- Volume 59, Issue 2, 2009

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA–DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

A yellow- to orange-pigmented, Gram-negative, rod-shaped, motile and non-spore-forming bacterium, strain FSW06-204dT, was isolated from subsurface water of the acidic bog lake, Lake Grosse Fuchskuhle (Brandenburg, Germany). Optimum growth of this strain occurred over a pH range from 5.5 to 6.0 and the growth rate strongly decreased at pH values above 6.5. In addition, the strain exhibited a low tolerance towards NaCl and grew only at a NaCl concentration of up to 0.5 %. 16S rRNA gene sequence analysis of strain FSW06-204dT showed the highest sequence similarity to Novosphingobium hassiacum W-51T (96.7 %) and formed a distinct cluster with Novosphingobium nitrogenifigens DSM 19370T (96.4 %) within the genus Novosphingobium. Strain FSW06-204dT shared a 21 bp signature gap with the latter species, a feature that is absent in all other members of the family Sphingomonadaceae. DNA–DNA hybridization of strain FSW06-204dT and N. nitrogenifigens DSM 19370T showed a low relatedness value of 24 % (reciprocal: 39 %). The major respiratory quinone was ubiquinone Q-10 (91 %) and the predominant fatty acid was C18 : 1 ω7c (43.3 %). Two characteristic 2-hydroxy fatty acids, C14 : 0 2-OH (8.1 %) and C15 : 0 2-OH (6.5 %), were abundant. Polar lipids consisted mainly of phosphatidyldimethylethanolamine and phosphatidylethanolamine; however, only moderate amounts of sphingoglycolipids were present and phosphatidylcholine was lacking. Characterization by 16S rRNA gene sequence, physiological features, pigment analysis and polyamine, ubiquinone, polar lipid and fatty acid contents revealed that strain FSW06-204dT represents a novel species of the genus Novosphingobium within the class Alphaproteobacteria. The name Novosphingobium acidiphilum sp. nov. is proposed for this acidophilic and salt-sensitive species with the type strain FSW06-204dT (=DSM 19966T=CCM 7496T=CCUG 55538T).

Two strains of psychrotolerant bacteria, designated M7T and M5, isolated from Antarctic coastal marine environments were studied to determine their taxonomic position. The organisms comprised Gram-negative, rod-shaped, facultatively anaerobic cells that were motile by means of single polar flagella. Neither of the bacterial isolates had a requirement for Na+. These two psychrotolerant strains grew at temperatures ranging from −4 to 30 °C. Both strains were capable of producing H2S from thiosulfate and were able to use sodium nitrate and trimethylamine N-oxide as terminal electron acceptors during anaerobic growth. 16S rRNA gene sequence analysis placed M7T and M5 within the genus Shewanella; the strains showed the highest similarity (99.9 and 99.2 % respectively) with respect to the type strains of Shewanella livingstonensis and Shewanella frigidimarina. However the levels of gyrB sequence similarity between strain M7T and the type strains of S. livingstonensis and S. frigidimarina were 87.6 and 87.4 %, respectively. DNA–DNA hybridization experiments performed between the Antarctic isolate M7T and S. livingstonensis LMG 19866T and S. frigidimarina LMG 19475T revealed levels of relatedness of 32 and 35 %, respectively. Strain M5 showed 100 % DNA relatedness with respect to strain M7T. The DNA G+C content of these bacteria was 42 mol%. Several phenotypic characteristics, the cellular fatty acid compositions and the quinone content of strains M7T and M5 served to differentiate them from related shewanellae. On the basis of the data from this polyphasic taxonomic study, M7T and M5 constitute a single genospecies. They represent a novel species of the genus Shewanella, for which the name Shewanella vesiculosa sp. nov. is proposed. The type strain is M7T (=LMG 24424T =CECT 7339T).

