Genome of the marine alphaproteobacterium Hoeflea phototrophica type strain (DFL-43T)
© The Author(s) 2013
Published: 25 February 2013
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.
Keywordsaerobic rod-shaped motile photoheterotroph Phenotype MicroArray bacteriochlorophyll a symbiosis dinoflagellates Prorocentrum lima Phyllobacteriaceae
Strain DFL-43T (= DSM 17068 = NCIMB 14078) is the type strain of Hoeflea phototrophica, a marine member of the Phyllobacteriaceae (Rhizobiales, Alphaproteobacteria) . The genus, which was named in honor of the German microbiologist Manfred Höfle , contains four species, with H. marina as type species ; the name of a fifth member of the genus, ‘Hoeflea siderophila’, is until now only effectively published . H. phototrophica DFL-43T and strain DFL-44 were found in the course of a screening program for marine bacteria containing the photosynthesis reaction-center genes pufL and pufM . The species epithet ‘phototrophica’ refers to the likely ability of H. phototrophica strains to use light as an additional energy source . Strain DFL-43T was isolated from single cells of a culture of the toxic dinoflagellate Prorocentrum lima maintained at the Biological Research Institute of Helgoland, Germany . Here we present a summary classification and a set of features for H. phototrophica DFL-43T including so far undiscovered aspects of its phenotype, together with the description of the complete genomic sequencing and annotation.
This work is part of the Marine Microbial Initiative (MMI) which enabled the J. Craig Venter Institute (JCVI) to sequence the genomes of approximately 165 marine microbes with funding from the Gordon and Betty Moore Foundation. These microbes were contributed by collaborators worldwide, and represent an array of physiological diversity, including carbon fixers, photoautotrophs, photoheterotrophs, nitrifiers, and methanotrophs. The MMI was designed to complement other ongoing research at JCVI and elsewhere to characterize the microbial biodiversity of marine and terrestrial environments through metagenomic profiling of environmental samples.
Classification and features
16S rRNA analysis
A representative genomic 16S rRNA sequence of H. phototrophica DFL-43T was compared using NCBI BLAST [5,6] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database  and the relative frequencies of taxa and keywords (reduced to their stem ) were determined, weighted by BLAST scores. The most frequently occurring genera were Rhizobium (53.7%), Sinorhizobium (24.0%), Hoeflea (4.5%), Bartonella (4.5%) and Ahrensia (3.7%) (132 hits in total). Regarding the two hits to sequences from members of the species, both, the average identity within HSPs and the average coverage by HSPs were 100.0%. Regarding the single hit to sequences from other members of the genus, the average identity within HSPs was 98.2%, whereas the average coverage by HSPs was 100.0%. Among all other species, the one yielding the highest score was H. marina (AY598817), which corresponded to an identity of 98.2% and an HSP coverage of 100.0%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was AY922224 (Greengenes short name ‘whalefall clone 131720’), which showed an identity of 98.1% and an HSP coverage of 97.5%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were ‘bee’ (3.1%), ‘singl’ (3.0%), ‘abdomen, bumbl, distinct, honei, microbiota, simpl’ (2.9%), ‘microbi’ (2.8%) and ‘structur’ (1.8%) (118 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found, indicating that H. phototrophica is rarely found in environmental samples.
Morphology and physiology
The utilization of carbon compounds by H. phototrophica DFL-43T was also determined for this study using PM01 microplates in an OmniLog phenotyping device (BIOLOG Inc., Hayward, CA, USA). The microplates were inoculated at 28°C with a cell suspension at a cell density of 85% Turbidity and dye D. Further additives were artificial sea salts, vitamins, trace elements and NaHCO3. The exported measurement data were further analyzed with the opm package for R , using its functionality for statistically estimating parameters from the respiration curves such as the maximum height, and automatically translating these values into negative, ambiguous, and positive reactions. The strain was studied in two independent biological replicates, and reactions with a different behavior between the two repetitions were regarded as ambiguous and are not listed below.
