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Tar spot is a devasting corn disease caused by the obligate fungal pathogen Phyllachora maydis. Since its initial identification in the United States in 2015, P. maydis has become an increasing threat to corn production. Despite this, P. maydis has remained largely understudied at the molecular level due to difficulties surrounding its obligate lifestyle. Here, we generated a significantly improved P. maydis nuclear and mitochondrial genome using a combination of long- and short-read technologies and also provide the first transcriptomic analysis of primary tar spot lesions. Our results show that P. maydis is deficient in inorganic nitrogen utilization, is likely heterothallic, and encodes for significantly more protein coding genes, including secreted enzymes and effectors, than previous determined. Furthermore, our expression analysis suggests that following primary tar spot lesion formation, P. maydis might reroute carbon flux away from DNA replication and cell division pathways and towards pathways previously implicated in having significant roles in pathogenicity, such as autophagy and secretion. Together, our results identified several highly expressed unique secreted factors that likely contribute to host recognition and subsequent infection, greatly increasing our knowledge of the biological capacity of P. maydis, which have much broader implications for mitigating tar spot of corn. 

ABSTRACT Cyanobacteria are the prokaryotic group of phytoplankton responsible for a significant fraction of global CO 2 fixation. Like plants, cyanobacteria use the enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco) to fix CO 2 into organic carbon molecules via the Calvin-Benson-Bassham cycle. Unlike plants, cyanobacteria evolved a carbon-concentrating organelle called the carboxysome—a proteinaceous compartment that encapsulates and concentrates Rubisco along with its CO 2 substrate. In the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942, we recently identified the McdAB system responsible for uniformly distributing carboxysomes along the cell length. It remains unknown what role carboxysome positioning plays with respect to cellular physiology. Here, we show that a failure to distribute carboxysomes leads to slower cell growth, cell elongation, asymmetric cell division, and elevated levels of cellular Rubisco. Unexpectedly, we also report that even wild-type S. elongatus undergoes cell elongation and asymmetric cell division when grown at the cool, but environmentally relevant, growth temperature of 20°C or when switched from a high- to ambient-CO 2 environment. The findings suggest that carboxysome positioning by the McdAB system functions to maintain the carbon fixation efficiency of Rubisco by preventing carboxysome aggregation, which is particularly important under growth conditions where rod-shaped cyanobacteria adopt a filamentous morphology. IMPORTANCE Photosynthetic cyanobacteria are responsible for almost half of global CO 2 fixation. Due to eutrophication, rising temperatures, and increasing atmospheric CO 2 concentrations, cyanobacteria have gained notoriety for their ability to form massive blooms in both freshwater and marine ecosystems across the globe. Like plants, cyanobacteria use the most abundant enzyme on Earth, Rubisco, to provide the sole source of organic carbon required for its photosynthetic growth. Unlike plants, cyanobacteria have evolved a carbon-concentrating organelle called the carboxysome that encapsulates and concentrates Rubisco with its CO 2 substrate to significantly increase carbon fixation efficiency and cell growth. We recently identified the positioning system that distributes carboxysomes in cyanobacteria. However, the physiological consequence of carboxysome mispositioning in the absence of this distribution system remains unknown. Here, we find that carboxysome mispositioning triggers changes in cell growth and morphology as well as elevated levels of cellular Rubisco. 

Bacterial microcompartments (BMCs) confine a diverse array of metabolic reactions within a selectively permeable protein shell, allowing for specialized biochemistry that would be less efficient or altogether impossible without compartmentalization. BMCs play critical roles in carbon fixation, carbon source utilization, and pathogenesis. Despite their prevalence and importance in bacterial metabolism, little is known about BMC “homeostasis,” a term we use here to encompass BMC assembly, composition, size, copy-number, maintenance, turnover, positioning, and ultimately, function in the cell. The carbon-fixing carboxysome is one of the most well-studied BMCs with regard to mechanisms of self-assembly and subcellular organization. In this minireview, we focus on the only known BMC positioning system to date—the maintenance of carboxysome distribution (Mcd) system, which spatially organizes carboxysomes. We describe the two-component McdAB system and its proposed diffusion-ratchet mechanism for carboxysome positioning. We then discuss the prevalence of McdAB systems among carboxysome-containing bacteria and highlight recent evidence suggesting how liquid-liquid phase separation (LLPS) may play critical roles in carboxysome homeostasis. We end with an outline of future work on the carboxysome distribution system and a perspective on how other BMCs may be spatially regulated. We anticipate that a deeper understanding of BMC organization, including nontraditional homeostasis mechanisms involving LLPS and ATP-driven organization, is on the horizon. 

