Molecular and phenotypic analyses reveal the non-identity of the Phaeobacter gallaeciensis type strain deposits CIP 105210T and DSM 17395.

The marine genus Phaeobacter currently comprises six species, some of which were intensively studied mainly due to their ability to produce secondary metabolites. The type strain of the type species, Phaeobacter gallaeciensis BS107(T), has been deposited at several public culture collections worldwide. Based on differences in plasmid profiles, we detected that the alleged P. gallaeciensis type strains deposited at the Collection Institute Pasteur (CIP; Paris, France) as CIP 105210 and at the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany) as DSM 17395 are not identical. To determine the identity of these strains, we conducted DNA-DNA hybridization, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), 16S rRNA gene and internal transcribed spacer (ITS) sequence analyses, as well as physiological experiments. Based on the detailed 16S rRNA gene reanalysis we showed that strain CIP 105210 most likely corresponds to the original P. gallaeciensis type strain BS107(T). In contrast, the Phaeobacter strain DSM 17395 exhibits a much closer affiliation to Phaeobacter inhibens DSM 16374(T) ( = T5(T)) and should thus be allocated to this species. The detection of the dissimilarity of strains CIP 105210(T) and DSM 17395 will influence future comparative studies within the genus Phaeobacter.

Origin and evolution of a novel DnaA-like plasmid replication type in Rhodobacterales.

Large extrachromosomal elements are widespread among Alphaproteobacteria, but it is unclear how up to a dozen low-copy plasmids can stably coexist within the same cell. We systematically analyzed the distribution of different replicons in about 40 completely sequenced genomes of the Roseobacter clade (Rhodobacterales) and surprisingly identified a novel plasmid replicon type. The conserved replication module comprises the characteristic partitioning operon (parAB) and a hitherto unknown replicase. The latter shows a weak homology to the chromosomal replication initiator DnaA and was accordingly named "DnaA-like." Phylogenetic analyses of the adjacent parAB genes document a common ancestry with repA- and repB-type plasmids and moreover indicate the presence of two dnaA-like compatibility groups. This conclusion is supported by conserved palindrome sequences within the replication module that probably represent crucial centromeric anchors for plasmid partitioning. The functionality of dnaA-like replicons was proven by transformation experiments in Phaeobacter gallaeciensis BS107 (DSM 17395). This Roseobacter strain furthermore allows the phenotypical monitoring of plasmid incompatibility, based on a 262-kb dnaA-like replicon required for the brown pigmentation of the bacterium. Uptake of an incompatible construct induces its loss, hence resulting in white colonies. Accordingly, we could substantiate the in silico predictions about stable maintenance of dnaA-like plasmids and thereby functionally validate our approach of plasmid classification based on phylogenetic analyses.

Think pink: photosynthesis, plasmids and the Roseobacter clade.

Aerobic anoxygenic photosynthesis providing additional ATP for a photoheterotrophic lifestyle is characteristic for several representatives of the marine Roseobacter clade. The patchy distribution of photosynthesis gene clusters (PGCs) within this lineage probably results from horizontal transfers and this explanation is supported by two cases of plasmid-located PGCs. In this study sequencing of the three Sulfitobacter guttiformis plasmids (pSG4, pSG53, pSG118) was initiated with the objective to analyse the 118 kb-sized photosynthetic replicon, but our annotation revealed several additional important traits including key genes of the primary metabolism. The comparison of the two photosynthesis plasmids from S. guttiformis and Roseobacter litoralis showed that their replication modules are located at precisely the same position within the 45 kb-sized PGC. However, comprehensive phylogenetic analyses of the non-homologous replicases (RepB-III, DnaA-like I) and the two ParAB partitioning proteins unequivocally document an independent origin of their extrachromosomal replicons. The analogous positioning within the two photosynthesis super-operons can be explained by a two-step recombination scenario and seems to be the ultimate result of stabilizing selection. Our exemplary analyses of 'pink' plasmids document that chromosomal outsourcing is a common phenomenon in the Roseobacter clade and subsequent horizontal exchanges offer rapid access to the marine pan-genome.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters.

