PCR快速鉴定actinobacteria三种模板制备方法的比较

目的 本研究旨在建立准确、简便、快速的放线细菌鉴定技术，为普通和极端环境放线细菌资源的调查和开发利用创造条件。方法 从放线细菌固体培养基上挑取少量菌体，用微波炉法快速制备基因组DNA作为PCR模板，与液体培养法得到的菌体以超声波法或冻融法制备的模板进行了PCR扩增效果的比较研究。结果P CR检测结果表明微波炉法制备的模板可进行有效的体外扩增，目的条带特异，而超声波法或冻融法并不对所有菌株有效，并有非特异扩增产物产生。结论 组合微波炉法快速制备放线细菌基因组DNA技术和23S rRNA特异插入序列PCR扩增技术建立了准确、简便、快速的actinobacteria鉴别体系。     

Antibiotic discovery: history,methods and perspectives

Antimicrobial resistance is considered a major public-health issue. Policies recommended by the World Health Organization (WHO) include research on new antibiotics. No new class has been discovered since daptomycin and linezolid in the 1980s, and only optimisation or combination of already known compounds has been recently commercialised. Antibiotics are natural products of soil-living organisms. Actinobacteria and fungi are the source of approximately two-thirds of the antimicrobial agents currently used in human medicine; they were mainly discovered during the golden age of antibiotic discovery. This era declined after the 1970s owing to the difficulty of cultivating fastidious bacterial species under laboratory conditions. Various strategies, such as rational drug design, to date have not led to the discovery of new antimicrobial agents. However, new promising approaches, e.g. genome mining or CRISPR-Cas9, are now being developed. The recent rebirth of culture methods from complex samples has, as a matter of fact, permitted the discovery of teixobactin from a new species isolated from soil. Recently, many biosynthetic gene clusters were identified from human-associated microbiota, especially from the gut and oral cavity. For example, the antimicrobial lugdunin was recently discovered in the oral cavity. The repertoire of human gut microbiota has recently substantially increased, with the discovery of hundreds of new species. Exploration of the repertoire of prokaryotes associated with humans using genome mining or newer culture approaches could be promising strategies for discovering new classes of antibiotics.     

Actinobacterial enzyme inhibitors – A review

Abstract

Actinobacteria have potential as important new sources of enzyme inhibitors. Enzyme inhibitors have great demand in medicine, agriculture and biotechnology. In medicine, enzyme inhibitors can be used as therapeutic agents for bacterial, fungal, viral and parasitic diseases as well as treating cancer, neurodegenerative, immunological and cardiovascular diseases. Enzyme inhibitors are also valuable for the control of carbohydrate-dependent diseases such as diabetes, obesity and hyperlipidemia and melanogenesis in skin. They can be also involved in crop protection against plant pathogens, herbivorous pests and abiotic stresses such as drought. In this review, we discuss about several actinobacterial enzyme inhibitors with various industrial uses and biotechnological applications.     

Actinobacteria challenge the paradigm: A unique protein architecture for a well-known,central metabolic complex

α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

The α-oxoacid dehydrogenase complexes constitute a family of three-partite, ubiquitous metabolic complexes devoted to the oxidative decarboxylation of α-oxoacids and the concomitant production of reducing equivalents in form of NADH (1). Three such complexes are known: the pyruvate dehydrogenase (PDH), that feeds the tricarboxylic acid (TCA) cycle with carbon units in form of acetyl-CoA; the 2-oxoglutarate dehydrogenase (ODH), part of the oxidative branch of the TCA cycle; and the branched chain α-ketoacid dehydrogenase (BCKDH), involved in the catabolism of aliphatic amino acids. These large tripartite complexes share a common molecular architecture organized around a core made by the E2 component, a flexible, multidomain protein which bears the acyltransferase activity required to transfer the acyl group from the decarboxylated substrate to the CoA-SH acceptor; the number of E2 subunits and the symmetry of the core depend on the complex and the species (1–5). First shown by the crystal structure of Azotobacter vinelandii E2p, the E2 C-terminal catalytic core assumes an obligate homotrimeric state much similar to chloramphenicol acetyltransferase (6), with which it also shares the catalytic mechanism (7). The observed, higher-order oligomerization states are made possible by intermolecular trimer–trimer interactions (TTIs) mediated by a well-conserved, C-terminal 3₁₀ hydrophobic helix which makes intermolecular symmetric interactions (5), then confirmed on other E2 enzymes and sometimes described as “knobs and sockets” (3). These interactions make symmetric, highly oligomeric states which adopt, in most cases, either an octahedral 432 symmetry, eight E2 homotrimers being positioned at the vertexes of a cube, or an icosahedral 532 symmetry, with 20 trimers assembled as a dodecahedron; the number of subunits depends on the complex and the species (1). More recently, the presence of an irregularly shaped, 42-mer E2 assembly was described in the archaeon Thermoplasma acidophilum (8), although this peculiar oligomeric state is still based on the same kind of interactions between the C-terminal helices. Thus, the oligomeric state of the core, responsible for the large size of the complex, was observed in all analyzed complexes and is a trend commonly accepted to be universally conserved in Eubacteria, Archaea, and Eukarya. While the reasons for the presence (and evolutionary conservation) of such huge macromolecular scaffolds remain unclear (9), active site coupling (transfer of acyl groups between lipoyl domains within the core) has often been proposed as the major advantage (1, 10). Also, despite the tripartite organization of these complexes as separate E1/E2/E3 enzymes had always been considered as universal, Corynebacterium glutamicum was shown to be deprived of an E2o component (specific to the ODH complex) and to rather possess an E2o succinyl transferase domain fused to E1o, in a protein called OdhA (11, 12). The same situation has then been confirmed for the model Mycobacterium smegmatis (13). As the lipoyl binding domain of E2o is absent from the E2o-E1o fusion, the ODH activity depends on functional lipoyl groups provided in trans and proven to be supplied by E2p from the PDH complex (14), therefore suggesting the presence of a mixed PDH/ODH supercomplex. By using an integrative structural biology approach, we describe here how C. glutamicum E2p, that was expected to serve as the core of the mixed complex, breaks the rule about the oligomeric state of acyltransferase E2 enzymes, reducing its size to the minimal, catalytically active trimeric unit. We also provide evidence supporting these features as a common trait of Actinobacteria.     

