Evolution of symbiotic bacteria in the distal human intestine

The adult human intestine contains trillions of bacteria, representing hundreds of species and thousands of subspecies. Little is known about the selective pressures that have shaped and are shaping this community's component species, which are dominated by members of the Bacteroidetes and Firmicutes divisions. To examine how the intestinal environment affects microbial genome evolution, we have sequenced the genomes of two members of the normal distal human gut microbiota, Bacteroides vulgatus and Bacteroides distasonis, and by comparison with the few other sequenced gut and non-gut Bacteroidetes, analyzed their niche and habitat adaptations. The results show that lateral gene transfer, mobile elements, and gene amplification have played important roles in affecting the ability of gut-dwelling Bacteroidetes to vary their cell surface, sense their environment, and harvest nutrient resources present in the distal intestine. Our findings show that these processes have been a driving force in the adaptation of Bacteroidetes to the distal gut environment, and emphasize the importance of considering the evolution of humans from an additional perspective, namely the evolution of our microbiomes.     

Ultrastructural and molecular evidence for potentially symbiotic bacteria within the byssal plaques of the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study reports on the presence of a putatively symbiotic bacterial flora within the byssus plaque of the deep sea hydrothermal mussel Bathymodiolus azoricus, contributing to metal sequestration/deposition and testing positive to methane oxidizing symbiont-specific fluorescent probes. Combining an array of approaches including histology, electron microscopy, X-ray microanalysis, analytical chemistry, and microbiology we provide evidence for the frequently assumed, but rarely shown influence of prokaryotes on the biogeochemical cycling of metals as well as inorganic C sources (i.e., methane) at deep sea hydrothermal vents. Our results indicate that in spite of its antibacterial protective sheath, the byssus plaque gives access to a whole range of prokaryotic organisms which may be responsible for the extremely high concentration of metallic elements (Fe, Cu, Zn, Mn, Co, Mo, Cd, Pb and Hg) measured in this attachment organ. The very high levels of metals in byssus, together with its frequent renewal rate due to the dynamic nature of the habitat, suggest that intra-byssal bacteria may have a major influence on biomineralisation/deposition of metals. The presence of a methanotroph morphotype within the byssus plaque was confirmed by FISH and TEM. The implications of the biogeochemical cycling of metals and methane at hydrothermal vents are discussed.     

Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae)

Summary

Codakia orbicularis is a tropical lucinid harboring gill endosymbionts which are environmentally transmitted from a free living-symbiont form to the new host generation after metamorphosis. Structural changes occurring in the cellular organization from incomplete gill filaments in young aposymbiotic juveniles to full differentiated gill filaments containing bacterial endosymbionts in reared symbiotic juveniles, were analyzed for juveniles from 250 μm to 2 μm shell-length. Aposymbiotic juveniles possess differentiated gill filaments with ciliated, intermediary, and lateral zones similar to those described in wild juveniles, except for the bacteriocytes which are lacking. Granule cells, which progressively differentiate during the morphogenesis of the gill filament, do not appear as a consequence of symbiosis. Experimental colonization of aposymbiotic juveniles by the free-living symbiont form has been obtained through the addition of unsterilized sand collected from the natural habitat of C. orbicularis. Two days after exposure to crude sand, symbiosis-competent bacteria enter by endocytosis at the apical pole of undifferentiated cells which progressively differentiate into classical bacteriocytes similar to those found in the adult gill filaments. Undifferentiated cells of aposymbiotic gill filaments remain receptive to bacteria several months after metamorphosis, and become bacteriocytes when aposymbiotic juveniles get contact with the symbiont free-living form. Therefore, the environmental transmission of symbionts does not appear to be restrained to a defined period of time during post-larval development in C. orbicularis.     

Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process ofHypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH₂ were 0.9×10⁻³M (PE-1) and 1.2×10⁻³M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.     

Molecular phylogeny of the genus Dactylopius (Hemiptera: Dactylopiidae) and identification of the symbiotic bacteria

Phylogenetic analyses, from polymerase chain reaction (PCR)-amplified 12S rRNA and 18S rRNA gene sequences from cochineal insects of the genus Dactylopius present in Mexico, showed that D. ceylonicus, D. confusus, and D. opuntiae are closely related. D. coccus constitutes a separate clade, and D. tomentosus is the most distantly related. Bacterial 16S rRNA sequences from all the Dactylopius species sampled showed a common β-proteobacteria, related to Azoarcus, also found in eggs and in bacteriocytes in D. coccus. We propose the name "Candidatus Dactylopiibacterium carminicum" for this endosymbiont. Other bacterial sequences recovered from the samples were close to those from soil or plant associated bacteria, like Massilia, Herbaspirillum, Acinetobacter, Mesorhizobium, and Sphingomonas, suggesting a possible horizontal transmission from Cactaceae plant sap to Dactylopius spp. during feeding. This is the first molecular analysis of Dactylopius species and of their associated bacteria.     

Characterization of endophytic and symbiotic bacteria within plants of the endemic association Centaureetum horridae Mol

Abstract

We investigated the internal association of bacteria with Astragalus terraccianoi and Centaurea horrida, two endemic plants of the Mediterranean islands, forming the phytosociological association Centaureetum horridae, typical of windswept cliffs on the rocky shores of Asinara (Sardinia, Italy) and other limited locations. Sampling occurred in the protected natural park of the Asinara island. Roots and stems of the two plants and the root nodules of A. terraccianoi were surface sterilized in order to remove external and rhizospheric microbiota and to subsequently isolate the culturable bacterial communities. Plate counts revealed densities of endophytes between 3.7 × 10² and 2.8 × 10⁴ colony forming units per gram of fresh weight. 16S rDNA sequencing revealed the occurrence of bacteria displaying high similarity with Actinobacterium sp., Paenibacillus sp., Rhizobium sp., Methylobacterium sp., Pedobacter panaciterrae, Aerococcus viridans, Stenotrophomonas rhizophila, Bacillus sporothermodurans, Bacillus pumilus, Bacillus simplex, Bacillus flexus, Streptomyces ciscaucasicus and Dyella sp. The putative nitrogen-fixing rhizobium symbiont of A. terraccianoi was identified for the first time. It turned out to belong to the slow-growing Bradyrhizobium genus and to share a 97% similarity with Bradyrhizobium canariense. It was found to be nonculturable and to coexist in nodules with a number of different endophytes.     

Interrelationship of Bradyrhizobium sp. and plant growth-promoting bacteria in cowpea: Survival and symbiotic performance

The objective of this study was to evaluate the survival of cowpea during bacterial colonization and evaluate the interrelationship of the Bradyrhizobium sp. and plant growth-promoting bacteria (PGPB) as a potential method for optimizing symbiotic performance and cowpea development. Two experiments using the model legume cowpea cv. “IPA 206” were conducted. In the first experiment, cowpea seeds were disinfected, germinated and transferred to sterilized Gibson tubes containing a nitrogen-free nutritive solution. The experimental design was randomized blocks with 24 treatments [Bradyrhizobium sp. (BR 3267); 22 PGPB; absolute control (AC)] with three replicates. In the second experiment, seeds were disinfected, inoculated according to their specific treatment and grown in Leonard jars containing washed and autoclaved sand. The experimental design was randomized blocks with 24 treatments [BR 3267; 22 BR 3267 + PGPB; AC] with three replicates. Scanning electron microscopy demonstrated satisfactory colonization of the roots of inoculated plants. Additionally, synergism between BR 3267 and PGPB in cowpeas was observed, particularly in the BR 3267 + Paenibacillus graminis (MC 04.21) and BR 3267 + P. durus (C 04.50), which showed greater symbiotic performance and promotion of cowpea development.     

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.     