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

Genome organisation of the marine Roseobacter clade member Marinovum algicola.

The Roseobacter clade, belonging to the family Rhodobacteraceae of the class Alphaproteobacteria, is one of the major bacterial groups in marine environments. A remarkable wealth of diverse large plasmids has been detected in members of this lineage. Here, we analysed the genome structure and extrachromosomal DNA content of four strains of the roseobacter species Marinovum algicola by pulsed-field gel electrophoresis. They were originally isolated from toxic dinoflagellates and possess multireplicon genomes with sizes between 5.20 and 5.35 Mb. In addition to the single circular chromosomes (3.60-3.74 Mb), whose organisation seem to be conserved, 9 to 12 extrachromosomal replicons have been detected for each strain. This number is unprecedented for roseobacters and proposes a sophisticated regulation of replication and partitioning to ensure stable maintenance. The plasmid lengths range from 7 to 477 kb and our analyses document a circular conformation for all but one of them, which might represent a linear plasmid-like prophage. In striking contrast to other roseobacters, up to one-third of the genomic information (1.75 Mb) is plasmid borne in Marinovum algicola. The plasmid patterns of some strains are conspicuously different, indicating that recombination and conjugative gene transfer are dominant mechanisms for microevolution within the Roseobacter clade.

Taxonomic characterization of members of the genus Corallococcus: molecular divergence versus phenotypic coherency.

Corallococcus coralloides DSM 2259(T), Corallococcus exiguus DSM 14696(T), Corallococcus macrosporus DSM 14697(T) and more than 35 strains identified as members of Corallococcus on the basis of morphology were subjected to partial sequences analysis of three housekeeping genes (lepA, fusA and rpoB), complementing a recent phylogenetic analysis based on genes coding for 16S rRNA and gyrB. Phylogenetic analysis of each gene, generated by maximum likelihood and two different additive treeing algorithms, resulted in the separate position of C. macrosporus DSM 14697(T) and a few Corallococcus strains that were more closely related to Myxococcus xanthus than to the other members of Corallococcus. The latter strains formed three clearly separate clusters by 16S rRNA gene phylogeny. This relationship, however, was only partially recovered by the other gene trees. Group 1, embracing the type strains of C. coralloides and C. exiguus, only emerged as a coherent cluster in the 16S rRNA gene tree. In all other gene trees this cluster embraced organisms of cluster 3, which either formed coherent subclusters (gyrB, lepA) or which appeared polyphyletic (fusA, rpoB). Group 2 organisms consistently constituted a monophyletic cluster, though their branching within the gene trees differed. A concatenated tree, based on the analysis of about 5400 nucleotides of all five partial genes was most similar to the 16S rRNA gene tree. In order to determine whether the individual clusters that emerged by 16S rRNA gene analysis (>99.1% intracluster similarities) show phenetic properties that would allow their description as new species, a few strains of each group were subjected to the analysis of whole cell fatty acid and physiological properties. Riboprint patterns were generated for some members of group 1. While the DNA-DNA reassociation values and riboprint patterns confirmed the genomic heterogeneity of members of cluster 1, none of the other properties investigated were sufficiently discriminative to allow the formal description of strain clusters as new species.

Gene Flow Across Genus Barriers - Conjugation of Dinoroseobacter shibae's 191-kb Killer Plasmid into Phaeobacter inhibens and AHL-mediated Expression of Type IV Secretion Systems.

Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient both individually but also together documenting the efficiency of conjugation. We then studied the influence of externally added quorum sensing (QS) signals on the expression of the T4SS located on the sister plasmids. A QS deficient D. shibae null mutant (ΔluxI1 ) lacking synthesis of N-acyl-homoserine lactones (AHLs) was cultivated with a wide spectrum of chemically diverse long-chain AHLs. All AHLs with lengths of the acid side-chain ≥14 reverted the ΔluxI1 phenotype to wild-type. Expression of the T4SS was induced up to log2 ∼3fold above wild-type level. We hypothesize that conjugation in roseobacters is QS-controlled and that the QS system may detect a wide array of long-chain AHLs at the cell surface.

