Culture-dependent and -independent methods revealed an abundant myxobacterial community shaped by other bacteria and pH in Dinghushan acidic soils.

Myxobacteria are one of the most promising secondary metabolites producers. However, they are difficult to isolate and cultivate. To obtain more myxobacteria and know the effects of environmental factors on myxobacterial community, we characterized myxobacterial communities in Dinghushan acidic forest soils of pH 3.6-4.5 with culture-dependent and -independent techniques, and analyzed environmental factors shaping myxobacterial communities. A total of 21 myxobacteria were isolated using standard cultivation methods, including eleven isolates of Corallococcus, nine isolates of Myxococcus and one isolate of Archangium, and contained three potential novel species. In addition, a total of 67 unknown myxobacterial operational taxonomic units (OTUs) were obtained using high-throughput sequencing method. The abundance of Myxococcales account for 0.9-2.2% of bacterial communities, and Sorangium is the most abundant genus (60.1%) in Myxococcales. Correlation analysis demonstrated that bacterial diversity and soil pH are the key factors shaping myxobacterial community. These results revealed an abundant myxobacterial community which is shaped by other bacteria and pH in Dinghushan acidic forest soils.

Comparison of chemical and microbiological changes during the aerobic composting and vermicomposting of green waste.

This research was conducted to compare chemical and microbiological properties during aerobic composting (AC) and vermicomposting (VC) of green waste. Relative to AC, VC significantly decreased the pH and lignin and cellulose contents, and significantly increased the electrical conductivity and total N and available P contents. For AC, BIrii41_norank (order Myxococcales) was the major bacterial genus at 30 d and again became dominant genus from 90-150 d, with relative abundances of 2.88% and 4.77-5.19%, respectively; at 45 d and 60 d, the dominant bacterial genus was Nitrosomonadaceae_uncultured (order Nitrosomonadales) with relative abundances of 2.83-7.17%. For VC, the dominant bacterial genus was BIrii41_norank (except at 45 d), which accounted for 2.11-7.96% of the total reads. The dominant fungal class was Sordariomycetes in AC (relative abundances 39.2-80.6%) and VC (relative abundances 42.1-69.5%). The abundances of microbial taxa and therefore the bacterial and fungal community structures differed between VC and AC. The quality of the green waste compost product was higher with VC than with AC. These results will also help to achieve further composting technology breakthroughs in reducing the composting time and improving compost quality.

Transcriptional changes when Myxococcus xanthus preys on Escherichia coli suggest myxobacterial predators are constitutively toxic but regulate their feeding.

Predation is a fundamental ecological process, but within most microbial ecosystems the molecular mechanisms of predation remain poorly understood. We investigated transcriptome changes associated with the predation of Escherichia coli by the myxobacterium Myxococcus xanthus using mRNA sequencing. Exposure to pre-killed prey significantly altered expression of 1319 predator genes. However, the transcriptional response to living prey was minimal, with only 12 genes being significantly up-regulated. The genes most induced by prey presence (kdpA and kdpB, members of the kdp regulon) were confirmed by reverse transcriptase quantitative PCR to be regulated by osmotic shock in M. xanthus, suggesting indirect sensing of prey. However, the prey showed extensive transcriptome changes when co-cultured with predator, with 40 % of its genes (1534) showing significant changes in expression. Bacteriolytic M. xanthus culture supernatant and secreted outer membrane vesicles (OMVs) also induced changes in expression of large numbers of prey genes (598 and 461, respectively). Five metabolic pathways were significantly enriched in prey genes up-regulated on exposure to OMVs, supernatant and/or predatory cells, including those for ribosome and lipopolysaccharide production, suggesting that the prey cell wall and protein production are primary targets of the predator's attack. Our data suggest a model of the myxobacterial predatome (genes and proteins associated with predation) in which the predator constitutively produces secretions which disable its prey whilst simultaneously generating a signal that prey is present. That signal then triggers a regulated feeding response in the predator.