Repression of competition favours cooperation: experimental evidence from bacteria

Repression of competition (RC) within social groups has been suggested as a key mechanism driving the evolution of cooperation, because it aligns the individual’s proximate interest with the interest of the group. Despite its enormous potential for explaining cooperation across all levels of biological organization, ranging from fair meiosis, to policing in insect societies, to sanctions in mutualistic interactions between species, there has been no direct experimental test of whether RC favours the spread of cooperators in a well‐mixed population with cheats. To address this, we carried out an experimental evolution study to test the effect of RC upon a cooperative trait – the production of iron‐scavenging siderophore molecules – in the bacterium Pseudomonas aeruginosa. We found that cooperation was favoured when competition between siderophore producers and nonsiderophore‐producing cheats was repressed, but not in a treatment where competition between the two strains was permitted. We further show that RC altered the cost of cooperation, but did not affect the relatedness among interacting individuals. This confirms that RC per se, as opposed to increased relatedness, has driven the observed increase in bacterial cooperation.     

Impact of plant nutrients on the relationship between a herbivorous insect and its symbiotic bacteria

The interactions between herbivorous insects and their symbiotic micro-organisms can be influenced by the plant species on which the insects are reared, but the underlying mechanisms are not understood. Here, we identify plant nutrients, specifically amino acids, as a candidate factor affecting the impact of symbiotic bacteria on the performance of the phloem-feeding aphid Aphis fabae. Aphis fabae grew more slowly on the labiate plant Lamium purpureum than on an alternative host plant Vicia faba, and the negative effect of L. purpureum on aphid growth was consistently exacerbated by the bacterial secondary symbionts Regiella insecticola and Hamiltonella defensa, which attained high densities in L. purpureum-reared aphids. The amino acid content of the phloem sap of L. purpureum was very low; and A. fabae on chemically defined diets of low amino acid content also grew slowly and had elevated secondary symbiont densities. It is suggested that the phloem nutrient profile of L. purpureum promotes deleterious traits in the secondary symbionts and disturbs insect controls over bacterial abundance.     

Scavenger deterrent factor (SDF) from symbiotic bacteria of entomopathogenic nematodes

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are symbiotically associated with bacteria in the genera Xenorhabdus and Photorhabdus, respectively. The symbiotic bacteria produce a chemical compound(s) that deterred ants from feeding on nematode-killed insects (i.e., cadavers) and has been previously referred to as an Ant Deterrent Factor (ADF). We studied the response of different arthropod scavenger species which included the ant Lepisiota frauenfeldi, cricket Gryllus bimaculatus, wasps Vespa orientalis and Paravespula sp., and calliphorid fly Chrysomya albiceps, to ADF. These scavengers (ants, crickets, and wasps) were exposed to cadavers with and without the nematode/bacterium complex or to Photorhabdus luminescens cultures of different ages on different substrates. The ant, cricket, and wasp species did not feed on nematode-killed insects containing the nematode/bacterium complex that were 2 days old and older but fed on 1-day-old nematode-killed and freeze -killed insects. Crickets consumed 2- to 7-day-old axenic nematode-killed insects, 1-, 4-, and 5-day-old insects killed by the bacterium, Serratia marcescens, and freeze-killed, putrid insects that were up to 10 days old. The crickets only partially consumed 2- and 3-day-old insects killed by S. marcescens which differed significantly from the 1-, 4-, and 5-day-old killed insects by this bacterium. Ants fed only on 5% sucrose solution (control) and 1- to 3- day old cultures of P. luminescens containing 5% sucrose but not on older cultures of P. luminescens. Wasps did not feed on meat treated with P. luminescens supernatant, whereas they fed on meat treated with Escherichia coli supernatant and control meat. Calliphorid flies did not oviposit on meat treated with P. luminescens supernatant but did oviposit on untreated meat. Based on the response of these scavengers, the chemical compound(s) responsible for this deterrent activity should be called "scavenger deterrent factor" (SDF).     

