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. 2021 Jul;116(1):277-297.
doi: 10.1111/mmi.14708. Epub 2021 Mar 8.

The McdAB system positions α-carboxysomes in proteobacteria

Joshua S MacCready  1 , Lisa Tran  2 , Joseph L Basalla  1 , Pusparanee Hakim  1 , Anthony G Vecchiarelli  1
Affiliations

The McdAB system positions α-carboxysomes in proteobacteria

Joshua S MacCready et al. Mol Microbiol. 2021 Jul.

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 cyanobacterium Synechococcus elongatus PCC 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 proteobacterium Halothiobacillus 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 the mcdAB locus. 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 SUMMARY: Cyanobacteria are well known to fix atmospheric CO2 into 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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Overview of α‐ and β‐carboxysome composition, operon structure, and prevalence in proteobacteria. (a) Cartoon illustration of internal reactions (left) and known components (right) of β‐carboxysomes and (b) α‐carboxysomes. (c) α‐carboxysome‐containing proteobacteria display diverse metabolic capacities. (d) H. neapolitanus α‐carboxysome operon. Red = Rubisco or Rubisco associated, green = CsoS2 which mediates Rubisco/shell interaction, blue = carbonic anhydrase, purple = putative mcdA gene, yellow = putative mcdB gene, dark grey = shell component, light grey = hypothetical protein (Hyp). Gene colors are matched to the proteins shown in panel B and functionally equivalent proteins for β‐carboxysomes in panel A [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 2
FIGURE 2
An McdAB system positions α‐carboxysomes. (a) The carboxysome reporter CbbS‐mTQ is distributed in WT H. neapolitanus cells. Scale bar: 2 µm. (b) Electron micrograph showing α‐carboxysomes confined to the nucleoid in WT cells. Scale bar: 200 nm. (c) Homogenous distribution of α‐carboxysomes is lost in the absence of McdA. Scale bar: 2 µm. (d) Polar localization of assembled α‐carboxysomes in the absence of McdA (yellow arrow). Scale bar: 200 nm. (e) Homogenous distribution of α‐carboxysomes is lost in the absence of McdB. Scale bar: 2 µm. (f) Polar localization of assembled α‐carboxysomes in the absence of McdB (yellow arrow). Scale bar: 200 nm. (g) Quantification of carboxysome distributions. In ∆mcdA (n = 210 cells), 76% of cells had a single polar focus, 7% displayed two foci at opposing poles, and 17% lacked a focus. In ∆mcdB (n = 220 cells), 66% of cells had a single polar focus, 5% displayed two foci at opposing poles, and 29% lacked a focus [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 3
FIGURE 3
Aggregated α‐carboxysomes are nucleoid excluded. (a) The carboxysome reporter CbbS‐mTQ colocalizes with the DAPI‐stained nucleoid in WT H. neapolitanus cells. In (b) ∆mcdA and (c) ∆mcdB mutants, aggregated α‐carboxysomes are nucleoid excluded. PCC values were calculated from n ≥ 100 cells per cell population. (d) In WT cells, carboxysomes colocalize with the DAPI‐stained nucleoid. (e) After treatment with ciprofloxacin, the nucleoid condenses and carboxysomes remain colocalized with the DAPI‐stained nucleoid. In ∆mcdA (f) and ∆mcdB (h) strains, aggregated carboxysomes are excluded from the DAPI‐stained nucleoid. After ciprofloxacin treatment of ∆mcdA (g) and ∆mcdB (i) strains, the nucleoid condenses and carboxysomes remain aggregated despite the increased cytoplasmic space. Scale bar: 2 µm [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 4
FIGURE 4
α‐McdB loads onto α‐carboxysomes and interacts with α‐McdA. (a) mNG‐McdB (magenta) colocalizes with the carboxysome reporter CbbS‐mTQ (cyan) in WT H. neapolitanus. (b) In the absence of McdA, McdB strongly colocalizes with carboxysome aggregates. PCC values were calculated from n ≥ 100 cells per cell population. Scale bar: 2 µm. (c) Bacterial‐2‐Hybrid (B2H) analysis of α‐McdA and α‐McdB. α‐McdA was positive for self‐association. α‐McdA directly interacts with α‐McdB. α‐McdB did not self‐associate. B2H image is representative of three independent trials. (d) SEC‐MALS plot for H. neapolitanus α‐McdB; monomer MW = 10 kDa [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 5
FIGURE 5