Myxobacteria in high moor and fen: An astonishing diversity in a neglected extreme habitat.

Increasing antibiotic resistances of numerous pathogens mean that myxobacteria, well known producers of new antibiotics, are becoming more and more interesting. More than 100 secondary metabolites, most of them with bioactivity, were described from the order Myxococcales. Especially new myxobacterial genera and species turned out to be reliable sources for novel antibiotics and can be isolated from uncommon neglected habitats like, for example, acidic soils. Almost nothing is known about the diversity of myxobacteria in moors, except some information from cultivation studies of the 1970s. Therefore, we evaluated the myxobacterial community composition of acidic high moor and fen both with cultivation-independent 16S rRNA clone bank analysis and with cultivation. Phylogenetic analyses of clone sequences revealed a great potential of undescribed myxobacteria in high moor and fen, whereby all sequences represent unknown taxa and were detected exclusively by cultivation-independent analyses. However, many clones were assigned to sequences from other cultivation-independent studies of eubacterial diversity in acidic habitats. Cultivation revealed different strains exclusively from the genus Corallococcus. Our study shows that the neglected habitat moor is a promising source and of high interest with regard to the cultivation of prospective new bioactive secondary metabolite producing myxobacteria.

Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ.

UNLABELLED: Bacteria capable of reduction of nitrous oxide (N2O) to N2 separate into clade I and clade II organisms on the basis of nos operon structures and nosZ sequence features. To explore the possible ecological consequences of distinct nos clusters, the growth of bacterial isolates with either clade I (Pseudomonas stutzeri strain DCP-Ps1, Shewanella loihica strain PV-4) or clade II (Dechloromonas aromatica strain RCB, Anaeromyxobacter dehalogenans strain 2CP-C) nosZ with N2O was examined. Growth curves did not reveal trends distinguishing the clade I and clade II organisms tested; however, the growth yields of clade II organisms exceeded those of clade I organisms by 1.5- to 1.8-fold. Further, whole-cell half-saturation constants (Kss) for N2O distinguished clade I from clade II organisms. The apparent Ks values of 0.324 ± 0.078 µM for D. aromatica and 1.34 ± 0.35 µM for A. dehalogenans were significantly lower than the values measured for P. stutzeri (35.5 ± 9.3 µM) and S. loihica (7.07 ± 1.13 µM). Genome sequencing demonstrated that Dechloromonas denitrificans possessed a clade II nosZ gene, and a measured Ks of 1.01 ± 0.18 µM for N2O was consistent with the values determined for the other clade II organisms tested. These observations provide a plausible mechanistic basis for why the relative activity of bacteria with clade I nos operons compared to that of bacteria with clade II nos operons may control N2O emissions and determine a soil's N2O sink capacity. IMPORTANCE: Anthropogenic activities, in particular fertilizer application for agricultural production, increase N2O emissions to the atmosphere. N2O is a strong greenhouse gas with ozone destruction potential, and there is concern that nitrogen may become the major driver of climate change. Microbial N2O reductase (NosZ) catalyzes N2O reduction to environmentally benign dinitrogen gas and represents the major N2O sink process. The observation that bacterial groups with clade I nosZ versus those with clade II nosZ exhibit distinct affinities to N2O has implications for N2O flux models, and these distinct characteristics may provide opportunities to curb N2O emissions from relevant soil ecosystems.

How Myxobacteria Cooperate.

Prokaryotes often reside in groups where a high degree of relatedness has allowed the evolution of cooperative behaviors. However, very few bacteria or archaea have made the successful transition from unicellular to obligate multicellular life. A notable exception is the myxobacteria, in which cells cooperate to perform group functions highlighted by fruiting body development, an obligate multicellular function. Like all multicellular organisms, myxobacteria face challenges in how to organize and maintain multicellularity. These challenges include maintaining population homeostasis, carrying out tissue repair and regulating the behavior of non-cooperators. Here, we describe the major cooperative behaviors that myxobacteria use: motility, predation and development. In addition, this review emphasizes recent discoveries in the social behavior of outer membrane exchange, wherein kin share outer membrane contents. Finally, we review evidence that outer membrane exchange may be involved in regulating population homeostasis, thus serving as a social tool for myxobacteria to make the cyclic transitions from unicellular to multicellular states.

