Lessons from studies of Symbiobacterium thermophilum, a unique syntrophic bacterium

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a cognate Bacillus sp. We have been studying the unique features of S. thermophilum in terms of taxonomy, ecology, genome biology, and physiology. Here we overview current knowledge of this bacterium. Although S. thermophilum shows several physiological properties of Gram-negative bacteria, 16S rRNA gene-based phylogeny indicated that it represents a distinct lineage of Gram-positive bacteria with deep branching between the clades for the high-G+C (Actinobacteria) and the low-G+C (Firmicutes) groups. Ecological study has revealed that S. thermophilum and its relatives are widely distributed in the natural environment, including soil, animal intestines and seawater. A whole genome sequencing study uncovered its unusual features, which overall indicate that this bacterium is a member of Firmicutes despite of its high G+C content (68.7%). The genome appeared to retain fully the genes for primary metabolism, except for carbonic anhydrase. We discovered that carbon dioxide is a marked inducer of the mono-growth of S. thermophilum, and speculated that this is due to a lack of carbonic anhydrase. The lines of evidence suggest that S. thermophilum requires additional conditions for full growth, including not only the supply of an unknown positive factor but also the elimination of oxygen and self-growth inhibitory substances. We conclude that the role of the cognate Bacillus is to establish a complex environment suitable for the growth of S. thermophilum, which is achieved by supplying and removing multiple factors. Understandings of this type of mutualism should provide new insight into microbial physiology as well as the issue of uncultivability.     

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.     

CO2 supply induces the growth of Symbiobacterium thermophilum, a syntrophic bacterium

Symbiobacterium thermophilum is a unique syntrophic bacterium that exhibits marked growth only in coculture with a cognate Bacillus sp. In this study, we found that the bacterium is capable of marked mono-growth when supplied with CO2 or bicarbonate. The evidence suggests that the genetic defect for carbonic anhydrase in this bacterium is a reason for the syntrophic property based on CO2 requirement.     

Identification of Indole Derivatives as Self-Growth Inhibitors of Symbiobacterium thermophilum, a Unique Bacterium Whose Growth Depends on Coculture with a Bacillus sp.

Symbiobacterium thermophilum is a syntrophic bacterium whose growth depends on coculture with a Bacillus sp. Recently, we discovered that CO₂ generated by Bacillus is the major inducer for the growth of S. thermophilum; however, the evidence suggested that an additional element is required for its full growth. Here, we studied the self-growth-inhibitory substances produced by S. thermophilum. We succeeded in purifying two substances from an ether extract of the culture supernatant of S. thermophilum by multiple steps of reverse-phase chromatography. Electron ionization mass spectrometry and nuclear magnetic resonance analyses of the purified preparation identified the substances as 2,2-bis(3′-indolyl)indoxyl (BII) and 1,1-bis(3′-indolyl)ethane (BIE). The pure growth of S. thermophilum was inhibited by authentic BII and BIE with MICs of 12 and 7 μg/ml, respectively; however, its growth in coculture with Bacillus was not inhibited by BII at the saturation concentration and was inhibited by BIE with an MIC of 14 μg/ml. Both BII and BIE inhibited the growth of other microorganisms. Unexpectedly, the accumulation levels of both BII and BIE in the pure culture of S. thermophilum were far lower than the MICs (<0.1 μg/ml) while a marked amount of BIE (6 to 7 μg/ml) equivalent to the MIC had accumulated in the coculture. An exogenous supply of surfactin alleviated the sensitivities of several BIE-sensitive bacteria against BIE. The results suggest that Bacillus benefits S. thermophilum by detoxifying BII and BIE in the coculture. A similar mechanism may underlie mutualistic relationships between different microorganisms.     

Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain.

Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.     

