16S-23S Internal Transcribed Spacer Research Articles

Taxonomic interpretation of non-heterocystous Cyanobacteria (Oscillatoriales) from eastern India with particular emphasis on Lyngbya Plectonema complex

Filamentous non-heterocystous cyanobacterial taxa from 8 genera were collected from different ecological niches like high altitudes, plains and estuaries of eastern India. The systematic accounts of 23 taxa were investigated with a polyphasic approach considering morpho taxonomy, cultural behavior, and molecular phylogenetic analysis with 16S and 16S-23S Internal Transcribed Spacer (ITS) regions as molecular markers. The collected taxa were from the families Oscillatoriaceae, Phormidiaceae and Pseudanabaenaceae with 8 representative genera viz. Lyngbya, Plectonema, Oscillatoria, Limnothrix, Leptolyngbya, Planktothrix, Desertifilum and Phormidium. The 16S-23S ITS region-based molecular characterization of 13 species from Oscillatoriaceae, 6 species from Phormidiaceae, and 4 species from Pseudanabaenaceae were found to be congruent with earlier phylogenetic studies using other markers. Phylogenetic tree analysis revealed habitat-specific clustering of ITS sequences of the investigated taxa. The 16S molecular marker-based phylogenetic analysis, along with cultural studies of the Lyngbya-Plectonema clade, highlighted the need for morphotaxonomic reconsideration of Lyngbya birgei and Plectonema tomasinianum related to the formation of false branching. The present study affirmed that 98% sequence similarity in the ITS region can be considered as a threshold percentage for conspecificity determination in the Lyngbya genus. Keywords: Cyanobacteria; ITS; Oscillatoriaceae; Phormidiaceae; Phylogenetic tree; Pseudanabaenaceae.

Evidence for Bartonella quintana in Lice Collected from the Clothes of Ethiopian Homeless Individuals.

Human lice, Pediculus humanus, can transmit various pathogens, including Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii. Xenosurveillance is an epidemiological approach to assessing human infection risks performed by screening vectors of infectious disease agents. In the proof-of-principle study reported herein, the DNA of 23 human lice was collected from the clothes of 30 homeless Ethiopian individuals. These samples were assessed using 16S rRNA gene-specific pan-eubacterial PCR for screening, followed by Bartonella genus 16S-23S internal transcribed spacer (ITS) sequence-specific PCR, Bartonella genus gltA gene-specific PCR, and 16S rRNA gene PCR with specificity for relapsing-fever-associated Borrelia spp. with subsequent sequencing of the amplicons. In one sample, the pan-eubacterial 16S rRNA gene-specific screening PCR, the Bartonella genus 16S-23S ITS sequence-specific PCR, and the Bartonella genus gltA gene-specific PCR allowed for the sequencing of B. quintana-specific amplicons. In two additional samples, Bartonella genus gltA gene-specific PCR also provided sequences showing 100% sequence identity with B. quintana. In total, 3/23 (13.0%) of the assessed lice were found to be positive for B. quintana. Correlating clinical data were not available; however, the assessment confirmed the presence of B. quintana in the local louse population and thus an associated infection pressure. Larger-sized cross-sectional studies seem advisable to more reliably quantify the infection risk of lice-infested local individuals. The need for prevention by providing opportunities to maintain standard hygiene for Ethiopian homeless individuals is stressed by the reported findings, especially in light of the ongoing migration of refugees.