Geobacter, Anaeromyxobacter and Anaerolineae populations are enriched on anodes of root exudate-driven microbial fuel cells in rice field soil.

Plant-based sediment microbial fuel cells (PMFCs) couple the oxidation of root exudates in living rice plants to current production. We analysed the composition of the microbial community on anodes from PMFC with natural rice field soil as substratum for rice by analysing 16S rRNA as an indicator of microbial activity and diversity. Terminal restriction fragment length polymorphism (TRFLP) analysis indicated that the active bacterial community on anodes from PMFCs differed strongly compared with controls. Moreover, clones related to Deltaproteobacteria and Chloroflexi were highly abundant (49% and 21%, respectively) on PMFCs anodes. Geobacter (19%), Anaeromyxobacter (15%) and Anaerolineae (17%) populations were predominant on anodes with natural rice field soil and differed strongly from those previously detected with potting soil. In open circuit (OC) control PMFCs, not allowing electron transfer, Deltaproteobacteria (33%), Betaproteobacteria (20%), Chloroflexi (12%), Alphaproteobacteria (10%) and Firmicutes (10%) were detected. The presence of an electron accepting anode also had a strong influence on methanogenic archaea. Hydrogenotrophic methanogens were more active on PMFC (21%) than on OC controls (10%), whereas acetoclastic Methanosaetaceae were more active on OC controls (31%) compared with PMFCs (9%). In conclusion, electron accepting anodes and rice root exudates selected for distinct potential anode-reducing microbial populations in rice soil inoculated PMFC.

Enhanced production of undecylprodigiosin in Streptomyces coelicolor by co-cultivation with the corallopyronin A-producing myxobacterium, Corallococcus coralloides.

Prolific producers of natural products like streptomycetes and myxobacteria live in complex natural frameworks consisting of many microorganisms. Presumably intricate physiological and metabolic regulatory networks have evolved to enable the organisms to respond to intra- and interspecies interactions, e.g. biosynthesis of specific natural products is up-regulated due to competitors in the surrounding area. The soil-dwelling bacterium, Streptomyces coelicolor, produces the biologically-active compound, undecylprodigiosin (Red). Co-incubation with the corallopyronin A-producer, Corallococcus coralloides, was performed to explore the hypothesis that Red production can be enhanced by a myxobacterial competitor. Co-cultivation resulted in earlier onset and increased production of Red (60-fold increase of the intra-cellular concentration). Using different Corallococcus-derived extracts for elicitation, revealed that water-soluble factors triggered the enhanced production of Red which shows antimicrobial, immunosuppressive and anticancer properties.

[Isolation and identification of cultivable myxobacteria in the rhizosphere soils of medicinal plants].

OBJECTIVE: We isolated myxobacteria and explored their diversity from the rhizosphere soils of some medicinal plants. METHODS: We used the helper bacteria baiting technique to isolate myxobacteria from the rhizosphere soils collected in South China Botanical Garden and Nanling National Forest Park. The myxobacteria were identified by morphological characteristics and 16S rDNA gene sequences analysis. RESULTS: A total of 50 strains were isolated from 22 soil samples, which were identified into 7 genera, Myxococcus (18), Corallococcus (11), Cystobacter (7), Archangium (8), Stigmatella (1), Chondromyces (4) and Pyxidicoccus (1). The dominant genera were Myxococcus and Corallococcus. CONCLUSION: Environmental factors were associated with the diversity of myxobactria. Myxobacteria better adapt in high organic matter content and neutral pH environments. The strains of Myxococcus and Corallococcus had a good adaptability for different pH. Meanwhile, the dependence of the strains of Myxococcus and Cystobacter on organic carbon content was not too obvious, and they can also be found in the poor soils. Our findings provided an important scientific base for the development and utilization of myxobacteria resources.

