Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase

Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds 1–6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase (ACC) is a target for drug discovery against diabetes, cancer and other diseases 7–9. We report here the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kD), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active toward short- and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase (LCC). The holoenzyme is a homo-hexamer with molecular weight of 720 kD. The 3.0 Å crystal structure of Mycobacterium avium subspecies paratuberculosis LCC (MapLCC) holoenzyme revealed an architecture that is strikingly different compared to those of related biotin-dependent carboxylases 10,11. In addition, the domains of each monomer have no direct contacts with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence

R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes. PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection

Convergent Evolution of Hyperswarming Leads to Impaired Biofilm Formation in Pathogenic Bacteria

Most bacteria in nature live in surface-associated communities rather than planktonic populations. Nonetheless, how surface-associated environments shape bacterial evolutionary adaptation remains poorly understood. Here, we show that subjecting Pseudomonas aeruginosa to repeated rounds of swarming, a collective form of surface migration, drives remarkable parallel evolution toward a hyperswarmer phenotype. In all independently evolved hyperswarmers, the reproducible hyperswarming phenotype is caused by parallel point mutations in a flagellar synthesis regulator, FleN, which locks the naturally monoflagellated bacteria in a multiflagellated state and confers a growth rate-independent advantage in swarming. Although hyperswarmers outcompete the ancestral strain in swarming competitions, they are strongly outcompeted in biofilm formation, which is an essential trait for P. aeruginosa in environmental and clinical settings. The finding that evolution in swarming colonies reliably produces evolution of poor biofilm formers supports the existence of an evolutionary trade-off between motility and biofilm formation

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence [preprint]

Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes with the potential to confer production of R-bodies (i.e., a “reb cluster”). R-bodies are large, extendable protein polymers best known for their role in killing of paramecia by the bacterium Caedibacter taeniospiralis, and genes in the reb cluster have been implicated in PA14 virulence. Here, we present evidence that PA14 expresses reb cluster genes during colonization of plant and nematode hosts. We identify products of the reb cluster that are R-body-associated and that control stochastic expression of R-body structural genes. We also show that R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. These observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection

Facultative control of matrix production optimizes competitive fitness in <i>Pseudomonas aeruginosa</i> PA14 biofilm models

As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O(2). We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O(2)-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities