Many animals and plants possess symbiotic microorganisms inside their body, wherein intimate interactions occur between the partners. The Insecta, often rated as the most diverse animal group, show various types of endosymbiotic associations, ranging from obligate mutualism to facultative parasitism. Although technological advancements in culture-independent molecular techniques, such as quantitative PCR, molecular phylogeny and in situ hybridization, as well as genomic and metagenomic analyses, have allowed us to directly observe endosymbiotic associations in vivo, the molecular mechanisms underlying insect-microbe interactions are not well understood, because most of these insect endosymbionts are neither culturable nor genetically manipulatable. However, recent studies have succeeded in the isolation of several facultative symbionts by using insect cell lines or axenic media, revolutionizing studies of insect endosymbiosis. This article reviews the amazing diversity of bacterial endosymbiosis in insects, focusing on several model systems with culturable endosymbionts, which provide a new perspective towards understanding how intimate symbiotic associations may have evolved and how they are maintained within insects.
Cultivation-independent molecular surveys have shown members of the bacterial phylum Chloroflexi to be ubiquitous in various natural and artificial ecosystems. Among the subphylum-level taxa of the Chloroflexi known to date, the formerly uncultured 'subphylum I' had well been recognized as a typical group that contains a number of environmental gene clones with no culture representatives. In order to reveal their ecophysiology, attempts were made over the past decade to domesticate them into laboratory cultures, and significant advances have been made in cultivating strains belonging to the group. The microorganisms characterized so far include seven species in six genera, i.e., Anaerolinea, Levilinea, Leptolinea, Bellilinea, Longilinea, and Caldilinea, and were proposed to represent two classes, Anaerolineae and Caldilineae, providing solid insights into the phenotypic and genetic properties common to the group. Another subphylum-level uncultured group of the Chloroflexi, i.e., the class Ktedonobacteria, has also been represented recently by a cultured strain. In addition to the results from these tangible cultures, data obtained from functional analyses of uncultured Chloroflexi populations by assessing substrate uptake patterns are accumulating at an encouraging rate. In this review, recent findings on the ecological significance and possible ecophysiological roles of 'Chloroflexi subphylum I' are discussed based on findings from both the characteristics of the cultured Chloroflexi and molecular-based analyses.
To investigate changes in bacterial communities associated with a fungal foliar disease, epiphytic bacteria from powdery mildew-infected and uninfected leaves of cucumber and Japanese spindle were analyzed using both culture-dependent and -independent methods. Dilution plate counting suggested that powdery mildew-infected leaves likely accommodated larger populations of phyllosphere bacteria than uninfected leaves. Community-level physiological profiles (CLPP) also indicated that functional diversity, richness, and evenness of bacterial communities were significantly greater in the phyllosphere of powdery mildew-infected leaves. Genotype diversity and richness based on band patterns of denaturing gradient gel electrophoresis (DGGE) of the phyllosphere bacterial community were greater for leaves infected by powdery mildew. A principle component analysis of CLPP and DGGE patterns revealed a clear difference between infected and uninfected leaves of both plant species. These results suggest that powdery mildew-infection results in larger bacterial populations, and greater diversity and richness, and also changes the structure of the phyllosphere bacterial community. Furthermore, DNA sequences of the DGGE bands that showed greater intensity in the infected than uninfected leaves, differed between cucumber and Japanese spindle. This suggests that specific bacteria are associated with the plant species accompanying this fungal infection.
The tracer 15N2 was used to investigate sites of N2 fixation and the possible translocation of the fixed N. Young sugarcane plants (Saccharum officinarum L.) from a stem cutting were exposed to 15N2-labeled air in a 500 mL plastic cylinder. Plants fed 15N2 for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for growth. After 3 days of feeding, the percentage of N derived from 15N2 was higher in the roots (2.22%) and stem cutting (0.271%) than the shoot (0.027%). Most of the fixed N was distributed in the 80% ethanol-insoluble fractions in each plant part, and the 15N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot. The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.
Frankia is a nitrogen-fixing actinobacterium that establishes root nodule symbiosis with actinorhizal plants. The molecular basis of the symbiosis is largely unknown because genetic manipulation of Frankia has not been feasible. In this study we made novel technical attempts to transform Frankia strain CcI3. We generated fusion marker genes consisting of a tetracycline resistance gene with a high codon usage similarity to Frankia's and promoters of the strain's translation initiation factor 3 gene. We flanked the fusion genes with genomic sequences from strain CcI3 in the expectation that they would be integrated into the targeted site by homologous recombination. We introduced the transformation constructs into Frankia cells by electroporation and selected transformants in liquid media. The growth of antibiotic resistant cells was dependent on the presence of construct DNA. PCR analysis of the genome and reverse transcription-PCR analysis confirmed that the marker genes were introduced into the cells. Integration of the marker genes into the chromosome by homologous recombination did occur, but at a low frequency. Most of the constructs were not integrated into the chromosome and existed as degraded molecules in the cells. Marker genes declined in the transformant population during maintenance, showing that the transformation was unstable.
