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1.  Characterization of the Bacterial Community Associated with Larvae and Adults of Anoplophora chinensis Collected in Italy by Culture and Culture-Independent Methods 
BioMed Research International  2013;2013:420287.
The wood-boring beetle Anoplophora chinensis Forster, native to China, has recently spread to North America and Europe causing serious damage to ornamental and forest trees. The gut microbial community associated with these xylophagous beetles is of interest for potential biotechnological applications in lignocellulose degradation and development of pest-control measures. In this study the gut bacterial community of larvae and adults of A. chinensis, collected from different host trees in North Italy, was investigated by both culture and culture-independent methods. Larvae and adults harboured a moderately diverse bacterial community, dominated by Proteobacteria, Actinobacteria, and Firmicutes. The gammaproteobacterial family Enterobacteriaceae (genera Gibbsiella, Enterobacter, Raoultella, and Klebsiella) was the best represented. The abundance of such bacteria in the insect gut is likely due to the various metabolic abilities of Enterobacteriaceae, including fermentation of carbohydrates derived from lignocellulose degradation and contribution to nitrogen intake by nitrogen-fixing activity. In addition, bacteria previously shown to have some lignocellulose-degrading activity were detected at a relatively low level in the gut. These bacteria possibly act synergistically with endogenous and fungal enzymes in lignocellulose breakdown. The detection of actinobacterial symbionts could be explained by a possible role in the detoxification of secondary plant metabolites and/or protection against pathogens.
doi:10.1155/2013/420287
PMCID: PMC3771249  PMID: 24069601
2.  Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae) 
BMC Microbiology  2012;12(Suppl 1):S4.
Background
Bacteria of the genus Asaia have been recently recognized as secondary symbionts of different sugar-feeding insects, including the leafhopper Scaphoideus titanus, vector of Flavescence dorée phytoplasmas. Asaia has been shown to be localized in S. titanus gut, salivary glands and gonoducts and to be maternally transmitted to the progeny by an egg smearing mechanism. It is currently not known whether Asaia in S. titanus is transmitted by additional routes. We performed a study to evaluate if Asaia infection is capable of horizontal transmission via co-feeding and venereal routes.
Results
A Gfp-tagged strain of Asaia was provided to S. titanus individuals to trace the transmission pathways of the symbiotic bacterium. Co-feeding trials showed a regular transfer of bacterial cells from donors to recipients, with a peak of frequency after 72 hours of exposure, and with concentrations of the administrated strain growing over time. Venereal transmission experiments were first carried out using infected males paired with uninfected females. In this case, female individuals acquired Gfp-labelled Asaia, with highest infection rates 72-96 hours after mating and with increasing abundance of the tagged symbiont over time. When crosses between infected females and uninfected males were conducted, the occurrence of “female to male” transmission was observed, even though the transfer occurred unevenly.
Conclusions
The data presented demonstrate that the acetic acid bacterial symbiont Asaia is horizontally transmitted among S. titanus individuals both by co-feeding and venereal transmission, providing one of the few direct demonstrations of such a symbiotic transfer in Hemiptera. This study contributes to the understanding of the bacterial ecology in the insect host, and indicates that Asaia evolved multiple pathways for the colonization of S. titanus body.
doi:10.1186/1471-2180-12-S1-S4
PMCID: PMC3287515  PMID: 22376056
3.  Bacterial Endosymbiont Localization in Hyalesthes obsoletus, the Insect Vector of Bois Noir in Vitis vinifera▿  
One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “Candidatus Phytoplasma solani” and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the “Candidatus Sulcia muelleri” cluster in the Bacteroidetes and in the “Candidatus Purcelliella pentastirinorum” group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name “Candidatus Vidania fulgoroideae.” Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and “Candidatus Cardinium hertigii.” Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As “Ca. Sulcia” is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca. Sulcia” and “Ca. Purcelliella,” “Ca. Vidania,” or both.
doi:10.1128/AEM.02121-10
PMCID: PMC3067237  PMID: 21183640
4.  Acetic Acid Bacteria, Newly Emerging Symbionts of Insects▿  
Applied and Environmental Microbiology  2010;76(21):6963-6970.