A novel alphaproteobacterium, strain LD81T, was isolated from the marine macroalga Laminaria saccharina. The bacterium is mesophilic and shows a typical marine growth response. It is a chemoheterotrophic aerobe with the potential for denitrification. Growth optima are 25 °C, pH 5.5 and 3 % NaCl. Strain LD81T has a unique phylogenetic position, not fitting any of the known families of the Alphaproteobacteria. The 16S rRNA gene sequence revealed a distant relationship to species of several orders of the Alphaproteobacteria, with less than 90 % sequence similarity. Phylogenetically, strain LD81T is related to the type strains of Terasakiella pusilla (88.4 % 16S rRNA gene sequence similarity) and the three Thalassospira species (88.9–89.2 %). It forms a cluster with these bacteria and a novel as-yet undescribed isolate (KOPRI 13522; 96.6 % sequence similarity). Strain LD81T has a relatively low DNA G+C content (51.1 mol%) and, due to its distant phylogenetic position from all other alphaproteobacteria, strain LD81T (=NCIMB 14374T =JCM 14845T) is considered as the type strain of a novel species within a new genus, for which the name Kiloniella laminariae gen. nov., sp. nov. is proposed. The genus Kiloniella represents the type of the new family Kiloniellaceae fam. nov. and order Kiloniellales ord. nov.

A group of exopolysaccharide-producing bacteria was isolated from the root environment of Arabidopsis thaliana. The genetic diversity revealed by REP-PCR fingerprinting indicated that the isolates correspond to different strains. 16S rRNA gene sequence analysis showed that the isolates are closely related to the strains Rhizobium sp. YAS34 and USDA 1920, respectively isolated from sunflower roots and Medicago ruthenica nodules. These bacteria belong to the Rhizobium lineage of the Alphaproteobacteria, and the closest known species was Rhizobium sullae. DNA–DNA hybridization experiments and biochemical analysis demonstrated that the nine strains isolated from A. thaliana and Rhizobium strains YAS34 and USDA 1920 constitute a novel species within the genus Rhizobium, for which the name Rhizobium alamii sp. nov. is proposed. The type strain is GBV016T (=CFBP 7146T =LMG 24466T).

A novel heterotrophic, marine, strictly aerobic, motile bacterium was isolated from the Red Sea at a depth of 1 m. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to the genera Oleispira, Oceanobacter and Thalassolituus, each of which contains a single species, within the class Gammaproteobacteria. Phenotypic, genotypic and phylogenetic analyses supported the creation of a novel genus and species to accommodate this bacterium, for which the name Bermanella marisrubri gen. nov., sp. nov. is proposed. The type strain of Bermanella marisrubri is RED65T (=CECT 7074T =CCUG 52064T).

An aerobic, Gram-negative, motile bacterium, strain B51T, was isolated from seawater obtained from Semarang Port in Indonesia. Cells of strain B51T were peritrichously flagellated and rod-shaped. Strain B51T was able to degrade alkanes, branched alkanes and alkylnaphthalenes. 16S rRNA gene sequence analysis revealed that strain B51T was affiliated with the family Rhodobacteraceae, and was related most closely to Thioclava pacifica TL 2T (94.6 % similarity). The DNA G+C content of strain B51T was 66.5 mol%. The major cellular fatty acids were C18 : 1 ω7c (84.9 %), C18 : 1 ω9c (13.8 %), C16 : 0 (8.7 %), C18 : 0 (6.4 %) and anteiso-C15 : 0 (5.8 %) and the major quinone was ubiquinone-10. Based on its phenotypic and phylogenetic characteristics, strain B51T is considered to represent a novel species of a new genus, for which the name Tropicimonas isoalcanivorans gen. nov., sp. nov. is proposed. The type strain of the type species is B51T (=JCM 14837T=DSM 19548T).

An aerobic, Gram-negative, motile bacterium, strain C02T, was isolated from seawater obtained from Semarang Port in Indonesia. Cells of strain C02T were peritrichously flagellated and rod-shaped. Strain C02T was able to degrade naphthalene, alkylnaphthalenes and phenanthrene. 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria and was related most closely to Marinovum algicola FF3T (95.7 % similarity) and Thalassobius aestuarii JC2049T (95.2 %). The DNA G+C content of strain C02T was 64.6 mol%. The major cellular fatty acids were C18 : 1 ω7c (50.9 % of the total), C16 : 0 (17.9 %), 11 methyl C18 : 1 ω7c (14.7 %), C18 : 1 ω9c (2.9 %) and C19 : 0 cyclo ω8c (2.4 %), and the predominant respiratory lipoquinone was ubiquinone-10. Based on physiological, chemotaxonomic and phylogenetic data, strain C02T is suggested to represent a novel species of a new genus, for which the name Tropicibacter naphthalenivorans gen. nov., sp. nov. is proposed. The type strain of Tropicibacter naphthalenivorans is C02T (=JCM 14838T=DSM 19561T).