Morphological and Molecular Evaluation of Pseudanabaena epilithica sp. nov. and P. suomiensis sp. nov. (Pseudanabaenaceae, Cyanobacteria) from Finland

Cyanobacteria represent a cosmopolitan group of oxyphototrophic bacteria. Although free-living cyanobacteria thriving in aquatic habitats as well as cyanobacteria in terrestrial symbiotic systems (lichens) have been extensively studied in Finland, the diversity of terrestrial rock-inhabiting cyanobacteria is overlooked. As part of an ongoing effort to study terrestrial epilithic cyanobacteria from Finland, we isolated two Pseudanabaena-like cyanobacterial strains and characterized them using a polyphasic approach. Although the two strains were firmly placed within the Pseudanabaena clade in the 16S rRNA phylogenetic analyses, relationships among species were better resolved when phylogenetic analyses were based on a concatenate alignment of 16S rRNA gene and 16S–23S Internal Transcribed Spacer (ITS) region. In addition, 16S–23S ITS percent dissimilarity proved to be more useful for species discrimination in Pseudanabaena compared to secondary structures of conserved 16S–23S ITS domains (D1–D1′, box B, V2 and V3 helices). Considering morphological, molecular and ecological information, we describe P. epilithica sp. nov. and P. suomiensis sp. nov. under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants. Neither toxins nor antimicrobial metabolites were detected during LC-MS analysis or antimicrobial susceptibility testing, respectively. Lastly, our phylogenetic analyses revealed that many Pseudanabaena strains are misidentified and highlight the need for taxonomic revision in this poorly studied cyanobacterial genus.

Identification of geosmin biosynthetic gene in geosmin-producing colonial cyanobacteria Coelosphaerium sp. and isolation of geosmin non-producing Coelosphaerium sp. from brackish Lake Shinji in Japan

Geosmin is an earthy-muddy smelling compound produced in aquatic ecosystems by microorganisms including cyanobacteria. An increase in geosmin levels affecting the local fishery occurred in May 2007 in Lake Shinji, Japan, and geosmin-producing colonial cyanobacterium, Coelosphaerium sp. G2, was isolated from a water sample from the lake and identified. Cyanobacteria Coelosphaerium sp. is commonly found in Lake Shinji; however, prior to 2007, earthy-muddy odors were not a frequent issue. Further, there was no information regarding the geosmin biosynthetic gene in colonial cyanobacteria. Here, the geosmin biosynthetic gene (geoA) in strain G2 was identified and its nucleotide sequence was determined. It was found that geoA had 79% and 78% identity with geoA from filamentous geosmin-producing cyanobacteria Fischerella sp. PCC 9431 and geoA2 from Phormidium sp. P2r, respectively. The deduced amino acid sequence of GeoA consisted of two domains that were annotated as terpene cyclase. In 2015, geosmin non-producing Coelosphaerium sp. S3C5 was isolated from Lake Shinji and identified by morphological and genetic analyses. There was no difference in morphology or nucleotide sequences of 16S rRNA and 16S-23S internal transcribed spacer (ITS) between geosmin-producing and non-producing strains, which are therefore closely related and can exist in Lake Shinji. Distinguishing the two strains by observation under a microscope and sequencing of 16S rRNA and 16S-23S ITS have proven difficult. Inconsistency between the appearance of Coelosphaerium cells and the detection of the odor in water samples could therefore be attributed to dominance by the geosmin-producing strain or the non-producing strain. The increase in earthy smell is assumed to be caused by an increase in the geosmin-producing strain in Lake Shinji. Genetic analysis of geoA in Coelosphaerium sp. and the relative abundances of geosmin-producing and non-producing Coelosphaerium strains in Lake Shinji can be used to mitigate the economic damages caused by geosmin. Development of a molecular method to monitor the geosmin-producing strain in water ecosystems is equally important to alleviate the earthy smell caused by this particular strain.

