AVibrio choleraeBolA-like protein is required for proper cell shape and cell envelope integrity

Abstract

25BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. 26 While diverse cellular phenotypes have been linked to this protein family, the molecular 27 pathways through which these proteins mediate their effects are not well-described. Here, 28 we investigated the role of BolA family proteins in Vibrio cholerae, the cholera pathogen. 29 Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, 30 in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. 31 cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the 32 generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-33 shaped, multi-polar, elongated and wide cells that predominated in exponential phase 34 ΔibaG V. cholerae cultures were not observed in stationary phase cultures. The V. 35 cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, 36 including cell wall acting antibiotics and bile, and was defective in intestinal colonization. 37 ΔibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane 38 lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope 39 stressors. Transposon-insertion sequencing analysis of ibaG's genetic interactions 40 suggested that ibaG is involved in several processes involved in the generation and 41 homeostasis of the cell envelope. Furthermore, co-purification studies revealed that IbaG 42 interacts with proteins containing iron-sulfur clusters or involved in their assembly. 43 Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell 44 envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins. 45 46 47 3 Importance 48BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins 49 have been linked to a variety of phenotypes, but the pathways and mechanisms through 50 which they act have not been extensively characterized. Here, we unraveled the role of 51 the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG 52 was associated with dramatic changes in cell morphology, sensitivity to envelope 53 stressors, and intestinal colonization defects. IbaG was found to be required for 54 biogenesis of several components of the V. cholerae cell envelope and to interact with 55 numerous iron-sulfur cluster containing proteins and factors involved in their assembly. 56 Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope 57 homeostasis through its effects on iron-sulfur proteins and associated pathways. The 58 diversity of processes involving iron-sulfur containing proteins is likely a factor underlying 59 the range of phenotypes associated with BolA family proteins. 60 61 4 62The BolA protein family is widely conserved across gram-negative bacteria and 63 eukaryotes (1). These proteins have been linked to a range ...