大震災が促進したコミュニティ・デベロップメント : 中華系コミュニティにみる「地域化」と「国際化」 (「被災外国人の研究」グループ)

The DNA recombination and repair machinery of Mycoplasma pneumoniae is composed of a limited set of approximately 11 proteins. Two of these proteins were predicted to be encoded by neighboring open reading frames (ORFs) MPN340 and MPN341. Both ORFs were found to have sequence similarity with genes that encode proteins belonging to the DNA helicase superfamily 1 (SF1). Interestingly, while a homolog of the MPN341 ORF is present in the genome of Mycoplasma genitalium (ORF MG244), MPN340 is an M. pneumoniae-specific ORF that is not found in other mycoplasmas. Moreover, the length of MPN340 (1590 base pairs [bp]) is considerably shorter than that of MPN341 (2148 bp). Examination of the MPN340-encoded amino acid sequence indicated that it may lack a so-called 2B subdomain, which is found in most SF1 DNA helicases. Also, the MPN340-encoded amino acid sequence was found to differ between subtype 1 strain M129 and subtype 2 strain FH at three amino acid positions. Both protein variants, which were termed PcrA(s) M129 and PcrA(s) FH, respectively, as well as the MPN341- and MG244-encoded proteins (PcrA Mpn and PcrA Mge , respectively), were purified, and tested for their ability to interact with DNA. While PcrA Mpn and PcrA Mge were found to bind preferentially to single-stranded DNA, both PcrA(s) M129 and PcrA(s) FH did not demonstrate significant DNA binding. However, all four proteins were found to have divalent cation- and ATP-dependent DNA helicase activity. The proteins displayed highest activity on partially double-stranded DNA substrates carrying 3' single-stranded extensions

Lugdunomycin, an Angucycline-Derived Molecule with Unprecedented Chemical Architecture

The angucyclines form the largest family of polycyclic aromatic polyketides, and have been studied extensively. Herein, we report the discovery of lugdunomycin, an angucycline-derived polyketide, produced by Streptomyces species QL37. Lugdunomycin has unique structural characteristics, including a heptacyclic ring system, a spiroatom, two all-carbon stereocenters, and a benzaza-[4,3,3]propellane motif. Considering the structural novelty, we propose that lugdunomycin represents a novel subclass of aromatic polyketides. Metabolomics, combined with MS-based molecular networking analysis of Streptomyces sp. QL37, elucidated 24 other rearranged and non-rearranged angucyclines, 11 of which were previously undescribed. A biosynthetic route for the lugdunomycin and limamycins is also proposed. This work demonstrates that revisiting well-known compound families and their producer strains still is a promising approach for drug discovery

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era

The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described

Unravelling key enzymatic steps in C-ring cleavage during angucycline biosynthesis

Abstract Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products

PcrA^s_FH preferentially unwinds DNA substrates with a 3′ ss terminus.

<p>(A) DNA helicase activity is not significantly influenced by the position of the fluorescent label in the DNA substrate. DNA helicase assays were performed with PcrA^s_FH and either substrate ‘a’ (6-FAM-labelled at either the 3′ end [lanes 1 and 2] or 5′ end [lanes 3 and 4] of strand 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone-0070870-g001" target="_blank">Fig. 1</a>]) or substrate ‘b’ (6-FAM-labelled at either the 3′ end [lanes 5 and 6] or 5′ end [lanes 7 and 8] of strand 1). The DNA helicase reactions were carried out in volumes of 10 µl and contained DNA substrate (at 8 nM) and either 0 nM (marked ‘−’, lanes 1, 3, 5 and 7) or 12 nM of protein (‘+’, lanes 2, 4, 6 and 8). After the reaction (10 min at 37°C), the samples were deproteinized, electrophoresed through a native 12% polyacrylamide gel, and analyzed by fluorometry. The positions of the substrates (s) and reaction products (p) are indicated at the right-hand side of the gels. (B) PcrA_s^FH preferentially unwinds branched DNA substrates that carry a 3′ ss terminus. The DNA helicase activity of PcrA^s_FH was tested on branched DNA substrates carrying either a 3′ ss terminus (substrate ‘d’, lanes 1 and 2), a 5′ ss terminus (substrate ‘e’, lanes 3 and 4) or blunt ends (substrate ‘f’, lanes 5 and 6). The sequences and structures of the different substrates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone-0070870-g001" target="_blank">Fig. 1</a>. The reactions were performed in a similar fashion as described in (A).</p