Development of a membrane dialysis bioreactor and its application to a large-scale culture of a symbiotic bacterium,Symbiobacterium thermophilum

A simple membrane dialysis bioreactor was developed for a large-scale axenic culture of Symbiobacterium thermophilum, a symbiotic thermophile that requires co-cultivation with an associating thermophilic Bacillus strain S for normal growth. The bioreactor consisted of an outer- and an inner-coaxial cylindrical compartment bordered across a dialyzing membrane, which enabled a 1 l-scale dialysis culture with exchange of low molecular metabolites between the two compartments to be performed. Using the bioreactor, growth characteristics of S. thermophilum and Bacillus strain S were assessed under two medium conditions. The growth of S. thermophilum was measured by quantitative PCR because the bacterium formed no visible colonies and gave abnormally low turbidity. In medium containing 2% tryptone peptone, S. thermophilum proliferated up to 4x10(7) cells/ml, and strict dependence on the co-culture with Bacillus strain S was observed. On the other hand, medium containing 0.5% yeast extract not only facilitated the growth of S. thermophilum in the co-culture (6x10(7) cells/ml), but also allowed limited pure growth independent of Bacillus strain S (1x10(7) cells/ml), implying that some component of yeast extract can partially replace the growth requirement of S. thermophilum supplied by Bacillus strain S. Both the oxidative redox potential values and the cell morphology in the independently growing culture suggested the occurrence of marked unbalanced growth possibly caused by significant metabolic changes. The bioreactor is applicable to the analyses of culturing characteristics in symbiotic systems between free-living microorganisms.     

Design,synthesis and evaluation of novel indole derivatives as AKT inhibitors

Herein, we describe the discovery and synthesis of a new series of 1,2,4,7-tetra-substituted indole derivatives as novel AKT inhibitors by optimization of a weak hit methyl 4-(2-aminoethoxy)-1H-indole-2-carboxylate (1). Both representative compounds 6a and 6o exhibited the most potent inhibitory activities against AKT1, with inhibition rates of 72.5% and 78.6%, respectively, at concentrations of 10 nM. In addition, compounds 6a and 6o also potently inhibited the phosphorylation of the downstream GSK3 protein and displayed slightly better anti-proliferative activities in a prostate cancer cell line.     

Design,synthesis and biological evaluation of indole derivatives as Vif inhibitors

The crystal structure of viral infectivity factor (Vif) was reported recently, which makes it possible to design new inhibitors against Vif by structure-based drug design. Through analysis of the protein surface of Vif, the C2 pocket located in the N-terminal was found, which is suit for developing small molecular inhibitors. Then, in our article, fragment-based virtual screening (FBVS) was conducted and a series of fragments was obtained, among which, Zif-1 bearing indole scaffold and pyridine ring can form H-bonds with Tyr148 and Ile155. Subsequently, 19 derivatives of Zif-1 were synthesized. Through the immune-fluorescence staining and Western blot assays, Zif-15 shows potent activity in inhibiting Vif-mediated A3G degradation. Further docking experiment shows that Zif-15 form H-bond interactions with residues His139, Tyr148 and Ile155. Therefore, Zif-15 is a promising lead compound against Vif that can be used to treat AIDS.     

Oxygen-dependent growth of the sulfate-reducing bacterium Desulfovibrio oxyclinae in coculture with Marinobacter sp. Strain MB in an aerated sulfate-depleted chemostat

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae and the facultatively aerobic heterotroph Marinobacter sp. strain MB was grown for 1 week under anaerobic conditions at a dilution rate of 0.05 h(-1). It was then exposed to an oxygen flux of 223 micromol min(-1) by gassing the growth vessel with 5% O(2). Sulfate reduction persisted under these conditions, though the amount of sulfate reduced decreased by 45% compared to the amount reduced during the initial anaerobic mode. After 1 week of growth under these conditions, sulfate was excluded from the incoming medium. The sulfate concentration in the growth vessel decreased exponentially from 4.1 mM to 2.5 microM. The coculture consumed oxygen effectively, and no residual oxygen was detected during either growth mode in which oxygen was supplied. The proportion of D. oxyclinae cells in the coculture as determined by in situ hybridization decreased from 86% under anaerobic conditions to 70% in the microaerobic sulfate-reducing mode and 34% in the microaerobic sulfate-depleted mode. As determined by the most-probable-number (MPN) method, the numbers of viable D. oxyclinae cells during the two microaerobic growth modes decreased compared to the numbers during the anaerobic growth mode. However, there was no significant difference between the MPN values for the two modes when oxygen was supplied. The patterns of consumption of electron donors and acceptors suggested that when oxygen was supplied in the absence of sulfate and thiosulfate, D. oxyclinae performed incomplete aerobic oxidation of lactate to acetate. This is the first observation of oxygen-dependent growth of a sulfate-reducing bacterium in the absence of either sulfate or thiosulfate. Cells harvested during the microaerobic sulfate-depleted stage and exposed to sulfate and thiosulfate in a respiration chamber were capable of anaerobic sulfate and thiosulfate reduction.     