Biogeography of cryoconite forming cyanobacteria on polar and Asian glaciers

AbstractAimCryoconite, a microbe‐mineral aggregate found on glaciers worldwide, is formed by microbial phototrophs, principally cyanobacteria. Despite their ecological importance in supraglacial environments, the phylogeographical distributions of supraglacial cyanobacteria are poorly understood. Here, we investigate the biogeographical distribution of cyanobacteria on glaciers in the Antarctic, Arctic and Asia.LocationGlaciers in the Antarctic, Arctic and Asia.MethodsWe analysed contiguous sequences of 16S rRNA genes and 16S–23S internal transcribed spacer (ITS) regions, determined by a long read strategy and single‐filament PCR analysis in 38 glacial samples. We analysed cyanobacterial distribution patterns and genetic differentiation.ResultsThe cyanobacterial 16S rRNA gene sequences were grouped into 20 operational taxonomic units (OTUs), and the six major OTUs that accounted for 88% of sequences were distributed broadly from polar to Asian glaciers, suggesting that they are cosmopolitan at the species level. However, analysis of the more variable ITS region revealed geographical differentiation at the strain level. Nineteen OTUs, including the six major OTUs, showed considerable genetic differentiation among geographical regions; at the population level, they are, thus, geographically restricted. Only one of the phylotype exhibits a population structure which does not show a relationship with geographical distribution, suggesting that is cosmopolitan, even at the strain level.Main conclusionsOur 16S rRNA gene analyses suggest a global distribution of species of cyanobacteria colonizing glacier surfaces; however, the 16S–23S ITS regions revealed that most of the phylotypes are fundamentally endemic to particular areas at the population level and indicate limited migration among regions. Our result suggests that selection pressures among geographical regions are strong driving forces shaping genetic structure in cyanobacteria.

A morphological and phylogenetic study of a filamentous cyanobacterium, Microcoleus vaginatus, associated with the moss Mnium cuspidatum

Mnium cuspidatum Hedw. from the Bashuigou landscape of the Guandi Mountains, Shanxi Province, China, found to be associated with a cyanobacterium that was successfully isolated and cultivated under axenic conditions and designated as Microcoleus sp. GD based on its morphology. Phylogenetic analysis of 16S rRNA alone and of the 16S rRNA combined with the 16S–23S internal transcribed spacer (ITS) region, and the presence of an 11-bp insert in the 16S rRNA gene, indicated that Microcoleus sp. GD was as Microcoleus vaginatus. Phylogenetic analysis of the 16S rRNA gene sequences revealed that Microcoleus sp. GD formed a well-supported clade with aquatic epipelic strains of M. vaginatus from the Czech Republic and with desert strains from the USA. Microcoleus sp. GD was more closely related to the aquatic strains living at the seciment/water interface. The secondary structure of the 16S–23S ITS showed that the Box-B helix of Microcoleus sp. GD was structurally was similar to these strains but quite different from the desert strains. Furthermore, the D1-D1′ helices of all strains varied but Microcoleus sp. GD was again most similar to the aquatic strains.

Desertifilum fontinale sp. nov. (Oscillatoriales, Cyanobacteria) from a warm spring in East Africa, based on conventional and molecular studies.

Desertifilum is a filamentous cyanobacterium of crusts and biofilms, recently described from the extreme hot and dry Thar Desert in north-western India. A new species was isolated from a warm spring near Lake Bogoria, Kenya and was characterised by light and electron microscopy and phylogenetically using the 16S rRNA gene, beta and alpha subunits including intergenic spacer (cpcBA-IGS) and 16S-23S internal transcribed spacer (ITS). The aquatic habitat of the new material from Kenya revealed a wider distribution of members of this genus. The new taxon was found to be closest to Desertifilum tharense (Oscillatoriales). The separation of the new species described here as D. fontinale sp. nov., using conventional and molecular traits, was based on differences in phenotypic features, 16S-23S ITS sequence and habitat.

Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis.

The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S–23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S–23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.

First Report of Acidovorax avenae subsp. avenae Causing Sugarcane Red Stripe in Gabon.

