Streptothricin-F Inhibition of FtsZ Function: A Promising Approach for Controlling Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) is a significant pathogenic bacterium affecting the kiwifruit industry. This study investigated the target sites of streptothricin-F (ST-F), produced by Streptomyces lavendulae gCLA4. The inhibition of ST-F on Psa was examined by the microscopic structural differences of Psa before and after treatment with ST-F, as well as the interaction between ST-F and cell division-related proteins. The results revealed filamentation of Psa after ST-F treatment, and fluorescence microscopy showed that ST-F inhibited the formation of the Z-ring composed of FtsZ protein. In vitro experiments and molecular docking demonstrated that ST-F can bind to FtsZ with a binding energy of 0.4 µM and inhibit FtsZ's GTP-dependent polymerization reaction. In addition, ST-F does not exert inhibitory effects on cell division in Psa strains overexpressing ftsZ. In conclusion, FtsZ is one of the target sites for ST-F inhibition of Psa, highlighting its potential as a therapeutic target for controlling Psa-induced kiwifruit bacterial canker.

Streptomyces sp. JCK-6131 Protects Plants Against Bacterial and Fungal Diseases via Two Mechanisms.

Plant bacterial and fungal diseases cause significant agricultural losses and need to be controlled. Beneficial bacteria are promising candidates for controlling these diseases. In this study, Streptomyces sp. JCK-6131 exhibited broad-spectrum antagonistic activity against various phytopathogenic bacteria and fungi. In vitro assays showed that the fermentation filtrate of JCK-6131 inhibited the growth of bacteria and fungi with minimum concentration inhibitory (MIC) values of 0.31-10% and 0.31-1.25%, respectively. In the in vivo experiments, treatment with JCK-6131 effectively suppressed the development of apple fire blight, tomato bacterial wilt, and cucumber Fusarium wilt in a dose-dependent manner. RP-HPLC and ESI-MS/MS analyses indicated that JCK-6131 can produce several antimicrobial compounds, three of which were identified as streptothricin E acid, streptothricin D, and 12-carbamoyl streptothricin D. In addition, the disease control efficacy of the foliar application of JCK-6131 against tomato bacterial wilt was similar to that of the soil drench application, indicating that JCK-6131 could enhance defense resistance in plants. Molecular studies on tomato plants showed that JCK-6131 treatment induced the expression of the pathogenesis-related (PR) genes PR1, PR3, PR5, and PR12, suggesting the simultaneous activation of the salicylate (SA) and jasmonate (JA) signaling pathways. The transcription levels of PR genes increased earlier and were higher in treated plants than in untreated plants following Ralstonia solanacearum infection. These results indicate that Streptomyces sp. JCK-6131 can effectively control various plant bacterial and fungal diseases via two distinct mechanisms of antibiosis and induced resistance.

Mosaic Ends Tagmentation (METa) Assembly for Highly Efficient Construction of Functional Metagenomic Libraries.

Functional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naive and cultivation-independent exploration of metagenomes. However, preparation of these libraries is often limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, mosaic ends tagmentation (METa) assembly, for highly efficient functional metagenomic library preparation. We applied tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergized with homology-based assembly cloning to result in a 300-fold increase in cloning efficiency compared to traditional blunt-cloning-based protocols. We show that compared to published libraries prepared by state-of-the-art protocols, METa assembly is on average ca. 20- to 200-fold more efficient and can prepare gigabase-sized libraries with as little as 200 ng of input DNA. We show the usefulness of METa assembly first by using a normative 5-µg mass of soil metagenomic DNA to prepare a 700-Gb library that allowed us to discover novel nourseothricin resistance genes and a potentially new mode of resistance to this antibiotic and second by using only 300 ng of goose fecal metagenomic DNA to prepare a 27-Gb library that captured numerous tetracycline and colistin resistance genes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low-biomass or scarce microbiomes. IMPORTANCE Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing, but precise annotation is often limited to characterized genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes. The process of preparing these libraries is DNA greedy and inefficient, however. Here, we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and a goose fecal microbiome and allowed us to discover new antibiotic resistance genes and mechanisms. This library preparation method will potentially allow the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such as biomedical swabs or exotic samples.

Production of a broad spectrum streptothricin like antibiotic from halotolerant Streptomyces fimbriatus isolate G1 associated with marine sediments.

Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.

Draft genome sequence reveals co-occurrence of multiple antimicrobial resistance and plant probiotic traits in rice root endophytic strain Burkholderia sp. LS-044 affiliated to Burkholderia cepacia complex.

OBJECTIVES: Members of the Burkholderia cepacia complex (Bcc) have been isolated from various environmental and clinical samples and reportedly pose a threat to human health. Here we examine the draft genome sequence of Burkholderia sp. LS-044, an antibiotic-resistant endophytic strain affiliated to the Bcc (ST895) inhabiting rice (Oryza sativa ssp. japonica cv. Tainung 71) root. METHODS: Antimicrobial susceptibility of LS-044 was evaluated comparatively with other Burkholderia sp. (CC-Al74 and CC-3XP9) using commercial ATB PSE 5 test strips. The genome of LS-044 was sequenced using an Illumina MiSeq platform. Plant probiotic and antimicrobial resistance genes were screened by Rapid Annotation using Subsystem Technology (RAST), CARD 2017, NCBI and/or UniProt. RESULTS: Plant-associated members of Bcc (LS-044 and CC-Al74) exhibited greater resistance to the majority of antibiotics tested. The draft genome sequence of LS-044 contained 8.78 Mbp in 62 contigs having a G + C content of 66.5%, 8868 coding sequences and 75 RNAs. The genome harboured genes coding for LysR-type ß-lactamase transcription regulator, classes A, C and D ß-lactamases, several metal-dependent ß-lactamases, antibiotic efflux proteins, and proteins conferring resistance to colistin, streptothricin, colicin and fluoroquinolones. Similarly, it also possessed genes for copper homeostasis, copper-cobalt-zinc-cadmium-chromium resistance and reduction of mercury. Genes involved in flagellar motility, hydrolysis of murein and chitin, production of siderophore and auxin, and metabolism of aromatic compounds were also found. CONCLUSION: Genome sequence data revealed an interlinked occurrence of plant probiotic traits and antimicrobial resistance in the rice root endophyte LS-044.

