An Enzyme Assay Kit for GABA Quantification in Plant Tissues.

Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200 mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues.

Intracellular Phage Tail-Like Nanostructures Affect Susceptibility of Streptomyces lividans to Osmotic Stress.

Contractile injection systems (CISs) are a large group of phage tail-like nanostructures conserved among bacteria. Despite their wide distribution, the biological significance of CISs in bacteria remains largely unclear except for a few unicellular bacteria. Here, we show that Streptomyces lividans-a model organism of filamentous Gram-positive bacteria with highly conserved CIS-related gene clusters-produces intracellular CIS-like nanostructures (Streptomyces phage tail-like particles [SLPs]) that affect phenotypes of this bacterium under hyperosmotic conditions. In contrast to typical CISs released from the cells, SLPs are localized in the cytoplasm of S. lividans. In addition, loss of SLPs leads to (i) delayed erection of aerial mycelia on hyperosmotic solid medium and (ii) decreased growth during the transition from exponential growth phase to stationary phase in hyperosmotic liquid medium. Localization of fluorescent protein-tagged SLPs showed partial correlation with cell wall synthesis-related proteins, including MreB, an actin-like cytoskeleton protein. Our pulldown assay and subsequent quantitative proteome analysis also suggest that 30S ribosomal proteins and cell wall-related proteins, including MreB, are coeluted with SLPs. Furthermore, an interaction assay using the recombinant proteins revealed a direct interaction between a sheath protein of SLP and ribosomal protein S16. Results of cross-linking experiments show indirect interactions between SLPs and translation elongation factors. These findings collectively suggest that SLPs are directly or indirectly associated with a protein interaction network within the cytoplasm of S. lividans and that SLP loss ultimately affects the susceptibility of the bacterium to certain stress conditions. IMPORTANCE Recent bioinformatic analyses have revealed that CIS-related gene clusters are highly conserved in Gram-positive actinomycetes, especially members of the genus Streptomyces known for their ability to produce therapeutic antibiotics. While typical CISs are released from the cells and can act as protein translocation systems that inject effector proteins into the target cells, our results indicate the unique intracellular localization of SLPs, CIS-related nanostructures produced by S. lividans. In addition, the direct and indirect interactions of SLPs with cytoplasmic proteins and SLP localization within specific regions of mycelia suggest that the biological significance of SLPs is related to intracellular processes. Further, SLP loss leads to increased susceptibility of S. lividans to osmotic stress, suggesting that production of these phage tail-like nanostructures ultimately affects the fitness of the bacterium under certain stress conditions. This work will provide new insight into the phage tail-like nanostructures highly conserved in Streptomyces species.

Characterization of 5'-nucleotidases secreted from Streptomyces.

To study the ability of Streptomyces to utilize environmental nucleotides, we screened for strains exhibiting extracellular 5'-inosine monophosphate (IMP)-dephosphorylating activity in our collection of soil isolates and obtained two producers: NE5-10 and Y2F8-2. The enzyme responsible for the activity was purified from the culture supernatant of each strain, and its mass spectral data were used to identify the coding sequence. The gene was successfully identified in the whole genome sequence of each strain; it was located in a conserved gene cluster of phosphate-related functions and encoded an approximately 600-amino acid long protein containing an N-terminal secretion signal. The mature part of the protein exhibited similarity to a known bacterial 5'-nucleotidase. The locus of the 5'-nucleotidase gene contained genes encoding proteins involved in phosphate utilization. The conserved gene arrangement of the locus in various Streptomyces genomes suggested the genetic region to be involved in phosphate-scavenging in this group of bacteria. Phylogenetic analysis demonstrated that the isolated Streptomyces enzymes represent an uncharacterized group of bacterial 5'-nucleotidases. Enzymatic characterization of the two Streptomyces enzymes demonstrated that both enzymes exhibited 5'-nucleotidase activity but differed in terms of optimal temperature and pH, dependence on divalent cations, and substrate specificity. The Km and Vmax values of the 5'-IMP-dephosphorylating activity were 0.239 mM and 9.47 U/mg, respectively, for NE5-10 and 0.221 mM and 38.17 U/mg, respectively, for Y2F8-2. Enzyme activity in the culture broth of the two Streptomyces producers occurred in a phosphate-limitation-dependent manner, supporting their involvement in the acquisition of phosphorus. KEY POINTS: • We purified and characterized nucleotidases from two Streptomyces. • Two nucleotidases were presumed to be involved in phosphate acquisition. • It showed diversity in phosphate acquisition among microorganisms.

Genome Sequence of "Caldinitratiruptor microaerophilus," a Unique Microaerophilic Bacterium Closely Related to Symbiobacterium.

We present the genome sequence of "Caldinitratiruptor microaerophilus" JCM16183, isolated by Fardeau et al. from a French hot spring. This microaerophilic bacterium represents a novel taxon related to the genus Symbiobacterium. The high (71%) G+C content of its 3.60-Mb circular genome supports the divergence of this bacterium from Clostridia.

Organocatalytic activity of granaticin and its involvement in bactericidal function.

