Molecular Identification and Localization of Filamentous Symbiotic Bacteria Associated with the Hydrothermal Vent Annelid Alvinella pompejana

Alvinella pompejana is a polychaetous annelid that inhabits high-temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.     

昆虫病原线虫的共生细菌

昆虫病原线虫与其共生细菌二者互惠共生 :共生细菌需要昆虫病原线虫作为载体以寄生寄主昆虫并做为自己的营养来源 ,而昆虫病原线则需要依靠共生细菌来杀死昆虫。综述了共生细菌的病原作用、抗菌作用与杀虫作用 ,评述了共生细菌的基因工程进展 ,讨论了昆虫共生细菌在昆虫病原线虫致病性的作用。     

Composition and Biophysical Properties of Lipids in Xenorhabdus nematophilus and Photorhabdus luminescens, Symbiotic Bacteria Associated with Entomopathogenic Nematodes

Primary and secondary forms of Photorhabdus luminescens Hm and Xenorhabdus nematophilus N2-4 were grown at 18 and 28(deg)C for 24 to 96 h, and we made determinations of the fatty-acid compositions of total lipids and of the fluidity measured by 5-doxyl-stearic acid embedded in liposomes made from total lipids. The levels of the unsaturated fatty acids 16:1 and 18:1 (those with chain lengths of 16 or 18 and one double bond) generally were higher in primary-phase variants of P. luminescens grown at 18(deg)C than in those grown at 28(deg)C. Prolonged culture at 18(deg)C caused the level of 18:1 to fall and reach that observed at 28(deg)C. The ratio of saturated to unsaturated fatty acids rose with prolonged culture times in variants of each species at both phases. When grown at 18(deg)C, the proportion of 16:1 in X. nematophilus was lower than in P. luminescens; the patterns of temperature-induced changes were similar in these species. X. nematophilus contained a greater percentage of short-chain fatty acids (i.e., with chain lengths of <14.0) than P. luminescens. Lipid liposomes from primary and secondary cultures of both bacterial species grown at 18(deg)C were more ordered (i.e., less fluid) than those grown at 28(deg)C. This result suggests the surprising absence of homeoviscous adaptation of membranes to temperature. Also, liposomes from primary cultures were more ordered than those from secondary cultures and membranes from primary cultures of P. luminescens were more ordered at both culture temperatures than membranes from X. nematophilus. The biological significance of the effect of growth conditions on membrane biophysical properties in these bacteria is discussed.     

Genotyping of Human Lice Suggests Multiple Emergences of Body Lice from Local Head Louse Populations

Background

Genetic analyses of human lice have shown that the current taxonomic classification of head lice (Pediculus humanus capitis) and body lice (Pediculus humanus humanus) does not reflect their phylogenetic organization. Three phylotypes of head lice A, B and C exist but body lice have been observed only in phylotype A. Head and body lice have different behaviours and only the latter have been involved in outbreaks of infectious diseases including epidemic typhus, trench fever and louse borne recurrent fever. Recent studies suggest that body lice arose several times from head louse populations.

Methods and Findings

By introducing a new genotyping technique, sequencing variable intergenic spacers which were selected from louse genomic sequence, we were able to evaluate the genotypic distribution of 207 human lice. Sequence variation of two intergenic spacers, S2 and S5, discriminated the 207 lice into 148 genotypes and sequence variation of another two intergenic spacers, PM1 and PM2, discriminated 174 lice into 77 genotypes. Concatenation of the four intergenic spacers discriminated a panel of 97 lice into 96 genotypes. These intergenic spacer sequence types were relatively specific geographically, and enabled us to identify two clusters in France, one cluster in Central Africa (where a large body louse outbreak has been observed) and one cluster in Russia. Interestingly, head and body lice were not genetically differentiated.

Conclusions

We propose a hypothesis for the emergence of body lice, and suggest that humans with both low hygiene and head louse infestations provide an opportunity for head louse variants, able to ingest a larger blood meal (a required characteristic of body lice), to colonize clothing. If this hypothesis is ultimately supported, it would help to explain why poor human hygiene often coincides with outbreaks of body lice. Additionally, if head lice act as a reservoir for body lice, and that any social degradation in human populations may allow the formation of new populations of body lice, then head louse populations are potentially a greater threat to humans than previously assumed.     