Design,synthesis and biological evaluation of lapachol derivatives possessing indole scaffolds as topoisomerase I inhibitors

A series of novel lapachol derivatives possessing indole scaffolds was designed and synthesized. The in vitro anti-proliferative activity of these novel compounds was evaluated in Eca109 and Hela cell lines. Almost all the tested compounds showed manifested potent inhibitory activity against the two tested cancer cell lines. Topo I-mediated DNA relaxation activity indicated that these novel compounds have potent Topoisomerase I inhibition activity. The most potent compounds 4n and 4k demonstrated more cytotoxicity than camptothecin and was comparable to camptothecin in inhibitory activities on Topoisomerase I in our biological assay. In addition, the Hoechst 33342 staining method also showed that the complex can induce Hela cell apoptosis.     

Substituted derivatives of indole acetic acid as aldose reductase inhibitors with antioxidant activity: structure-activity relationship

Although multiple biochemical pathways are likely to be responsible for the pathogenesis of diabetic complications, substantial evidence suggests a key role for the polyol pathway and oxidative stress initiated by hyperglycemia. Thus aldose reductase, the first enzyme of the polyol pathway, has been identified as a potential target of pharmacological intervention to prevent diabetic complications. Aldose reductase inhibitors endowed with antioxidant activity would be dually beneficial. The aim of the study was to evaluate the structure-activity relationship of commercially available indole derivatives supported by the molecular modeling of their interaction with the enzyme aldose reductase from the viewpoint of the inhibitory effect on the enzyme and their antioxidant activity. The partially purified aldose reductase was prepared from rabbit eye lenses. In vitro inhibiton of the aldose reductase was determined by a conventional method. Antioxidant action of the compounds was documented in a DPPH test. Marked differences were recorded in the aldose reductase inhibition activities of 1- and 3-indole acetic acid derivatives. The interaction energies of the inhibitor vs. enzyme-NADP(+) complexes, calculated by computer aided molecular modeling, were in agreement with the higher inhibitory efficacy of 1-indole acetic acid in contrast with 3-indole acetic acid. The more efficient 1-indole acetic acid was proved to create stronger electrostatic interaction with NADP(+). However, the order of the antioxidant activities of the compounds studied was not in agreement with that of the inhibitory efficacies.     

Synthesis and biological evaluations of oleanolic acid indole derivatives as hyaluronidase inhibitors with enhanced skin permeability

Oleanolic acid (OA) is a natural cosmeceutical compound with various skin beneficial activities including inhibitory effect on hyaluronidase but the anti-hyaluronidase activity and mechanisms of action of its synthetic analogues remain unclear. Herein, a series of OA derivatives were synthesised and evaluated for their inhibitory effects on hyaluronidase. Compared to OA, an induction of fluorinated (6c) and chlorinated (6g) indole moieties led to enhanced anti-hyaluronidase activity (IC₅₀ = 80.3 vs. 9.97 and 9.57 µg/mL, respectively). Furthermore, spectroscopic and computational studies revealed that 6c and 6g can bind to hyaluronidase protein and alter its secondary structure leading to reduced enzyme activity. In addition, OA indole derivatives showed feasible skin permeability in a slightly acidic environment (pH = 6.5) and 6c exerted skin protective effect by reducing cellular reactive oxygen species in human skin keratinocytes. Findings from the current study support that OA indole derivatives are potential cosmeceuticals with anti-hyaluronidase activity.     

