The Small-Molecule Language of Dynamic Microbial Interactions.

Although microbes are routinely grown in monocultures in the laboratory, they are almost never encountered as single species in the wild. Our ability to detect and identify new microorganisms has advanced significantly in recent years, but our understanding of the mechanisms that mediate microbial interactions has lagged behind. What makes this task more challenging is that microbial alliances can be dynamic, consisting of multiple phases. The transitions between phases, and the interactions in general, are often mediated by a chemical language consisting of small molecules, also referred to as secondary metabolites or natural products. In this microbial lexicon, the molecules are like words and through their effects on recipient cells they convey meaning. The current review highlights three dynamic microbial interactions in which some of the words and their meanings have been characterized, especially those that mediate transitions in selected multiphasic associations. These systems provide insights into the principles that govern microbial symbioses and a playbook for interrogating similar associations in diverse ecological niches.

Structural Elucidation of Cryptic Algaecides in Marine Algal-Bacterial Symbioses by NMR Spectroscopy and MicroED.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions.

Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme.

Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl coenzyme A (CoA) mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria.IMPORTANCE Cobamides, including vitamin B12, are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti, largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.

Anti-inflammatory mechanism of PPARγ on LPS-induced pulp cells: role of the ROS removal activity.

OBJECTIVES: PPARγ has an anti-inflammatory effect on LPS-induced pulpal inflammation by decreasing the expression of MMPs, ICAM-1 and VCAM-1. However, the anti-inflammatory mechanism of PPARγ on the cell adhesion molecules and their upper signal pathways has not been clarified in pulp cells. The aim of this study is to investigate the anti-inflammatory mechanism of PPARγ in pulpal inflammation. METHODS: Human dental pulp cells (HDPCs) were isolated from freshly extracted third molar and cultured. The over-expression of PPARγ was used by adenoviral PPARγ (Ad/PPARγ). The formation of ROS was analysed using DCFH-DA with FACS, and NO was analysed using colorimetric bioassay. The expression of inflammatory molecules and inflammatory mechanism of PPARγ involved signal pathway were determined by immunoblotting. RESULTS: LPS-induced HDPC decreased PPARγ expression gradually and strongly activated the ERK1/2 signals amongst the MAPK, and induced NF-κB translocation from the cytosol to the nucleus. On the other hand, the cells to restore PPARγ with Ad/PPARγ were inhibited ERK1/2 despite being stimulated with LPS. In addition, the cells treated with rosiglitazone (PPARγ agonist) also were inhibited ERK1/2 activation, and the expression of ICAM-1, VCAM-1 and NF-κB translocation under LPS stimulation. The GW9667 (PPARγ antagonist)-treated HDPC did not affect the adhesion molecules and signal activation. LPS-induced HDPC produced significant NO and ROS levels, but their production was attenuated in the PPARγ over-expressed cells. Overall, the PPARγ effect under LPS stimulation is due to the removal activity of cellular NO and ROS formation. CONCLUSION: These results suggest that anti-inflammatory mechanism of PPARγ is due to the removal activity of NO and ROS, and its removal effect suppressed ERK1/2 signal activation and NF-κB translocation. Therefore, the NO and ROS removal activity of PPARγ suggests major anti-inflammatory mechanism in HDPC, and it might offer us a possible molecule for various types of inflammatory inhibition.