Cathrin Spröer

Characterisation, genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56

Abstract. In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain Soxa0ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16Sxa0rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCCxa025531 and the epothilone producer S.xa0cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2xa0Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.

Cereulide-producing strains of Bacillus cereus show diversity

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600–1,800xa0ngxa0cereulidexa0mg−1 wet weight biomass), seven average producers (180–600xa0ngxa0cereulidexa0mg−1 wet weight biomass), four low cereulide producers (20–160xa0ngxa0cereulidexa0mg−1 wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.

Reclassification and emended description of Caulobacter leidyi as Sphingomonas leidyi comb. nov., and emendation of the genus Sphingomonas.

Caulobacter leidyi DSM 4733(T) has been shown to be affiliated with the family Sphingomonadaceae instead of the Caulobacteraceae, and due to its poor characterization has been omitted from the current edition of Bergeys Manual of Systematic Bacteriology and removed to limbo. We isolated a novel sphingoglycolipid-containing dimorphic prosthecate bacterium, designated strain 247, from a pre-alpine freshwater lake. Strain 247 and Caulobacter leidyi DSM 4733(T) were characterized in detail. The rod-shaped cells were Gram-stain-negative, aerobic, catalase- and oxidase-positive, and formed a stalk or polar flagellum. Both strains grew optimally at 28-30 °C, and pH 6.0-8.0. The major fatty acids were C(18 : 1)ω7c, C(16 : 0) and 11-methyl C(18 : 1)ω7c. C(14 : 0) 2-OH represents the major 2-hydroxy fatty acid. Q-10 was the major respiratory quinone and the major polar lipids were diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylcholine, three glycolipids, two phosphoaminolipids and two unidentified sphingoglycolipids. The major polyamine was sym-homospermidine. The G+C content of genomic DNA of strains 247 and DSM 4733(T) was 67.6 mol% and 67.0 mol%, respectively. According to 16S rRNA gene sequence analysis and DNA-DNA hybridization, strains DSM 4733(T) and 247 were phylogenetically closely related (99.6 % 16S rRNA gene sequence similarity, 82.9 % DNA-DNA hybridization value) and affiliated to the genus Sphingomonas. The closest recognized species was Sphingomonas aquatilis DSM 15581(T) (98.1 % sequence similarity). In addition, the presence of cystine arylamidase, absence of β-galactosidase, and the inability to utilize l-arabinose, galactose and sucrose distinguished strains DSM 4733(T) and 247 from most other members of the family Sphingomonadaceae. So far, the dimorphic life cycle that involves a prosthecate and a flagellated stage is unique for strains DSM 4733(T) and 247 among all members of the family Sphingomonadaceae. Therefore, Caulobacter leidyi is reclassified as Sphingomonas leidyi, with the type strain DSM 4733(T) (u200a= ATCC 15260(T) = CIP 106443(T) = VKM B-1368(T)) and strain 247 (DSM 25078 = LMG 26658) as an additional strain of this species.

Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species.

Streptococcus tigurinus sp. nov., isolated from blood of patients with endocarditis, meningitis and spondylodiscitis

Four Gram-stain-positive, catalase-negative, coccus-shaped bacterial strains were isolated from multiple blood cultures of patients with endocarditis, meningitis and spondylodiscitis. The isolates were tentatively identified as viridans streptococci on the basis of phenotypic characteristics. Comparative 16S rRNA gene sequencing studies showed that the organisms were members of the Streptococcus mitis group but did not correspond to any recognized species. The nearest phylogenetic relative was S. mitis ATCC 49456(T), with 98.6% sequence similarity. The representative strain AZ_3a(T) showed less than 96.8, 97.6, 94.5 and 95.5% similarity to the phylogenetically most closely related species by recA, rpoB, sodA and groEL gene sequence analysis, respectively. DNA-DNA hybridization analyses showed a low reassociation value of 32.2% between strain AZ_3a(T) and S. mitis DSM 12643(T). Reassociation values with members of other S. mitis group species ranged from 27.3 to 49.7%. The G+C content of the DNA was 40.0 mol%. Based on our biochemical and molecular analyses, the isolates represent a novel species, for which the name Streptococcus tigurinus sp. nov. is proposed. The type strain is AZ_3a(T) (u200a= CCOS 600(T) u200a= DSM 24864(T)).

Polynucleobacter acidiphobus sp. nov., a representative of an abundant group of planktonic freshwater bacteria.

