Actinomycete Bacteria Research Articles

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies.

Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

Cellulose degradation by polysaccharide monooxygenases.

Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity.

Interaction specificity between leaf-cutting ants and vertically transmitted Pseudonocardia bacteria.

BackgroundThe obligate mutualism between fungus-growing ants and microbial symbionts offers excellent opportunities to study the specificity and stability of multi-species interactions. In addition to cultivating fungus gardens, these ants have domesticated actinomycete bacteria to defend gardens against the fungal parasite Escovopsis and possibly other pathogens. Panamanian Acromyrmex echinatior leaf-cutting ants primarily associate with actinomycetes of the genus Pseudonocardia. Colonies are inoculated with one of two vertically transmitted phylotypes (Ps1 or Ps2), and maintain the same phylotype over their lifetime. We performed a cross-fostering experiment to test whether co-adaptations between ants and bacterial phylotypes have evolved, and how this affects bacterial growth and ant prophylactic behavior after infection with Escovopsis.ResultsWe show that Pseudonocardia readily colonized ants irrespective of their colony of origin, but that the Ps2 phylotype, which was previously shown to be better able to maintain its monocultural integrity after workers became foragers than Ps1, reached a higher final cover when grown on its native host than on alternative hosts. The frequencies of major grooming and weeding behaviors co-varied with symbiont/host combinations, showing that ant behavior also was affected when cuticular actinomycete phylotypes were swapped.ConclusionThese results show that the interactions between leaf-cutting ants and Pseudonocardia bear signatures of mutual co-adaptation within a single ant population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0308-2) contains supplementary material, which is available to authorized users.

Control of Tick Infestations in Oryctolagus cuniculus (Lagomorpha: Leporidae) With Spinosad Under Laboratory and Field Conditions.

Because of great economic loss in the world's livestock industry, and the serious risks to human health, the control of ticks and tick-borne diseases is one of the most important health management issues today. Current methodology involves integrated tick control for preventing the development of resistance. Rabbits are hosts for immature stages of the three-host tick Hyalomma lusitanicum Koch; so, we focus on this host as a strategy to interrupt the tick life cycle. Spinosad is an insecticide-acaricide, produced by the fermentation of metabolites of the actinomycete bacterium Saccharopolyspora spinosa. We administered spinosad orally by force-feeding naturally and artificially infested rabbits, and under field conditions by administering treated food via a hopper during the period of peak infestation and reinfestation risk for rabbits. No living larvae were recovered from treated laboratory rabbits. In naturally infested rabbits, the number of live ticks collected from treated rabbits (mean = 0.62 ticks per ear) was significantly lower than those recovered from untreated rabbits (mean = 7.27; P < 0.001), whereas the number of dead ticks collected from untreated rabbits (mean = 6.53) was significantly lower than those recovered from treated rabbits (mean = 18.62; P < 0.001). In addition, free and continually reinfested rabbits freely ingested low doses of spinosad, reducing the tick burden from 48.00 (Day 0) to 26.09 ticks per ear in treated rabbits (Day 16), whereas controls maintained the infection (46.64). This strategy could be useful as an alternative or supplement to traditional acaricides in tick control programs.

Soil microbial community response and recovery following group selection harvest: Temporal patterns from an experimental harvest in a US northern hardwood forest

Forest harvesting and the associated loss of nutrients and carbon has the potential to negatively affect the soil microbial community, which plays a significant role in the health and productivity of the forest ecosystem. We used an experiment to evaluate the effects of group selection using whole-tree harvesting on the soil microbial community in a second growth northern hardwood forest dominated by sugar maple (Acer saccharum Marsh.) in northern Wisconsin, USA. We compared the response of the soil microbial community in 200m2 and 380m2 harvested gaps to unharvested controls during the spring and summer in the first two years post-harvest, and continued to monitor changes in the soil microbial communities and microenvironment in 380m2 gaps in years four, five, and seven post-harvest. Changes in community size and composition were assessed using phospholipid fatty acid (PLFA) analysis. We found that the abundance of arbuscular mycorrhizal fungi initially decreased following harvest, while abundance of anaerobic and gram positive bacteria, and the cyclo/pre-cyclo microbial stress ratio increased; responses that can be linked to microclimatic variability and resource accessibility. Neither actinomycete bacteria nor saprotrophic fungi exhibited an initial response to harvest, but in later years, we observed a decrease in actinomycetes and an increase in fungal abundance, suggesting a competitive interaction between the two main complex carbon-utilizing microbial groups. Canopy gap size had a minimal effect on the soil microbial community, resulting in a higher microbial stress ratio in 200m2 gaps. The microbial community exhibited seasonal and yearly fluctuation, which reinforces the need for repeated sampling over multiple seasons to correctly interpret management effects. Despite the large amount of seasonal and yearly variability, we began to see signs of recovery in the soil microbial community between two and four years post-harvest. We conclude that group selection accomplished via whole-tree harvesting of this size and scale does not result in long-term effects on the soil microbial community in this temperate northern hardwood forest.

