Resequencing and annotation of the Nostoc punctiforme ATTC 29133 genome: facilitating biofuel and high-value chemical production

Cyanobacteria are capable of converting carbon dioxide into oxygen via photosynthesis and have been proposed as significant players in the evolution of current atmospheric oxygen levels during the Precambrian era (Meeks et al. 2001; Schirrmeister et al. 2013) and as origin of chloroplasts via endosymbiosis with eukaryotic cells (Agapakis et al. 2011; Chu et al. 2004). The economic significance of cyanobacteria centers on their potential as synthesis platform for biofuels and other natural products (Machado and Atsumi 2012; Rosgaard et al. 2012); and gene clusters coding for novel enzymes that catalyze unique chemical reactions have been identified from several cyanobacterial genomes (Kleigrewe et al. 2016; Zarzycki et al. 2013). Access to cyanobacterial genomes with up-to-date annotation will provide an improved framework for studying and understanding the biology of this phylogenetic group that is essential for the global carbon and nitrogen cycle and to develop genetic tools that will facilitate the engineering of cyanobacteria for natural products synthesis. Nostoc punctiforme is a filamentous cyanobacterium found in the majority of illuminated environments and it plays a key role in the global nitrogen cycle through its facultative diazotrophic traits. Its life cycle is complex with vegetative cells differentiating into heterocysts, hormogonia and akinetes. The genome of N. punctiforme has been described previously as a large bacterial genome approaching 10 million (M) base pairs (bp) and the most recent genome sequence of the type strain N. punctiforme ATCC 29133 was released into the National Center for Biotechnology Information’s (NCBI’s) public nonredundant (nr) database more than 15 years ago (Meeks et al. 2001). Since then, this article has been cited more than 170 times and the initial annotation of this genome has been extensively utilized to identify genes involved in the carbon and nitrogen fixation process mediated by N. punctiforme and related species. This highlights the importance of this microorganism as a model system and the value of an up-to-date annotation of its genome. The N. punctiforme ATCC 29133 genome that was available through NCBI’s nr database (GenBank ID: CP001037.1) prior to this study contained 9,059,191 bp with approximately three quarters (77.44%) of its genome sequence coding for genes. Of the 6690 protein-coding genes, 2601 (38.3%) had no functional annotation, which subsequently renders a comprehensive understanding of their physiological role and the metabolic capability of the complete genome rather challenging. The rapid improvements of sequencing technologies and sequence analysis tools in the past decade has extended our ability of generating and mining genomic information to several levels beyond what was possible more than a decade ago (Gomez-Escribano et al. 2016; Shendure and Lieberman Aiden 2012) when the first version of the N. punctiforme ATCC 29133 genome was released. For instance, sequencing of bacterial genomes using Pacific Biosciences’s (PacBio’s) single molecule sequencing approach that simultaneously allows the detection of deoxyribonucleic acid (DNA) methylation patterns in bacterial genomes is now routinely performed in many laboratories (Flusberg et al. 2010; Gomez-Escribano et al. 2016; Koren et al. 2013). Moreover, the ample development of bioinformatics tools with various strategies for genome assembly and annotation has dramatically increased our ability to understand the biology of prokaryotes and eukaryotes at the genomic level and to mine their genomes for natural products (Weber et al. 2015; Medema et al. 2015; Yandell and Ence 2012). In this context, the objective of this study is to provide an update of the N. punctiforme genome through resequencing and reannotating of its genome with state-of-the-art technologies. The data presented here were generated and analyzed as part of Community Science Program (Project ID 1393) at the Department of Energy’s Joint Genome Institute (DOE’s JGI) and the sequenced and annotated genome are publically available through the Joint Genome Institute’s Integrated Microbial Genomes and Microbiomes (IMG/M; https://img.jgi.doe.gov/cgi-bin/mer/main.cgi) system (Markowitz et al. 2012) samples warehouse under the IMG Submission Identifier (ID) 62757. The data can be explored and analyzed using the IMG/M system or downloaded for further analyses using stand-alone tools.

We selected N. punctiforme ATCC 29133 for genome resequencing and reannotation due to its importance in the global carbon and nitrogen cycle, its potential biotechnological applications and the value and impact of the first publicly available version of its genome on the scientific community. It is very likely that an updated annotation of the N. punctiforme ATTC 29133 genome will benefit future genome explorations that target genes associated with carbon and nitrogen fixation and the production of biofuels and value-added chemicals.