An aerobic, moderately halophilic, Gram-negative, motile, non-sporulating rod-shaped bacterium, designated strain A10T, was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain A10T was able to grow at NaCl concentrations in the range 5–17.5 % (w/v) with optimum growth at 10 % (w/v) NaCl. Growth occurred at temperatures of 20–40 °C (optimal growth at 37 °C) and pH 5.5–8.5 (optimal growth at pH 7.0–8.0). The major respiratory quinone was ubiquinone 9. The major fatty acids were C16 : 0, C18 : 1 ω7c, C19 : 0 cyclo ω8c and C12 : 0 3-OH. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipids, glycolipids and an aminoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain A10T is closely related to Halomonas avicenniae MW2aT (95.7 % sequence similarity), Halomonas marisflavi SW32T (95.2 %) and Halomonas indalinina GC2.1T (95.0 %). Strain A10T formed a coherent phylogenetic branch with these three species, separated from other species of Halomonas and closely related genera (with 16S rRNA gene sequence similarities below 94.0 %). A complete 23S rRNA gene sequence comparison of strain A10T with closely related species confirmed the phylogenetic position of the novel isolate, forming a branch with the species Halomonas avicenniae, Halomonas indalinina and Halomonas marisflavi, separated from other species of the genera belonging to the family Halomonadaceae (showing sequence similarities below 91.7 %). DNA–DNA hybridization studies between strain A10T and Halomonas avicenniae MW2aT, Halomonas marisflavi DSM 15357T and Halomonas indalinina CG2.1T were 21, 17 and 10 %, respectively. These levels of DNA–DNA relatedness were low enough to classify strain A10T as representing a genotypically distinct species. Overall, the phenotypic, genotypic, chemotaxonomic and phylogenetic results demonstrated that strain A10T represents a new genus and species. The name Kushneria aurantia gen. nov., sp. nov. is proposed, with strain A10T (=CCM 7415T=CECT 7220T) as the type strain. This is the type species of the new proposed genus, which belongs to the family Halomonadaceae. In addition, our data support the placement of the species Halomonas marisflavi, Halomonas indalinina and Halomonas avicenniae within this new genus, as Kushneria marisflavi comb. nov., Kushneria indalinina comb. nov. and Kushneria avicenniae comb. nov., respectively.

A pink-coloured bacterial strain, designated 5715S-9T, was isolated from an air sample collected in Suwon, Republic of Korea. The cells were strictly aerobic, Gram-negative, non-spore-forming, non-flagellated and short-rod-shaped. On the basis of 16S rRNA gene sequence analysis, the organism was a member of the genus Rubellimicrobium, showing the highest sequence similarities with Rubellimicrobium mesophilum MSL-20T (96.2 % sequence similarity) and Rubellimicrobium thermophilum DSM 16684T (93.9 %). The major polar lipids were phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid. The major fatty acids (>10 %) were C18 : 1 ω7c, C16 : 0 and 11-methyl C18 : 1 ω7c. The G+C content of the DNA was 69 mol%. On the basis of both phylogenetic and phenotypic evidence, strain 5715S-9T represents a novel species of the genus Rubellimicrobium, for which the name Rubellimicrobium aerolatum sp. nov. is proposed. The type strain is 5715S-9T (=KACC 12504T=DSM 19297T).

Two strains of marine, heterotrophic, alkaliphilic bacteria, designated A3F-7T and ssthio04PA2-7c, were isolated from hard coral and marine sand, respectively, collected in the Republic of Palau. A phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were related to members of the genus Moritella. However, the sequence similarities between the isolates and the type strains of the Moritella species were less than 93 %. The G+C contents of the isolates were around 57 mol%, the major respiratory quinone was Q-8 and the predominant cellular fatty acids were 16 : 1ω7c, 16 : 0, 18 : 1ω7c and 14 : 0. On the basis of the phylogenetic data, phenotypic characteristics and DNA–DNA hybridization results, strains A3F-7T and ssthio04PA2-7c represent a novel species of a novel genus, for which the name Paramoritella alkaliphila gen. nov., sp. nov. is proposed. The type strain of Paramoritella alkaliphila is A3F-7T (=MBIC06429T =DSM 19956T).