Deuteromethylactin B from a Freshwater-derivedStreptomycessp.

Compared to their terrestrial and marine counterparts, little is known about the capacity of freshwater-derived actinomycete bacteria to produce novel secondary metabolites. In the current study, we highlight the disparities that exist between cultivation-independent and -dependent analyses of actinomycete communities from four locations in Lake Michigan sediment. Furthermore, through phylogenetic analysis of strains isolated from these locations, we identified a Streptomyces sp., strain B025, as being distinct from other Streptomyces spp. isolated from sediment. Upon fermentation this strain produced a rare class of eightmembered lactone secondary metabolites, which have been for their antitumor properties. We used spectroscopic and chemical derivitization techniques to characterize octalactin B (1) in addition to its corresponding novel, unnatural degradation product (2).

Tetrameric c-di-GMP Mediates Effective Transcription Factor Dimerization to Control Streptomyces Development

SummaryThe cyclic dinucleotide c-di-GMP is a signaling molecule with diverse functions in cellular physiology. Here, we report that c-di-GMP can assemble into a tetramer that mediates the effective dimerization of a transcription factor, BldD, which controls the progression of multicellular differentiation in sporulating actinomycete bacteria. BldD represses expression of sporulation genes during vegetative growth in a manner that depends on c-di-GMP-mediated dimerization. Structural and biochemical analyses show that tetrameric c-di-GMP links two subunits of BldD through their C-terminal domains, which are otherwise separated by ∼10 Å and thus cannot effect dimerization directly. Binding of the c-di-GMP tetramer by BldD is selective and requires a bipartite RXD-X8-RXXD signature. The findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions.

Toxic pyrene metabolism in Mycobacterium gilvum PYR-GCK results in the expression of mammalian cell entry genes as revealed by transcriptomics study.

Mycobacterium gilvum PYR-GCK is a bacterial strain under study for its bioremediation use on heavy hydrocarbon pollutants in the environment. During the course of our study, mammalian cell entry (mce) genes, known to facilitate pathogenicity in M. tuberculosis, were highly expressed during a comparative and substrate-related cultural global transcriptomic study. RNA sequencing of the global transcriptome of the test strain in two different substrates, pyrene and glucose, showed high expression of the mce genes based on the differential results. After validating the expression of these genes with quantitative real-time PCR, we arrived at the conclusion that the genes were expressed based on the pyrene substrate (a phytosterol compound), and sterol metabolism is said to activate the expression of the mce genes in some actinomycetes bacteria, M. gilvum PYR-GCK in this case. This study is believed to be important based on the fact that some mycobacterial strains are undergoing a continuous research as a result of their use in practical bioremediation of anthropogenic exposure of toxic organic wastes in the environment.

Tumoural form of Whipple's disease simulating carcinomatosis

Whipple's disease is a rare disease caused by the actinomycete bacteria Tropheryma whipplei, which cause intestinal infection. The most common symptoms are chronic diarrhoea, weight loss, abdominal pain, arthritis and neurological abnormalities, which can be fatal. This paper reports a case of a 57-year-old Brazilian woman with diarrhoea, vomiting, abdominal pain, appetite loss, intermittent fever, malaise, weight loss and malnutrition. Migratory polyarthralgia and recurrent visual scotomas preceded the symptoms. The retroperitoneal pseudotumour formation finding was associated with prolonged wasting syndrome, which did not respond to usual therapies, thus leading to the investigation of carcinomatosis disease. After laparotomy, biopsy and histochemical study of the lesions with negative results for malignancy, we proceeded to the investigation of Whipple's disease, which was then confirmed. The patient improved clinically and started gaining weight after using ceftriaxone (IV).

Legume nodulation

Legume nodulation

Novel Phialophora species from leaf-cutting ants (tribe Attini)

Ants in the tribe Attini (Hymenoptera, Formicidae) maintain a 50 million-year-old lifestyle of co-evolution with symbiotic basidiomycetous fungi which they cultivate as essential source of nutrition. However, other microorganisms have been reported from ant habitats indicating a higher diversity of consistently associated species than established to date. Recently, black yeast-like fungi have been reported as a possible symbiont, like a competitor of the actinomycete bacteria that grow in the integument of the insects. During the mating season, gynes of Atta capiguara and A. laevigata were collected from nests located in Botucatu, SP, Brazil and sampled using flotation technique. Phylogenetic analysis based on ITS, partial 28S (LSU) and β-tubulin sequences revealed the occurrence of two novel species of Phialophora among the melanized fungi isolated.

Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factorσAntA

Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant) gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF) RNA polymerase sigma factor named σAntA which represents a new sub-family of ECF σ factors that is only found in antimycin producing strains. We show that σAntA controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. σAntA is highly conserved in antimycin producing strains and the −10 and −35 elements at the σAntA regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all σAntA proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.

Antagonism of marineActinomycetesbacteria against virulence and bio-luminescence disease causingVibrio harveyi

Luminescence is caused by Vibrio harveyi, which is responsible for causing mass mortality (80 - 100%) among Penaeus monodon hatcheries. An alternative strategy is the use of marine Actinomycetes to control V. harveyi. Eighty three marine Actinomycetes bacteria were isolated from marine and brackishwater of Muttukadu, Chennai and treated against growth and virulence factors produced by V. harveyi. Among 83 Actinomycetes isolates, 2 (CBWA3 and CBWA10) showed higher zone of inhibition against V. harveyi. When the Actinomycetes isolates were co-cultured with V. harveyi, the growth of V. harveyi was reduced to 4.66 log cfu mL−1 by CBWA3 and 3.75 log cfu mL−1 by CBWA10. The virulence factors like luminescence, crude bacteriocin, protease production and phospholipase activity were determined during co-culture and it was observed that phospholipase activity was very high in control and virulence was weak in the treatment. This study confirms that marine Actinomycetes bacterial isolates can be effectively utilized to control luminescence disease in the aquaculture system

Anti-microbial and cytotoxic 1,6-dihydroxyphenazine-5,10-dioxide (iodinin) produced by Streptosporangium sp. DSM 45942 isolated from the fjord sediment

Phenazine natural products/compounds possess a range of biological activities, including anti-microbial and cytotoxic, making them valuable starting materials for drug development in several therapeutic areas. These compounds are biosynthesized almost exclusively by eubacteria of both terrestrial and marine origins from erythrose 4-phosphate and phosphoenol pyruvate via the shikimate pathway. In this paper, we report isolation of actinomycete bacteria from marine sediment collected in the Trondheimfjord, Norway. Screening of the isolates for biological activity produced several "hits", one of which was followed up by identification and purification of the active compound from the actinomycete bacterium Streptosporangium sp. The purified compound, identified as 1,6-dihydroxyphenazine-5,10-dioxide (iodinin), was subjected to extended tests for biological activity against bacteria, fungi and mammalian cells. In these tests, the iodinin demonstrated high anti-microbial and cytotoxic activity, and was particularly potent against leukaemia cell lines. This is the first report on the isolation of iodinin from a marine-derived Streptosporangium.

Interspecies Interactions Stimulate Diversification of the Streptomyces coelicolor Secreted Metabolome

ABSTRACTSoils host diverse microbial communities that include filamentous actinobacteria (actinomycetes). These bacteria have been a rich source of useful metabolites, including antimicrobials, antifungals, anticancer agents, siderophores, and immunosuppressants. While humans have long exploited these compounds for therapeutic purposes, the role these natural products may play in mediating interactions between actinomycetes has been difficult to ascertain. As an initial step toward understanding these chemical interactions at a systems level, we employed the emerging techniques of nanospray desorption electrospray ionization (NanoDESI) and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry to gain a global chemical view of the model bacterium Streptomyces coelicolor interacting with five other actinomycetes. In each interaction, the majority of secreted compounds associated with S. coelicolor colonies were unique, suggesting an idiosyncratic response from S. coelicolor. Spectral networking revealed a family of unknown compounds produced by S. coelicolor during several interactions. These compounds constitute an extended suite of at least 12 different desferrioxamines with acyl side chains of various lengths; their production was triggered by siderophores made by neighboring strains. Taken together, these results illustrate that chemical interactions between actinomycete bacteria exhibit high complexity and specificity and can drive differential secondary metabolite production.