Symbiotic Deficiencies Associated with a coxWXYZ Mutant of Bradyrhizobium japonicum

The terminal oxidase complexes encoded by coxMNOP and coxWXYZ were studied by analysis of mutations in each of the two oxidases. Carbon monoxide difference spectra obtained from membranes of coxMNOP mutant bacteroids were like those obtained for the wild type, whereas bacteroid membranes of a coxWXYZ mutant were deficient in CO-reactive cytochrome b. Experiments involving cyanide inhibition of oxidase activity were consistent with the conclusion that the coxX mutant is deficient in a membrane-associated O₂-binding component. The viable cell number (bacteria that could be recultured from crushed nodules) was 20 to 29% lower for the coxX mutant than for the wild-type or the CoxN⁻ strain. In three separate greenhouse studies, nodules of a coxX mutant had significantly lower (28 to 34% less) acetylene reduction rates than the wild-type nodules did, and plants inoculated with a double mutant (coxMNOP coxWZYZ) had rates 30% lower than those of wild-type-inoculated plants.     

Bacteria Associated with Immunoregulatory Cells in Mice

This study examined bacteria-immune interactions in a mouse model possessing microbiota-dependent immune regulatory features similar to those occurring in human atopy, colitis, and immune regulation. Associations between the abundance of several bacterial phylotypes and immunoregulatory target cell types were identified, suggesting that they may play a role in these phenotypes.Bacteria are involved in critical aspects of immune system development and regulation (5, 23, 26, 29). Mice raised under germfree conditions exhibit a variety of abnormalities, including hypoplastic Peyer''s patches, reduced numbers of IgA-producing cells, relatively unstructured spleen and lymph nodes, and hypogammaglobulinemic serum (23). Remarkably, after several weeks of exposure to standard intestinal microbiota, normal immune structure and function are restored. Mechanistic details underlying microbe-immune interactions have been recently elucidated for two common intestinal bacteria. Bacteroides thetaiotaomicron was shown to induce the angiogenin Ang4, a component of innate immunity possessing microbicidal activity against a wide range of intestinal microbes, including both bacterial and fungal pathogens (16). In addition, studies of the Bacteroides fragilis zwitterionic capsular polysaccharide A have established it as a cognate antigen of certain CD4⁺ T cells, which programs immune effector polarization (24) and protection of mice from infection by Helicobacter hepaticus through several immune-mediated mechanisms (25). Resident microbiota also modify the interaction of dendritic cells with regulatory T-cell populations, with resultant susceptibility to chronic inflammatory disease, like colitis (15, 28).Recent work by Braun and colleagues has characterized a mouse model with unique immunologic features linking resident microbiota with levels of regulatory CD8⁺ T cells (13, 17, 39). This model is comprised of two physically isolated colonies of isogenic mice harboring distinct microbial communities: conventional floras (CF) and restricted floras (RF). CF refers to C57BL/6 mice housed in a standard specific-pathogen-free facility, while RF refers to C57BL/6 mice containing a different complement of intestinal microorganisms (13, 30), originally created by transferring several nonpathogenic anaerobic bacteria into antibiotic-treated mice (13). RF mice differ from CF mice in several immunologic phenotypes, including selective reduction of marginal zone (MZ) B cells (39), plasmacytoid dendritic cells (pDC) (13), and invariant natural killer (iNK) T cells (38a), as well as naïve CD4⁺ and CD8⁺ T cells (17). In addition, RF mice were shown to be resistant to colitis under genetic or adoptive transfer conditions that permit disease activity in CF mice (2). RF mice also cleared experimental infections by Campylobacter jejuni more slowly than did their CF counterparts (6). The resulting concept is that certain resident microbiota, which may be more abundant in RF mice than in CF mice, induce invariant Qa-1 T cells, with resultant changes in host immunoregulation and microbial surveillance (2).An important issue raised by the foregoing observations is the identity of resident microbiota responsible for this host immunoregulatory response. The objective of this study was to develop a methodology, based on bacteria-immune interactions in the RF/CF mouse model, to identify candidate microbiota. In this study, we employed a series of experiments examining associations between the population densities of bacterial rRNA genes and several immunologic features that differ between CF and RF mice.     

