A Single-Cell Genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.     

Ectopic positioning of the cell division plane is associated with single amino acid substitutions in the FtsZ-recruiting SsgB in Streptomyces

In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium Streptomyces, cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here, we show that various amino acid (aa) substitutions in the highly conserved SsgB protein result in ectopically placed septa that sever spores diagonally or along the long axis, perpendicular to the division plane. Fluorescence microscopy revealed that between 3.3% and 9.8% of the spores of strains expressing SsgB E120 variants were severed ectopically. Biochemical analysis of SsgB variant E120G revealed that its interaction with FtsZ had been maintained. The crystal structure of Streptomyces coelicolor SsgB was resolved and the key residues were mapped on the structure. Notably, residue substitutions (V115G, G118V, E120G) that are associated with septum misplacement localize in the α2–α3 loop region that links the final helix and the rest of the protein. Structural analyses and molecular simulation revealed that these residues are essential for maintaining the proper angle of helix α3. Our data suggest that besides altering FtsZ, aa substitutions in the FtsZ-recruiting protein SsgB also lead to diagonally or longitudinally divided cells in Streptomyces.     

Rapid and Specific Method for Evaluating Streptomyces Competitive Dynamics in Complex Soil Communities

Quantifying target microbial populations in complex communities remains a barrier to studying species interactions in soil environments. Quantitative PCR (qPCR) assays were developed for quantifying pathogenic Streptomyces scabiei and antibiotic-producing Streptomyces lavendulae strains in complex soil communities. This assay will be useful for evaluating the competitive dynamics of streptomycetes in soil.Streptomyces spp. are ubiquitous soil bacteria that are noted for their capacity to produce a vast array of bioactive compounds, including antibiotics (10). Antibiotic-mediated species interactions are believed to be important to Streptomyces fitness and plant disease biocontrol in soil, and yet quantitative data on Streptomyces interactions in soil are limited. Moreover, because the impacts of one species on another can be mediated through interactions with other microbes in the community, detecting these impacts requires a sensitive and accurate method for quantifying the target populations within a complex community. Here, we describe a sensitive and specific assay that targets a short hypervariable region of the 16S rRNA gene to distinguish among Streptomyces organisms in complex soil communities. Streptomyces strains DL93 (Streptomyces lavendulae, an antibiotic producer that is effective in plant disease biocontrol [9]) and DL87 (Streptomyces scabiei, a plant pathogen) were studied in the present work. This approach has significant potential to shed light on the diversity and complexity of Streptomyces species interactions in soil.     

Heavy metal resistant strains are widespread along Streptomyces phylogeny

The genus Streptomyces comprises a group of bacteria species with high economic importance. Several of these species are employed at industrial scale for the production of useful compounds. Other characteristic found in different strains within this genus is their capability to tolerate high level of substances toxic for humans, heavy metals among them. Although several studies have been conducted in different species of the genus in order to disentangle the mechanisms associated to heavy metal resistance, little is known about how they have evolved along Streptomyces phylogeny. In this study we built the largest Streptomyces phylogeny generated up to date comprising six genes, 113 species of Streptomyces and 27 outgroups. The parsimony-based phylogenetic analysis indicated that (i) Streptomyces is monophyletic and (ii) it appears as sister clade of a group formed by Kitasatospora and Streptacidiphilus species, both genera also monophyletic. Streptomyces strains resistant to heavy metals are not confined to a single lineage but widespread along Streptomyces phylogeny. Our result in combination with genomic, physiological and biochemical data suggest that the resistance to heavy metals originated several times and by different mechanisms in Streptomyces history.     