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 1- to 3-mm wide and several dm long dark red stripes were observed on sugarcane (Saccharum spp.) leaves of many plants of cultivar R570. These plants were 5.5 months old in the first ratoon crop. Additionally, spindle leaves of several stalks were rotted and could be easily pulled out of the top of the stalk. Longitudinal sections of diseased stalks showed reddish-brown discoloration of the upper stem and the rotted spindle had an unpleasant odor. Circular, convex, smooth, yellow-cream pigmented bacterial colonies with 2 to 3 mm diameter were isolated after 3 days at 28°C from young leaf lesions on YPGA (yeast extract 7 g/L, peptone 7 g/L, glucose 7 g/L, agar 15 g/L, pH 6.8 to 7.0). The 16S-23S internal transcribed spacer (ITS) of two representative colonies was PCR amplified, and the nucleotide sequences were shown to be 99% identical to the 16S-23S ITS sequence from the genome of Acidovorax avenae subsp. avenae strain ATCC 19860 (GenBank: CP002521.1). One of these A. avenae subsp. avenae isolates from Gabon was inoculated into greenhouse grown plants of sugarcane cultivar R570. Plants were inoculated by injection into the sheath of spindle leaves above the meristem with the bacterial strain (12 plants) or with a water control (six plants). In this method, the bacteria (108 CFU/ml) were injected using a syringe through the leaf sheath until filling the leaf whorl. Three weeks post-inoculation, one to several cm long red-brown stripes were observed on leaves of 11 of 12 inoculated plants. Seven weeks post-inoculation, all plants exhibited symptoms, from red, brown, or black stripes to leaf necrosis, rotting, and death of the spindle leaves (six plants). All six control plants were symptomless. In a second experiment, 6 of 12 plants showed symptoms 3 weeks post inoculation, and the pathogen was successfully re-isolated from all six symptomatic plants with YPGA medium. The 16S-23S ITS of three single colonies obtained each from different symptomatic plants were PCR amplified and the nucleotide sequences were again found 99% identical to the 16S-23S ITS sequence from the genome of A. avenae subsp. avenae ATCC 19860. To our knowledge, this is the first report of A. avenae subsp. avenae, the causal agent of sugarcane red stripe (also reported as top rot), in Gabon. It is also the first description of the occurrence of the top rot form of the disease in R570, a cultivar that is grown in several locations of Africa, the Mascarene Islands, and the French West Indies. A large-scale survey needs to be undertaken to determine the distribution of red stripe in Gabon, a disease for which several outbreaks have been reported recently worldwide (1,2).

Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains

The rumen is the fermentative forestomach of ruminant animals, and is host to a wide range of anaerobic bacteria whose primary function is to facilitate forage degradation. Butyrivibrio and Pseudobutyrivibrio are closely related proteolytic and fibrolytic genera within the family Lachnospiraceae, and are commonly isolated from the rumens of animals fed fibrous diets. The ribosomal RNA (rRNA) operon is an important phylogenetically informative locus that is present in multiple copies in bacterial genomes. Ribosomal RNA genes are typically arranged in the order 16S-23S-5S, with internal transcribed spacer (ITS) regions located between the genes. However, in the rumen bacterium, Butyrivibrio proteoclasticus B316, rRNA operons have a 16S-5S-23S rRNA gene arrangement, and analysis of bacterial genome projects revealed that this configuration was present in all publicly available complete genomes from members of the family Lachnospiraceae. The 16S-23S ITS region is commonly used to identify bacterial strains, thus we sought to determine the utility of this region from rumen Butyrivibrio and Pseudobutyrivibrio isolates for their rapid molecular identification. Polymerase chain reaction was used to amplify 16S-23S ITS regions, which were assessed for length polymorphism (ITS-LP), and restriction fragment length polymorphism (ITS-RFLP) using AluI, HaeIII and HhaI on a panel of 13 Butyrivibrio and Pseudobutyrivibrio reference strains. Cluster analysis of the resulting banding patterns revealed that while the ITS-LP method did not group the strains according to major Butyrivibrio and Pseudobutyrivibrio clades identified via 16S rRNA gene sequences, ITS-RFLP was more discriminative, and able to rapidly delineate the strains into these clades.