The streptothricin acetyltransferase (sat) gene as a positive selectable marker for methanogenic archaea.

A repertoire of sophisticated genetic tools has significantly enhanced studies of Methanosarcina genera, yet the lack of multiple positive selectable markers has limited the types of genetic experiments that can be performed. In this study, we report the development of an additional positive selection system for Methanosarcina that utilizes the antibiotic nourseothricin and the Streptomyces rochei streptothricin acetyltransferase (sat) gene, which may be broadly applicable to other groups of methanogenic archaea. Nourseothricin was found to inhibit growth of four different methanogen species at concentrations ≤300 µg/ml in liquid or on solid media. Selection of nourseothricin resistant transformants was possible in two genetically tractable Methanosarcina species, M. acetivorans and M. barkeri, using the sat gene as a positive selectable marker. Additionally, the sat marker was useful for constructing a gene deletion mutant strain of M. acetivorans, emphasizing its utility as a second positive selectable marker for genetic analyses of Methanosarcina genera. Interestingly, two human gut-associated methanogens Methanobrevibacter smithii and Methanomassillicoccus luminyensis were more sensitive to nourseothricin than either Methanosarcina species, suggesting the nourseothricin-sat gene pair may provide a robust positive selection system for development of genetic tools in these and other methanogens.

Insights into the Function of the N-Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis.

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

Promotion Effect of Streptothricin on a Glucose Oxidase Enzymatic Reaction and Its Application to a Colorimetric Assay.

Previously, we reported that ε-poly-L-lysine (25 - 35 residues) significantly promoted a glucose oxidase enzymatic (GOx) reaction using ferricyanide ion as the oxidant, and that the effect was due to the formation of a polyion complex between anionic GOx and protonated (polycationic) ε-poly-L-lysine. Here, we show that streptothricins (STs), which have an L-ß-lysine oligomer (1 - 7 residues) and possess only several positive charges at most, also effectively promote the GOx enzymatic reaction. Interestingly, the promotion effect increased with the size of the lysine oligomer of STs, suggesting that the ionic valence is a key factor determining the degree of the promotion effect. The GOx enzymatic reaction is accompanied by a color change due to the reduction of yellow ferricyanide ion to a colorless reductant. A more distinctive color change can be achieved by the addition of Fe(III) ions due to the formation of Prussian blue. Thus, the promotion effect allowed for colorimetric detection of STs at the 1 mg/L level. The detection method was simple and easy to carry out, and would become a helpful tool for the detection of STs.

Functional analysis of methyltransferases participating in streptothricin-related antibiotic biosynthesis.

Streptothricin (ST) and its related compounds produced by Streptomyces strains are broad-spectrum antibiotics that consist of carbamoylated d-gulosamine, amino-acid side chain, and streptolidine lactam moieties. BD-12, a streptothricin-related antibiotic, has a glycine-derived side chain and two N-methyl groups, whereas ST-F carrying the l-ß-lysine side chain has no methyl group. In our previous studies, we identified and characterized the BD-12 and ST biosynthetic gene clusters. Here we report the functional analysis of two methyltransferase genes (orf 6 and orf 13) in the BD-12 biosynthetic gene cluster. Combinatorial biosynthesis using these two methyltransferase genes and the ST biosynthetic gene cluster resulted in the production of three methylated forms of ST-F. Among them, N,N'-dimethyl-ST-F, a novel compound generated in the present study, showed bacteria-specific antibiotic activities, although ST-F exhibits antibiotic activities against both prokaryotes and eukaryotes. Our findings also demonstrated that the orf 6 and orf 13 genes are responsible for the N-methylations of the amide bonds in the streptolidine lactam and in the amino-acid side chain linkage, respectively, and that N-methyl modification of the streptolidine lactam confers resistance in part against an ST hydrolase, SttH.

In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation.

Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA+ restored streptothricin resistance to B. subtilissatA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 µM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilissatA+ in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis The initial characterization of the enzyme provides insights into its mechanism of catalysis.

Streptothricin resistance as a novel selectable marker for transgenic plant cells.

Streptothricins are known as antimicrobial agents produced by Streptomyces spp. Bacterial resistance to streptothricin is mediated by specific enzymes exhibiting an acetyltransferase activity which renders the drug non-toxic for bacteria. The nucleotide sequence of several streptothricin resistance genes from bacteria have been described. Certain cells of eukaryotic parasites (such as Ustilago maydis or Leishmania spp.) are sensitive to streptothricin and the introduction of the bacterial resistance gene sat2 renders them resistant. We show that numerous species of plants are sensitive to low concentrations of streptothricin. Moreover, introduction of the bacterial resistance gene sat3 under the control of the 35S cauliflower mosaic virus promoter protects these cells from the toxic action of streptothricin. Therefore, sat3-mediated streptothricin resistance appears to be a promising selective marker for genetic manipulation of plant cells.