Widespread predatory abilities in the genus Streptomyces

The natural role of antibiotics in the ecology of Streptomyces is debated and still largely unknown. The predatory myxobacteria and many other genera of prokaryotic epibiotic and wolfpack predators across different taxa possess secondary metabolites with antimicrobial action, and these compounds have a role in predation. If all epibiotic predators are antibiotic producers, it is worth testing whether all antibiotic producers are predators too. We show here that Streptomyces are non-obligate epibiotic predators of other microorganisms and that predatory abilities are widespread in this genus. We developed a test for predatory activity which revealed that a large proportion of traditionally isolated Streptomyces strains and all oligophilic Streptomyces isolates show predatory activity. Those that did not show predatory ability on first challenge could do so after many generations of selection or acclimation. Using time-lapse photomicrography, we demonstrate that the growth of the tips of Streptomyces hyphae is accompanied by disappearance of cells of other bacteria in the vicinity presumably due to lysis. Predatory activity is restricted to surface growth and is not obligately associated with antibiotic production in conventional culture. However, some of the genes crucial to the regulation of secondary metabolite pathways are differentially expressed during predatory growth on different prey species as compared to saprophytic growth. Our findings strengthen the association between epibiotic predation and antibiotic production.     

Analysis of bacterial communities associated with spores of Gigaspora margarita and Gigaspora rosea

The spores of arbuscular mycorrhizal fungi (AMF) form a unique microhabitat that is suitable for the colonization by many species of bacteria. The aim of the current study was to analyze the bacterial communities associated with the surface of spores of the AMF species Gigaspora margarita MAFF 520054 and Gigaspora rosea JP1. The two AMF species were propagated with tobacco (Nicotiana tabacum) grown in a mixture of sand and soil. In another experiment, G. margarita was propagated with tobacco or alfalfa (Medicago sativa) grown in vermiculite or a mixture of sand and soil. The bacterial community composition of the new-formed spores and sand/soil substrate was analyzed using PCR of 16S rDNA fragments and denaturing gradient gel electrophoresis (DGGE). Clustering analysis revealed that the bacterial communities on the surface of G. margarita spores was different form that in the substrate or on the surface of the G. rosea spores, and both the host plant and the substrate could influence the composition of spore-associated bacterial populations of the G. margarita. Sequence analysis of the major DGGE bands of G. margarita spore samples revealed that most of the bacterial sequences were affiliated with the phyla Proteobacteria (Azospirillum, Azovibrio, Polyangium, Ramlibacter, Rubrivivax, Sphingomonas, and Rhizobium) and Actinobacteria (Streptomyces, Amycolatopsis, and Pseudonocardia).     

Threats and opportunities of plant pathogenic bacteria

Plant pathogenic bacteria can have devastating effects on plant productivity and yield. Nevertheless, because these often soil-dwelling bacteria have evolved to interact with eukaryotes, they generally exhibit a strong adaptivity, a versatile metabolism, and ingenious mechanisms tailored to modify the development of their hosts. Consequently, besides being a threat for agricultural practices, phytopathogens may also represent opportunities for plant production or be useful for specific biotechnological applications. Here, we illustrate this idea by reviewing the pathogenic strategies and the (potential) uses of five very different (hemi)biotrophic plant pathogenic bacteria: Agrobacterium tumefaciens, A. rhizogenes, Rhodococcus fascians, scab-inducing Streptomyces spp., and Pseudomonas syringae.     

Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed “social motility,” based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies.     

Acanthamoeba release compounds which promote growth of Listeria monocytogenes and other bacteria

Listeria monocytogenes can grow as a saphrophyte in diverse habitats, e.g., soil, rivers, lakes, and on decaying plant material. In these environments, the bacteria are frequently exposed to predatory protozoa such as Acanthamoeba. Although L. monocytogenes is a facultative intracellular pathogen it does not infect or survive intracellular in Acanthamoeba castellanii, unlike several other facultative intracellular bacteria. Instead, motile L. monocytogenes can form large aggregates on amoebal cells and are effectively phagocytosed and eventually digested by Acanthamoeba. Here, we demonstrate that non-motile L. monocytogenes represent a less preferred prey in co-cultures with A. castellanii. Moreover, we found that the presence of Acanthamoeba strongly promotes growth of the bacteria in non-nutrient saline, by releasing nutrients or other growth promoters. Thus, the lack of motility and ability to utilize amoebal metabolites may aid to avoid eradication by amoebal predation in low-nutrient environments.     