Isolation and identification of myxobacteria from saline-alkaline soils in Xinjiang, China.

Fifty-eight terrestrial and salt-tolerant myxobacteria were isolated from the saline-alkaline soils collected from Xinjiang, China. Based on the morphologies and the 16S rRNA gene sequences, these isolates were assigned into 6 genera, Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium. All the strains grew better with 1% NaCl than without NaCl. Some Myxococcus strains were able to grow at 2% NaCl concentration, suggesting that these strains may be particular type of terrestrial myxobacteria.

Co-cultivation of Sorangium cellulosum strains affects cellular growth and biosynthesis of secondary metabolite epothilones.

Sorangium cellulosum, a cellulolytic myxobacterium, is capable of producing a variety of bioactive secondary metabolites. Epothilones are anti-eukaryotic secondary metabolites produced by some S. cellulosum strains. In this study, we analyzed interactions between 12 strains of S. cellulosum consisting of epothilone-producers and non-epothilone producers isolated from two distinct soil habitats. Co-cultivation on filter papers showed that different Sorangium strains inhibited one another's growth, whereas epothilone production by the producing strains changed markedly for most (73%) pairwise mixtures. Using a quantitative polymerase chain reaction, we demonstrated that the expression of epothilone biosynthetic genes in the epothilone producers typically changed significantly when these bacteria were mixed with non-producing strains. The results indicated that intraspecies interactions between different S. cellulosum strains not only inhibited the growth of partners, but also could change epothilone production.

Myxobacteria: natural pharmaceutical factories.

Myxobacteria are amongst the top producers of natural products. The diversity and unique structural properties of their secondary metabolites is what make these social microbes highly attractive for drug discovery. Screening of products derived from these bacteria has revealed a puzzling amount of hits against infectious and non-infectious human diseases. Preying mainly on other bacteria and fungi, why would these ancient hunters manufacture compounds beneficial for us? The answer may be the targeting of shared processes and structural features conserved throughout evolution.

Cell flexibility affects the alignment of model myxobacteria.

Myxobacteria are social bacteria that exhibit a complex life cycle culminating in the development of multicellular fruiting bodies. The alignment of rod-shaped myxobacteria cells within populations is crucial for development to proceed. It has been suggested that myxobacteria align due to mechanical interactions between gliding cells and that cell flexibility facilitates reorientation of cells upon mechanical contact. However, these suggestions have not been based on experimental or theoretical evidence. Here we created a computational mass-spring model of a flexible rod-shaped cell that glides on a substratum periodically reversing direction. The model was formulated in terms of experimentally measurable mechanical parameters, such as engine force, bending stiffness, and drag coefficient. We investigated how cell flexibility and motility engine type affected the pattern of cell gliding and the alignment of a population of 500 mechanically interacting cells. It was found that a flexible cell powered by engine force at the rear of the cell, as suggested by the slime extrusion hypothesis for myxobacteria motility engine, would not be able to glide in the direction of its long axis. A population of rigid reversing cells could indeed align due to mechanical interactions between cells, but cell flexibility impaired the alignment.

Expanded phylogeny of myxobacteria and evidence for cultivation of the 'unculturables'.

An expanded neighbour-joining tree of myxobacteria is presented based on the analysis of 16S rRNA gene sequences of 101 strains (including types) representing 3 suborders, 6 families, 20 genera, 46 species, and 12 other novel taxa. The distinctions amongst members of the three suborders (Sorangiineae, Cytobacterineae and Nannocystineae) are reaffirmed. The positions of anaerobic myxobacteria, novel groups (Pyxidicoccus and several Cystobacter species) in Cystobacterineae, the marine genera (Plesiocystis, Haliangium, Enhygromyxa), and two additional novel taxa ('Paraliomyxa miuraensis', brackish-water isolate) were together revealed for the first time. Changes in the nomenclature of several isolates (Polyangium vitellinum Pl vt1(T), Polyangium thaxteri Pl t3, Polyangium cellulosum, NOSO-1, NOCB-2, NOCB-4) are also highlighted. Suborders Sorangiineae and Nannocystineae hold great promise for novel strain discovery. In Sorangiineae, the new family Phaselicystidaceae, with a monotypic genus, was added. Nine additional novel taxa were discovered in this suborder for which new genera or even families may be erected in the near future. These taxa appear to represent the so-called viable but not culturable (VBNC) group of myxobacteria. Based on at least 4% phylogenetic distance, new clades were formed comprising of novel Nannocystineae and Sorangiineae isolates. Overall, the myxobacteria, on the basis of bracket distance, could be divided into 16 clusters, as supported by tree topology and a morphology-based approach.