The advent of affordable second and third-generation DNA sequencing resulted in a significant reduction of costs per sequence supporting the democratization of genome sequencing (Caporaso et al. 2012). Sequencing microbial genomes is now a standard procedure performed in many laboratories and third-generation sequencing technologies, such as PacBio’s SMRT Sequencing platform, facilitate to generate almost complete high-quality genomes from archaea and bacteria in only a few hours (Eid et al. 2009; Rice et al. 2016). This development resulted in a vast amount of genomic information that is now available through public database such as NCBI’s GenBank (https://www.ncbi.nlm.nih.gov/genome/browse/) or JGI’s IMG (https://img.jgi.doe.gov/). These two databases alone contained a total of 89971 and 47009 prokaryotic genome submissions, including 5590 and 26707 non-redundant complete prokaryotic genomes, when accessed on December 27th 2016 respectively. With this almost infinite and continuously growing amount of sequence information, identification and extraction of relevant and accurate data becomes a major challenge. Microbiologists are nowadays limited mostly by the relatively low throughput of molecular experiments that remain necessary to test the functionality and biological role of proteins newly identified using in silico approaches. This renders accurate functional prediction as key to an enhanced understanding of biological processes (Jiang et al. 2016). The importance of an accurate function prediction is even more pronounced in organisms that function as model systems and whose genomes serve as reference for the analyses of phylogenetically related genomes (Cormier et al. 2016); which explains the growing interest in improving the genome annotation of model system microorganisms such as Bacillus pumilus (Gioia et al. 2007; Stepanov et al. 2016) and Pichia pastoris (Komagataella phaffi) (Valli et al. 2016; De Schutter et al. 2009), whose genomes were published during the early phase of the genomics era.

Nostoc punctiforme is an important model system for examining genomic and phenotypic properties of cyanobacteria and their biological carbon and nitrogen fixation capabilities (Sandh et al. 2014). An additional interest in the enhanced understanding of the N. punctiforme genome has been sparked by the increased utilization of cyanobacteria as synthesis platform of biofuels and other natural products (Machado and Atsumi 2012; Rosgaard et al. 2012). For these reasons we selected the type strain N. punctiforme ATCC 29133 for resequencing and reannotation with the assumption that an improved genome sequence and annotation would be of significant value to the scientific community. The newly sequenced, assembled and annotated N. punctiforme ATCC 29133 genome presented here, consists of 984 more genes and 987 more genes with predicted function compared to the previously available N. punctiforme ATCC 29133 genome. The improved gene calling and ability to predict gene function is most likely the result of combination of improved sequence quality and gene annotation algorithm that are now available. It can be anticipated that increasing the repertoire of genes with assigned function within the new N. punctiforme ATCC 29133 genome by ~24% might allow to fillin some of the knowledge-gaps that exist in our current understanding of the molecular processes during microbial carbon and nitrogen fixation. The new N. punctiforme genome also contained ~79% more biosynthetic clusters when compared to the previous version, opening new opportunities to explore the capability of N. punctiforme and its phylogenetically related neighbors for secondary metabolite synthesis.

Access to a reannotated N. punctiforme ATCC 29133 genome through a versatile genome browser that allows to explore DNA sequence data without any or only limited bioinformatics skills will enhance the ability of the scientific community to mine this genome for new insights into genes and gene pathways associated with carbon and nitrogen fixation and secondary metabolite synthesis. It might furthermore facilitate the identification of the genetic properties that might be responsible for some of the metabolic functions and phenotypes associated with N. punctiforme and close relatives. In the case presented here, we opted to load and provide the genome sequence and annotation through IMG/M, a system that has been maintained by the Department of Energy for over a decade and that facilitates comparative analysis and visualization of multilayered omics data (Chen et al. 2016). Subsequent analysis can be performed within the system using a variety of the visualization and analysis tools that have been implemented since the first release of this genome data analysis system in 2005 (Markowitz et al. 2008). Of particular interest for users might be the gene neighborhood function within the Biosynthetic Cluster option, through which the global structure as well as the nucleotide sequence of individual genes and gene clusters can be retrieved for subsequent wet-lab experiments. As new omics data, such as proteomic and transcriptomic data, become available, they can also be integrated easily into the IMG/M structure to complement the reannotated N. punctiforme ATCC 29133 genome data generated during this study. To increase the value of the genome data presented here, sequence data and annotation can also be downloaded through IMG/M and the JGI’s website and analyzed with other genome analysis software, including genome annotation pipelines and analysis systems such as SEED, RAST (Overbeek et al. 2014; Aziz et al. 2008) and the Department of Energy Systems Biology Knowledgebase (KBase; http://kbase.us).

To maximize the benefit of genomes generated with federal funding and to provide access to up-to-date sequence information without the need of in-depth bioinformatics skills, we advocate to resequence and annotate microbial type strains that were sequenced in the early stage of the genomics era, with up-to-date sequencing platforms and improved annotation software and to make these genomes and their annotations available to the scientific community through user-friendly genome browsers, such as the JGI’s IMG/M, at the time of publication.