H. phototrophica DFL-43T was positive for D,L-malic acid, D-cellobiose, D-fructose, D-galactonic acid-γ-lactone, D-galactose, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D-malic acid, D-mannitol, D-melibiose, D-sorbitol, D-trehalose, D-xylose, L-alanine, L-arabinose, L-glutamic acid, L-glutamine, L-lactic acid, L-lyxose, L-malic acid, L-proline, L-serine, acetic acid, adonitol, α-D-glucose, α-keto-glutaric acid, α-methyl-D-galactoside, β-methyl-D-glucoside, bromo-succinic acid, citric acid, ethanolamine, fumaric acid, m-inositol, maltose, maltotriose, mono-methyl succinate, propionic acid, pyruvic acid, succinic acid, sucrose and uridine. The strain was negative for 1,2-propanediol, 2′-deoxy-adenosine, D,L-α-glycerol-phosphate, D-alanine, D-aspartic acid, D-fructose-6-phosphate, D-glucosaminic acid, D-glucose-1-phosphate, D-glucose-6-phosphate, D-mannose, D-psicose, D-serine, D-threonine, L-alanyl-glycine, L-aspartic acid, L-fucose, L-galactonic acid-γ-lactone, L-rhamnose, L-threonine, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, acetoacetic acid, adenosine, α-D-lactose, α-hydroxy-butyric acid, α-hydroxy-glutaric acid-γ-lactone, α-keto-butyric acid, β-phenylethylamine, dulcitol, glycolic acid, glycyl-L-aspartic acid, glyoxylic acid, inosine, m-hydroxy-phenylacetic acid, m-tartaric acid, mucic acid, thymidine, tricarballylic acid, tween 40, tween 80 and tyramine.
Phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine were the predominant polar lipids of the membrane. The most frequent cellular fatty acids in strain DFL-43T are the mono-unsaturated straight chain acids C18:1 ω7 (62.8%) and its methylated form C18:1 ω7 11Me (21%), followed by C16:0 (6.3%) and C19:1 (3.4%) . The absorption spectrum of an acetone/methanol extract showed the presence of bacteriochlorophyll a and an additional carotenoid (possibly spheroidenone) in small amounts . Further experiments indicated that the pigment production depends on the concentration of sea salts in the medium .
Genome sequencing and annotation
Genome project history
Classification and general features of H. phototrophica DFL-43T according to the MIGS recommendations .
Species Hoeflea phototrophica
Subspecific genetic lineage (strain)
Reference for biomaterial
Biebl et al. 2006
Relationship to oxygen
Prorocentrum lima ME130
Health status of Host
from a culture of Prorocentrum lima ME130
Time of sample collection
April 1, 2002
Genome sequencing project information
High quality draft
Two genomic libraries: 40 kb fosmid library and 3 kb pUC18 plasmid library
10.3 × Sanger
Gene calling method
Prodigal 2.0, Infernal 1.0.2
Final ID pending; previous version ABIA00000000
Genbank Date of Release
final version not yet available
NCBI project ID
Source Material Identifier
Environmental, Marine Microbial Initiative
Growth conditions and DNA extractions
Cells of strain DFL-43T were grown for two to three days on a LB & sea-salt agar plate, containing (l-1) 10 g tryptone, 5 g yeast extract, 10 g NaCl, 17 g sea salt (Sigma-Aldrich S9883) and 15 g agar. A single colony was used to inoculate LB & sea-salt liquid medium and the culture was incubated at 28°C on a shaking platform. The genomic DNA was isolated using the Qiagen Genomic 500 DNA Kit (Qiagen 10262) as indicated by the manufacturer. DNA quality and quantity were in accordance with the instructions of the genome sequencing center. DNA is available through the DNA Bank Network .
Genome sequencing and assembly
The genome was sequenced with the Sanger technology using a combination of two libraries. All general aspects of library construction and sequencing performed at the JCVI can be found on the JCVI website. Base calling of the sequences were performed with the phredPhrap script using default settings. The reads were assembled and assemblies analyzed using the phred/phrap/consed pipeline . The last gaps were closed by adding new reads produced by recombinant PCR and PCR primer walks. In total 21 Sanger reads were required for gap closure and improvement of low quality regions. The final consensus sequence was built from 46,086 Sanger reads (10.3 × coverage).
Gene prediction was carried out using GeneMark as part of the genome annotation pipeline in the Integrated Microbial Genomes Expert Review (IMG-ER) system . To identify coding genes, Prodigal  was used, while ribosomal RNA genes within the genome were identified using RNAmmer . Other non-coding genes were predicted using Infernal . Manual functional annotation was performed within the IMG platform  and the Artemis Genome Browser .
% of Total
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of replicons
Genes with function prediction
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with the general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
Intracellular trafficking, secretion, and vesicular transport
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Sequencing, assembly, annotation and data analysis for the first draft version were supported by the Gordon and Betty Moore Foundation Marine Microbiology Initiative, as part of its Marine Microbial Sequencing Project (http://www.moore.org/marinemicro). Support for the subsequent gap closure and analysis via the German Research Foundation (DFG) SFB/TRR 51 is gratefully acknowledged. We also thank the European Commission which supported phenotyping via the Microme project 222886 within the Framework 7 program.
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