Bacteria Associated with Butterfly Stain of Chilean Tepa

Butterfly stain, common in Chilean tepa (Laureliopsis philippiana[Looser] Schodde) trees, appears in cross-section of the stem as a series of partially overlapping orange-brown arcs that in early stages resembles a butterfly. Bacterial isolates from stained tepa wood samples cultured under aerobic and anaerobic conditions were identified using several commercially available identification systems. Pseudomonas sp. were most common in the cultures handled aerobically, and Clostridium sp. were found in the cultures handled anaerobically. A pectinolytic Bacillus sp. and a Gram-negative rod-shaped bacterium that emitted a strong naphthalene-like odor similar to that of stained wood were also isolated. Not all isolates could be identified. This is the first report of Clostridium from tepa. Received: 4 June 1999 / Accepted: 25 August 1999     

Diversity of Bacteria Associated with Natural Aphid Populations

The bacterial communities of aphids were investigated by terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments generated by PCR with general eubacterial primers. By both methods, the γ-proteobacterium Buchnera was detected in laboratory cultures of six parthenogenetic lines of the pea aphid Acyrthosiphon pisum and one line of the black bean aphid Aphis fabae, and one or more of four previously described bacterial taxa were also detected in all aphid lines except one of A. pisum. These latter bacteria, collectively known as secondary symbionts or accessory bacteria, comprised three taxa of γ-proteobacteria (R-type [PASS], T-type [PABS], and U-type [PAUS]) and a rickettsia (S-type [PAR]). Complementary analysis of aphids from natural populations of four aphid species (A. pisum [n = 74], Amphorophora rubi [n = 109], Aphis sarothamni [n = 42], and Microlophium carnosum [n = 101]) from a single geographical location revealed Buchnera and up to three taxa of accessory bacteria, but no other bacterial taxa, in each aphid. The prevalence of accessory bacterial taxa varied significantly among aphid species but not with the sampling month (between June and August 2000). These results indicate that the accessory bacterial taxa are distributed across multiple aphid species, although with variable prevalence, and that laboratory culture does not generally result in a shift in the bacterial community in aphids. Both the transmission patterns of the accessory bacteria between individual aphids and their impact on aphid fitness are suggested to influence the prevalence of accessory bacterial taxa in natural aphid populations.     

昆虫病原线虫共生菌杀虫蛋白的研究进展

昆虫病原线虫共生菌杀虫蛋白对许多农业害虫都有很明显的毒杀作用,作为一种新型的具有开发潜力和应用前景的生物防治资源,已经引起科学家们的关注并成为国内外研究的热点之一.综述了昆虫病原线虫共生菌杀虫蛋白基因的结构和功能及其杀虫机制方面的研究进展.     

Identification and Characterization of a Symbiotic Bacterium Associated with Steinernema carpocapsae in Korea

A symbiotic bacterium was identified from the entomopathogenic nematode, Steinernema carpocapsae, collected in Korea and its biological significance was examined for insecticidal and antibiotic effects. The symbiotic bacterium was isolated from the nematode-infected hemolymph of the fifth instar larvae of Spodoptera exigua. The intra-hemocoelic injection of the bacterial isolates killed the insect hosts within 24h. Several biochemical and morphological characters of the bacterium were identical to those of Xenorhabdus nematophilus. The bacterial growth was not inhibited on the media containing 4,000 ppm of streptomycin sulfate or 2,000 ppm of penicillin. They showed antibacterial effects on Escherichia coli, Ralstonia solanacearum and Pseudomonas aeruginosa, but not on Pseudomonas putida and Pseudomonas fluorescens.     