Evidence that preimmunization with a heat-killed preparation of Mycobacterium vaccae reduces corticotropin-releasing hormone mRNA expression in the extended amygdala in a fear-potentiated startle paradigm

Posttraumatic stress disorder (PTSD) is a trauma and stressor-related disorder that is characterized by dysregulation of glucocorticoid signaling, chronic low-grade inflammation, and impairment in the ability to extinguish learned fear. Corticotropin-releasing hormone (Crh) is a stress- and immune-responsive neuropeptide secreted from the paraventricular nucleus of the hypothalamus (PVN) to stimulate the hypothalamic-pituitary-adrenal (HPA) axis; however, extra-hypothalamic sources of Crh from the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) govern specific fear- and anxiety-related defensive behavioral responses. We previously reported that preimmunization with a heat-killed preparation of the immunoregulatory environmental bacterium Mycobacterium vaccae NCTC 11659 enhances fear extinction in a fear-potentiated startle (FPS) paradigm. In this follow-up study, we utilized an in situ hybridization histochemistry technique to investigate Crh, Crhr1, and Crhr2 mRNA expression in the CeA, BNST, and PVN of the same rats from the original study [Fox et al., 2017, Brain, Behavior, and Immunity, 66: 70–84]. Here, we demonstrate that preimmunization with M. vaccae NCTC 11659 decreases Crh mRNA expression in the CeA and BNST of rats exposed to the FPS paradigm, and, further, that Crh mRNA expression in these regions is correlated with fear behavior during extinction training. These data are consistent with the hypothesis that M. vaccae promotes stress-resilience by attenuating Crh production in fear- and anxiety-related circuits. These data suggest that immunization with M. vaccae may be an effective strategy for prevention of fear- and anxiety-related disorders.     

In silico prediction of potential metallothioneins and metallohistins in actinobacteria

Metallothioneins and metallohistins are short peptides with a high cysteine and/or histidine content able to coordinate metals intracellularly, thereby increasing the tolerance against elevated concentrations of metals. Because of their features, they can be detected by in silico prediction from proteomes annotated from sequenced genomes. Here, we analyzed 73 sequenced actinobacterial genomes for peptides (≤ 100 amino acids) with a high content of cysteine and histidine (≥ 15%) and identified 103 putative metallothioneins and metallohistins. For 45 of these peptides, we found similarities to metal binding protein domains, including zinc fingers, heavy metal transporters or eukaryotic metallothioneins, which can serve as proof-of-principle in underscoring a potential function as metal binding peptides. An evolutionary origin from metal containing domains of enzymes is discussed and metallohistins not containing cysteine are described for the first time for bacteria.     

