Ultrastructural characterization and multilocus sequence analysis (MLSA) of ‘Candidatus Rickettsiella isopodorum’, a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda)

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation ‘Candidatus Rickettsiella isopodorum’ is introduced for this new lineage of isopod-associated Rickettsiella bacteria.     

Genetic and electron-microscopic characterization of Rickettsiella tipulae, an intracellular bacterial pathogen of the crane fly, Tipula paludosa

Rickettsiella tipulae is an intracellular bacterial pathogen of larvae of the crane fly, Tipula paludosa (Diptera: Tipulidae) and has previously been claimed to represent an independent species within the genus Rickettsiella. Recently, this taxon has been reorganized and transferred as a whole from the α-proteobacterial order Rickettsiales to the γ-proteobacterial order Legionellales. Here we present the electron-microscopic identification of this rickettsial pathogen together with the first DNA sequence information for R. tipulae. The results of our 16S rDNA-based phylogenetic analysis demonstrate that the transfer to the order Legionellales is justified for R. tipulae. However, there is no phylogenetic basis to consider R. tipulae an independent species, but instead conclusive evidence substantiating its species level co-assignment with Rickettsiella melolonthae. Furthermore, implications of our results for a possible reorganization of the internal structure of the genus Rickettsiella are discussed.     

Multilocus Sequence Analysis (MLSA) of ‘Rickettsiella agriotidis’, an Intracellular Bacterial Pathogen of Agriotes Wireworms

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as ‘Rickettsiella agriotidis’. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of ‘R. agriotidis’ to the pathotypes ‘Rickettsiella melolonthae’ and ‘Rickettsiella tipulae’, i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. ‘R. agriotidis’ forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of ‘R. agriotidis’ and further R. popilliae synonyms has been critically evaluated.     

Genetic and Electron-Microscopic Characterization of ‘<Emphasis Type="Italic">Rickettsiella agriotidis</Emphasis>’, a new <Emphasis Type="Italic">Rickettsiella</Emphasis> Pathotype Associated with Wireworm, <Emphasis Type="Italic">Agriotes</Emphasis> sp. (Coleoptera: Elateridae)

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae), are severe and widespread agricultural pests affecting numerous crops. A previously unknown intracellular bacterium has been identified in a diseased Agriotes larva. Microscopic studies revealed the subcellular structures characteristic of Rickettsiella infections. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the wireworm pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the new pathotype designation ‘R. agriotidis’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that—on the basis of the currently accepted organization of the genus Rickettsiella—this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.     

Multilocus sequence analysis (MLSA) of ‘Rickettsiella costelytrae' and ‘Rickettsiella pyronotae’, intracellular bacterial entomopathogens from New Zealand

Aims

Larvae of scarab beetles live in the soil and are frequently hosts for microbial pathogens. In New Zealand, larvae of the grass grub, Costelytrae zealandica (Coleoptera: Scarabaeidae), and manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae), have been collected from field populations showing loss of vigour and a whitened appearance. Diagnosis indicated an intracellular infection of fat body tissues by Rickettsiella‐like micro‐organisms. Rickettsiella bacteria are under evaluation as a possible new source of insect bio‐control agents for important agricultural pests as, e.g. scarabaeid and elaterid larvae. The present study aimed at the unequivocal molecular taxonomic identification and comparison of the bacteria associated with Costelytra and Pyronota.

Methods and Results

Electron microscopy and phylogenetic reconstruction using a multilocus sequence analysis approach based on the 16S ribosomal RNA gene together with four protein‐encoding markers (ftsY, gidA, rpsA, and sucB) demonstrated that both bacteria from New Zealand are phylogenetically closely related, but not identical, and belong to the taxonomic genus Rickettsiella.

Conclusions

The bacteria under study should be referred to as pathotypes ‘Rickettsiella costelytrae’ and ‘Rickettsiella pyronotae’, respectively. Moreover, on the basis of the currently accepted systematic organization of the genus Rickettsiella, both pathotypes should be considered synonyms of the nomenclatural type species, Rickettsiella popilliae.

Significance and Impact of the Study

The study demonstrates that Rickettsiella bacteria are geographically widespread pathogens of scarabaeid larvae. Implications of the phylogenetic findings presented for the stability of host adaptation by Rickettsiella bacteria are critically discussed.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.     