A Gram-negative strain, B11T, was isolated from tidal flat sediment in Yeosu, Republic of Korea. Strain B11T did not require NaCl for growth and grew between 18 and 44 °C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B11T was associated with the genus Alishewanella and was closely related to the type strain of Alishewanella fetalis (98.3 % similarity). Within the phylogenetic tree, the novel isolate shared a branching point with A. fetalis. Analysis of 16S rRNA gene sequences and DNA–DNA relatedness, as well as physiological and biochemical tests, indicated genotypic and phenotypic differences between strain B11T and the type strain of A. fetalis. Thus, strain B11T is proposed as a representative of a novel species, Alishewanella aestuarii sp. nov.; the type strain is B11T (=KCTC 22051T =DSM 19476T).

Analyses of nucleotide sequences from the D1/D2 domains of the large-subunit rDNA and phenotypic characteristics showed that the genera Moniliella and Trichosporonoides are members of a single, monophyletic clade that would be best represented by a single anamorphic genus. On the basis of taxonomic priority, we propose the transfer of the five species of the genus Trichosporonoides to the genus Moniliella. The description of the genus Moniliella is emended and the following new combinations are proposed: Moniliella madida comb. nov., Moniliella megachiliensis comb. nov., Moniliella nigrescens comb. nov., Moniliella oedocephalis comb. nov. and Moniliella spathulata comb. nov. In addition, ten strains representing a novel yeast species belonging to the Moniliella clade were isolated from flowers in Thailand, Cuba and Brazil. Analysis of the internal transcribed spacer and D1/D2 large-subunit rDNA sequences indicated that the isolates represent a single species that was distinct from other species of the Moniliella clade. The name Moniliella fonsecae sp. nov. is proposed to accommodate these strains. The type strain is BCC 7726T (=CBS 10551T).

A population of Kiitricha minuta Xu et al., 2008 , a small kiitrichid ciliate, was isolated from a brackish water sample in Jiaozhou Bay, Qingdao, northern China. After comparison of its morphology and infraciliature, it is believed that this morphotype should be assigned to the genus Caryotricha; hence, a new combination is suggested, Caryotricha minuta ( Xu et al., 2008 ) nov. comb. The small-subunit (SSU) rRNA gene sequence was determined in order to elucidate the phylogenetic position of this poorly known, ambiguous genus. The organism can be clearly separated from its congener, Caryotricha convexa Kahl, 1932, by the extremely shortened ventral cirral rows in the posterior ends. Based on the data available, an improved diagnosis is given for the genus: marine Kiitrichidae with prominent buccal field; two highly developed undulating membranes; non-grouped, uniform cirral rows on both ventral and dorsal sides; enlarged transverse cirri present, which are the only differentiated cirri; marginal cirri not present; one short migratory row located posterior to buccal field; structure of dorsal kineties generally in Kiitricha pattern. The sequence of the SSU rRNA gene of C. minuta differs by 13 % from that of Kiitricha marina. Molecular phylogenetic analyses (Bayesian inference, least squares, neighbour joining, maximum parsimony) indicate that Caryotricha, together with Kiitricha, diverges at a deep level from all other spirotrichs. Its branching position is between Phacodiniidia and Licnophoridia. The results strongly support the distinct separation of the Kiitricha–Caryotricha clade, which always branches basal to the Stichotrichia–Hypotrichia–Oligotrichia–Choreotrichia assemblage. These results also confirm the previous hypothesis that the Kiitricha–Caryotricha group, long assumed to be a close relation to the euplotids, represents a taxon at subclass level within the spirotrichs.

The genus Kiitricha was long assumed to be the most primordial taxon in the Stichotrichia [hypotrichs sensu lato (s. l.)] based on its morphological features and was considered to be an intermediate between heterotrichs and the traditional hypotrichous assemblage. In order to evaluate the phylogenetic position of Kiitricha within the Hypotrichia, we sequenced the small-subunit rRNA gene and the alpha-tubulin gene for a Qingdao population of Kiitricha marina. Phylogenetic trees were constructed and compared to morphological and morphogenetic data. The results show that (i) Kiitricha is positioned near Phacodinium, both of which always form a sister clade to the assemblage including Stichotrichia, Hypotrichia, Oligotrichia and Choreotrichia, (ii) Kiitricha, which may represent an intermediate between heterotrichs (s. l.) and the Stichotrichia–Hypotrichia complex, is probably an ancestor-like form of the latter group and (iii) in contrast to morphological characters, both molecular and ontogenetic data support the separation of Kiitricha from the hypotrichs (s. l.). Thus, Kiitricha might be placed in the class Spirotrichea at about subclass level, next to Phaconidiidia, Hypotrichia and Stichotrichia, which supports the establishment of a new subclass Protohypotrichia n. subclass within the class Spirotrichea, with characterizations including slightly differentiated somatic ciliature (i.e. cirri on the ventral side generally uniform and non-grouped, no clearly defined marginal cirral rows, ciliature on the dorsal side mixed with cirri and dikinetids, no clearly differentiated dorsal kineties) and a unique but intermediate morphogenetic pattern of cortical structures between Hypotrichia and Stichotrichia.