ATPase rates of the PcrA helicases from <i>M. pneumoniae</i> and <i>M. genitalium</i>.

a<p>The ATPase rates were determined either in the presence (+ ssDNA) or absence (- ssDNA) of φX174 virion DNA, as described in Materials and methods. The data represent averages (with standard deviation [SD]) from two independent measurements. Basal rates of ATP hydrolysis, which were determined in the absence of protein, were subtracted from the data.</p>b<p>The average stimulation shows the average fold stimulation of the ATPase rate by ssDNA for each protein.</p>c<p>ND, not determined.</p

Sequences and structures of the DNA substrates used in this study.

<p>(A) Numbers, names and sequences of the oligonucleotides that were used to generate the DNA substrates shown in (B). (B) Schematic structures of the DNA substrates. Oligonucleotides are indicated as numbered lines. The numbers (in circles) correspond to the numbers of the oligonucleotides shown in (A). Substrates ‘a’ and ‘d’ contain a 3′ 24-nt ss extension, substrates ‘b’ and ‘e’ contain a 5′ 24-nt ss extension, and substrates ‘c’ and ‘f’ are blunt-ended. The black dots represent 6-FAM labels at the 5′ end of the oligonucleotides.</p

Protein concentration- and time-dependence of the DNA helicase activity of PcrA^s_M129.

<p>(A) Protein concentration-dependence of the DNA helicase activity. The DNA unwinding reactions were performed as described in Materials and Methods, using 4 nM of substrate ‘a’ and a range of PcrA^s_M129 concentrations (0–100 nM, as indicated above the lanes). Lane 1 shows a control reaction in which protein was omitted, and incubation was performed for 5 min at 100°C instead of at 37°C. (A) Time course of the DNA helicase activity of PcrA^s_M129 (at 0.1 µM) using substrate ‘a’ (at 4 nM). Reactions were performed at 37°C for the times indicated above the lanes. The control reactions shown in lanes 9 (‘No protein’) and 10 (‘5′ 100°C’) were incubated for 5 min at 37°C before deproteinization of the samples.</p

Relative rate of the DNA helicase activities of PcrA^s_M129, PcrA^s_FH, PcrA<i>_Mpn</i> and PcrA<i>_Mge</i>.

a<p>The data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone-0070870-g006" target="_blank">Fig. 6</a> were fitted to estimate the time required to displace 50% of either substrate ‘a’ or substrate ‘b’. The results were then expressed as relative rates by comparison with the results obtained from the most efficient DNA helicase reaction, which included PcrA^s_FH and substrate ‘a’. The procedure used to calculate the relative rates was described by Soultanas et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone.0070870-Soultanas2" target="_blank">[48]</a>.</p

Protein concentration-dependence of the DNA helicase activities of the PcrA proteins.

<p>Reactions were carried out in a similar fashion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone-0070870-g004" target="_blank">Fig. 4</a>, and contained 4 nM of either substrate ‘a’ (□) or ‘b’ (▪) and a range of concentrations of either PcrA^s_M129 (A), PcrA^s_FH (B), PcrA<i>_Mpn</i> (C) or PcrA<i>_Mge</i> (D). The protein concentrations tested were 0 pM, 50 pM, 0.2 nM, 0.8 nM, 3.2 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. DNA unwinding was quantified by determination of the percentage of free (labeled) oligonucleotide that was displaced from the DNA substrates. Quantification was performed using Quantity One software (Bio-Rad), after separation of reaction products on polyacrylamide gels. Data shown are the average of two independent experiments. Error bars indicate the standard deviation of the mean. In the inset at the right-hand corner of each graph, a detail is shown of the DNA helicase activity (%) at the lowest protein concentrations tested.</p