First Report of Sugarcane Leaf Scald in Gabon Caused by a Highly Virulent and Aggressive Strain of Xanthomonas albilineans.

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 0.5 to 1 cm wide chlorotic stripes covered with many small red streaks were observed on sugarcane (Saccharum spp.) leaves of a single plant of cultivar R581. After removal of the leaves covering the base of the stalks, abnormal development of basal side shoots was also observed. Transversal sections of a diseased stalk showed reddening of the vessels near the nodes. Circular, convex, smooth, shiny, translucent, non-mucoid, honey-yellow pigmented bacterial colonies were isolated from stalk pieces and side shoots on XAS selective agar medium (1). The nucleotide sequence of the 16S-23S internal transcribed spacer (ITS) of a representative colony was shown to be 100% identical to the 16S-23S ITS sequence from the genome of Xanthomonas albilineans strain GPE PC73 (GenBank: FP565176.1). This strain from Gabon was named GAB266. Sugarcane stalks of greenhouse grown cultivar CP68-1026 were inoculated with X. albilineans strains XaFL07-1 from Florida, GPE PC73 from Guadeloupe, and GAB266. Five stalks were inoculated by the modified decapitation method (3) with each strain or with a water control. One month post-inoculation (MPI), white pencil lines and severe necrosis were observed on leaves inoculated with strains XaFL07-1 and GPE PC73, and no disease symptoms appeared on non-inoculated leaves that developed 2 to 3 MPI. These results are in agreement with those generally obtained after inoculation of susceptible sugarcane cultivars with X. albilineans strains from various geographical locations under greenhouse conditions (Rott, unpublished results). In contrast, 1 MPI, only discrete white to red pencil lines were observed along with necrosis on leaves inoculated with strain GAB266, and by 2 to 3 MPI, all five inoculated plants were wilted. The pathogen was successfully reisolated by the stalk blot inoculation technique (3) with XAS medium, from all five inoculated stalks and from 98 of 114 internodes. In a second greenhouse experiment, the same three strains of X. albilineans were inoculated as described above into five sugarcane cultivars differing in resistance to leaf scald in Guadeloupe (2) (CP68-1026, highly susceptible; B69566, susceptible; R570, tolerant; B8008, resistant; Co6415, highly resistant). The same symptoms as those described above were again observed on inoculated leaves of the five sugarcane cultivars 1 MPI. Strains XaFL07-1 and GPE PC73 produced occasionally a single pencil line on non-inoculated leaves 2 to 3 MPI, but only strain GAB266 caused leaf scalding and/or plant death 2 to 3 MPI: cultivar CP68-1026 (5 of 5 plants), B69566 (5 of 5 plants), R570 (4 of 5 plants), B8008 (5 of 5 plants), and only non-inoculated leaves of cultivar Co6415 remained symptomless (5 plants). Strain GAB266 from Gabon appeared, therefore, more virulent and aggressive than the two strains of X. albilineans from Florida and Guadeloupe. To our knowledge, this is the first report of leaf scald of sugarcane in Gabon and the first description of an unusual highly virulent and aggressive strain of X. albilineans. A large-scale survey needs to be undertaken to determine the distribution of leaf scald disease and this new pathotype/race of X. albilineans in Gabon and other geographical locations.

Toxins and Antibiotic Resistance in Staphylococcus aureus Isolated from a Major Hospital in Lebanon.

