Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.

Bacteria have evolved numerous pathways to sense and respond to changing environmental conditions, including, within Gram-positive bacteria, the stressosome complex that regulates transcription of general stress response genes. However, the signalling molecules recognized by Gram-positive stressosomes have yet to be identified, hindering our understanding of the signal transduction mechanism within the complex. Furthermore, an analogous pathway has yet to be described in Gram-negative bacteria. Here we characterize a putative stressosome from the Gram-negative bacterium Vibrio brasiliensis. The sensor protein RsbR binds haem and exhibits ligand-dependent control of the stressosome complex activity. Oxygen binding to the haem decreases activity, while ferrous RsbR results in increased activity, suggesting that the V. brasiliensis stressosome may be activated when the bacterium enters anaerobic growth conditions. The findings provide a model system for investigating ligand-dependent signalling within stressosome complexes, as well as insights into potential pathways controlled by oxygen-dependent signalling within Vibrio species.

Globin coupled sensors (GCS) are O-sensing proteins used by bacteria to monitor the surrounding gaseous environment. To investigate the biphasic O dissociation kinetics observed for full-length GCS proteins, isolated globin domains from Pectobacterium carotovorum ssp. carotovorum (PccGlobin), and Bordetella pertussis (BpeGlobin), have been characterized. PccGlobin is found to be dimeric, while BpeGlobin is monomeric, indicating key differences in the globin domain dimer interface. Through characterization of wild type globin domains and globin variants with mutations at the dimer interface and within the distal pocket, dimerization of the globin domain is demonstrated to correlate with biphasic dissociation kinetics. Furthermore, a distal pocket tyrosine is identified as the primary hydrogen bond donor, while a secondary hydrogen bond donor within the distal heme pocket is involved in conformation(s) that lead to the second O dissociation rate. These findings highlight the role of the globin dimer interface in controlling properties of both the heme pocket and full-length GCS proteins.

The discovery of the globin-coupled sensor (GCS) family of haem proteins has provided new insights into signalling proteins and pathways by which organisms sense and respond to changing oxygen levels. GCS proteins consist of a sensor globin domain linked to a variety of output domains, suggesting roles in controlling numerous cellular pathways, and behaviours in response to changing oxygen concentration. Members of this family of proteins have been identified in the genomes of numerous organisms and characterization of GCS with output domains, including methyl accepting chemotaxis proteins, kinases, and diguanylate cyclases, have yielded an understanding of the mechanism by which oxygen controls activity of GCS protein output domains, as well as downstream proteins and pathways regulated by GCS signalling. Future studies will expand our understanding of these proteins both in vitro and in vivo, likely demonstrating broad roles for GCS in controlling oxygen-dependent microbial physiology and phenotypes.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.