Diversity of Chemotactic Heterotrophic Bacteria Associated with Arctic Cyanobacteria

The abundance and diversity of chemotactic heterotrophic bacteria associated with Arctic cyanobacteria was determined. The viable numbers ranged between 10⁴ and 10⁶ cell g^?1 cyanobacterial biomass. A total of 112 morphotypes, representing 22 phylotypes based on their 16S rRNA sequence similarity were isolated from the samples. All the phylotypes were Gram-negative with affiliation to the proteobacterial and bacteroidetes divisions. Among the 22 phylotypes, 14 were chemotactic to glucose. Majority of the phylotypes were psychrotolerant showing growth up to 30 °C. Representatives of Alphaproteobacteria, the genus Flavobacterium and the gammaproteobacterial Alcanivorax sp, were psychrophilic with growth at or below 18 °C. A significant percentage of phylotypes were pigmented (~68 %), rich in unsaturated membrane fatty acids and tolerated pH values and NaCl concentrations between 5.0–8.0 and 0.15–1.0 M, respectively. The percentages of phylotypes producing extracellular cold-active enzymes at 4 °C were amylase (18.18 %), lipase and urease (45.45 %), caseinase (59.09 %) and gelatinase (31.8 %).     

Barophilic Bacteria Associated with Digestive Tracts of Abyssal Holothurians

Abyssal holothurians and sediment samples were collected at depths of 4,430 to 4,850 m in the Demerara abyssal plain. Bacterial concentrations in progressive sections of the holothurian digestive tract, as well as in surrounding surface sediments, were determined by epifluorescence microscopy. Total bacterial counts in sediments recently ingested by the animals were 1.5- to 3-fold higher than in surrounding sediments at the deepest station. Lowest counts were observed consistently in the foregut, where the digestive processes of the holothurian are believed to occur. In most animals, counts increased 3- to 10-fold in the hindgut. Microbial activity at 3°C and in situ and atmospheric pressure were determined for gut and sediment samples by measuring the utilization of [¹⁴C]glutamic acid, the doubling time of the mixed-population of culturable bacteria, and the percentage of the total bacterial count responsive to yeast extract in the presence of nalidixic acid, using epifluorescence microscopy. A barophilic microbial population, showing elevated activity under deep-sea pressure, was detected by all three methods in sediments removed from the hindgut. Transmission electron micrographs revealed intact bacteria directly associated with the intestinal lining only in the hindgut. The bacteria are believed to be carried as an actively metabolizing, commensal gut flora that transforms organic matter present in abyssal sediments ingested by the holothurian. Using data obtained in this study, it was calculated that sediment containing organic matter altered by microbial activity cleared the holothurian gut every 16 h, suggesting that abyssal holothurians and their associated gut flora are important participants in nutrient cycles of the abyssal benthic ocean.     

七种我国昆虫病原线虫共生菌的分离与鉴定

昆虫病原线虫共生菌是一类具有广阔发展前景的生物防治资源, 由于这类菌的分类鉴定工作起步晚, 存在分类混乱和不系统的现象, 在本土资源十分丰富的我国尤为突出。本文对本实验室分离的7个共生菌菌株进行了分类描述, 建立了以生理形态, 生化特征为分类基础, 结合16S rDNA序列分析的有效鉴定方法。     