Molecular characterization of Staphylococcus aureus is of both clinical and infection control importance. Virulence determinants using PCR and multiple drug resistance profiles were studied in 130 S. aureus isolates. PCR-RFLP analysis of the 16S–23S DNA spacer region was done to investigate the level of 16S–23S ITS (internal transcribed spacer) polymorphism. Methicillin-resistant S. aureus (MRSA), which represented 72% of the studied isolates, showed multiple drug resistance with 18% being resistant to 10–18 of the drugs used compared to a maximum resistance to 9 antibiotics with the methicillin sensitive S. aureus (MSSA) isolates. Exfoliative toxin A (ETA) was more prevalent than B (ETB) with virulent determinants being additionally detected in multiple drug-resistant isolates. 16S–23S ITS PCR-RFLP combined with sequencing of the primary product was successful in generating molecular fingerprints of S. aureus and could be used for preliminary typing. This is the first study to demonstrate the incidence of virulent genes, ACME, and genetic diversity of S. aureus isolates in Lebanon. The data presented here epitomize a starting point defining the major genetic populations of both MRSA and MSSA in Lebanon and provide a basis for clinical epidemiological studies.

Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria

To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) region for timely patient management. Two mPCR assays were developed: Slow-Growers (SG) mPCR was used for the detection of slow-growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast-Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species-specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S-23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005-2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.

Mycobacterium shinjukuense sp. nov., a slowly growing, non-chromogenic species isolated from human clinical specimens

Seven isolates of a slowly growing, non-chromogenic Mycobacterium species were obtained from sputum and bronchial lavage fluid samples from elderly patients in different regions of Japan. These isolates were distinguished from related non-tuberculous species by colony morphology, positive results for Tween hydrolysis, catalase at 68 °C, nitrate reductase and pyrazinamidase and negative results for semi-quantitative catalase, urease and arylsulfatase. The mycolic acid pattern obtained by HPLC revealed a single cluster of late-eluting mycolic acids similar to but different from those of Mycobacterium malmoense ATCC 29571(T). The 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), rpoB and hsp65 sequences were unique in comparison with those of other mycobacteria. Comparison of 16S rRNA gene sequences showed that the isolates were most closely related to Mycobacterium tuberculosis H37Rv(T) (21 base differences in 1508 bp; 98.6 % 16S rRNA gene sequence similarity). A representative strain, GTC 2738(T), showed 91.9 % rpoB sequence similarity with Mycobacterium marinum strain M, 95 % hsp65 sequence similarity with Mycobacterium kansasii CIP 104589(T) and 81.1 % 16S-23S ITS sequence similarity with Mycobacterium gordonae ATCC 14470(T). Phylogenetic analysis of concatenated sequences of the 16S rRNA, rpoB and hsp65 genes showed that strain GTC 2738(T) was located on a distinct clade adjacent to M. tuberculosis, M. ulcerans and M. marinum, with bootstrap values of 81 %. DNA-DNA hybridization demonstrated less than 70 % reassociation with type strains of genetically related species and supported the novel species status of the isolates. On the basis of this evidence, a novel species with the name Mycobacterium shinjukuense sp. nov. is proposed. The type strain, isolated from a sputum sample, is strain GTC 2738(T)( = JCM 14233(T) = CCUG 53584(T)).

Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences

Dadheech P.K., Ballot A., Casper P., Kotut K., Novelo E., Lemma B., Pröschold T. and Krienitz L. 2010. Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences. Phycologia 49: 361–372. DOI: 10.2216/09-71.1Arthrospira comprises multicellular, cylindrical, usually screwlike coiled trichomes and is cultivated commercially. In this study, 33 new strains of Arthrospira isolated from plankton samples collected in Mexico, East Africa and India were investigated and compared with 53 strains or samples of earlier considerations. The study included observations of morphological features and molecular phylogenetic analyses on the basis of nucleotide sequences of internal transcribed spacer (ITS) between 16S rRNA and 23S rRNA genes and partial sequences of beta and alpha subunits including intergenic spacer (cpcBA-IGS) of phycocyanin operon. Morphological traits of Arthrospira such as trichome width, type of coiling and apical cell were not always consistent in culture conditions. It was revealed that Arthrospira phylogeny on the basis of cpcBA-IGS locus was broadly comparable with the ITS region as both phylogenetic trees derived from nucleotide sequences could be divided into two main clusters. Cluster I comprised sequences from American strains mainly, whereas cluster II contained the sequences of the strains originating from Africa and Asia chiefly. Both genetic regions of the strains investigated in the present study coincidently showed a significant sequence divergence among Arthrospira strains from East Africa, India and Mexico indicating possible distinct evolutionary lineages.