Plasmid curing and the loss of grip--the 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae.

Surface colonization is characteristic for a broad range of marine roseobacters and many strains have been isolated from biofilms, microbial mats and dinoflagellates. Phaeobacter inhibens DSM 17395, one of the best-studied representatives of the Roseobacter group, is an effective colonizer of marine surfaces, but the genetic basis of this trait is unknown. Based on the composition of its 65-kb RepA-I type plasmid that contains more than 20 genes for polysaccharide metabolism, including a rhamnose operon, which is required for O-antigen formation in Escherichia coli, it was hypothesized that this replicon was essential for surface attachment. Accordingly, a holistic approach was taken and the functional role of this extrachromosomal element in P. inhibens was investigated. Plasmid curing was performed with the homologous RepA-I replication system of Dinoroseobacter shibae DSM 16493(T). The Δ65-kb mutant completely lost its stickiness and could neither attach to artificial (glass, polystyrene) nor to natural surfaces (algae) and, consequently, its ability to form biofilms was impaired. Surprisingly, the mutant also lost the capacity for flagellar swimming motility required for surface colonization and the dispersal of biofilms. The data clearly showed that the 65-kb replicon of P. inhibens DSM 17395 was a genuine biofilm plasmid-mediating surface attachment. Homologous replicons are widely distributed among Rhodobacterales thus indicating the general importance of extrachromosomal elements for biofilm formation.

Genome of the R-body producing marine alphaproteobacterium Labrenzia alexandrii type strain (DFL-11(T)).

Labrenzia alexandrii Biebl et al. 2007 is a marine member of the family Rhodobacteraceae in the order Rhodobacterales, which has thus far only partially been characterized at the genome level. The bacterium is of interest because it lives in close association with the toxic dinoflagellate Alexandrium lusitanicum. Ultrastructural analysis reveals R-bodies within the bacterial cells, which are primarily known from obligate endosymbionts that trigger "killing traits" in ciliates (Paramecium spp.). Genomic traits of L. alexandrii DFL-11(T) are in accordance with these findings, as they include the reb genes putatively involved in R-body synthesis. Analysis of the two extrachromosomal elements suggests a role in heavy-metal resistance and exopolysaccharide formation, respectively. The 5,461,856 bp long genome with its 5,071 protein-coding and 73 RNA genes consists of one chromosome and two plasmids, and has been sequenced in the context of the Marine Microbial Initiative.

Genome of the marine alphaproteobacterium Hoeflea phototrophica type strain (DFL-43(T)).

Hoeflea phototrophica Biebl et al. 2006 is a member of the family Phyllobacteriaceae in the order Rhizobiales, which is thus far only partially characterized at the genome level. This marine bacterium contains the photosynthesis reaction-center genes pufL and pufM and is of interest because it lives in close association with toxic dinoflagellates such as Prorocentrum lima. The 4,467,792 bp genome (permanent draft sequence) with its 4,296 protein-coding and 69 RNA genes is a part of the Marine Microbial Initiative.

Flavobacterium aquidurense sp. nov. and Flavobacterium hercynium sp. nov., from a hard-water creek.