Abstract Carboxysomes are protein-based organelles that are essential for allowing cyanobacteria to fix CO2. Previously, we identified a two-component system, McdAB, responsible for equidistantly positioning carboxysomes in the model cyanobacterium Synechococcus elongatus PCC 7942 (MacCready JS, Hakim P, Young EJ, Hu L, Liu J, Osteryoung KW, Vecchiarelli AG, Ducat DC. 2018. Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria. eLife 7:pii:e39723). McdA, a ParA-type ATPase, nonspecifically binds the nucleoid in the presence of ATP. McdB, a novel factor that directly binds carboxysomes, displaces McdA from the nucleoid. Removal of McdA from the nucleoid in the vicinity of carboxysomes by McdB causes a global break in McdA symmetry, and carboxysome motion occurs via a Brownian-ratchet-based mechanism toward the highest concentration of McdA. Despite the importance for cyanobacteria to properly position their carboxysomes, whether the McdAB system is widespread among cyanobacteria remains an open question. Here, we show that the McdAB system is widespread among β-cyanobacteria, often clustering with carboxysome-related components, and is absent in α-cyanobacteria. Moreover, we show that two distinct McdAB systems exist in β-cyanobacteria, with Type 2 systems being the most ancestral and abundant, and Type 1 systems, like that of S. elongatus, possibly being acquired more recently. Lastly, all McdB proteins share the sequence signatures of a protein capable of undergoing liquid–liquid phase separation. Indeed, we find that representatives of both McdB types undergo liquid–liquid phase separation in vitro, the first example of a ParA-type ATPase partner protein to exhibit this behavior. Our results have broader implications for understanding carboxysome evolution, biogenesis, homeostasis, and positioning in cyanobacteria. 

Cyanobacteria are tiny organisms that can harness the energy of the sun to power their cells. Many of the tools required for this complex photosynthetic process are packaged into small compartments inside the cell, the carboxysomes. In Synechococcus elongatus, a cyanobacterium that is shaped like a rod, the carboxysomes are positioned at regular intervals along the length of the cell. This ensures that, when the bacterium splits itself in half to reproduce, both daughter cells have the same number of carboxysomes. Researchers know that, in S. elongatus, a protein called McdA can oscillate from one end of the cell to the other. This protein is responsible for the carboxysomes being in the right place, and some scientists believe that it helps to create an internal skeleton that anchors and drags the compartments into position. Here, MacCready et al. propose another mechanism and, by combining various approaches, identify a new partner for McdA. This protein, called McdB, is present on the carboxysomes. McdB also binds to McdA, which itself attaches to the nucleoid – the region in the cell that contains the DNA. McdB forces McdA to release itself from DNA, causing the protein to reposition itself along the nucleoid. Because McdB attaches to McdA, the carboxysomes then follow suit, constantly seeking the highest concentrations of McdA bound to nearby DNA. Instead of relying on a cellular skeleton, these two proteins can organize themselves on their own using the nucleoid as a scaffold; in turn, they distribute carboxysomes evenly along the length of a cell. Plants also obtain their energy from the sun via photosynthesis, but they do not carry carboxysomes. Scientists have tried to introduce these compartments inside plant cells, hoping that it could generate crops with higher yields. Knowing how carboxysomes are organized so they can be passed down from one generation to the next could be important for these experiments. 

Abstract Carboxysomes are protein‐based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is used to equally space out β‐carboxysomes across cell lengths by a two‐component system (McdAB) in the model cyanobacteriumSynechococcus elongatusPCC 7942. More recently, we found that McdAB systems are widespread among β‐cyanobacteria, which possess β‐carboxysomes, but are absent in α‐cyanobacteria, which possess structurally and phyletically distinct α‐carboxysomes. Cyanobacterial α‐carboxysomes are thought to have arisen in proteobacteria and then horizontally transferred into cyanobacteria, which suggests that α‐carboxysomes in proteobacteria may also lack the McdAB system. Here, using the model chemoautotrophic proteobacteriumHalothiobacillus neapolitanus, we show that a McdAB system distinct from that of β‐cyanobacteria operates to position α‐carboxysomes across cell lengths. We further show that this system is widespread among α‐carboxysome‐containing proteobacteria and that cyanobacteria likely inherited an α‐carboxysome operon from a proteobacterium lacking themcdABlocus. These results demonstrate that McdAB is a cross‐phylum two‐component system necessary for positioning both α‐ and β‐carboxysomes. The findings have further implications for understanding the positioning of other protein‐based bacterial organelles involved in diverse metabolic processes. Plain language summaryCyanobacteria are well known to fix atmospheric CO₂into sugars using the enzyme Rubisco. Less appreciated are the carbon‐fixing abilities of proteobacteria with diverse metabolisms. Bacterial Rubisco is housed within organelles called carboxysomes that increase enzymatic efficiency. Here we show that proteobacterial carboxysomes are distributed in the cell by two proteins, McdA and McdB. McdA on the nucleoid interacts with McdB on carboxysomes to equidistantly space carboxysomes from one another, ensuring metabolic homeostasis and a proper inheritance of carboxysomes following cell division. This study illuminates how widespread carboxysome positioning systems are among diverse bacteria. Carboxysomes significantly contribute to global carbon fixation; therefore, understanding the spatial organization mechanism shared across the bacterial world is of great interest.