Development of a real-time PCR for detection of the oyster pathogen Nocardia crassostreae based on its homogeneous 16S–23S rRNA intergenic spacer region

Nocardia crassostreae, the causative agent of Pacific oyster nocardiosis (PON), is a Gram-positive actinomycete bacterium associated with Pacific oyster (Crassostrea gigas) mortalities. Oysters infected with this bacterium have been reported previously from the west coast of North America and Japan. More recently, N. crassostreae was reported in oyster culture areas in the Netherlands. In this study, a sensitive real-time PCR for specific detection of N. crassostreae was developed, and the intra-species divergence of N. crassostreae from different geographical locations was studied. The 16S–23S rRNA intergenic spacer (ITS) region of N. crassostreae was sequenced for a number of infected oysters originating from the Netherlands, Japan and Canada. The sequence analyses showed an absence of genetic variation in the ITS region between N. crassostreae from different geographical locations. Based on these ITS sequences a species-specific and highly sensitive SYBR Green real-time PCR assay was developed to facilitate detection of N. crassostreae in oyster tissue. To evaluate this new detection tool for N. crassostreae a preliminary validation was carried out and real-time PCR results were compared with other detection methods (histology, conventional PCR and bacterial isolation) using field samples from Lake Grevelingen, the Netherlands. The genetic homogeneity in the ITS region between N. crassostreae from different geographical locations might be explained by the recent spread of the organism via the international trade in Pacific oysters for aquaculture purposes. However, the lack of genetic variation could also suggest that N. crassostreae is a genetically monomorphic species.

Dynamic Disease Management inTrachymyrmexFungus-Growing Ants (Attini: Formicidae)

Multipartner mutualisms have potentially complex dynamics, with compensatory responses when one partner is lost or relegated to a minor role. Fungus-growing ants (Attini) are mutualistic associates of basidiomycete fungi and antibiotic-producing actinomycete bacteria; the former are attacked by specialized fungi (Escovopsis) and diverse generalist microbes. Ants deploy biochemical defenses from bacteria and metapleural glands (MGs) and express different behaviors to control contaminants. We studied four Trachymyrmex species that differed in relative abundance of actinomycetes to understand interactions among antimicrobial tactics that are contingent on the nature of infection. MG grooming rate and actinomycete abundance were negatively correlated. The two species with high MG grooming rates or abundant actinomycetes made relatively little use of behavioral defenses. Conversely, the two species with relatively modest biochemical defenses relied heavily on behavior. Trade-offs suggest that related species can evolutionarily diverge to rely on different defense mechanisms against the same threat. Neither bacterial symbionts nor MG secretions thus appear to be essential for mounting defenses against the specialized pathogen Escovopsis, but reduced investment in one of these defense modes tends to increase investment in the other.

Analysis of the bacterial communities associated with two ant–plant symbioses

Insect fungiculture is practiced by ants, termites, beetles, and gall midges and it has been suggested to be widespread among plant–ants. Some of the insects engaged in fungiculture, including attine ants and bark beetles, are known to use symbiotic antibiotic-producing actinobacteria to protect themselves and their fungal cultivars against infection. In this study, we analyze the bacterial communities on the cuticles of the plant–ant genera Allomerus and Tetraponera using deep sequencing of 16S rRNA. Allomerus ants cultivate fungus as a building material to strengthen traps for prey, while Tetraponera ants cultivate fungus as a food source. We report that Allomerus and Tetraponera microbiomes contain >75% Proteobacteria and remarkably the bacterial phyla that dominate their cuticular microbiomes are very similar despite their geographic separation (South America and Africa, respectively). Notably, antibiotic-producing actinomycete bacteria represent a tiny fraction of the cuticular microbiomes of both Allomerus and Tetraponera spp. and instead they are dominated by γ-proteobacteria Erwinia and Serratia spp. Both these phyla are known to contain antibiotic-producing species which might therefore play a protective role in these ant–plant systems.

Extrachromosomal genetic elements in Micrococcus

Micrococci are Gram-positive G + C-rich, nonmotile, nonspore-forming actinomycetous bacteria. Micrococcus comprises ten members, with Micrococcus luteus being the type species. Representatives of the genus play important roles in the biodegradation of xenobiotics, bioremediation processes, production of biotechnologically important enzymes or bioactive compounds, as test strains in biological assays for lysozyme and antibiotics, and as infective agents in immunocompromised humans. The first description of plasmids dates back approximately 28years, when several extrachromosomal elements ranging in size from 1.5 to 30.2kb were found in Micrococcus luteus. Up to the present, a number of circular plasmids conferring antibiotic resistance, the ability to degrade aromatic compounds, and osmotolerance are known, as well as cryptic elements with unidentified functions. Here, we review the Micrococcus extrachromosomal traits reported thus far including phages and the only quite recently described large linear extrachromosomal genetic elements, termed linear plasmids, which range in size from 75kb (pJD12) to 110kb (pLMA1) and which confer putative advantageous capabilities, such as antibiotic or heavy metal resistances (inferred from sequence analyses and curing experiments). The role of the extrachromosomal elements for the frequently proven ecological and biotechnological versatility of the genus will be addressed as well as their potential for the development and use as genetic tools.