Phylogeny of the Order Bacillales inferred from 3’ 16S rDNA and 5’ 16S-23S ITS nucleotide sequences

A short 220 bp sequence was used to study the taxonomic organization of the bacterial Order Bacillales. The nucleotide sequences of the 3’ end of the 16S rDNA and the 16S-23S Internal transcribed spacer (ITS) were determined for 32 Bacillales species and strains. The data for 40 additional Bacillales species and strains were retrieved directly from Genbank. Together, these 72 Bacillales species and strains encompassed eight families and 21 genera. The 220 bp se- quence used here covers a conserved 150 bp sequence located at the 3’ end of the 16S rDNA and a conserved 70 bp sequence located at the 5’ end of the 16S-23S ITS. A neighbor-joining phylogenetic tree was inferred from comparative analyses of all 72 nucleotide sequences. Eight major Groups were revealed. Each Group was sub-divided into sub-groups and branches. In general, the neighbor-joining tree presented here is in agreement with the currently accepted phylogeny of the Order Bacillales based on phenotypic and genotypic data. The use of this 220 bp sequence for phylogenetic analyses presents several advantages over the use of the entire 16S rRNA genes or the generation of extensive phenotypic and genotypic data. This 220 bp sequence contains 150 bp at the 3’ end of the 16S rDNA which allows discrimination among distantly related species and 70 bp at the 5’ end of the 16S-23S ITS which, owing to its higher percentage of nucleotide sequence divergence, adds discriminating power among closely related species from same genus and closely related genera from same family. The method is simple, rapid, suited to large screening programs and easily accessible to most laboratories.

Phylogeny of γ-proteobacteria inferred from comparisons of 3’ end 16S rRNA gene and 5’ end 16S-23S ITS nucleotide sequences

The phylogeny of γ-proteobacteria was inferred from nucleotide sequence comparisons of a short 232 nucleotide sequence marker. A total of 64 γ-proteobacterial strains from 13 Orders, 22 families, 40 genera and 59 species were analyzed. The short 232 nucleotide sequence marker used here was a combination of a 157 nucleotide sequence at the 3’ end of the 16S rRNA gene and a 75 nucleotide sequence at the 5’ end of the 16S-23S Internal Transcribed Spacer (ITS) sequence. Comparative analyses of the 3’ end of the 16S rRNA gene nucleotide sequence showed that the last 157 bp were conserved among strains from same species and less conserved in more distantly related species. This 157 bp sequence was selected as the first part in the construction of our nucleotide sequence marker. A bootstrapped neighbor-joining tree based on the alignment of this 157 bp was constructed. This 157 bp could distinguish γ-proteobacterial species from different genera from same family. Closely related species could not be distinguished. Next, an alignment of the 16S-23S ITS nucleotide sequences of alleles from same bacterial strain was performed. The first 75 bp at the 5’ end of the 16S-23S ITS was highly conserved at the intra-strain level. It was selected as the second part in the construction of our nucleotide sequence marker. Finally, a bootstrapped neighbor-joining tree based on the alignment of this 232 bp sequence was constructed. Based on the topology of the neighbour-joining tree, four major Groups, Group I to IV, were revealed with several sub-groups and clusters. Our results, based on the 232 bp sequence were, in general, in agreement with the phylogeny of γ-proteobacteria based on the 16S rRNA gene. The use of this 232 bp sequence as a phylogenetic marker presents several advantages over the use of the entire 16S rRNA gene or the generation of extensive phenotypic and genotypic data in phylogenetic analyses. First, this marker is not allele-dependant. Second, this 232 bp marker contains 157 bp from the 3’ end of the 16S rRNA gene and 75 bp from the 5’ end of the 16S-23S ITS. The 157 bp allows discrimination among distantly related species. Owing to its higher rate of nucleotide substitutions, the 75 bp adds discriminating power among closely related species from same genus and closely related genera from same family. Because of its higher percentage of nucleotide sequence divergence than the 16S rRNA gene, the 232 bp marker can better discriminate among closely related γ-proteobacterial species. Third, the method is simple, rapid, suited to large screening programs and easily accessible to most laboratories. Fourth, this marker can also reveal γ-proteobacterial species which may appear misassigned and for which additional characterization appear warranted.