Landscape‐scale Variation in Pathogen‐suppressive Bacteria in Tropical Dry Forest Soils of Costa Rica

Bacteria in the genus Streptomyces are ubiquitous in soil and are well‐known for their production of diverse secondary metabolites, including antibiotics that can inhibit soil‐borne plant pathogens and suppress disease. Pathogen‐suppressive soil bacteria have the potential to influence plant community composition and diversity, but remain relatively unexplored in tropical forest soils. To estimate the potential for disease suppression among Streptomyces communities in tropical dry forests, we cultured soil‐borne Streptomyces from plots in two forests in northwestern Costa Rica (Santa Rosa and Palo Verde) and quantified antibiotic‐mediated pathogen inhibition against three plant pathogens. The potential for pathogen inhibition and disease suppression by Streptomyces was highly variable across the landscape. Densities of pathogen‐suppressive Streptomyces varied by over ten‐fold and were correlated with soil nutrients across the plots. In particular, Streptomyces communities became more pathogen‐suppressive as labile soil P decreased. Inhibitor densities were significantly higher in Santa Rosa than Palo Verde, which may be related to differences in soil texture and/or plant community composition between the two forests. Our findings suggest potential differences in the degree and specificity of antibiotic‐mediated disease suppression in tropical dry forest soils of Costa Rica, and highlight the need for further studies on the drivers of pathogen‐suppressive phenotypes as well as the consequences of spatially variable pathogen inhibition for plant community composition in tropical forest ecosystems.     

Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.     

DNA capturing machinery through spore-displayed proteins

Aims: The purpose of this study was to develop a general method for the facile development of a new DNA biosensor which utilizes streptavidin‐displayed spores as a molecular machinery. Methods and Results: Fluorescence spectroscopy was used as a monitoring tool for the streptavidin displayed on the surface of Bacillus thuringiensis spores and as a diagnosis method for DNA detection. As a proof‐of‐concept, four pathogenic bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumonia were used for the detection of pathogenic species. In addition, a set of mutant variants of Wilson’s disease were also used for the detection of single nucleotide polymorphism (SNP) in this system. Conclusions: This strategy, utilizing streptavidin‐displayed spores, is capable of capturing DNA targets for the detection of pathogenic bacteria and for mutation analysis in Wilson’s disease. Significance and Impact of the Study: This approach could be useful as a simple platform for developing sensitive spore‐based biosensors for any desired DNA targets in diagnostic applications.     

Spore dissemination by mycophagous adult drosophilids

Although most fungal spores are transported by wind, some remain on lamellae even after fungal fruiting bodies start to decay. This raises the question: are these remaining spores useless or instead transported by other means? In this study, we accordingly investigated whether adult dipteran insects transport fungal spores. Our examination revealed that fungal spores were present in intestines of most drosophilid adults but almost completely absent from those of other dipteran adults. At least some spores excreted by Drosophila angularis and D. brachynephros retained the capacity to germinate. Structural damage to spores that passed through the digestive tract of these two drosophilid species varied among fungal species, with a greater number of colorless spores generally damaged than colored ones. These results suggest that adult drosophilid flies, but not other dipterans, can transport fungal spores.     

Streptomyces tunisiensis sp. nov., a novel Streptomyces species with antibacterial activity

A novel actinomycete strain designated CN-207^T was isolated from northern Tunisian soil. This strain exhibited potent broad spectrum antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several other Gram-positive and Gram-negative bacteria. Strain CN-207^T developed greyish aerial mycelium and pale grey substrate mycelium on yeast extract/malt agar. The isolate produced branching vegetative mycelia with sporangiophores bearing sporangia developing at a late stage of growth. The sporangia contained smooth, non-motile spores. Chemotaxonomic characteristics of strain CN-207^T were typical of the Streptomyces genus. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CN-207^T belonged to the genus Streptomyces, and was most closely related to Streptomyces griseoincarnatus DSM 40274^T, Streptomyces variabilis DSM 40179^T, Streptomyces labedae DSM 41446^T and Streptomyces erythrogriseus DSM 40116^T. Low DNA–DNA relatedness values were recorded between strain CN-207^T and its closest phylogenetic neighbours. Strain CN-207^T was also distinguished from the nearest phylogenetic neighbours using a combination of morphological and phenotypic characteristics. On the basis of its phenotypic and molecular properties, strain CN-207^T is considered as a novel species of the Streptomyces genus, for which the name Streptomyces tunisiensis sp. nov. is proposed. The type strain is CN-207^T (=JCM 17589^T = DSM 42037^T).     