一株具霉菌抑制活性细菌的鉴定

为探讨菌株HB02对霉菌生长的影响,将HB02与霉菌孢子悬液同时加入到PYG肉汤,在28℃培养15d。在培养的第3、69、、12和15天测定培养液中的黄曲霉和禾谷镰刀菌菌丝重量。结果显示:与对照组相比,HB02可显著抑制黄曲霉和禾谷镰刀菌生长(P<0.01)。通过常规细菌学实验鉴定该菌株为一株乳酸杆菌。通过分子生物学方法测定了菌株的16S rRNA基因序列,进一步证实了HB02为一株乳酸杆菌。根据Berthier等的方法,通过HB0216S/23S rRNA基因间隔(Intergenic Spacer Regions,ISR)序列的扩增和测定,再通过基因文库中所有细菌基因序列比较分析,最终鉴定菌株HB02为一株弯曲乳酸杆菌(Lactobacillus curvatus)。     

Iantherans A and B, unique dimeric polybrominated benzofurans as Na,K-ATPase inhibitors from a marine sponge, Ianthella sp

Two novel tetrabrominated benzofuran derivatives, named iantherans A and B, were isolated from an Australian marine sponge of the genus lanthella. The unique structures comprised of 2,3-bis(sulfooxy)-1,3-butadiene and two brominated benzofuran moieties were determined by spectroscopic and chemical methods. Iantheran A has a (Z,Z)-1,3-butadiene moiety, whereas iantheran B is the geometric isomer possessing a (Z,E)-1,3-butadiene moiety. The inhibitory activities of the iantherans and their derivatives against Na,K-ATPase as well as the efficacy of iantheran A against other several enzymes were evaluated.     

Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors

A homogenous TR-FRET-based in vitro coupling assay for the MAP3Ks–MEK1–ERK2 kinase cascade was established and was used to screen for inhibitors of the ERK/MAPK pathway. A series of coumarin derivatives were identified from the screen. These compounds potently inhibit the activation of the unactivated human MEK1 by upstream MAP3Ks (including BRAF and COT), but do not inhibit the activity of the activated MEK1. In addition, the potency of these compounds in inhibiting MEK1 activation is not affected by varying the ATP concentration, suggesting that these inhibitors are not competitive with ATP. As expected, the coumarin compounds potently inhibit LPS-induced TNF production and ERK phosphorylation in THP-1 cells, with the most potent compound having an IC₅₀ of 90 nM. Molecular modeling studies suggest that these coumarins bind to an allosteric site in the inactive conformation of MEK1. This site has been shown to be utilized by the biarylamine series of MEK inhibitors such as PD318088. Very interestingly, the identified coumarin derivatives are almost identical to a series of inhibitors recently reported that block LPS-induced TNF production. Our findings have therefore raised the possibility that other naturally occurring or synthetic coumarins with anti-cancer and anti-inflammatory activities might exert their biological function through the inhibition of MEK1.     

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 degrees C were approximately 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by approximately 0.2 M and approximately 5 degrees C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 degrees C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families.     

Indeno[1,2-b]indole derivatives as a novel class of potent human protein kinase CK2 inhibitors

Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a convenient and straightforward synthesis protocol. The compounds were tested for inhibition of human protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC(50) in the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-dione) inhibited human CK2 with an IC(50) of 0.11 μM and did not significantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive inhibition by compound 4b was shown and a K(i) of 0.06 μM was determined. Our findings indicate that indeno[1,2-b]indoles are a promising starting point for further development and optimization of human protein kinase CK2 inhibitors.     

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).     

Identification of isothiazole-4-carboxamidines derivatives as a novel class of allosteric MEK1 inhibitors

The development of potent, orally bioavailable, and selective series of 5-amino-3-hydroxy-N(1-hydroxypropane-2-yl)isothiazole-4-carboxamidine inhibitors of MEK1 and MEK-2 kinase is described. Optimization of the carboxamidine and the phenoxyaniline group led to the identification of 55 which gave good potency as in vitro MEK1 inhibitors, and good oral exposure in rat.     

一株纤维蛋白溶解酶产生菌的鉴定及其产酶条件初步研究

从亚洲传统发酵食品--虾酱中筛选到一株产纤维蛋白溶解酶能力较强的菌株,通过形态和常规生理生化性质鉴定,发现该菌株与芽孢杆菌属细菌的特征很相近,结合16S rDNA序列分析,构建系统发育树,确定其分类地位,由中国典型培养物保藏中心定名为Bacillus sp.nov.SK006(CCTCC No.M 205071),并优化了发酵培养基组成及培养条件,本研究为该菌株的深入研究和广泛应用提供了理论依据.