Termite gas emissions select for hydrogenotrophic microbial communities in termite mounds

Organoheterotrophs are the dominant bacteria in most soils worldwide. While many of these bacteria can subsist on atmospheric hydrogen (H₂), levels of this gas are generally insufficient to sustain hydrogenotrophic growth. In contrast, bacteria residing within soil-derived termite mounds are exposed to high fluxes of H₂ due to fermentative production within termite guts. Here, we show through community, metagenomic, and biogeochemical profiling that termite emissions select for a community dominated by diverse hydrogenotrophic Actinobacteriota and Dormibacterota. Based on metagenomic short reads and derived genomes, uptake hydrogenase and chemosynthetic RuBisCO genes were significantly enriched in mounds compared to surrounding soils. In situ and ex situ measurements confirmed that high- and low-affinity H₂-oxidizing bacteria were highly active in the mounds, such that they efficiently consumed all termite-derived H₂ emissions and served as net sinks of atmospheric H₂. Concordant findings were observed across the mounds of three different Australian termite species, with termite activity strongly predicting H₂ oxidation rates (R² = 0.82). Cell-specific power calculations confirmed the potential for hydrogenotrophic growth in the mounds with most termite activity. In contrast, while methane is produced at similar rates to H₂ by termites, mounds contained few methanotrophs and were net sources of methane. Altogether, these findings provide further evidence of a highly responsive terrestrial sink for H₂ but not methane and suggest H₂ availability shapes composition and activity of microbial communities. They also reveal a unique arthropod–bacteria interaction dependent on H₂ transfer between host-associated and free-living microbial communities.

For most soil bacteria, organic rather than inorganic compounds serve as primary energy and carbon sources for growth (1, 2). Molecular hydrogen (H₂), while a major component of Earth’s early atmosphere and likely the first energy source for life (3, 4), currently has a secondary role in sustaining these bacteria (2). This reflects that contemporary concentrations of atmospheric H₂ (0.53 parts per million [ppm]) are thought to be insufficient for hydrogenotrophic growth to be thermodynamically favorable (5). Soil bacteria nevertheless consume much atmospheric H₂ (∼70 teragrams per year) and, as such, constitute the most important sink in the global H₂ cycle (6, 7). Bacteria from several dominant soil phyla consume atmospheric H₂ using high-affinity hydrogenases (group 1h [NiFe]-hydrogenases; apparent Michaelis-Menten half-saturation constant [K_m] < 100 nM) primarily to persist during organic carbon starvation (2, 8–12). In some soils, bacteria are exposed to elevated levels of H₂ produced as a result of microbial fermentation and nitrogen fixation, geological processes, and increasingly anthropogenic activities (13, 14). The effect of such H₂ exposure on community composition and activities has remained enigmatic. Within natural ecosystems, it has been reported that bacteria in close proximity to root nodules rapidly recycle nitrogenase-derived H₂ and use it to support hydrogenotrophic growth (15, 16). In microcosm experiments, elevated H₂ exposure stimulates the activity and growth of a small proportion of hydrogenotrophic bacteria (17–22). These bacteria encode lower-affinity hydrogenases (group 1d and 2a [NiFe]-hydrogenases; apparent K_m > 100 nM) with chemosynthetic RuBisCO lineages (type IC to IE) in order to use H₂ as an electron donor for aerobic respiration and CO₂ fixation (22, 23). Nevertheless, H₂ exposure has only minor effects on the abundance, diversity, and composition of communities during the moderate time courses of these experiments, indicating it remains a secondary energy source and weak selective pressure (19–22).Termite mounds are underexplored soil-derived environments where microbial communities are exposed to greatly elevated levels of gases such as H₂. In anoxic environments, such as animal gastrointestinal tracts or marine sediments, fermentation supplies sufficient H₂ for a multitude of intra- and interspecies metabolic pathways, including lithoautotrophy (14, 24, 25). However, H₂ accumulation is rarely observed due to rapid turnover and tightly coupled production and consumption (14, 26). The gastrointestinal tracts of termites are an exception. H₂ is the central intermediate during microbial digestion of lignocellulose and is produced at concentrations comparable to geothermal sources (27–29), such that termites have even been explored for biofuel production (30). Most H₂ is consumed by symbiotic gut bacteria, which produce volatile fatty acids absorbed by termites (reviewed in ref. 31), and some is used by methanogens and emitted as methane (CH₄) (32, 33). Yet considerable amounts of H₂ leak into the environment. Termites emit H₂ and CH₄ at rates of up to 1.5 µmol H₂ ⋅ g⁻¹ ⋅ h⁻¹ and up to 1 µmol CH₄ ⋅ g⁻¹ ⋅ h⁻¹, with vast differences between feeding groups and species (34). As such, termites are recognized as a globally relevant source of atmospheric CH₄ emissions (35) and may also contribute to H₂ emissions (36). However, termite mounds harbor bacterial communities that can mitigate emissions. Specialized mound-associated communities of methanotrophic Proteobacteria consume approximately half of all CH₄ produced by termites (37–39). One historical study indicates H₂ is also oxidized by mounds (40). Due to high rates of termite respiration, CO₂ concentrations of up to 8% are also observed in termite mounds (41) and may enhance lithoautotrophic growth.Microbial communities of termite mounds are entirely distinct from those of termite guts (42–46). Whereas primarily anaerobic fermentative Firmicutes, Bacteroidota, and Spirochaetota dominate in guts, potentially aerobic respiratory Actinobacteriota, Proteobacteria, and Acidobacteriota reside in mounds. Mound and soil communities appear to be closely related, reflecting mound material is primarily derived from surrounding soil. However, Actinobacteriota tend to be more abundant in mounds than soils (45, 46), albeit with some exceptions reported (47). A cultivation-based study suggests some of these bacteria may serve as defensive symbionts for termites, specifically fungus-growing species, by producing antimicrobial compounds (48). Yet the factors that select for actinobacterial growth have yet to be resolved (45). Members of this phylum have recently been recognized for their ability to scavenge atmospheric H₂ (9, 49, 50) and often grow during H₂-enriched microcosm experiments (19, 22). On this basis, we hypothesized that sustained H₂ and CO₂ emissions from termites may select for communities dominated by hydrogenotrophic Actinobacteriota. In this study, we addressed these knowledge gaps by investigating the composition, capabilities, and activities of mound-associated bacterial communities. We profiled mounds and surrounding soils of three common mound-building termite species, namely, the wood-feeding Microcerotermes nervosus (Mn), soil-interface feeding Macrognathotermes sunteri (Ms), and grass-feeding Tumulitermes pastinator (Tp), representing the dominant feeding groups of Australian termites. We show that termite emissions have selected for highly abundant and active hydrogenotrophic communities, whereas methanotrophic bacteria remain rare despite elevated substrate availability.     