The Evolution of an Osmotically Inducible dps in the Genus Streptomyces

Dps proteins are found almost ubiquitously in bacterial genomes and there is now an appreciation of their multifaceted roles in various stress responses. Previous studies have shown that this family of proteins assemble into dodecamers and their quaternary structure is entirely critical to their function. Moreover, the numbers of dps genes per bacterial genome is variable; even amongst closely related species - however, for many genera this enigma is yet to be satisfactorily explained. We reconstruct the most probable evolutionary history of Dps in Streptomyces genomes. Typically, these bacteria encode for more than one Dps protein. We offer the explanation that variation in the number of dps per genome among closely related Streptomyces can be explained by gene duplication or lateral acquisition, and the former preceded a subsequent shift in expression patterns for one of the resultant paralogs. We show that the genome of S. coelicolor encodes for three Dps proteins including a tailless Dps. Our in vivo observations show that the tailless protein, unlike the other two Dps in S. coelicolor, does not readily oligomerise. Phylogenetic and bioinformatic analyses combined with expression studies indicate that in several Streptomyces species at least one Dps is significantly over-expressed during osmotic shock, but the identity of the ortholog varies. In silico analysis of dps promoter regions coupled with gene expression studies of duplicated dps genes shows that paralogous gene pairs are expressed differentially and this correlates with the presence of a sigB promoter. Lastly, we identify a rare novel clade of Dps and show that a representative of these proteins in S. coelicolor possesses a dodecameric quaternary structure of high stability.     

Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection

Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces.     

Isolation and characterization of soil Streptomyces species as potential biological control agents against fungal plant pathogens

The use of antagonist microorganisms against fungal plant pathogens is an attractive and ecologically alternative to the use of chemical pesticides. Streptomyces are beneficial soil bacteria and potential candidates for biocontrol agents. This study reports the isolation, characterization and antagonist activity of soil streptomycetes from the Los Petenes Biosphere Reserve, a Natural protected area in Campeche, Mexico. The results showed morphological, physiological and biochemical characterization of six actinomycetes and their inhibitory activity against Curvularia sp., Aspergillus niger, Helminthosporium sp. and Fusarium sp. One isolate, identified as Streptomyces sp. CACIS-1.16CA showed the potential to inhibit additional pathogens as Alternaria sp., Phytophthora capsici, Colletotrichum sp. and Rhizoctonia sp. with percentages ranging from 47 to 90 %. This study identified a streptomycete strain with a broad antagonist activity that could be used for biocontrol of plant pathogenic fungi.     

Early endosomes motility in filamentous fungi: How and why they move

Elongate hyphae of filamentous fungi grow predominantly at their tips, whereas organelles are positioned in the subapical parts of the cell. Organelle positioning and long-distance intracellular communication involves active, energy-dependent transport along microtubules (MTs). This is mediated by specialized molecular motors, named kinesins and dynein, which utilize ATP hydrolysis to “walk” along the tubulin polymers. Work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans has shown that early endosomes (EEs) are one of the major cargos of MT-dependent motors in fungi. EEs are part of the early endocytic pathway, and their motility behavior and the underlying transport machinery is well understood. However, the physiological role of constant bi-directional EE motility remains elusive. Recent reports, conducted in the corn smut fungus U. maydis, have provided novel insights into the cellular function of EE motility. They show that EE motility is crucial for the distribution of the protein synthesis machinery, and also that EEs transmit signals during plant infection that trigger the production of fungal effector proteins, required for successful invasion into host plants.