversity, since recombination events are frequent in cyanobacteria.

Oligodeoxynucleotides involving a stable nitroxide radical, 5-(2,2,5,5-tetramethyl-3-ethynylpyrroline-1-oxyl)-2-deoxyuridine residue (TMP), and a fluorescence dye, quinacrine (Qu), on each complementary strand and those on the same strand in the duplexes were prepared. Three base-pair units locate between TMP and Qu. The interaction between TMP and the excited singlet Qu has been studied by picosecond transient absorption spectroscopy and fluorometry. From the biphasic fluorescence decay profile, it was suggested that there exist two sites of the dye in the oligodeoxynucleotide with the modifiers in the different complementary strands. In addition, no effective fluorescence quenching was observed for this oigodeoxynucleotide. On the other hand, the triphasic fluorescence decay profile was observed for the oligodeoxynucleotide having Qu and TMP in the same strand and the fluorescence was slightly quenched. Picosecond transient absorption spectroscopy revealed that the enhanced intersystem-crossing by TMP was responsible for the quenching of the fluorescent state of the dye. From these results, it has been concluded that the role of the DNA duplex in the quenching process of the dye by TMP was rather small.