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Proteins are prone to aggregate when expressed above their solubility limits. Aggregation may occur rapidly, potentially as early as proteins emerge from the ribosome, or slowly, following synthesis. However, in vivo data on aggregation rates are scarce. Here, we classified the Escherichia coli proteome into rapidly and slowly aggregating proteins using an in vivo image-based screen coupled with machine learning. We find that the majority (70%) of cytosolic proteins that become insoluble upon overexpression have relatively low rates of aggregation and are unlikely to aggregate co-translationally. Remarkably, such proteins exhibit higher folding rates compared to rapidly aggregating proteins, potentially implying that they aggregate after reaching their folded states. Furthermore, we find that a substantial fraction (~ 35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our study provides a global view of aggregation rates and hence solubility limits of proteins in a bacterial cell.

Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide–antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections. Multidrug-resistant Escherichia coli have a high frequency of collateral sensitivity to antimicrobial peptides, which may arise from changes in lipopolysaccharide regulation.