The association between Dioscorea sansibarensis and Orrella dioscoreae as a model for hereditary leaf symbiosis.

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.

Limited feeding on bacteria by two intertidal benthic copepod species as revealed by trophic biomarkers.

Harpacticoids can discriminate between biofilms of different bacterial strains. We investigated whether assimilation of bacteria is selective and whether harpacticoids select for the most nutritional bacteria. We specifically focused on the role of bacterial characteristics in copepod food selection. Trophic biomarkers (stable isotopes, fatty acids) were used to test selective assimilation of three bacteria by the harpacticoids Platychelipus littoralis and Delavalia palustris, all isolated from a salt marsh. The bacteria Gramella sp., Jannaschia sp. and Photobacterium sp. with contrasting ribosomal protein and fatty acid contents were (13)C-labelled and offered in a food patch choice experiment with monospecific and combination treatments (single and two strains per microcosm respectively). Low assimilation of bacterial carbon and lack of significant fatty acid transfer proved that bacteria were a poor food source for the harpacticoids. Assimilation was copepod species-specific and bacteria strain-specific (preference for Photobacterium). However, only a low degree of selective feeding occurred; it can partly be explained by bacterial extracellular metabolites rather than by biochemical content and densities. Finally, the energetic cost of differential bacterivory resulted in a negative fatty acid balance for Platychelipus, while Delavalia showed an improved fatty acid profile and thus a positive response to the low-quality bacterial food.

Carnobacterium iners sp. nov., a psychrophilic, lactic acid-producing bacterium from the littoral zone of an Antarctic pond.

Two lactic acid-producing, Gram-stain-positive rods were isolated from a microbial mat actively growing in the littoral zone of an Antarctic lake (Forlidas Pond) in the Pensacola mountains and studied using a polyphasic taxonomic approach. The isolates were examined by phylogenetic analysis of the 16S rRNA gene, multilocus sequence analysis of pheS, rpoA and atpA, and biochemical and genotypic characteristics. One strain, designated LMG 26641, belonged to Carnobacterium alterfunditum and the other strain, designated LMG 26642(T), could be assigned to a novel species, with Carnobacterium funditum DSM 5970(T) as its closest phylogenetic neighbour (99.2 % 16S rRNA gene sequence similarity). Carnobacterium iners sp. nov. could be distinguished biochemically from other members of the genus Carnobacterium by the lack of acid production from carbohydrates. DNA-DNA relatedness confirmed that strain LMG 26642(T) represented a novel species, for which we propose the name Carnobacterium iners sp. nov. (type strain is LMG 26642(T)  = CCUG 62000(T)).

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter.

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.

Evaluation of MALDI-TOF MS as a tool for high-throughput dereplication.

The present study examined the suitability of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid grouping of bacterial isolates, i.e. dereplication. Dereplication is important in large-scale isolation campaigns and screening programs since it can significantly reduce labor intensity, time and costs in further downstream analyses. Still, current dereplication techniques are time consuming and costly. MALDI-TOF MS is an attractive tool since it performs fast and cheap analyses with the potential of automation. However, its taxonomic resolution for a broad diversity of bacteria remains largely unknown. To verify the suitability of MALDI-TOF MS for dereplication, a total of 249 unidentified bacterial isolates retrieved from the rhizosphere of potato plants, were analyzed with both MALDI-TOF MS and repetitive element sequence based polymerase chain reaction (rep-PCR). The latter technique was used as a benchmark. Cluster analysis and inspection of the profiles showed that for 204 isolates (82%) the taxonomic resolution of both techniques was comparable, while for 45 isolates (18%) one of both techniques had a higher taxonomic resolution. Additionally, 16S rRNA gene sequence analysis was performed on all members of each delineated cluster to gain insight in the identity and sequence similarity between members in each cluster. MALDI-TOF MS proved to have higher reproducibility than rep-PCR and seemed to be more promising with respect to high-throughput analyses, automation, and time and cost efficiency. Its taxonomic resolution was situated at the species to strain level. The present study demonstrated that MALDI-TOF MS is a powerful tool for dereplication.

Identification of lactobacilli by pheS and rpoA gene sequence analyses.

The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.

Algibacter mikhailovii sp. nov., a novel marine bacterium of the family Flavobacteriaceae, and emended description of the genus Algibacter.