Bacterial species identification from MALDI-TOF mass spectra through data analysis and machine learning

At present, there is much variability between MALDI-TOF MS methodology for the characterization of bacteria through differences in e.g., sample preparation methods, matrix solutions, organic solvents, acquisition methods and data analysis methods. After evaluation of the existing methods, a standard protocol was developed to generate MALDI-TOF mass spectra obtained from a collection of reference strains belonging to the genera Leuconostoc, Fructobacillus and Lactococcus. Bacterial cells were harvested after 24 h of growth at 28 °C on the media MRS or TSA. Mass spectra were generated, using the CHCA matrix combined with a 50:48:2 acetonitrile:water:trifluoroacetic acid matrix solution, and analyzed by the cell smear method and the cell extract method. After a data preprocessing step, the resulting high quality data set was used for PCA, distance calculation and multi-dimensional scaling. Using these analyses, species-specific information in the MALDI-TOF mass spectra could be demonstrated. As a next step, the spectra, as well as the binary character set derived from these spectra, were successfully used for species identification within the genera Leuconostoc, Fructobacillus, and Lactococcus. Using MALDI-TOF MS identification libraries for Leuconostoc and Fructobacillus strains, 84% of the MALDI-TOF mass spectra were correctly identified at the species level. Similarly, the same analysis strategy within the genus Lactococcus resulted in 94% correct identifications, taking species and subspecies levels into consideration. Finally, two machine learning techniques were evaluated as alternative species identification tools. The two techniques, support vector machines and random forests, resulted in accuracies between 94% and 98% for the identification of Leuconostoc and Fructobacillus species, respectively.     

Meiothermus granaticius sp. nov., a new slightly thermophilic red-pigmented species from the Azores

Two red-pigmented isolates, with optimum growth temperatures between 45 and 50 °C, were recovered from a hot spring in the Furnas, Área da Fonte 1825 on the Island of São Miguel in the Azores. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented a new species of the genus Meiothermus. These new isolates could be distinguished from other strains of the species of the genus Meiothermus primarily by the fatty acid composition and polar lipid pattern, since they did not possess 2-OH fatty acids or glycolipid variant GL-1a. Moreover, the two new isolates had the lowest growth temperature range of any of the known species of the genus Meiothermus. On the basis of the results presented here we propose the name Meiothermus granaticius for the new species represented by strains AF-68^T (=DSM 23260^T = LMG 25524^T) and AF-49 (=DSM 23259 = LMG 25525).     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus)

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria'' any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.     

Microbial tolerance in secondary peritonitis is dose dependent

Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48 h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48 h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24 h (median: 5.04 log CFU) and 48 h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48 h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10⁵ CFU and 2 × 10⁸ CFU Klebsiella. Cells harvested from animals 48 h after infection, were unresponsive to an ex vivo stimulation with 10⁵ CFU Klebsiella, but fully responded to 10⁸ CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.     

p-Hydroxyphenylpyruvate dioxygenase is a herbicidal target site for beta-triketones from Leptospermum scoparium

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism and is the molecular target site of β-triketone pharmacophores used to treat hypertyrosinemia in humans. In plants, HPPD is involved in the biosynthesis of prenyl quinones and tocopherols, and is the target site of β-triketone herbicides. The β-triketone-rich essential oil of manuka (Leptospermum scoparium), and its components leptospermone, grandiflorone and flavesone were tested for their activity in whole-plant bioassays and for their potency against HPPD. The achlorophyllous phenotype of developing plants exposed to manuka oil or its purified β-triketone components was similar to that of plants exposed to the synthetic HPPD inhibitor sulcotrione. The triketone-rich fraction and leptospermone were approximatively 10 times more active than that of the crude manuka oil, with I₅₀ values of 1.45, 0.96 and 11.5 μg mL⁻¹, respectively. The effect of these samples on carotenoid levels was similar. Unlike their synthetic counterpart, steady-state O₂ consumption experiments revealed that the natural triketones were competitive reversible inhibitors of HPPD. Dose-response curves against the enzyme activity of HPPD provided apparent I₅₀ values 15.0, 4.02, 3.14, 0.22 μg mL⁻¹ for manuka oil, triketone-rich fraction, leptospermone and grandiflorone, respectively. Flavesone was not active. Structure-activity relationships indicate that the size and lipophilicity of the side-chain affected the potency of the compounds. Computational analysis of the catalytic domain of HPPD indicates that a lipophilic domain proximate from the Fe²⁺ favors the binding of ligands with lipophilic moieties.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Kneallhazia carolinensae sp. nov., a microsporidian pathogen of the thief ant, Solenopsis carolinensis

A new species of microsporidia is described from adults of the thief ant, Solenopsis carolinensis, collected in Florida, USA. Morphological and genetic characterization of this new species showed that it is most closely related to the genus Kneallhazia and is therefore formally designated, Kneallhazia carolinensae sp. nov. Masses of ovoid, binucleate spores were localized to fat body of adult workers and measured 6.2 ± 0.1 × 3.1 ± 0.1 μm (fresh) and 6.0 ± 0.1 × 3.4 ± 0.1 μm (fixed). These spores were in direct contact with the cell cytoplasm and contained an isofilar polar filament with 12-15 coils. Blastn analysis revealed that the K. carolinensae 16S rDNA sequence exhibited 91% identity with the 16S rDNA gene of K. solenopsae. The morphological and sequence data support the conclusion that K. carolinensae is a novel microsporidian species distinct from K. solenopsae.     

Phenotypic Effect of “Candidatus Rickettsiella viridis,” a Facultative Symbiont of the Pea Aphid (Acyrthosiphon pisum), and Its Interaction with a Coexisting Symbiont

A gammaproteobacterial facultative symbiont of the genus Rickettsiella was recently identified in the pea aphid, Acyrthosiphon pisum. Infection with this symbiont altered the color of the aphid body from red to green, potentially affecting the host''s ecological characteristics, such as attractiveness to different natural enemies. In European populations of A. pisum, the majority of Rickettsiella-infected aphids also harbor another facultative symbiont, of the genus Hamiltonella. We investigated this Rickettsiella symbiont for its interactions with the coinfecting Hamiltonella symbiont, its phenotypic effects on A. pisum with and without Hamiltonella coinfection, and its infection prevalence in A. pisum populations. Histological analyses revealed that coinfecting Rickettsiella and Hamiltonella exhibited overlapping localizations in secondary bacteriocytes, sheath cells, and hemolymph, while Rickettsiella-specific localization was found in oenocytes. Rickettsiella infections consistently altered hosts'' body color from red to green, where the greenish hue was affected by both host and symbiont genotypes. Rickettsiella-Hamiltonella coinfections also changed red aphids to green; this greenish hue tended to be enhanced by Hamiltonella coinfection. With different host genotypes, Rickettsiella infection exhibited either weakly beneficial or nearly neutral effects on host fitness, whereas Hamiltonella infection and Rickettsiella-Hamiltonella coinfection had negative effects. Despite considerable frequencies of Rickettsiella infection in European and North American A. pisum populations, no Rickettsiella infection was detected among 1,093 insects collected from 14 sites in Japan. On the basis of these results, we discuss possible mechanisms for the interaction of Rickettsiella with other facultative symbionts, their effects on their hosts'' phenotypes, and their persistence in natural host populations. We propose the designation “Candidatus Rickettsiella viridis” for the symbiont.     

Complete mitochondrial genome of the stonefly Cryptoperla stilifera Sivec (Plecoptera: Peltoperlidae) and the phylogeny of Polyneopteran insects

We present the complete mitogenome of a stonefly, Cryptoperla stilifera Sivec (Plecoptera; Peltoperlidae). The mitogenome was a circular molecule consisting of 15,633 nucleotides, 37 genes and a A + T-rich region. C. stilifera mitogenome was similar to Pteronarcys princeps mitogenome (Plecoptera; Pteronarcyidae). All transfer RNA genes (tRNAs) had typical cloverleaf secondary structures except for trnSer (AGN), where the stem-loop structure of the dihydrouridine (DHU) arm was missing. The A + T-rich region of C. stilifera had two stem-loops and each had two interlink. Three conserved sequence blocks (CSBs) were present in the A + T-rich regions of C. stilifera, Peltoperla tarteri and Peltoperla arcuata. Moreover, many polynucleotide stretches (Poly N, N = A, T and C) in the A + T-rich region of C. stilifera Phylogenetic relationships of Polyneopteran species were constructed based on the nucleotide sequences of 13 protein coding genes (PCGs). Both maximum likelihood (ML) and Bayesian inference (BI) analyses supported Grylloblattodea as the sister group to Plecoptera + Dermaptera and Embiidina and Phasmatodea as sister groups.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).     

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae

Background

Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity.

Results

We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99 % sequence identity in rDNA sequence and orthology across 85.6 % of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8 %) were present in Serratia while 33 (84.6 %) and 35 (89 %) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively.

Conclusion

The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result – killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1697-8) contains supplementary material, which is available to authorized users.     

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141 bp, 16236 bp and 16183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A + T-rich region. The genomes were highly compact with only 60–68 bp of non-coding intergenic spacer. However, considerable differences in the A + T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.