We previously discovered that actinorhodin, a benzoisochromanequinone antibiotic produced by Streptomyces coelicolor A3(2), serves as a catalyst facilitating the oxidation of ascorbic acid and cysteine (PNAS 48:17,152, 2014). In the present study, we screened for similar ascorbic acid-oxidizing activity in the culture broth of various Streptomyces spp., and discovered marked activity in the culture broth of Streptomyces vietnamensis. The principle active compound was granaticin, a pigmented antibiotic that is structurally related to actinorhodin. The absence of any metals in the purified granaticin fraction indicated that granaticin was an organocatalyst. Granaticin catalyzed the oxidation of L-ascorbic acid, generating L-dehydroascorbic acid and hydrogen peroxide (H2O2) at a 1:1 stoichiometric ratio, with 15 times higher reactivity than that of actinorhodin at an optimum pH of 7.0. Granaticin also oxidizes sulfhydryl compounds, including L-cysteine and glutathione. Growth inhibitory assays demonstrated that knockout mutants of the catalase gene exhibit high sensitivity to granaticin. The results suggest that the bactericidal activity of granaticin is exerted by the oxidation of sulfhydryl groups of cellular components and the toxicity of H2O2 generated during the oxidation reaction.

GABA enzymatic assay kit.

We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.

Extracytoplasmic diaphorase activity of Streptomyces coelicolor A3(2).

Metabolism and utilization of plant-derived aromatic substances are fundamental to the saprophytic growth of Streptomyces. Here, we studied an enzyme activity reducing 2,6-dichlorophenolindophenol and nitroblue tetrazolium in the culture supernatant of Streptomyces coelicolor A3(2). N-terminal amino acid sequencing of a nitroblue tetrazolium-reducing enzyme revealed that the enzyme corresponds to the SCO2180 product. The protein exhibited a marked similarity with dihydrolipoamide dehydrogenase, the E3 subunit of 2-oxo-acid dehydrogenase complex. A recombinant SCO2180 protein formed a homodimer and exhibited a diaphorase activity catalyzing NADH-dependent reduction of various quinonic substrates. Similar nitroblue tetrazolium-reducing activities were observed for other Streptomyces strains isolated from soil, implying that the diaphorase-catalyzed reduction of quinonic substances widely occurs in the extracytoplasmic space of Streptomyces.

Developmental defect of cytochrome oxidase mutants of Streptomyces coelicolor A3(2).

To study the link between energy metabolism and secondary metabolism/morphological development in Streptomyces, knockout mutants were generated with regard to the subunits of the cytochrome oxidase supercomplex (CcO) in Streptomyces coelicolor A3(2). All mutants exhibited an identical phenotype: viable but defective in antibiotic production and cell differentiation when grown in both complex and minimal media. The growth yield of the CcO mutant was about half of that of the WT strain on glucose medium while both strains grew similarly on maltose medium. Intracellular ATP measurement demonstrated that the CcO mutant exhibited high intracellular ATP level. A similar elevation of intracellular ATP level was observed with regard to the WT strain cultured in the presence of BCDA, a copper-chelating agent. Reverse transcriptase PCR analysis demonstrated that the transcription of ATP synthase operon is upregulated in the CcO mutant. Addition of carbonylcyanide m-chlorophenylhydrazone, an inhibitor of ATP synthesis, promoted antibiotic production and aerial mycelia formation in the CcO mutant and BCDA-treated WT cells. We hypothesize that the deficiency of CcO causes accumulation of intracellular ATP, and that the high ATP level inhibits the onset of development in S. coelicolor.

Streptomyces metabolites in divergent microbial interactions.

Streptomyces and related bacteria produce a wide variety of secondary metabolites. Of these, many compounds have industrial applications, but the question of why this group of microorganism produces such various kinds of biologically active substances has not yet been clearly answered. Here, we overview the results from our studies on the novel function and role of Streptomyces metabolites. The diverged action of negative and positive influences onto the physiology of various microorganisms infers the occurrence of complex microbial interactions due to the effect of small molecules produced by Streptomyces. The interactions may serve as a basis for the constitution of biological community.

Natural low-molecular mass organic compounds with oxidase activity as organocatalysts.

Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of L-ascorbic acid and L-cysteine as substrates by analytical methods for enzymes. Our findings were as follows: (i) oxidation reactions producing H2O2 proceeded upon addition of ACT to the reaction mixture; (ii) ACT was not consumed during the reactions; and (iii) a small amount (catalytic amount) of ACT consumed an excess amount of the substrates. Even at room temperature, atmospheric pressure, and neutral pH, ACT showed catalytic activity in aqueous solution, and ACT exhibited substrate specificity in the oxidation reactions. These findings reveal ACT to be an organocatalyst. ACT is known to show antibiotic activity, but its mechanism of action remains unknown. On the basis of our results, we propose that ACT kills bacteria by catalyzing the production of toxic levels of H2O2. We also screened various other natural products of bacterial, plant, and animal origins and found that several of the compounds exhibited catalytic activity, suggesting that living organisms produce and use these compounds as biocatalysts in nature.