The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle

Abstract

Unicellular cyanobacteria have recently been recognized for their contributions to nitrogen fixation in marine environments, a function previously thought to be filled mainly by filamentous cyanobacteria such as Trichodesmium. To begin a systems level analysis of the physiology of the unicellular N(2)-fixing microbes, we have sequenced to completion the genome of Cyanothece sp. ATCC 51142, the first such organism. Cyanothece 51142 performs oxygenic photosynthesis and nitrogen fixation, separating these two incompatible processes temporally within the same cell, while concomitantly accumulating metabolic products in inclusion bodies that are later mobilized as part of a robust diurnal cycle. The 5,460,377-bp Cyanothece 51142 genome has a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of a linear element in the genome of a photosynthetic bacterium. On the 429,701-bp linear chromosome is a cluster of genes for enzymes involved in pyruvate metabolism, suggesting an important role for the linear chromosome in fermentative processes. The annotation of the genome was significantly aided by simultaneous global proteomic studies of this organism. Compared with other nitrogen-fixing cyanobacteria, Cyanothece 51142 contains the largest intact contiguous cluster of nitrogen fixation-related genes. We discuss the implications of such an organization on the regulation of nitrogen fixation. The genome sequence provides important information regarding the ability of Cyanothece 51142 to accomplish metabolic compartmentalization and energy storage, as well as how a unicellular bacterium balances multiple, often incompatible, processes in a single cell.

Fig. 1.

Overview of processes involved in daily metabolic cycling in Cyanothece 51142. Photosynthesis fixes carbon during the day, which is stored in glycogen granules. Glycogen is rapidly consumed during a burst of respiration in the early dark period, which coincides with peak nitrogenase activity, fermentation, and a minimum of photosynthetic capacity (10). Fixed nitrogen is stored in cyanophycin granules, which are completely depleted during the following day. Phosphate is stored in polyphosphate bodies.

Fig. 2.

General genome features and distribution of gene functions within the Cyanothece 51142 genome. Labels are given in base pairs for the circular chromosome (A), linear chromosome (B), and plasmids (C). Genes are colored by functional category as follows: energy, fatty acid, and phospholipid metabolism (red), cell envelope (orange), cellular processes (steel blue), central intermediary metabolism (light green), photosynthesis and respiration (dark green), regulation (cyan), DNA, transcription, and translation (dark blue), small molecule biosynthesis (magenta), transport and binding (purple), unknown/hypothetical (gray), other (black), and noncoding RNA (yellow). The two rRNA operons on the circular chromosome are indicated in yellow at 3.95 Mb and 4.10 Mb in A. The glucose and pyruvate metabolism cluster on the linear chromosome is shown in red and light green between 375 kb and 400 kb in B.

Fig. 3.

Clusters of N₂-fixation-related genes. Shown are genes with conserved synteny between Cyanothece 51142 and other nitrogen-fixing cyanobacteria. Black arrows represent genes assigned to functional categories and white arrows correspond to hypothetical genes and genes of unknown function. Missing sequence information from the spheroid body of Rhopalodia gibba is indicated by dots. (∫∫) denotes gaps in the sequence, with the length of the omitted sequence in kb. (*) indicates the location of DNA insertion elements, with the size of the element in kb. A possible inversion event in Synechococcus sp. JA-3–3Ab is highlighted in brackets. GenBank accession numbers for the sequences used are as follows: Cyanothece sp. ATCC 51142, CP000806; spheroid body of Rhopalodia gibba, AY728387; Crocosphaera watsonii WH 8501, AADV02000024; Trichodesmium erythraeum IMS101, CP000393; Nostoc punctiforme PCC 73102, CP001037; Anabaena sp. PCC 7120, BA000019; and Synechococcus sp. JA-3–3Ab, CP000239.

Fig. 4.

Genes encoding enzymes in glucose metabolism pathways in Cyanothece 51142. Pathways were generated by mapping Cyanothece 51142 genes onto known fermentative pathways (29, 40). (A) Each arrow shows the direction of the reaction. Broken arrows indicate that more than one catalytic step is involved. The numbers correspond to the enzymes involved: (1) enzymes for glycogen synthesis, (2) enzymes for glycogen degradation, (3) glucose 6-P isomerase pgi, (4) 6-phosphofructokinase pfkA, (5) fructose-bisphosphate aldolase fba, (6) triosephosphate isomerase tpi, (7) glyceraldehyde-3-P dehydrogenase gap, (8) phosphoglycerate kinase pgk, (9) phosphoglycerate mutase gpm, (10) enolase eno, (11) pyruvate kinase pykF, (12) lactate dehydrogenase ldh, (13) pyruvate dehydrogenase pdhA, (14) phosphotransacetylase pta, (15) acetate kinase ackA, (16) glucose-6-P dehydrogenase zwf, (17) 6-phosphogluconate dehydrogenase gnd, (18) xylulose-5-P phosphoketolase xpk, (19) aldehyde dehydrogenase ald, (20) alcohol dehydrogenase adh, and (21) pyruvate decarboxylase pdc. One star illustrates enzymes with a gene copy present on the linear chromosome. Two stars signify uniqueness to the linear chromosome. Red stars correspond to genes organized in the gene cluster shown in B. (B) Cluster of genes on the linear chromosome that encodes various enzymes involved in glucose metabolism.