The biosynthesis of branched dialkylpyrazines in myxobacteria.

The biosynthesis of the volatiles 2,5- and 2,6-diisopropylpyrazine (2 and 3, resp.) released by the myxobacteria Nannocystis exedens subsp. cinnabarina (Na c29) and Chondromyces crocatus (strains Cm c2 and Cm c5) was studied. Isotopically labeled precursors and proposed pathway intermediates were fed to agar plate cultures of the myxobacteria. Subsequently, the volatiles were collected by use of a closed loop stripping apparatus (CLSA), and incorporation into the pyrazines was followed by GC/MS analysis. [(2)H(8)]Valine was smoothly incorporated into both pyrazines clearly establishing their origin from the amino acid pool. The cyclic dipeptide valine anhydride (16)--a potential intermediate on the biosynthetic pathway to branched dialkylpyrazines--was synthesized containing (2)H(1) labels in specific positions. Feeding of [(2)H(16)]-16 and [(2)H(12)]-16 in both valine subunits mainly resulted in the formation of pyrazines derived from only one labeled amino acid, whereas only traces of the expected pyrazines with two labeled subunits were found. To investigate the origin of nitrogen in the pyrazines, a feeding experiment with [(15)N]valine was performed, resulting in the incorporation of the (15)N label. The results contradict a biosynthetic pathway via cyclic dipeptides, but rather point to a pathway on which valine is reduced to valine aldehyde. Its dimerization to 2,5-diisopropyldihydropyrazine 36 and subsequent oxidation results in 2. The proposed biosynthetic pathway neatly fits the results of earlier labeling studies and also explains the formation of the regioisomer 2,6-diisopropylpyrazine 3 by isomerization during the first condensation step of two molecules valine aldehyde. A general biosynthetic pathway to different classes of pyrazines is presented.

Correlations between the role, sequence conservation, genomic location and severity of phenotype in myxobacterial developmental genes.

Myxobacterial development requires the coordinated action of both intracellular and intercellular signalling pathways. A dataset of myxobacterial developmental gene properties suggests that genes encoding components of intracellular pathways tend to be less conserved, yield less severe phenotypes upon deletion and lie closer to the chromosomal origin than intercellular signalling genes. It would seem that there is a stronger negative selection affecting the mutation of intercellular signalling pathway genes than intracellular genes. Presumably, this is because the loss of social behaviour (and consequently sporulation) upon mutation of an intercellular gene is profoundly detrimental to the perpetuation of the organism. Conversely, mutation of an intracellular gene would typically result in a socially capable mutant. The correlations presented here between the severity of phenotype, genomic location and the degree of sequence conservation should aid rational exploration of the genomics of social development in the myxobacteria.

Dynamic-energy-budget-driven fruiting-body formation in myxobacteria.

We develop an interacting particle model to simulate the life cycle of myxobacteria, which consists of two main stages--the swarming stage and the development (fruiting body formation) stage. As experiments have shown that the phase transition from swarming to development stage is triggered by starvation, we incorporate into the simulation a system of ordinary differential equations (ODEs) called the dynamic energy budget, which controls the uptake and use of energy by individuals. This inclusion successfully automates the phase transition in our simulation. Only one parameter, namely, the food density, controls the entire simulation of the life cycle.