Mycobacterium mantenii sp. nov., a pathogenic, slowly growing, scotochromogenic species

Slowly growing, scotochromogenic bacteria of a novel Mycobacterium species were isolated from lymph node samples in two children and pulmonary samples in two elderly patients from different regions in the Netherlands as well as from a surface water sample in Zambia. Its 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences are unique in comparison with other mycobacteria. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that these micro-organisms are most closely related to Mycobacterium scrofulaceum ATCC 19981(T) (8 differences; 0.6 % divergence). The hsp65 sequence shows 96 % similarity to that of Mycobacterium saskatchewanense MB54784 and the rpoB sequence shows 95 % similarity to that of Mycobacterium chimaera CIP 107892(T). The 16S-23S ITS sequence places these micro-organisms within the Mycobacterium avium complex, as a novel ITS sequevar. This is not supported by analysis of the 16S rRNA, hsp65 or rpoB gene sequences. Their scotochromogenicity, combined with mostly positive urease, positive semiquantitative catalase and negative tellurite reduction tests, set these isolates apart from related species. The mycolic acid patterns, obtained by HPLC, are similar to that of Mycobacterium scrofulaceum, though the peak heights and distribution present minor differences. We propose the name Mycobacterium mantenii sp. nov. for this novel species. The type strain, isolated from a lymph node biopsy sample, is strain 04-1474(T) (=NLA000401474(T) =CIP 109863(T) =DSM 45255(T)).

Crenarchaeal community assembly and microdiversity in developing soils at two sites associated with deglaciation

Non-thermophilic Crenarchaeota are recognized as ubiquitous and abundant components of soil microbial communities. Previous studies of the foreland of the receding Rotmoosferner glacier in the Austrian Central Alps have demonstrated that crenarchaeal communities in soil at different stages of development are distinct from each other, with Group 1.1b crenarchaeal populations dominating throughout the successional gradient, while Group 1.1c crenarchaea are present in mature soils only. To determine whether this highly structured succession was unique to the Rotmoosferner glacier foreland, 1.1b and 1.1c communities were compared with those present along a successional gradient at Odenwinkelkees glacier, 125 km away, by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA reverse transcription polymerase chain reaction products. Similarities in community structure were observed; 1.1b communities were present throughout both successional gradients (though lacking the defined structure at Odenwinkelkees) and 1.1c communities were present only in mature soil. Comigration of bands on DGGE gels indicated that a number of similar crenarchaeal populations were present at both sites. To compare populations, and examine microscale diversity, 16S rRNA genes and complete 16S-23S internal transcribed spacer (ITS) regions representing six major band positions in DGGE analysis were amplified, cloned and sequenced and represented four 1.1b and two 1.1c lineages. The data provide no evidence of endemism, but large differences in the rate of sequence divergence in the ITS region (relative to that in 16S rRNA genes) were observed. Two of the 1.1b lineages (each possessing > 98% 16S rRNA gene similarity) had relatively long and highly divergent ITS sequences. In contrast, two other 1.1b and two 1.1c lineages (each possessing > 99% 16S rRNA gene similarity) exhibited markedly less variation in their respective 16S-23S ITS regions. The results reveal common patterns in the ecology and assembly of crenarchaeal communities in spatially separated soil systems and may indicate different evolutionary rates between soil crenarchaea lineages.

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera.