Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogenetic relationships among strains of the entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) isolated from Japan

The fungus Beauveria bassiana (Balsamo) Vuillemin has previously been classified using morphological characteristics, but morphology cannot reveal the phylogenetic relationships among conventionally classified strains. High levels of homology have been found in gene sequences among various B. bassiana strains, complicating the determination of their evolutionary relationships. To elucidate phylogenetic relationships among conventionally known Beauveria species, we analyzed 57 major strains of B. bassiana and 3 strains of B. brongniartii (Saccardo) Petch isolated from Japan by analysis of internal transcribed spacer (ITS) sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The ITS sequence analysis placed the 57 conventional B. bassiana strains into two clusters, B. bassiana and Beauveria pseudobassiana Rehner et Humber. In contrast, GP analysis produced five clusters of B. bassiana strains that included B. pseudobassiana clusters. These results suggested that GP was more accurate than ITS sequence analysis for determining phylogenetic relationships within B. bassiana. In addition, our findings suggested that conventional strains of B. bassiana isolated from Japan include both B. bassiana and B. pseudobassiana groups.     

Interspecies relations between <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains studied by AP-PCR and sequence analysis of ribosomal operon regions

The interspecies relationships between Bacillus thuringiensis strains producing different types of δ-endotoxins were studied using a range of molecular-biological methods. Analysis of the 16S rRNA nucleotide sequence, the 16S to 23S rRNA intergenic spacer sequence, and the 5′-terminal region of 23S rRNA allowed the studied strains to be subdivided into three groups based on the pattern of nucleotide substitutions. In terms of the pattern of substitutions, the strains of the first group are similar to the B. thuringiensis type strain ATCC 10792^T, the strains of the second group are practically identical to B. anthracis and the B. cereus type strain ATCC 14579^T, whereas the third group combines strains of B. thuringiensis subsp. morrisoni with the cry2 gene and strains of B. thuringiensis subsp. tenebrionis with the cry3 gene. PCR fingerprinting with the use of six different primer systems ((GTG)₅, REP, ERIC, and DIR) confirmed the presence of three statistically relevant groups, whose structure correlated with that suggested by the analysis of ribosomal operon regions.     

Fluorescent Heteroduplex Assay for Monitoring Bacillus anthracis and Close Relatives in Environmental Samples

A fluorescent heteroduplex method was developed to assess the presence of 16S rRNA gene (rDNA) sequences from Bacillus anthracis and close relatives in PCR-amplified 16S rDNA sequence mixtures from environmental samples. The method uses a single-stranded, fluorescent DNA probe, 464 nucleotides in length, derived from a B. anthracis 16S rRNA gene. The probe contains a unique, engineered deletion such that all probe-target duplexes are heteroduplexes with an unpaired G at position 343 (ΔG343). Heteroduplex profiles of sequences ≥85% similar to the probe were produced using an ABI 377 sequencer in less than 3 h. The method divides strains of the Bacillus cereus-Bacillus thuringiensis-B. anthracis group into two subgroups. Each subgroup is defined by a specific 16S rRNA gene sequence type. Sequence type A, containing one mismatch with the probe, occurs in B. anthracis and a small number of closely related clonal lineages represented mostly by food-borne pathogenic isolates of B. cereus and B. thuringiensis. Sequence type B, containing two mismatches with the probe, is found in the majority of B. cereus and B. thuringiensis strains examined to date. Sequence types A and B, when hybridized to the probe, generate two easily differentiated heteroduplexes. Thus, from heteroduplex profiles, the presence of B. cereus-B. thuringiensis-B. anthracis subgroups in environmental samples can be inferred unambiguously. The results show that fluorescent heteroduplex analysis is an effective profiling technique for detection and differentiation of sequences representing small phylogenetic or functional groups in environmental samples.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

A phylogenetic reconsideration of suctorian ciliates (Protista,Ciliophora, Phyllopharyngea) based on small subunit rRNA gene sequences

Compared with other ciliated protozoa, molecular studies of phylogenetic relationships within the subclass Suctoria are rare. In this work, phylogenetic analyses focusing on this group were performed based on all data available. In addition, the small subunit ribosomal RNA (SSU rRNA) genes of three suctorian ciliates (Acineta compressa, Acineta tuberosa and Paracineta limbata) were newly sequenced. Furthermore, the putative secondary structures of the variable region 2 of the SSU rRNA gene were predicted and compared within the Suctoria. Our results show that (i) there is support for the monophyly of the subclass Suctoria, which is a sister clade to the cyrtophorids; (ii) based on combined morphologic and molecular features, we propose the following evolutionary routine within the Suctoria: Exogenina – Evaginogenina – Endogenina; (iii) the similarities of the secondary structures of the V2 region and the SSU rRNA gene sequences within the subclass Suctoria are consistent with the branching of the phylogenetic lineages.     

Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.     

Distribution Analysis of IS<Emphasis Type="Italic">231</Emphasis>-like Sequences Among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Serovars Inferred from Restriction Fragment Length Polymorphisms

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.     

Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.     

Multilocus sequence analysis of Bacillus thuringiensis serovars navarrensis, bolivia and vazensis and Bacillus weihenstephanensis reveals a common phylogeny

The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.     

Characterization of an Environmental Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> from a Hot Spring in Western Himalayas

Bacillus anthracis, the etiological agent of anthrax, is responsible for a serious and often fatal disease of mammalian livestock and humans and is an important biological warfare agent. Bacillus sp. AKG was isolated from a hot spring in western Himalayas and species-specific primers targeting gyrB gene identified the strain as B. anthracis within cereus-group. Cloning, sequencing, and phylogenetic analysis of the partial gyrB sequence from strain AKG indicated a close affiliation with B. anthracis and a few recently isolated strains of B. thuringiensis (e.g., strain Al Hakam and serovar konkukian). Phylogenetic analysis of two other housekeeping genes, clpC and gdpD yielded similar results. This observation is further substantiated by phylogenetic reconstruction using concatenated sequences (1680 bases) of the three genes (gyrB, clpC, and gdpD). Phenotypic features indicated a non-anthracis affiliation for the strain AKG. A novel strategy to distinguish among strains of B. anthracis, B. cereus, and B. thuringiensis based on whole proteome comparison was developed and tested for the identification of this environmental strain. Proteome comparison was used to establish the identity of this unknown environmental strain. Group of replicate 2DE gels for whole cell proteome were generated for each of the three species and strain AKG. Protein spots unique to each group and those showing match between the groups, in a pair-wise comparison, indicated strain AKG as a member of B. thuringiensis. This strategy can be used to assign strains of B. cereus group to their respective species.     

INTRAGENOMIC NUCLEOTIDE POLYMORPHISM AMONG SMALL SUBUNIT (18S) RDNA PARALOGS IN THE DIATOM GENUS SKELETONEMA (BACILLARIOPHYTA)1

Morphological features of the siliceous cell wall traditionally have been used to diagnose and classify species of diatoms, though an increasing number of studies distinguish new species, in part, by phylogenetic analysis of rDNA sequences. Intragenomic sequence variation is common among the hundreds to thousands of rDNA cistrons present within a genome, and this variation has strong potential to obscure species boundaries based on rDNA sequences. We screened six Skeletonema culture strains for intragenomic nucleotide polymorphisms in the small subunit (SSU) rDNA gene and found that all strains had polymorphic sites, with proportions ranging from 0.57% to 1.81%. In all cases, transitions accounted for more than 70% of nucleotide differences at polymorphic sites. Polymorphic sites were split nearly evenly in the SSU rRNA molecule between the base‐paired regions of helices (52%) and the unpaired regions of loops and bulges (48%). Phylogenetic analysis showed that SSU rDNA genotypes were monophyletic for two of the six culture strains examined. Genotypes from the other four culture strains either showed little or no phylogenetic structure compared with genotypes of other conspecific culture strains or had phylogenetic structure that was incongruent with existing species boundaries. Moderate to strong support for monophyly was recovered for four of the seven species included in the analysis. Phylogenetic results combined with the low sequence divergence of SSU rDNA genotypes within species suggest that concerted evolution has not proceeded to completion in these species and/or that the rate at which variation is being generated exceeds the rate at which concerted evolution is expunging variation.     

Amoebae of the genera Vannella Bovee, 1965 and Platyamoeba isolated from fish and their phylogeny inferred from SSU rRNA gene and ITS sequences

Twenty strains of flattened amoebae including 17 isolated from fish were characterised morphologically both at light microscopical and ultrastructural levels and assigned to either the genus Vannella Bovee, 1965 or the genus Platyamoeba Page, 1969. Sequence-based phylogenetic analyses of SSU rRNA genes from a data set representing a total of 29 strains of flattened amoebae strongly indicated that morphological features discriminating between these genera do not reflect phylogenetic relationships of representative strains. Contrary to a previous study, strains of this expanded assemblage formed clusters that did not reflect their environmental origin. Monophyletic groups were of mixed origins and contained freshwater as well as marine strains of both genera isolated in geographically distant localities of various continents. These findings were supported by results of phylogenetic analyses of selected strains based on ITS sequences. However, topologies of acquired ITS trees were not congruent with results inferred from SSU rRNA analyses.     

A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae), in Vietnam

In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.     

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.     

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.     

Isolation of spore-forming bacilli from mosquitoes in natural breeding habitats in Iraq

New isolates of spore-forming bacilli from larvae and pupae of 3 species of mosquitoes are recorded in central Iraq.Bacillus sphaericus Meyer and Neide was isolated fromCulex pipiens (L.) larva.Bacillus carotarum Koch andBacillus cereus Frankland & Frankland were isolated fromTheobaldia longiareolata (Macquart) pupae.Bacillus laterosporus Laubach andBacillus thuringiensis (H 18) were isolated fromAedes caspius (Pallas) larvae. In addition, unidentifiedBacillus spp. were isolated fromCx. pipiens, T. longiareolata andAe. caspius larvae. Examination of soil samples collected from mosquito natural breeding habitats revealed isolates ofB. cereus, Bacillus thuringiensis H4a 4b; H 12 and H 16 and an unidentifiedBacillus sp.

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Résumé Des souches bactériennes sporogènes sont isolées de moustiques qui se trouvent dans la région centrale de l'Irak. Les résultats obtenus sont les suivants:Bacillus sphaericus Meyer & Neide [Culex pipiens (L.), larve],Bacillus carotarum Koch etBacillus cereus Frankland & Frankland [Theobaldia longiareolata (Macquart), nymphe],Bacillus laterosporus Laubach etBacillus thuringiensis (H 18) [Aedes caspius (Pallas), larvel]. L'examen des larves deCulex pipiens. T. longiareolata etAe. caspius, ainsi que l'analyse des échantillons du sol prélevés dans la région montrent la présence deBacillus cereus, Bacillus thuringiensis H4a 4b; H12 plus H16 et d'autresBacillus non identifiés.