The recovery of benthic foraminifera and bacteria after disturbance: experimental evidence

In experiments with living meiofauna, sediment is often sieved prior to incubation. This is primarily to remove macrofauna and to increase reproducibility among replicates. At the onset of the experiment, the bacteria are severely disturbed. The effects of these disturbances are ill-known but might affect the outcome of the experimental meiofaunal and biogeochemical studies substantially.We compared the disturbance induced by sieving with the disturbance in microcosms from which meiofauna was removed by flushing with argon. Both experimental situations were compared with untreated cores and the field situation. Neither sieving nor flushing induced changes in the composition of the foraminiferal community compared with the natural situation; the four most abundant species found in the field remained dominant during the experiment. Sieving led to a pronounced disturbance in both bacterial as well as foraminiferal abundance patterns. The depth distribution of some species seems to be related to food, although bacteria might play a regulating role as well.     

Experimental and mathematical simulation of plant growth promoting rhizobacteria and plant interaction under cadmium stress

Bacterial inoculants of the commercially available plant growth promoting rhizobacteria (PGPR) Arthrobacter mysorens 7, Flavobacterium sp. L30, and Klebsiella mobilis CIAM 880 were selected to obtain ecologically safe barley crop production on cadmium (Cd) polluted soils. All the PGPR immobilized 24–68% soluble cadmium from soil suspension. A. mysorens 7 and K. mobilis CIAM 880 were highly resistant to Cd and grew in up to 1 and 3 mmol CdCl₂ on DAS medium respectively. All PGPR were able to fix nitrogen (276–1014 nmol mg^–1 bacterial DW) and to produce indole acetic acid (IAA) (126–330 nmol mg^–1 bacterial DW) or ethylene (4.6–13.5 nmol bacterial DW). All the PGPR actively colonized barley root system and rhizosphere and significantly stimulated root elongation of barley seedlings (up to 25%), growing on soil containing 5 or 15 mg Cd kg^–1 of soil. Created in the simulation mathematical model confirms our hypothesis that PGPR beneficial effect on barley growing under Cd-stress is a complex process. One of mechanisms underlying this effect might be increase of bacterial migration from rhizoplane to rhizosphere, where PGPR bind soluble free Cd ions in biologically unavailable complex forms. Among the studied PGPR K. mobilis CIAM 880 was the most effective inoculant. Inoculation with K. mobilis CIAM 880 of barley plants growing on Cd contaminated soil (5 mg Cd kg^–1 of soil) under field conditions increased by 120% grain yield and 2-fold decreased Cd content in barley grain. The results suggest that the using K. mobilis CIAM 880 is an effective way to increase the plant yield on poor and polluted areas.     

Gill ultrastructure and symbiotic bacteria in Codakia orbicularis (Bivalvia,Lucinidae)

Summary The cellular organization of the gill, which harbors symbiotic bacteria, is described in juveniles and adults of Codakia orbicularis, a large tropical Lucinidae. The ciliary zone is similar in every species of Lucinidae described and includes the large clear cell which has been previously described as an intermediary cell. The intermediary zone is composed of a few narrow unciliated cells, which bind adjacent filaments together and constitute channels through which sea water circulates along the abfrontal part of the filaments. The lateral zone is more complex in C. orbicularis than in other Lucinidae, being composed of four cell types and differentiated into two distinct regions. The bacteriocytes and intercalary cells occupy the outermost bacteriocyte zone, while mucocytes and numerous cells crowded with proteinic, cystine-rich granules constitute the innermost secretory zone which has not been described in other species. The newly described granule cells are considered to be a key factor in the storage and metabolic conversion of sulfur compounds.     