Conserved features among α‐McdAB proteins within cso operons. (a) Features conserved or distinct (boxed) among identified α‐McdA proteins encoded within the cso operon of α‐carboxysome‐containing proteobacteria compared to classical ParA‐type proteins involved in the positioning of diverse cargoes. The deviant‐Walker A (blue), A’ (red), and B (purple) boxes, are conserved among all ParA family proteins. ParA‐type proteins shown: Escherichia coli phage P1 ParA (plasmid partitioning—YP_006528) (Abeles et al., 1985), Escherichia coli F plasmid SopA (plasmid partitioning—NP_061425) (Mori et al., 1986), Caulobacter crescentus ParA (chromosome segregation—AAB51267) (Mohl & Gober, 1997), Caulobacter crescentus MipZ (cell‐division positioning—NP_420968) (Thanbichler & Shapiro, 2006), Rhodobacter sphaeroides PpfA (chemotaxis cluster distribution—EGJ21499) (Roberts et al., 2012), and Bacillus subtilis Soj (chromosome segregation—NP_391977) (Marston & Errington, 1999). (b) General features of α‐McdB proteins encoded within the cso operon of α‐carboxysome‐containing proteobacteria. Percent composition of the amino acids alanine (A), proline (P), lysine (K), serine (S), and threonine (T) are presented to illustrate the strong bias for these amino acids (center). All α‐McdB proteins identified are highly hydrophilic across the entire primary sequence (bottom) [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 6
FIGURE 6
McdAB systems are widespread among carboxysome‐containing proteobacteria. (a) Table highlighting the prevalence and genomic context of all identified α‐McdAB sequences within α‐carboxysome‐containing proteobacteria. (b) Genomic arrangement when α‐McdAB are encoded within the cso operon. (c) Genomic arrangement when only α‐McdB is encoded within the cso operon. (d) Genomic arrangement when one copy of α‐McdB is encoded within the cso operon and a second copy of α‐McdB is encoded next to α‐mcdA at a distant locus. (e) α‐McdAB systems are widely distributed among proteobacterial taxonomic classes (left), orders (center), and metabolisms (right) [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 7
FIGURE 7
α‐carboxysome evolution among proteobacteria and α‐cyanobacteria. (a) Inferred phylogeny of α‐carboxysome‐containing proteobacteria and α‐cyanobacteria. Line colors: α‐McdAB found within cso operon (black), only α‐McdB found within cso operon (red), and neither α‐McdAB found within cso operon (blue). Black dot represents >70% bootstrap support (500 replicates). Green asterisk represents a shared cso operon ancestor among α‐cyanobacteria and proteobacteria that lacks the α‐mcdA gene. (b) Genomic arrangement of the H. neapolitanus cso operon. (c) Genomic arrangement of the T. halophila cso operon. (D) Genomic arrangement of the C. gracile cso operon, which lacks a McdAB system [Colour figure can be viewed at wileyonlinelibrary.com]
FIGURE 8
FIGURE 8
Similarities and differences among all known McdA and McdB proteins. (a) Features conserved or distinct among α‐McdA (green), β‐McdA Type 1 (red), β‐ McdA Type 2 (blue), and diverse classical ParA‐type proteins. All Walker‐boxes are well‐conserved among all classic ParA family proteins. ParA‐type proteins shown: Escherichia coli phage P1 ParA (plasmid partitioning—YP_006528) (Abeles et al., 1985), Escherichia coli F plasmid SopA (plasmid partitioning—NP_061425) (Mori et al., 1986), Caulobacter crescentus ParA (chromosome segregation—AAB51267) (Mohl & Gober, 1997), Caulobacter crescentus MipZ (cell‐division positioning—NP_420968) (Thanbichler & Shapiro, 2006), Rhodobacter sphaeroides PpfA (chemotaxis cluster distribution—EGJ21499) (Roberts et al., 2012), and Bacillus subtilis Soj (chromosome segregation—NP_391977) (Marston & Errington, 1999). (b) α‐McdB proteins lack the predicted central coiled‐coil and glutamine‐rich regions found in β‐McdB proteins. α‐McdB Type 2 proteins lack the charged N‐terminus conserved in all other McdB types. (c) PONDR disorder scatter plot for all McdB protein types. (d) DIC microscopy images showing purified H. neapolitanus McdB undergoes LLPS in vitro in the presence of the crowders PEG or Ficoll. Droplets exhibit liquid‐like properties such as fusion (yellow arrows). (e) SEC‐MALS plot for a representative β‐McdB Type 1 (S. elongatus McdB; monomer MW = 17 kDa), β‐McdB Type 2 (Synechococcus sp. PCC 7002 McdB; monomer MW = 21 kDa), and α‐McdB (H. neapolitanus McdB; monomer MW = 10 kDa). (f) McdA/B‐like sequences genomically neighbor BMC components across diverse microbes [Colour figure can be viewed at wileyonlinelibrary.com]
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