A novel marine bacterium, designated strain KMM 6171(T), was subjected to taxonomic analysis by using a polyphasic approach. Colonies were yellow-pigmented and cells were Gram-negative, heterotrophic rods displaying slow gliding motility. 16S rRNA gene sequence analysis indicated that strain KMM 6171(T) was closely related to the genus Algibacter, a member of the family Flavobacteriaceae, with sequence similarity of 96.7-96.8 %. The predominant cellular fatty acids were iso-C15 : 1, iso-C15 : 0, anteiso-C15 : 0, C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH and summed feature 3, comprising C16 : 1omega7c and/or iso-C15 : 0 2-OH. The DNA G+C content was 35.1 mol%. On the basis of the phenotypic, genotypic, chemotaxonomic and phylogenetic data, strain KMM 6171(T) represents a novel species of the genus Algibacter, for which the name Algibacter mikhailovii sp. nov. is proposed. The type strain is KMM 6171(T) (=KCTC 12710(T)=LMG 23988(T)). An emended description of the genus Algibacter based on the new data is also given.

Echinicola vietnamensis sp. nov., a member of the phylum Bacteroidetes isolated from seawater.

The taxonomic position of a novel marine, heterotrophic, gliding, halotolerant and light-pink-pigmented bacterium, designated strain KMM 6221(T), was examined by using a polyphasic approach. 16S rRNA gene sequence analysis indicated that strain KMM 6221(T) is affiliated with the genus Echinicola, a member of the phylum Bacteroidetes, with levels of similarity of 94.7-95.0 % to strains of Echinicola pacifica. Growth of strain KMM 6221(T) was observed with 0-15 % NaCl and at 6-44 degrees C. The DNA G+C content of strain KMM 6221(T) was 45.9 mol%. On the basis of molecular distinctiveness supported by phenotypic and chemotaxonomic data, strain KMM 6221(T) is considered to represent a novel species of the genus Echinicola, for which the name Echinicola vietnamensis sp. nov. is proposed. The type strain is KMM 6221(T) (=DSM 17526(T)=LMG 23754(T)).

Reclassification of Lactobacillus amylophilus LMG 11400 and NRRL B-4435 as Lactobacillus amylotrophicus sp. nov.

The taxonomic position of six Lactobacillus amylophilus strains isolated from swine waste-corn fermentations was reinvestigated. All strains were included in a multilocus sequence analysis (MLSA) study for species identification of Lactobacillus using the genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA). Partial pheS and rpoA gene sequences showed that strains LMG 11400 and NRRL B-4435 represent a separate lineage that is distantly related to the type strain of L. amylophilus, LMG 6900T, and to three other strains of the species. The MLSA data showed that the two strains LMG 11400 and NRRL B-4435 constituted a distinct cluster, sharing 100% pheS and rpoA gene sequence similarity. The other reference strains clustered together with the type strain of L. amylophilus, LMG 6900T, and were clearly differentiated from strains LMG 11400 and NRRL B-4435 (80 and 89% pheS and rpoA gene sequence similarity, respectively). The 16S rRNA gene sequences of the latter two strains are 100% identical, with the nearest phylogenetic neighbour L. amylophilus LMG 6900T showing only 97.2% 16S rRNA gene sequence similarity. Further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the two strains represent a single, novel Lactobacillus species, for which the name Lactobacillus amylotrophicus sp. nov. is proposed. The type strain is LMG 11400T (=NRRL B-4436T=DSM 20534T).

Reclassification of Lactobacillus brevis strains LMG 11494 and LMG 11984 as Lactobacillus parabrevis sp. nov.

A polyphasic study revealed taxonomic heterogeneity among reference strains of the species Lactobacillus brevis. Representative strains of L. brevis and related taxa were investigated by partial sequence analysis of the housekeeping gene encoding the alpha-subunit of phenylalanyl-tRNA synthase (pheS). Species-specific clusters were delineated for all taxa studied except for two L. brevis strains, LMG 11494 and LMG 11984, respectively isolated from cheese and wheat, which occupied a distinct position. Their phylogenetic affiliation was determined using 16S rRNA gene sequence analysis and it was found that both strains (with 99.9 % gene sequence similarity between them) belonged to the Lactobacillus buchneri group, with nearest neighbours Lactobacillus hammesii and L. brevis (gene sequence similarities of 99.2 and 98.1 %, respectively). Further genotypic and phenotypic studies, including fluorescent amplified fragment length polymorphism, DNA-DNA hybridization and DNA G+C content, clearly demonstrated that the two strains represent a single novel taxon for which the name Lactobacillus parabrevis sp. nov. is proposed (type strain LMG 11984(T)=ATCC 53295(T)).

Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.

The applicability of a multilocus sequence analysis (MLSA)-based identification system for lactobacilli was evaluated. Two housekeeping genes that code for the phenylalanyl-tRNA synthase alpha-subunit (pheS) and RNA polymerase alpha-subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the alpha-subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8-100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA-DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.

Enterococcus silesiacus sp. nov. and Enterococcus termitis sp. nov.

Three enterococci constituted two aberrant branches after numerical analysis of (GTG)5-PCR fingerprints: analogous patterns were found for two water isolates, strains W213 and W442T, and a separate position was found for an isolate from the gut of a termite, strain LMG 8895T. 16S rRNA gene sequence analysis classified all three strains in the Enterococcus faecalis species group. Further sequencing analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase alpha-subunit) and whole-cell-protein analysis confirmed a distinct position for the two water isolates and the termite strain, respectively. DNA-DNA hybridization experiments and distinct phenotypic features between the strains studied and representatives of the E. faecalis species group confirmed novel species status, respectively, for the two water isolates, strains W213 and W442T, and for strain LMG 8895T. The names Enterococcus silesiacus sp. nov. and Enterococcus termitis sp. nov. are proposed for the novel taxa, with W442T (= CCM 7319T = LMG 23085T) and LMG 8895T (= CCM 7300T) as the respective type strains.

Pediococcus stilesii sp. nov., isolated from maize grains.

A Gram-positive, coccus-shaped, lactic acid bacterium, strain LMG 23082T, was isolated from steeped maize grains. The organism is homofermentative and produces D- and L-lactic acid from glucose. 16S rRNA gene sequence analysis revealed that the organism belongs to the genus Pediococcus, with Pediococcus pentosaceus and Pediococcus acidilactici as nearest neighbours. Genotypic fingerprinting, whole-cell protein electrophoresis, DNA-DNA hybridizations and physiological and biochemical tests allowed differentiation of strain LMG 23082T from other established Pediococcus species. A remarkable feature was that, unlike other pediococci, this bacterium was capable of growth at pH 9.0. The strain studied represents a novel species for which the name Pediococcus stilesii sp. nov. is proposed with the type strain LMG 23082T (=BFE 1652T=FAIR-E 180T=CCUG 51290T), the only currently known isolate of the species.

Reclassification of Enterococcus flavescens Pompei et al. 1992 as a later synonym of Enterococcus casseliflavus (ex Vaughan et al. 1979) Collins et al. 1984 and Enterococcus saccharominimus Vancanneyt et al. 2004 as a later synonym of Enterococcus italicus Fortina et al. 2004.

The taxonomic relatedness between the species Enterococcus casseliflavus and Enterococcus flavescens and between Enterococcus italicus and Enterococcus saccharominimus was investigated. Literature data had already indicated the synonymy between E. casseliflavus and E. flavescens, but this observation had not been formally published. Additional evidence that the two taxa represent a single species was provided by comparison of the partial sequences for three housekeeping genes, phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the alpha subunit of ATP synthase (atpA). Additional genomic data derived from DNA-DNA hybridization demonstrated that the two species are synonymous. For E. italicus and E. saccharominimus, two recently described taxa, a high 16S rRNA gene sequence similarity of >99% and analogous phenotypic features indicated a close taxonomic relatedness. The same multilocus sequence analysis scheme for the three housekeeping genes was also applied for E. italicus and E. saccharominimus and indicated possible conspecificity, an observation that was also confirmed by a high DNA-DNA hybridization value (>or=78%). Data from the present study led to the proposal that E. flavescens should be reclassified as a later synonym of E. casseliflavus and that E. saccharominimus should be reclassified as a later synonym of E. italicus.

Reclassification of Leuconostoc argentinum as a later synonym of Leuconostoc lactis.