异小杆线虫XJZL1409(Heterorhabditis bacteriophora)共生菌的分离及致病性研究

本研究分离了来自新疆和田地区间作绿豆的4年枣0-40 cm根际土壤中的昆虫病原线虫XJZL1409Heterorhabditis bacteriophora的共生菌,并进行致病性测定。通过侵染期线虫和线虫致死的大蜡螟幼虫血腔直接分离,采用传统形态学观察结合分子生物学方法对菌株进行鉴定。同时大蜡螟Galleria mellonella 5龄幼虫接种菌株细胞发酵液测定致病性。菌株形态学特性结合16S r DNA序列系统发育分析,线虫XJZL1409的共生菌被鉴定为发光杆菌属Photorhabdus luminescens subsp. laumondii。室内菌液注射毒性测定结果显示,侵染致死的幼虫体色呈灰绿色;接种48 h后,每头幼虫接种菌液量为500 CFU时校正死亡率就可以达到100%; 48 h时致死中浓度LC50为10. 74 CFU/m L。同时菌液口服杀虫毒性结果表明,第5天时待测菌株菌液对大蜡螟幼虫的校正死亡率为21. 21%,体重抑制率为20. 02%。因此,昆虫病原线虫XJZL1409的共生菌为P. luminescens subsp. laumondii对大蜡螟幼虫具有很强的注射毒性,为研究线虫的致病机制及发掘新抗性基因奠定基础。     

Genomic and phylogenetic characterization of luminous bacteria symbiotic with the deep-sea fish Chlorophthalmus albatrossis (Aulopiformes: Chlorophthalmidae)

Bacteria forming light-organ symbiosis with deep-sea chlorophthalmid fishes (Aulopiformes: Chlorophthalmidae) are considered to belong to the species Photobacterium phosphoreum. The identification of these bacteria as P. phosphoreum, however, was based exclusively on phenotypic traits, which may not discriminate between phenetically similar but evolutionarily distinct luminous bacteria. Therefore, to test the species identification of chlorophthalmid symbionts, we carried out a genomotypic (repetitive element palindromic PCR genomic profiling) and phylogenetic analysis on strains isolated from the perirectal light organ of Chlorophthalmus albatrossis. Sequence analysis of the 16S rRNA gene of 10 strains from 5 fish specimens placed these bacteria in a cluster related to but phylogenetically distinct from the type strain of P. phosphoreum, ATCC 11040(T), and the type strain of Photobacterium iliopiscarium, ATCC 51760(T). Analysis of gyrB resolved the C. albatrossis strains as a strongly supported clade distinct from P. phosphoreum and P. iliopiscarium. Genomic profiling of 109 strains from the 5 C. albatrossis specimens revealed a high level of similarity among strains but allowed identification of genomotypically different types from each fish. Representatives of each type were then analyzed phylogenetically, using sequence of the luxABFE genes. As with gyrB, analysis of luxABFE resolved the C. albatrossis strains as a robustly supported clade distinct from P. phosphoreum. Furthermore, other strains of luminous bacteria reported as P. phosphoreum, i.e., NCIMB 844, from the skin of Merluccius capensis (Merlucciidae), NZ-11D, from the light organ of Nezumia aequalis (Macrouridae), and pjapo.1.1, from the light organ of Physiculus japonicus (Moridae), grouped phylogenetically by gyrB and luxABFE with the C. albatrossis strains, not with ATCC 11040(T). These results demonstrate that luminous bacteria symbiotic with C. albatrossis, together with certain other strains of luminous bacteria, form a clade, designated the kishitanii clade, that is related to but evolutionarily distinct from P. phosphoreum. Members of the kishitanii clade may constitute the major or sole bioluminescent symbiont of several families of deep-sea luminous fishes.     

Transmission of symbiotic bacteria Buchnera to parthenogenetic embryos in the aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea)

All phloem-feeding aphids have an absolute requirement for their primary bacterial symbionts Buchnera sp. The bacteria are transmitted vertically to either embryos in the viviparous morph or to eggs in the oviparous morph, with the implication that the symbiont population regularly passes through a genetic 'bottleneck', i.e. only a small proportion of the maternal symbiont population is transmitted to offspring. In this paper, we visualise this process in viviparous aphids using a specific immunolabelling technique for Buchnera. The images show a stream of bacteria originating from a single mycetocyte and entering the embryo, possibly via a membranous conduit, and individual bacterial cells free in the haemocoel of the aphid. Staining within the embryo blastoderm suggests over expression of antigen, perhaps indicative of rapid bacterial division immediately following infection.