Recent research in microbe-insect symbiosis has shown that acetic acid bacteria (AAB) establish symbiotic relationships with several insects of the orders Diptera, Hymenoptera, Hemiptera, and Homoptera, all relying on sugar-based diets, such as nectars, fruit sugars, or phloem sap. To date, the fruit flies Drosophila melanogaster and Bactrocera oleae, mosquitoes of the genera Anopheles and Aedes, the honey bee Apis mellifera, the leafhopper Scaphoideus titanus, and the mealybug Saccharicoccus sacchari have been found to be associated with the bacterial genera Acetobacter, Gluconacetobacter, Gluconobacter, Asaia, and Saccharibacter and the novel genus Commensalibacter. AAB establish symbiotic associations with the insect midgut, a niche characterized by the availability of diet-derived carbohydrates and oxygen and by an acidic pH, selective factors that support AAB growth. AAB have been shown to actively colonize different insect tissues and organs, such as the epithelia of male and female reproductive organs, the Malpighian tubules, and the salivary glands. This complex topology of the symbiosis indicates that AAB possess the keys for passing through body barriers, allowing them to migrate to different organs of the host. Recently, AAB involvement in the regulation of innate immune system homeostasis of Drosophila has been shown, indicating a functional role in host survival. All of these lines of evidence indicate that AAB can play different roles in insect biology, not being restricted to the feeding habit of the host. The close association of AAB and their insect hosts has been confirmed by the demonstration of multiple modes of transmission between individuals and to their progeny that include vertical and horizontal transmission routes, comprising a venereal one. Taken together, the data indicate that AAB represent novel secondary symbionts of insects.
doi:10.1128/AEM.01336-10
PMCID: PMC2976266  PMID: 20851977
5.  Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco▿ †  
Applied and Environmental Microbiology  2009;75(10):3314-3322.
Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between plants and bacteria. Plant DNA released during the degradation of plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or plant-associated bacteria could be in direct contact with plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying plant tissues, to develop a competence state, and to possibly acquire exogenous plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413(rbcL-ΔPaadA::gfp) carrying a promoterless aadA::gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic plant DNA. Opportunistic growth occurred on decaying plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for plant tissues.
doi:10.1128/AEM.02632-08
PMCID: PMC2681637  PMID: 19329660
6.  Acetobacter tropicalis Is a Major Symbiont of the Olive Fruit Fly (Bactrocera oleae)▿  
Applied and Environmental Microbiology  2009;75(10):3281-3288.
Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae. Using an A. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.
doi:10.1128/AEM.02933-08
PMCID: PMC2681620  PMID: 19304818
7.  Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events▿ †  
A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.
doi:10.1128/AEM.02185-07
PMCID: PMC2258602  PMID: 18165369
8.  Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis†  
Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.
doi:10.1128/AEM.72.2.1295-1301.2006
PMCID: PMC1392923  PMID: 16461679
9.  Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities 
Applied and Environmental Microbiology  2004;70(10):6147-6156.
ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng μl−1, while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.
doi:10.1128/AEM.70.10.6147-6156.2004
PMCID: PMC522057  PMID: 15466561
10.  Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of Bacillus and Related Genera 
The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus. We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.
doi:10.1128/AEM.69.9.5128-5137.2003
PMCID: PMC194986  PMID: 12957895
11.  Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes 
Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the Bacillus cereus group showed that B. anthracis Davis TE702 and B. mycoides G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the B. cereus group. Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced. Two main types of ITS were found, either with two tRNA genes (tRNAIle and tRNAAla) or without any at all. Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNAIle and tRNAAla genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS. Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences. Neighbor-joining analysis of tDNA-containing long ITS indicated that B. cereus and B. thuringiensis represent a single clade. Three signature sequences discriminated B. anthracis from B. cereus and B. thuringiensis, indicating that the anthrax agent started evolving separately from the related clades of the B. cereus group. B. mycoides and B. weienstephanensis were very closely related, while B. pseudomycoides appeared the most distant species.
doi:10.1128/AEM.69.1.33-40.2003
PMCID: PMC152393  PMID: 12513974

Results 1-11 (11)