Biology subjects

Mullineaux, C. W.

Publications and source records attributed to Mullineaux, C. W..

4 recordsLinked to original sources

bioRxiv2025

Flocculation of a cyanobacterium confers defense against bacterial predation

Many cyanobacteria, including unicellular species, are capable of flocculation: the formation of floating linked assemblages of many thousands of cells. Flocculation is a highly regulated process requiring both type IV pilus activity and the production of extracellular polysaccharides. Under standard laboratory conditions flocculation often slows culture growth, and its physiological advantages remain unclear. Proposed benefits include self-shading as protection from excess light exposure and flotation in the water column. Here, we determine whether flocculation can serve as a method of defense against bacterial predation. Using the unicellular cyanobacterium Synechocystis sp. PCC 6803, we show that flocculation is strongly triggered by exposure to live cells of "foreign" bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. The established P. aeruginosa virulence arsenal includes bacterial warfare systems for competition for resources and acquisition of nutrients by way of interbacterial competition. Here, we establish the use of these strategies for direct predation, via the use of Type VI Secretion Systems. Comparisons of P. aeruginosa co-cultures with either wild type Synechocystis or a non-flocculating mutant revealed that Synechocystis flocculation minimizes both growth of P. aeruginosa and cell lysis of Synechocystis. This in turn reduces the impact of P. aeruginosa on Synechocystis growth by mechanistically limiting the photosynthetic products that P. aeruginosa can access. From these data, we propose that the type VI secretion system of P. aeruginosa can be used for predation and that the primary function of cyanobacterial flocculation is for defense against microbial predation. IMPORTANCEBacteria in the environment rarely, if ever, grow in isolation. Some phenotypic traits can only be understood in the context of interspecies interactions. Here, we examine interactions between two bacterial species with very different modes of metabolism. Synechocystis sp. PCC 6803 is a freshwater cyanobacterium that grows photosynthetically using light, water, CO2 and minerals. Pseudomonas aeruginosa is a soil bacterium and an opportunistic pathogen that depends on a supply of organic molecules for survival. When P. aeruginosa is co-cultured with Synechocystis in mineral medium, it lyses Synechocystis cells, using its Type VI secretion system to access nutrients via predation. The presence of P. aeruginosa (and other foreign bacteria) triggers a Synechocystis response in which it aggregates to form floating assemblages or flocs. Floc formation creates a defensive barrier to P. aeruginosa predation, and we suggest this complex behavior is best understood as a defense against microbial predation in the wild.

microbiology↗

bioRxiv2024

The RRM domain-containing protein Rbp3 interacts with ribosomes and the 3' ends of mRNAs encoding photosynthesis proteins

RNA recognition motif (RRM) domain proteins are crucial RNA-binding proteins (RBPs) across all domains of life. In cyanobacteria, single RRM domain proteins are involved in mRNA targeting to the thylakoid membrane and acclimation to certain stress conditions, but many details of their physiological functions and molecular targets have remained unknown. The model cyanobacterium Synechocystis sp. PCC 6803 has a family of three genes encoding the RRM domain-containing proteins Rbp1, Rbp2 and Rbp3. Here, we verified the RNA-binding activity of Rbp3 in vivo and show that cells of a {Delta}rbp3 deletion strain had a lower PSI:PSII ratio and decreased pigment content and were significantly smaller than wild-type cells. To identify the set of interacting molecules, co-immunoprecipitation experiments were performed with a strain expressing a C-terminally FLAG-tagged Rbp3. Mass spectrometry of the elution fraction suggested physical proximity between Rbp3, ribosomes, and a very small number of other proteins. The most highly enriched transcript in the co-eluting RNA fraction was the psaAB mRNA. This was corroborated by fluorescent in situ hybridization (FISH) analyses showing decreased psaA mRNA signals in {Delta}rbp3, and colocalization with Rbp3-GFP in the wild type. Other mRNAs co-enriched with Rbp3 encode thylakoid, plasma membrane and carboxysome proteins. Binding assays using Bio-layer Interferometry validated the Rbp3-psaAB mRNA interaction, indicating a preference for folded RNA segments near or overlapping the respective stop codons. Significance statementThe mechanisms by which proteins are produced at specific sites and inserted into the intricate membrane systems of photosynthetic cyanobacteria are only partially understood. While RRM domain proteins are well-studied RNA-binding proteins in eukaryotes, their functions in bacteria remain underexplored. This study reveals that the RRM domain protein Rbp3 in the cyanobacterium Synechocystis sp. PCC 6803 binds mRNAs encoding photosynthetic proteins, plasma membrane proteins and carboxysome proteins and localizes near ribosomes. The bound RNA segments are typically near the ends of coding regions, or in 5' untranslated regions. These findings suggest that Rbp3 is involved in targeting mRNAs to various intracellular locations by interacting with structural elements within these mRNA molecules.

microbiology↗

bioRxiv2024

mRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, we find that the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that chaperoning of photosynthetic mRNAs by RbpG is important for the correct co-ordination of thylakoid protein translation and assembly. IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Some filamentous cyanobacteria show additional levels of complexity, with the differentiation of heterocysts, specialized nitrogen fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA Fluorescent in situ Hybridization as a way to probe cell-specific gene expression in multicellular cyanobacteria.

microbiology↗

bioRxiv2021

Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further. ImportanceProtein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

microbiology↗