Ten new Flavobacterium-like strains were isolated from freshwater of the hard-water creek Westerhöfer Bach, northern Germany. These strains formed two phylogenetic groups: strains WB 1.1-56T, WB 1.1-04, WB 1.1-14, WB 1.1-57 and WB 1.1-63; and strains WB 4.2-33T, WB 4.1-86, WB 4.2-34, WB 4.2-32 and WB 4.2-78. Cells were Gram-negative, yellow-pigmented, chemoheterotrophic rods. Their major fatty acid profiles were similar, consisting of iso-C(15 : 0), iso-C(15 : 0) 3-OH, iso-C(17 : 0) 3-OH and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH). DNA G+C contents for strains WB 1.1-56T and WB 4.2-33T were 33.5 and 37.5 mol%, respectively. Phylogenetic analysis based on almost complete 16S rRNA gene sequences indicated that strain WB 1.1-56T was phylogenetically most closely related to Flavobacterium frigidimaris KUC-1T, and that strain WB 4.2-33T was related most closely to F. frigidimaris KUC-1T and Flavobacterium saccharophilum DSM 1811T. Levels of 16S rRNA gene sequence similarity between strains WB 1.1-56T and WB 4.2-33T and the type strains of recognized members of the genus Flavobacterium were below 98 %. DNA-DNA hybridization experiments confirmed the separate genomic status of strains WB 1.1-56T and WB 4.2-33T. Strains WB 1.1-56T and WB 4.2-33T and their respective relatives differed from phylogenetically related Flavobacterium species based on several phenotypic characteristics. On the basis of their phenotypic and phylogenetic distinctiveness, the two groups of strains are considered to represent two novel species, for which the names Flavobacterium aquidurense sp. nov. (type strain WB 1.1-56T=DSM 18293T=CIP 109242T) and Flavobacterium hercynium sp. nov. (type strain WB 4.2-33T=DSM 18292T=CIP 109241T) are proposed.

Pedobacter duraquae sp. nov., Pedobacter westerhofensis sp. nov., Pedobacter metabolipauper sp. nov., Pedobacter hartonius sp. nov. and Pedobacter steynii sp. nov., isolated from a hard-water rivulet.

Five isolates that were related phylogenetically to members of the genus Pedobacter were isolated from freshwater of the hard-water creek Westerhöfer Bach, North Germany. The five strains (WB 2.1-25(T), WB 2.3-71(T), WB 3.3-3(T), WB 3.3-22(T) and WB 2.3-45(T)) were Gram-negative and chemoheterotrophic, with rod-shaped cells. Most of their metabolic properties matched those given in the description of the genus Pedobacter. Consistent with the genus description, their fatty acids included mainly iso-C(15 : 0) and summed feature 3 (C(16 : 1)omega7c, iso-C(15 : 0) 2-OH or both); C(16 : 1)omega5c, C(16 : 0), iso-C(15 : 0) 3-OH, C(16 : 0) 3-OH and iso-C(17 : 0) 3-OH were present in smaller amounts. The major isoprenoid quinone was menaquinone 7. With one exception, binary similarity values of the almost complete 16S rRNA gene sequences determined among the isolates as well as between the isolates and type strains of Pedobacter species were lower than 98.5 %. The only exception was the close relationship between Pedobacter caeni DSM 16990(T) and strain WB 2.3-45(T) (99.2 % similarity). DNA-DNA reassociation values determined for this pair of strains was 29.8 %, indicating that strain WB 2.3-45(T) represents a unique genospecies. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains WB 2.1-25(T) and WB 2.3-71(T) form a group that is moderately related to P. caeni and strain WB 2.3-45(T) (98.5 % similarity). Strains WB 3.3-3(T) and WB 3.3-22(T) (98.5 % similarity) branched separately from these four organisms. The five phylogenetically isolated strains differed from each other as well as from the type strain of the type species (Pedobacter heparinus DSM 2366(T)) and some related representatives of the genus in several metabolic reactions and cultural parameters. On the basis of phenotypic and phylogenetic distinctiveness, five novel species are proposed: Pedobacter duraquae sp. nov., with WB 2.1-25(T) (=DSM 19034(T)=CIP 109481(T)) as the type strain; Pedobacter westerhofensis sp. nov., with WB 3.3-22(T) (=DSM 19036(T)=CIP 109479(T)) as the type strain; Pedobacter metabolipauper sp. nov., with WB 2.3-71(T) (=DSM 19035(T)=CIP 109480(T)) as the type strain; Pedobacter hartonius sp. nov., with WB 3.3-3(T) (=DSM 19033(T)=CIP 109468(T)) as the type strain; and Pedobacter steynii sp. nov., with WB 2.3-45(T) (=DSM 19110(T)=CIP 109507(T)) as the type strain.

Deefgea rivuli gen. nov., sp. nov., a member of the class Betaproteobacteria.