New Method for Counting Bacteria Associated with Coral Mucus

The ability to count bacteria associated with reef-building corals in a rapid, reliable, and cost-effective manner has been hindered by the viscous and highly autofluorescent nature of the coral mucus layer (CML) in which they live. We present a new method that disperses bacterial cells by trypsinization prior to 4′,6-diamidino-2-phenylindole (DAPI) staining and quantification by epifluorescence microscopy. We sampled seawater and coral mucus from Porites lobata from 6 reef sites influenced by wastewater intrusion and 2 reef sites unaffected by wastewater in Hawaii. Bacterial and zooxanthella abundances and cell sizes were quantified for each sample. Bacteria were more abundant in coral mucus (ranging from 5.3 × 10⁵ ± 1.0 × 10⁵ cells ml⁻¹ to 1.8 × 10⁶ ± 0.2 × 10⁶ cells ml⁻¹) than in the surrounding seawater (1.9 × 10⁵ ± 0.1 × 10⁵ cells ml⁻¹ to 4.2 × 10⁵ ± 0.2 × 10⁵ cells ml⁻¹), and the mucus-associated cells were significantly smaller than their seawater counterparts at all sites (P < 0.0001). The difference in cell size between mucus- and seawater-associated bacteria decreased at wastewater-influenced sites, where simultaneously mucus bacteria were larger and seawater bacteria were smaller than those at uninfluenced sites. The abundance of zooxanthellae in mucus ranged from 1.1 × 10⁵ ± 0.1 × 10⁵ cells ml⁻¹ to 3.4 × 10⁵ ± 0.3 × 10⁵ cells ml⁻¹. The frequency of dividing cells (FDC) was higher in the surrounding seawater than in mucus, despite finding that a 1,000-fold-higher zooxanthella biovolume than bacterial biovolume existed in the CML. Establishment of a standardized protocol for enumeration will provide the field of coral microbial ecology with the urgently needed ability to compare observations across studies and regions.The extremely viscous and highly autofluorescent nature of coral mucus has been a major challenge in developing enumeration techniques and has limited our ability to study the ecological interactions among coral mucus layer (CML)-associated microbial communities. Only a few studies have used direct counts to quantify bacteria in the CML, and the methods and subsequent results vary widely. The techniques have included scanning electron microscopy (SEM) (34), phase-contrast microscopy (27), and epifluorescent microscopy using a variety of stains (acridine orange staining [8], SYBR gold [20], and 4′,6-diamidino-2-phenylindole [DAPI] [3]). Bacterial abundances reported from these studies spanned more than 5 orders of magnitude (from 1.6 × 10² cells [cm²]⁻¹ using acridine orange [8] to 6.2 × 10⁷ cells [cm²]⁻¹ using SYBR gold [20]), and some of the studies are difficult to compare to each other because different units were used, such as cells ml⁻¹ of mucus and cells (cm²)⁻¹ of coral. Some variation in abundance is likely due to differences in mucus sampling methods and differences among coral species. However, the enormous quantity of autofluorescence emitted in green and red wavelengths found in most coral species creates a substantial challenge for reliably counting fluorescently stained cells in that portion of the spectrum, because many of the particles are bacterium sized. Many of these same particles could be visible with phase-contrast microscopy as well. Thus far, researchers quantifying CML-associated bacteria using epifluorescence microscopy have prepared their samples by following well-established protocols that were developed for seawater. We suggest that the viscous and autofluorescent nature of coral mucus may require some modifications from standard seawater protocols for epifluorescence microscopy to be most effective.SEM is an alternative to fluorescence-dependent techniques. It has the advantage of acquiring images with sufficient detail to distinguish among particles and cells, but this method is time-consuming, visualizes only the surface of the sample, and is not widely available or affordable enough for it to be a standard field protocol. An additional limitation is that most studies that have employed SEM for CML observation have found bacteria to be too dispersed to count in a reasonable number of micrographs (8, 19).Here we present a new method that disperses bacterial cells by enzymatically digesting the mucus with trypsin (an adaptation of routine cellular biology cell line culture procedures) and subsequently staining the cells with DAPI for rapid quantification using epifluorescence microscopy. DAPI fluoresces in the blue end of the spectrum, and its emission does not overlap with the autofluorescence of the mucus samples. This method is rapid, uses reagents and equipment readily available in microbial ecology laboratories, and can provide necessary information for studies of the ecology of microbial cells associated with mucus. It may also be helpful for studies of other aquatic gel-associated microbial communities.This visualization capability revealed that bacteria living with the reef-building coral Porites lobata were significantly smaller than their water-associated counterparts and that this difference is reduced in reefs heavily influenced by anthropogenic impacts. There is only one other report that we are aware of that observed small bacterial cell size in mucus from corals (of the genus Fungia), but that study did not quantify cell size (34). Given that mucus is a carbon-rich environment (6, 11, 12, 18, 24, 25, 31), this discovery is counterintuitive. It highlights questions regarding the ecological interactions that must occur in situ to select for small cell size in such a rich environment (3, 4, 7, 8, 11, 25, 34).