The heterotrophic, aerobic, facultatively anaerobic, catalase- and oxidase-positive, non-motile strain MWH-PoolGreenA3(T), isolated from a rock pool filled with freshwater, was characterized. The strain grew on NSY medium over a NaCl range of 0.0-0.3u200a% (w/v). Whole-cell fatty acids were dominated by C(16u200a:u200a1)ω7c (feature 3), C(18u200a:u200a1)ω7c and straight-chain C(16u200a:u200a0); furthermore, the components C(12u200a:u200a0) and C(14u200a:u200a0) 2-OH were present. The DNA G+C content was 48.3 mol%. Phylogenetic analysis as well as strong similarities in phenotypic and chemotaxonomic traits indicated the affiliation with the genus Polynucleobacter. 16S rRNA gene similarity values with the three described species of the genus Polynucleobacter ranged from 96.7 to 97.8u200a%. DNA-DNA hybridization experiments did not reveal that the strain belongs to a previously described species of the genus Polynucleobacter. The strain can be discriminated from previously established species of the genus Polynucleobacter by chemotaxonomic and phenotypic traits. The bacterium possesses a free-living lifestyle and represents a group of planktonic freshwater bacteria occurring with high cell numbers in many freshwater lakes. Based on the phylogeny revealed and the chemotaxonomic and phenotypic differences from previously described species of the genus Polynucleobacter, we propose to establish the novel species Polynucleobacter acidiphobus sp. nov. with the type strain MWH-PoolGreenA3(T) (u200a=u200aDSM 21994(T) u200a=u200aCIP 110079(T)).

Arthrobacter phenanthrenivorans sp. nov., to accommodate the phenanthrene-degrading bacterium Arthrobacter sp. strain Sphe3.

A novel phenanthrene-degrading bacterium, designated strain Sphe3(T), was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod- to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 degrees C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3(T) consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(17 : 0), representing >86 % of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3(T) was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3(T) showed 99 and 98.9 % similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3(T) showed 91 % similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3(T) (=DSM 18606(T) =LMG 23796(T)).

Micromonospora cremea sp. nov. and Micromonospora zamorensis sp. nov., isolated from the rhizosphere of Pisum sativum.

Three actinobacterial strains, CR30(T), CR36 and CR38(T), were isolated from rhizosphere soil of Pisum sativum plants collected in Spain. The strains were filamentous, Gram-stain-positive and produced single spores. Phylogenetic, chemotaxonomic and morphological analyses confirmed that the three strains belonged to the genus Micromonospora. 16S rRNA gene sequence analysis of strains CR30(T) and CR36 showed a close relationship to Micromonospora coriariae NAR01(T) (99.3% similarity) while strain CR38(T) had a similarity of 99.0% with Micromonospora saelicesensis Lupac 09(T). In addition, gyrB gene phylogeny clearly differentiated the novel isolates from recognized Micromonospora species. DNA-DNA hybridization, BOX-PCR and ARDRA profiles confirmed that these strains represent novel genomic species. The cell-wall peptidoglycan of strains CR30(T) and CR38(T) contained meso-diaminopimelic acid. Both strains had MK-10(H(4)) as the main menaquinone and a phospholipid type II pattern. An array of physiological tests also differentiated the isolates from their closest neighbours. Considering all the data obtained, it is proposed that strains CR30(T) and CR36 represent a novel species under the name Micromonospora cremea sp. nov. (type strain CR30(T) = CECT 7891(T) = DSM 45599(T)), whereas CR38(T) represents a second novel species, for which the name Micromonospora zamorensis sp. nov. is proposed, with CR38(T) (u200a= CECT 7892(T) = DSM 45600(T)) as the type strain.

Propionispora vibrioides, nov. gen., nov. sp., a new gram-negative, spore-forming anaerobe that ferments sugar alcohols

Abstract. Anaerobic enrichment cultures, with erythritol as substrate, resulted in the isolation of a strain with properties not yet found in an existing genus in this combination. The strain, FKBS1, was strictly anaerobic, stained gram-negative and formed spores. Cells were small motile vibrios with flagella inserted at the concave side of the cell. Spores were located terminally and caused only slight swelling of the cells if compared to related spore-forming genera. FKBS1 fermented fructose, mannitol, sorbitol, xylitol and erythritol to propionic acid, acetic acid, CO2 and small amounts of H2 to balance the difference in the oxidation-reduction value between substrate and cell mass. The 16S rDNA sequence revealed relationship to the Sporomusa-Pectinatus-Selenomonas group. However, the phylogenetic distance to any of its members was too great to allow it to be placed in one of the existing genera. Morphologically the strain resembled Sporomusa, which, however, performs an acetogenic type of fermentation. The propionic-acid-forming genera of the group are either not spore-formers or, in the case of Dendrosporobacter quercicolus (syn. Clostridium quercicolum), morphologically different. It is therefore proposed to classify strain FKBS1 as a new genus and species, Propionispora vibrioides.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).