Comprehensive proteome profiling of the Fe(III)-reducing myxobacterium Anaeromyxobacter dehalogenans 2CP-C during growth with fumarate and ferric citrate.

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta-proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2-D reference map of the soluble A. dehalogenans proteome in order to provide a sound basis for further proteomic studies as well as to gain first global insights into the metabolic activities of this bacterium. Using a combination of 2-DE and MALDI-TOF-MS, a total of 720 proteins spots were identified, representing 559 unique protein species. Using the proteome data, altogether 50 metabolic pathways were found to be expressed during growth with fumarate as primary electron acceptor. An analysis of the pathways revealed an extensive display of enzymes involved in the catabolism and anabolism of a variety of amino acids, including the unexpected fermentation of lysine to butyrate. Moreover, using the reference gel as basis, a semi-quantitative analysis of protein expression changes of A. dehalogenans during growth with ferric citrate as electron acceptor was conducted. The adaptation to Fe(III) reducing conditions involved the expression changes of a total of 239 proteins. The results suggest that the adaptation to Fe(III) reductive conditions involves an increase in metabolic flux through the tricarboxylic acid cycle, which is fueled by an increased catabolism of amino acids.

Unique ecophysiology among U(VI)-reducing bacteria as revealed by evaluation of oxygen metabolism in Anaeromyxobacter dehalogenans strain 2CP-C.

Anaeromyxobacter spp. respire soluble hexavalent uranium, U(VI), leading to the formation of insoluble U(IV), and are present at the uranium-contaminated Oak Ridge Integrated Field Research Challenge (IFC) site. Pilot-scale in situ bioreduction of U(VI) has been accomplished in area 3 of the Oak Ridge IFC site following biostimulation, but the susceptibility of the reduced material to oxidants (i.e., oxygen) compromises long-term U immobilization. Following oxygen intrusion, attached Anaeromyxobacter dehalogenans cells increased approximately 5-fold from 2.2x10(7)+/-8.6x10(6) to 1.0x10(8)+/-2.2x10(7) cells per g of sediment collected from well FW101-2. In the same samples, the numbers of cells of Geobacter lovleyi, a population native to area 3 and also capable of U(VI) reduction, decreased or did not change. A. dehalogenans cells captured via groundwater sampling (i.e., not attached to sediment) were present in much lower numbers (<1.3x10(4)+/-1.1x10(4) cells per liter) than sediment-associated cells, suggesting that A. dehalogenans cells occur predominantly in association with soil particles. Laboratory studies confirmed aerobic growth of A. dehalogenans strain 2CP-C at initial oxygen partial pressures (pO2) at and below 0.18 atm. A negative linear correlation [micro=(-0.09xpO2)+0.051; R2=0.923] was observed between the instantaneous specific growth rate micro and pO2, indicating that this organism should be classified as a microaerophile. Quantification of cells during aerobic growth revealed that the fraction of electrons released in electron donor oxidation and used for biomass production (fs) decreased from 0.52 at a pO2 of 0.02 atm to 0.19 at a pO2 of 0.18 atm. Hence, the apparent fraction of electrons utilized for energy generation (i.e., oxygen reduction) (fe) increased from 0.48 to 0.81 with increasing pO2, suggesting that oxygen is consumed in a nonrespiratory process at a high pO2. The ability to tolerate high oxygen concentrations, perform microaerophilic oxygen respiration, and preferentially associate with soil particles represents an ecophysiology that distinguishes A. dehalogenans from other known U(VI)-reducing bacteria in area 3, and these features may play roles for stabilizing immobilized radionuclides in situ.

Myxobacteria--'microbial factories' for the production of bioactive secondary metabolites.

In this article, we briefly review the potential of myxobacteria as 'natural product factories' by highlighting results from the recently sequenced myxobacterial model strain Myxococcus xanthus. We will focus on the production of polyketides, non-ribosomally-made peptides, and their hybrids, and discuss the evaluation of biosynthetic potential using genome-based methods, as well as biosynthetic process engineering.