Complete genome sequence of bacteriochlorophyll-synthesizing bacterium Porphyrobacter neustonensis DSM 9434

The genus Porphyrobacter belongs to aerobic anoxygenic phototrophic bacteria cluster. Porphyrobacter neustonensis DSM 9434 was isolated from a eutrophic freshwater pond in Australia, and is able to synthesize Bacteriochlorophyll a as well as grow under aerobic conditions. It is the type species of the genus Porphyrobacter. Here we describe the characteristics of the strain DSM 9434, including the genome sequence and annotation, synthesis of BChl a, and metabolic pathways of the organism. The genome of strain DSM 9434 comprises 3,090,363 bp and contains 2,902 protein-coding genes, 47 tRNA genes and 6 rRNA genes. Strain DSM 9434 encodes 46 genes which participate in BChl a synthesis and this investigation shed light on the evolution and functional implications regarding bacteriochlorophyll synthesis. Electronic supplementary material The online version of this article (doi:10.1186/s40793-017-0243-5) contains supplementary material, which is available to authorized users.


Introduction
Aerobic anoxygenic phototrophic bacteria probably evolved after the accumulation of oxygen in the earth's biosphere [1]. They are widely distributed in the euphotic zone of the ocean as well as terrestrial water, and play an ecologically and biogeochemical important role in aquatic systems, especially marine carbon cycling [2][3][4]. AAP bacteria harvest light by Bacteriochlorophyll a and possess various carotenoids as auxiliary pigments [5]. They derive a significant portion of their energy requirements from light but perform photoheterotrophic metabolism based on an obligatory supply of organic substrates for growth [6]. Until now, all the AAP bacteria that have been discovered belong to the Proteobacteria, and the majority of cultured AAP strains are members of the Alphaproteobacteria [5].
Porphyrobacter has been proposed as a genus along with four Porphyrobacter strains being isolated from a eutrophic freshwater pond in Australia [7]. They are obligate aerobes in the AAP bacteria cluster. Porphyrobacter neustonensis strain DSM 9434 is the type strain of the genus Porphyrobacter [7]. To get insight into the capability of Porphyrobacter in adapt to harvest energy photosynthetically, recently, we obtained the complete genome of P. neustonensis strain DSM 9434 and detected key genes for synthesizing BChl a and mediating aerobic anoxygenic phototrophic metabolism. We also describe the genomic sequencing related to its annotation for understanding their physiological, metabolic and ecological functions in the environments.

Organism information
Classification and features P. neustonensis DSM 9434 was purified from a peptoneyeast extract alga plate after being isolated from the euphotic freshwater pond in Australia [7]. The strain grew with temperature between 10 and 37°C [7]. The cell is rod-shaped, and occasionally coccoid and ovoid (Fig. 1). The strain produced BChl a and carotenoid, analyzed by extracting cells with ethanol (Additional file 1: Figure S1). It grew aerobically in the dark and used a series of organic carbon, such as galactose, glucose, maltose, mannose, sucrose, xylose, arginine, as sole sources of carbon and energy [7]. Analysis of cell wall materials isolated from strain DSM 9434 detected muramic acid and diaminopimelic acid, the major components of peptidoglycan cell wall layer [7]. A high proportion of fatty acids identified as octadecenoic acids (18:1, 84%) is present in the cell with minor components of fatty acids, such as octadecadienoic acid (18:2, 6.1%), 2-hydroxytetradecanoic acid (2OH14:0, 2.7%) and hexadecanoic acid (16:0, 2.6%) [7]. Based on phylogenetic analysis of 16S rRNA gene sequence, the strain belongs to the Alphaproteobacteria class and falls into the cluster comprising the Porphyrobacter species (Fig. 2). The classification and features of P. neustonensis DSM 9434 are summarized in Table 1.  Table 2.

Genome sequencing information
Growth conditions and genomic DNA preparation P. neustonensis DSM 9434 was aerobically cultivated in Luria-Bertani medium at 28°C. High-quality genomic DNA was extracted using Qiagen DNA extraction kit based on its protocol. DNA sequencing of P. neustonensis DSM 9434 was performed using SMRT technology. One Library with insert size of 10 kb was constructed according to the large SMRTbell gDNA protocol (Pacific Biosciences, USA).   Evidence codes -IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project . These reads were assembled using HGAP Assembly version 2 (Pacific Biosciences, USA). The final contigs were checked for circularization and the overlapping ends were trimmed.

Genome annotation
The tRNA genes were identified using tRNAscan-SE 1.21 [8] with bacterial model, and rRNA genes were found via RNAmmer 1.2 Server [9]. The open reading frames (ORFs) and the functional annotation of translated ORFs were predicted and achieved by using the RAST server online [10]. Classification of some predicted genes and pathways were analyzed using COG database [11] and KEGG database [12,13].

Genome properties
The genome of strain DSM 9434 contains a single circular chromosome (Fig. 3). The complete genome of strain DSM 9434 comprises 3,090,363 bp with an average G + C content of 65.3%. The contig contains 2,902 coding sequences of total 2955 genes, 47 tRNAs and 2 operons of 16S-23S-5S rRNA gene. The summary of features and statistics of the genome is shown in Table 3 and genes belonging to COG functional categories are listed in Table 4.

Metabolism of P. neustonensis DSM 9434
The complete genome of P. neustonensis DSM 9434 was annotated for understanding the major metabolic pathways of carbon, nitrogen, sulfur and phosphorus based on the key genes it processes. As we mentioned, although it has bacteriochlorophyll-synthesis genes and acquires energy from light, the absence of carbon fixation and COoxidizing genes indicates that strain DSM 9434 is not able to grow autotrophically. They can only use organic carbon sources. It does not have a complete glycolysis pathway but processes key genes for the Entener-Doudoroff, the pentose phosphate pathway, and the tricarboxylic acid cycle. The genome of P. neustonensis DSM 9434 harbors a variety of transporter genes for ammonium (amtB) and other organic nitrogen substrates (e.g. amino acids, polyamines). It is lack of genes involved in nitrate/nitrite reduction, nitrogen fixation or anaerobic ammonium oxidation, thus strain DSM 9434 only relies on reduced nitrogen sources. The genes encoding urea transporter and urease (ureABC) are absent in the genome of DSM 9434, suggesting its incapability of utilizing urea as a C or N source in the environment. The lack of urea uptake and degradation may reflect the environmental adaption of strain DSM 9434 from a eutrophic pond, where ammonium and algae-derived organic N (e.g. amino acids and polyamines) are usually enriched [18,19]. P. neustonensis DSM 9434 processes genes involved in assimilatory SO 4 reduction (e.g. sulP encoding sulfate permease). Sulfate can be reduced to sulfide (cys), subsequently being incorporated into amino acids. The strain DSM 9434 is also able to utilize organic sulfur compounds (e.g. amino acids, alkanesulfonates); however, it missed the transporter genes (ssuACB) for uptake of extracellular alkanesulfonates. Strain DSM 9434 possesses the high-affinity phosphate transporter (pstSCAB) and regulatory genes (phoUBR), and genes for inorganic P storage as polyphosphate (ppk), a signal of using an alternative strategy for maintaining a phosphate supply [20]. The presence of genes encoding alkaline phosphatase in the genome of strain DSM 9434 indicates that it is capable of using both inorganic and organic forms of phosphorus.

Conclusion
The complete genome sequence of the BChl a synthesizing bacteria P. neustonensis DSM 9434 provide an insight into the genomic basis of its metabolic characteristics and bacteriochlorophyll-synthesis pathway. This investigation sheds light on the evolution of PGCs of aerobic anoxygenic phototrophs and provides the possibility for comparative genomics of AAP bacteria isolated from marine, freshwater and terrestrial environments.