Future potential for anti-infectives from bacteria – How to exploit biodiversity and genomic potential

The early stages of antibiotic development include the identification of novel hit compounds. Since actinomycetes and myxobacteria are still the most important natural sources of active metabolites, we provide an overview on these producers and discuss three of the most promising approaches toward finding novel anti-infectives from microorganisms. These are defined as the use of biodiversity to find novel producers, the variation of culture conditions and induction of silent genes, and the exploitation of the genomic potential of producers via “genome mining”. Challenges that exist beyond compound discovery are outlined in the last section.     

Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method

The Actinobacterium Gordonia rubripertincta CWB2 (DSM 46758) produces hydroxamate-type siderophores (188 mg L⁻¹) under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth which was confirmed by the Fe-CAS assay. Elution profile and its absorbance spectrum were similar to those of commercial (des)ferrioxamine B which was used as reference compound. This confirms previously made assumptions and shows for the first time that the genus Gordonia produces desferrioxamine-like siderophores. The reversed-phase HPLC protocol was optimized to separate metal-free and -loaded oxamines. This allowed to determine siderophore concentrations in solutions as well as metal affinity. The metal loading of oxamines was confirmed by ICP-MS. As a result, it was demonstrated that desferrioxamine prefers trivalent metal ions (Fe³⁺ > Ga³⁺ > V³⁺ > Al³⁺) over divalent ones. In addition, we aimed to show the applicability of the newly established reversed-phase HPLC protocol and to increase the re-usability of desferrioxamines as metal chelators by immobilization on mesocellular silica foam carriers. The siderophores obtained from strain CWB2 and commercial desferrioxamine B were successfully linked to the carrier with a high yield (up to 95%) which was verified by the HPLC method. Metal binding studies demonstrated that metals can be bound to non-immobilized and to the covalently linked desferrioxamines, but also to the carrier material itself. The latter was found to be unspecific and, therefore, the effect of the carrier material remains a field of future research. By means of a reversed CAS assay for various elements (Nd, Gd, La, Er, Al, Ga, V, Au, Fe, As) it was possible to demonstrate improved Ga³⁺- and Nd³⁺-binding to desferrioxamine loaded mesoporous silica carriers. The combination of the robust reversed-phase HPLC method and various CAS assays provides new avenues to screen for siderophore producing strains, and to control purification and immobilization of siderophores.     

Actinobacteria分离株的多相分类学研究

Actinobacteria classnov．一般包括具有超过50%G＋C的DNA碱基组成的微生物，其中一些菌种在生物技术和医药方面具有重要的意义。为了分离能够产生有用化合物的微生物菌种，我们将分离对象从已进行过深入研究的Streptomyces及其以外的放线菌扩大到所有的Actinobacteria，其中包括那些具有普通细菌形态的高G＋C含量革兰阳性细菌。为了对Actinobacteria这一较新的研究领域有详细的了解，我们又对两株有弱生物活性的IMB02B-165和IMB02B-172进行了多相分类学研究。化学分类和系统分类的结果表明IMB02B-165、IMB02B-172菌株均属于Arthrobacter属中Arthrobacter globiformis／Arthrobacter citreus组。