Previously, we have produced a phylogeny of species type strains from the plant-pathogenic genus Xanthomonas based on gyrB sequences. To evaluate this locus further for species and infraspecies identification, we sequenced an additional 203 strains comprising all the pathovar reference strains (which have defined plant hosts), 67 poorly characterized pathovars, currently classified as Xanthomonas campestris, and 59 unidentified xanthomonads. The well-characterized pathovars grouped either in clades containing their respective species type strain or in clades containing species related to Xanthomonas axonopodis. The Xanthomonas euvesicatoria, Xanthomonas perforans and Xanthomonas alfalfae species complex, Xanthomonas fuscans and Xanthomonas citri were discriminated as X. axonopodis-related clades and comprised a large proportion of unidentified strains as well as 80 pathovars representing all the X. axonopodis pathovars and many poorly characterized pathovars, greatly increasing the plant host ranges of the constituent species. Most xanthomonads from these three large clades were isolated from a taxonomically diverse range of plant hosts, including many weed species, from field systems in India, suggesting that these lineages became established and diversified in agricultural areas in this region. The majority of these xanthomonads had minimal sequence diversity, consistent with rapid and highly extensive pathovar diversification that has occurred in relatively recent times. Low-intensity farming practices may have provided conditions conducive to pathovar development, and evidence for pathovar diversification within other regional angiosperm floras is discussed. The gyrB locus was sufficiently discriminating to identify diversity within many species. Seven branches or clades were sufficiently distinct to be considered as potential novel species. This study has provided a comprehensive xanthomonad classification framework and has firmly established gyrB sequencing as a rapid and efficient identification tool.

The species of Clostridium comprise a very heterogeneous assemblage of bacteria that do not form a phylogenetically coherent group. It has been proposed previously that only a subset of the species of Clostridium that form a distinct cluster in the 16S rRNA tree (cluster I) should be regarded as the true representatives of the genus Clostridium (i.e. Clostridium sensu stricto). However, this cluster is presently defined only in phylogenetic terms, and no biochemical, molecular or phenotypic characteristic is known that is unique to species from this cluster. We report here phylogenomic and comparative analyses based on sequenced clostridial genomes in an attempt to bridge this gap and to clarify the evolutionary relationships among species of clostridia. In phylogenetic trees for species of clostridia based on concatenated sequences for 37 highly conserved proteins, the species of Clostridium cluster I formed a strongly supported clade that was separated from all other clostridia by a long branch. Several other Clostridium species that are not part of this cluster grouped reliably with other species of clostridia in a number of well-resolved clades. Our comparative genomic analyses have identified three conserved indels in three highly conserved proteins (a 4 aa insert in DNA gyrase A, a 1 aa deletion in ATP synthase beta subunit and a 1 aa insert in ribosomal protein S2) that are unique to the species of Clostridium cluster I and are not found in any other bacteria. blastp searches on various proteins in the genomes of Clostridium tetani E88 and Clostridium perfringens SM101 have also identified more than 10 proteins that are found uniquely in the cluster I species. These results provide evidence that the species of Clostridium cluster I not only are phylogenetically distinct but also share many unique molecular characteristics. These newly identified molecular markers provide useful tools to define and circumscribe the genus Clostridium sensu stricto in more definitive terms. We have also identified a 7–9 aa conserved insert in the enzyme phosphoglycerate dehydrogenase that is uniquely found in the Clostridium thermocellum, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter tengcogensis and Caldicellulosiruptor saccharolyticus homologues, and is absent from all other bacteria. These species form a well-defined clade in the phylogenetic trees and this indel provides a potential molecular marker for this clostridial cluster.