Biology subjects

MacCready, J. S.

Publications and source records attributed to MacCready, J. S..

5 recordsLinked to original sources

bioRxiv2024

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

Bacterial microcompartments (BMC) are protein-based organelles broadly distributed across all bacterial phyla and subclassified into [≥]60 functional variants. Despite their evolutionary and metabolic diversity, shell proteins that structurally compose the BMC surface are closely related across BMC classes. Herein, we sought to identify molecular and physiological features that could promote independent operation of more than one BMC type within the same cell by reducing inter-organelle cross-talk of shell proteins. We heterologously expressed shell proteins from the structurally well-defined BMC of Haliangium ochraceum (HO) within Synechococcus elongatus PCC 7942, a model cyanobacterium containing the {beta}-carboxysome. We find considerable cross-reactivity of the HO hexameric shell protein (HO BMC-H) with components of the {beta}-carboxysome; HO BMC-H can integrate into carboxysomes, disrupt its ultrastructural organization, and impair its associated CO2 fixation reactions. S. elongatus is unable to maintain the integrity of the {beta}-carboxysome over time when HO BMC-H is expressed in the absence of one or more of three broad strategies that act to increase the orthogonality between HO and carboxysome BMC shell proteins: i) reduced expression of promiscuous shell proteins; ii) sequestration of free HO BMC-H proteins via co-expression of other members of the same HO shell protein class, or; iii) heterologous expression of BMC positional system proteins McdAB (Maintenance of carboxysome distribution AB), revealing a putative moonlighting function of the McdAB protein family. Our results have implications for bacteria that encode more than one BMC within their genome and may have translational implications for the use of engineered BMCs for biotechnological applications.

synthetic biology↗

bioRxiv2020

The McdAB system positions α-carboxysomes in proteobacteria

Carboxysomes are protein-based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is utilized as a surface for the equidistant-spacing of {beta}-carboxysomes across cell lengths by a two-component system (McdAB) in the model cyanobacterium Synechococcus elongatus PCC 7942. More recently, we found that McdAB systems are widespread among {beta}-cyanobacteria, which possess {beta}-carboxysomes, but are absent in -cyanobacteria, which possess structurally distinct -carboxysomes. Since cyanobacterial -carboxysomes are thought to have arisen in proteobacteria and were subsequently horizontally transferred into cyanobacteria, this raised the question whether -carboxysome containing proteobacteria possess a McdAB system for positioning -carboxysomes. Here, using the model chemoautotrophic proteobacterium H. neapolitanus, we show that a McdAB system distinct from that of {beta}-cyanobacteria operates to position -carboxysomes across cell lengths. We further show that this system is widespread among -carboxysome containing proteobacteria and that cyanobacteria likely inherited an -carboxysome operon from a proteobacterium lacking the mcdAB locus. These results demonstrate that McdAB is a cross-phylum two-component system necessary for positioning - and {beta}-carboxysomes. The findings have further implications for understanding the positioning of other bacterial protein-based organelles involved in diverse metabolic processes.

microbiology↗

bioRxiv2020

Cyanobacterial growth and morphology are influenced by carboxysome positioning and temperature

Cyanobacteria are the prokaryotic group of phytoplankton responsible for a significant fraction of global CO2 fixation. Like plants, cyanobacteria use the enzyme Ribulose 1,5-bisphosphate Carboxylase/Oxidase (RuBisCO) to fix CO2 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 CO2 substrate. In the rod-shaped cyanobacterium Synechococcus elongatus PCC7942, 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 for the first time that a failure to distribute carboxysomes leads to a temperature-dependent decrease in cell growth rate, cell division arrest, cell elongation, asymmetric cell division, and a significant reduction in cellular levels of RuBisCO. Unexpectedly, we also report that even wild-type S. elongatus undergoes filamentous growth at the cool, but environmentally-relevant, growth temperature of 20{degrees}C. 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 at temperatures where rod-shaped cyanobacteria adopt a filamentous morphology. IMPORTANCEPhotosynthetic cyanobacteria are responsible for almost half of global CO2 fixation. Due to eutrophication, rising temperatures, and increasing atmospheric CO2 concentrations, cyanobacteria have recently 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 CO2 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 temperature-dependent changes in cell growth and morphology as well as a significant reduction in cellular levels of RuBisCO.

cell biology↗

bioRxiv2019

The McdAB Carboxysome Positioning System is Widespread Among β-cyanobacteria

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. McdA, a ParA-type ATPase, non-specifically 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 towards 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 used neighborhood analysis to show that the McdAB system is widespread among {beta}-cyanobacteria and often clusters near carboxysome-related components. Moreover, we show that two distinct McdAB systems exist in {beta}-cyanobacteria, with Type 2 systems being the most abundant (>98% of {beta}-cyanobacteria) and Type 1 systems, like that of S. elongatus, possibly being acquired more recently. Surprisingly, our analysis suggests that the McdAB system is completely absent in -cyanobacteria. Lastly, all McdB proteins we identified share the sequence signatures of a protein capable of undergoing Liquid-Liquid Phase Separation (LLPS). Indeed, we find that S. elongatus McdB undergoes LLPS in vitro, the first example of a ParA-type ATPase partner protein exhibiting this behavior. This is an intriguing finding given the recent demonstration of LLPS activity by {beta}-carboxysome core components. Our results have broader implications for understanding carboxysome biogenesis and positioning across all {beta}-cyanobacteria.\n\nIn BriefWe found that the McdAB carboxysome positioning system is widespread among {beta}-cyanobacteria, absent in -cyanobacteria, exists in two distinct forms, and that S. elongatus McdB undergoes liquid-liquid phase separation.

microbiology↗

bioRxiv2018

Protein Gradients on the Nucleoid Position the Carbon-fixing Organelles of Cyanobacteria

In BriefWe describe a novel positioning system that utilizes the cyanobacterial nucleoid to segregate, transport and equidistantly position the carbon-fixation machinery (carboxysomes).\n\nSummaryCarboxysomes are protein-based bacterial organelles that encapsulate a key enzyme of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we show that a small protein, termed McdB, localizes to carboxysomes through interactions with carboxysome shell proteins to drive emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly interacts to stimulate McdA ATPase activity, and indicate that carboxysome-dependent McdA depletion zone formation on the nucleoid is required for directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a new class of self-organizing proteins that follow a Brownian-ratchet mechanism, challenging the cytoskeletal model of organelle transport, for equidistant positioning of carboxysomes in cyanobacteria. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria more broadly.

microbiology↗