Two strains, designated WB 3.4-79(T) and WB 3.3-25, were isolated from a hard-water sample collected from the Westerhöfer Bach, Lower Saxony, Germany. The strains shared 100 % DNA-DNA relatedness, indicating membership of the same genospecies. This close relationship was supported by identical 16S rRNA gene sequences and high similarities in fatty acid composition and biochemical characteristics. The G+C content of the genomic DNA of strain WB 3.4-79(T) was 48.5 mol% and the predominant ubiquinone was Q-8. Major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Major fatty acids (>10 %) were C(16 : 0) and C(16 : 1)omega7c. Polyhydroxybutyrate and polyphosphate granules as well as unidentified enterosomes and a polar organelle are visible by electron microscopy. Comparative 16S rRNA gene sequence analysis indicated that the isolates were placed within the class Betaproteobacteria, remotely related to Chitinibacter tainanensis DSM 15459(T), Silvimonas terrae KCTC 12358(T), Formivibrio citricus DSM 6150(T) and Iodobacter fluviatilis DSM 3764(T). On the basis of phylogenetic and phenotypic distinctness, we propose a novel genus, Deefgea gen. nov., with Deefgea rivuli sp. nov. as the type species. The type strain of Deefgea rivuli is strain WB 3.4-79(T) (=DSM 18356(T)=CIP 109326(T)).

Gene sequence heterogeneity of Corallococcus coralloides strains isolated from geographically diverse locations.

Thirty-three strains classified as Corallococcus coralloides isolated from mostly soil samples in 14 countries of four continents, were subjected to phylogenetic analyses. Based on 16S rDNA analyses the strains form a highly related cluster, sharing above 98.7% sequence similarity. Four groups were recognized within this cluster, only one of which, containing two strains from St. Lucia, Lower Antilles, was exclusively defined by strains from the same sample. The other groups contained members from different countries, even continents. The largest group embraced the type strains of C. coralloides DSM 2259(T) and Corallococcus exiguus 14696(T) which were almost indistinguishable in their 16S rRNA gene sequence. Corallococcus macrosporus DSM 14697(T) grouped outside the C. coralloides cluster, showing a higher relationship to a member of Myxococcus. The topology of the tree generated on the basis of the partial gyrase B (gyrB) gene sequence supports the rRNA gene tree, though some differences in the order of branching were observed. As judged by the binary similarity values the higher resolution power of gyrB sequences was confirmed. From a taxonomic standpoint, the size of myxospores is not a valuable taxonomic criterion, as small- and medium-sized myxospores are members of the same group. If the species status of C. coralloides and C. exiguus is verified by other methods (e.g. DNA-DNA hybridisation, RiboTyping), the genus Corallococcus may embrace a broad range of yet-to-be described novel species. The presence of strains within the same sample displaying higher relatedness to strains from other locations points towards an intensive dispersal of myxospores across continents.

Grouping myxococci (Corallococcus) strains by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI TOF) mass spectrometry: comparison with gene sequence phylogenies.

Nine Corallococcus isolates and three type strains of Corallococcus species were characterized by Intact Cell Mass Spectrometry using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The resulting phenetic clustering was compared to the phylogenetic grouping based upon sequences of two housekeeping genes. The three dendrograms of relatedness resembled each other in that the isolates were highly similar to the type strains of Corallococcus exiguus and Corallococcus coralloides, while Corallococcus macrosporus and Myxococcus xanthus were more distantly related. While certain pairs of organisms were recovered by spectrometry and genes sequence analysis, others were detected by two of the three approaches. The degree of similarity determined by sequence analysis of the two genes was not higher than that revealed by MALDI-TOF analysis. The results show that the spectral profile, consisting of about 25 to 45 masses ranging between 2 and 20 kDa, have indeed taxonomic significance, confirming literature data that ribosomal proteins and certain housekeeping proteins are responsible for the masses obtained. Provided the availability of a database of type strains, MALDI-TOF analysis of unknown strains appears to be a rapid and inexpensive method to taxonomically cluster environmental isolates, expanding the spectrum to strains other than those of medical importance predominantly investigated so far.

Genome organization and localization of the pufLM genes of the photosynthesis reaction center in phylogenetically diverse marine Alphaproteobacteria.

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996(T) to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.