Leuconostoc argentinum, Leuconostoc lactis and ten related strains from Romanian dairy products formed a single cluster, clearly separated from other Leuconostoc species, after numerical analysis of repetitive extragenic palindromic-PCR patterns, whole-cell protein profiles (SDS-PAGE) and fluorescent amplified fragment length polymorphism (FAFLP) band patterns. 16S rRNA gene sequence analysis confirmed a very high similarity between both type strains and representative dairy isolates (>99.6 %). DNA-DNA hybridization experiments revealed high relatedness values between the type strains of L. argentinum and L. lactis and between these strains and representative Romanian strains. These data and the lack of phenotypic distinctive characteristics demonstrate that L. argentinum and L. lactis are synonymous.

Larkinella insperata gen. nov., sp. nov., a bacterium of the phylum 'Bacteroidetes' isolated from water of a steam generator.

A Gram-negative bacterium, designated strain LMG 22510T, was isolated from water of a pharmaceutical company steam generator. The cells had a ring-like and horseshoe-shaped morphology and possessed gliding motility. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain was a member of the Flexibacter group within the phylum 'Bacteroidetes'; its nearest neighbour was Spirosoma linguale (88.8 % sequence similarity). DNA base content, fatty acid composition and biochemical characteristics were determined. Genotypic and phenotypic data indicated that strain LMG 22510T could not be assigned to any recognized genus; therefore, a novel genus and species is proposed, Larkinella insperata gen. nov., sp. nov., with LMG 22510T (= NCIMB 14103T) as the type strain.

Surface microflora of four smear-ripened cheeses.

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.

Enterococcus devriesei sp. nov., associated with animal sources.

The taxonomic position of two bovine strains, LMG 13603 and LMG 14595, assigned to the species Enterococcus raffinosus on the basis of biochemical features, was reinvestigated. Both reference strains and two other isolates, 6/1 (=LMG 22829) originating from a charcoal-broiled river lamprey and IE38.4 (=LMG 22830) from the air of a poultry slaughter by-product processing plant, occupied a clearly separate position, on the basis of sequence analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase alpha-subunit), relative to the type strain of E. raffinosus and all other enterococcal species with validly published names. 16S rRNA gene sequencing of strains LMG 13603, LMG 14595, 6/1 and IE38.4 confirmed their phylogenetic position in the Enterococcus avium species group, there being more than 99 % 16S rRNA gene sequence similarity to most members of the group, including E. raffinosus, and revealed Enterococcus pseudoavium as the closest phylogenetic relative (99.8-99.9 %). Further phenotypic and genotypic analyses using whole-cell-protein electrophoresis, (GTG)(5)-PCR fingerprinting, ribotyping and DNA-DNA hybridization experiments demonstrated that all four strains represent a novel enterococcal species, for which the name Enterococcus devriesei sp. nov. is proposed. The type strain is LMG 14595T (=CCM 7299T).

Enterococcus canintestini sp. nov., from faecal samples of healthy dogs.

The taxonomic position of strain LMG 13590(T), originally isolated from dog faeces and classified as Enterococcus dispar in the BCCM/LMG Bacteria Catalogue, was reinvestigated. This strain and 12 recent isolates from faecal samples of healthy dogs occupied a clearly separate position when investigated with multilocus sequence analysis (MLSA) of the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS). The 16S rRNA gene sequence of one representative strain showed highest similarities of 98-99% with E. dispar LMG 13521(T), Enterococcus canis LMG 12316(T) and Enterococcus asini LMG 18727(T). A further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the 13 enterococcal dog faecal strains represent a single, novel Enterococcus species for which the name Enterococcus canintestini sp. nov. is proposed. The type strain is LMG 13590(T) (=CCM 7285(T)).

Enterococcus aquimarinus sp. nov., isolated from sea water.

Two enterococcal strains LMG 16607(T) and LMG 16612 originating from sea water were analysed in a polyphasic taxonomic study. Both strains, assigned as Enterococcus sp. in the BCCM/LMG culture collection, possessed analogous protein profiles, but these were different from all other enterococcal species. 16S rRNA gene sequence analysis of one strain showed the highest similarity, 96.9-96.1%, with its closest phylogenetic neighbours Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus saccharominimus and Enterococcus italicus. Further genomic analysis by (GTG)(5)-PCR fingerprinting and sequence analysis of the housekeeping gene phenylalanyl-tRNA synthase (pheS) and distinct biochemical features confirmed that the two strains represent a novel enterococcal species for which the name Enterococcus aquimarinus sp. nov. is proposed. The type strain is LMG 16607(T) (=CCM 7283(T)).