We showed that the photocatalytic effect of a coating of TiO2 greatly reduces the formation of a biofilm by Phormidium tenue (P. tenue), a filamentous cyanobacterium, on glass plates. Sample plates were immersed in P. tenue culture solution (OD730=0.3) under concurrent illumination with white fluorescent (WF) and UV light (0.3 mW cm-2, each) for 11 days. TiO2-coated glass plates showed greatly reduced adhesion of P. tenue over 11 days compared to bare plates. The number of P. tenue adhering to bare glass plates increased to over 106 cells cm-2 in 6 days. The photocatalytic anti-biofilm effect was also observed under WF light, although it was small and lasted only a few days. The addition of 1 mM mannitol, a scavenger for the hydroxyl radical (·OH), suppressed the effect. The surface of TiO2-coated plates was maintained in a highly hydrophilic state for 11 days, regardless of the addition of mannitol. Therefore, we conclude that the photocatalytic oxidation of P. tenue is effective in preventing the formation of a biofilm.
The effects of a nitrogen-fixing tree, black locust (Robinia pseudoacacia), on the distribution of bacterial species were examined in a Japanese black pine (Pinus thunbergii) and black locust-dominated area. DNA was extracted from the soil at depths of 0-5 and 5-10 cm, collected at the border between a Japanese black pine-dominated forest and a black locust-dominated forest, and the distribution of bacterial species was investigated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities did not differ between the two forests. The distribution of some bacterial species correlated significantly with soil pH, soil carbon [C], soil nitrogen [N], and soil N/phosphate [P], but not with soil C/N or soil P. The distributional relationships between ectomycorrhizal (ECM) fungal species and bacterial species were also analyzed. A positive correlation was observed between the distribution of some ECM fungi and bacterial species. These bacteria may have some interactions with ECM fungi in the field.
Endophytic bacteria (247 isolates) were randomly isolated from surface-sterilized stems of non-nodulated (Nod-), wild-type nodulated (Nod+), and hypernodulated (Nod++) soybeans (Glycine max [L.] Merr) on three agar media (R2A, nutrient agar, and potato dextrose agar). Their diversity was compared on the basis of 16S rRNA gene sequences. The phylogenetic composition depended on the soybean nodulation phenotype, although diversity indexes were not correlated with nodulation phenotype. The most abundant phylum throughout soybean lines tested was Proteobacteria (58-79%). Gammaproteobacteria was the dominant class (21-72%) with a group of Pseudomonas sp. significantly abundant in Nod+ soybeans. A high abundance of Alphaproteobacteria was observed in Nod- soybeans, which was explained by the increase in bacterial isolates of the families Rhizobiaceae and Sphingomonadaceae. A far greater abundance of Firmicutes was observed in Nod- and Nod++ mutant soybeans than in Nod+ soybeans. An impact of culture media on the diversity of isolated endophytic bacteria was also observed: The highest diversity indexes were obtained on the R2A medium, which enabled us to access Alphaproteobacteria and other phyla more frequently. The above results indicated that the extent of nodulation changes the phylogenetic composition of culturable bacterial endophytes in soybean stems.
A one-step multi-probe FISH method of detecting viable Vibrio parahaemolyticus was developed. Three candidate regions, corresponding to Helix 440+441, Helix 588, and Helix 1241 in 16S rRNA, were selected for detection, the thermodynamic parameters (ΔGoverall) of the probes were optimized, and VP437, VP612 and VP1253, whose fluorescence were 1.7 to 11.3 times that of ΔGoverall-unadjusted sequences, were designed. The addition of competitive oligonucleotides to reactions with VP612 and VP1253 strengthened the specificity of the probes. The three probes were labeled with FITC, TAMRA, and Cy5, respectively, and using a mixture of the probes and six competitive oligonucleotides, one-step FISH was applied to the species-specific detection of V. parahaemolyticus including epidemic strains of O3:K6 and O4:K68 serotypes. V. alginolyticus, V. rotiferianus, and V. campbellii were not detected in the reaction. Microcolonies (30-80 μm in diameter) of V. parahaemolyticus were observed within 6 hours at 37°C on seawater agar plates in both fresh and heat-damaged V. parahaemolyticus. Viable bacterial counts based on the proposed method were significantly different from those measured with typical vibrio selective media (CHROMagar Vibrio and TCBS).
The process by which a biofilm forms on the surface of the aquatic macrophyte Phragmites australis was investigated over a period of about two months (from mid-May to late-July, 2008) in Lake Biwa. The biofilm formed relatively quickly, its wet weight per unit area after seven day being that of a mature biofilm. This speed can be attributed to the many active bacteria in the early stage of its formation and the extracellular polymeric substances (EPS) they produce. The EPS carried electric charges that attracted nutrient ions from surrounding lake water, which, by electrostatic interaction, reached a high concentration as early as day 7 of the formation process. This significantly affected the biofilm community, which differed greatly from that of the lake water even at the beginning of biofilm formation. Brown amorphous compounds (a complex of organic and inorganic substances), covered the biofilm in the second half of its formation process producing a different community structure from that initially. This study revealed a fast and dynamic process of biofilm formation on the reed surface of reed.
We developed a new method that rapidly and specifically enumerates only viable Listeria monocytogenes in food using fluorescence in situ hybridization in combination with filter cultivation (FISHFC). Viable L. monocytogenes could be specifically quantified within 16 h using an Alexa647-labeled mRL-2 probe. The coefficient of the correlation between the new method and the conventional plating method was 0.959.
A monobromoacetic acid-resistant bacterium, Bacillus strain I37c, was isolated from a marine sediment core. The strain grew in a medium containing 1.8 mg mL-1 of monobromoacetic acid. It produced constitutively a 2-haloacid dehalogenase that catalyzed the dehalogenation of monobromoacetic acid, monochloroacetic acid, and both L- and D-2-chloropropionic acid. The optimal pH and temperature for the activity measured using a partly purified enzyme were similar to those of known group I haloacid dehalogenases.