Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

Many microbial communities are characterized by intense competition for nutrients and space. One way for an organism to gain control of these resources is by eliminating nearby competitors. The Type VI Secretion System (T6) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6-mediated toxicity, little is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6. Here we use directed evolution to examine the evolution of T6 resistance, subjecting eight replicate populations of Escherichia coli to T6 attack by Vibrio cholerae. Over ~500 generations of competition, the E. coli evolved to survive T6 attack an average of 27-fold better than their ancestor. Whole genome sequencing reveals extensive parallel evolution. In fact, we found only two pathways to increased T6 survival: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. Synthetic reconstruction of individual and combined mutations demonstrate that yejM and yjeP are synergistic, with yejM requiring the mutation in yejP to provide a benefit. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs underlie the effectiveness of T6 as a bacterial weapon, and help us understand how the T6 shapes the evolution of bacterial interactions.

word count: 229 18 Text word count: 3540 19 20 21 22 23 24 25 26 27 28 2 ABSTRACT 29Evolutionary arms races among organisms are broadly prevalent and bacteria have evolved 30 defensive strategies against various attackers. A common microbial aggression mechanism is the 31 Type VI Secretion System (T6SS), a contact-dependent bacterial weapon used to deliver toxic 32 effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins 33 that neutralize effectors. However, less is known about mechanisms that allow non-sibling bacteria 34 to respond to external cues and survive T6SS attacks independently of immunity proteins. In this 35 study, we show that resistance to T6SS attacks is promoted by a genetically controlled response to 36 exogenous glucose. We observe that multiple human Escherichia coli commensal strains lacking 37 immunity proteins are sensitive to T6SS attacks from pandemic Vibrio cholerae on nutrient-rich 38 media. By contrast, E. coli cells become resistant to attacks when co-cultured on the same media 39 with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find 40 that cAMP receptor protein (CRP), which alters expression of hundreds of genes in response to 41 glucose, controls resistance to T6SS attacks in E. coli cells. Consistent with the observed resistance 42 on media with glucose, an E. coli crp disruption mutant survives significantly better against V. 43 cholerae T6SS attacks even in the absence of glucose. Finally, we also show that resistance to 44 T6SS attacks depends on the pH of the medium and varies based on the target and killer strains. 45 IMPORTANCE 46Many Gram-negative bacteria, including important pathogens, encode T6SS genes to deliver toxic 47 effectors and eliminate competitors. Our results uncover a novel defense mechanism against T6SS 48 attacks that is triggered by an external stimulus and mediated by a metabolic response in non-kin 49 target cells. In microbiomes such as those in gastrointestinal tracts where T6SS activity is known 50 to occur, signaling by metabolites like glucose may affect the efficacy of T6SS attacks and alter 51 3 microbial community composition. Our findings could have vast implications for microbial 52 interactions during pathogen colonization of hosts and survival of bacterial cells in environmental 53 communities. Furthermore, the glucose-mediated resistance observed here might provide a novel 54 example of an evolutionary arms race between killer T6SS cells and target bacteria. 55 INTRODUCTION 56 Vibrio cholerae is the waterborne enteric pathogen that causes serious, often fatal cholera diarrheal 57 disease when ingested by humans. This ubiquitous microbe is found in dense polymicrobial marine 58 communities on chitinous surfaces and in animal reservoirs like fish or zooplankton (1-3). To 59 compete with other cells in densely-populated microbial environments, V. cholerae employs a 60 harpoon-like structure called the Type VI Secretion System (T6SS) (4-7). The T6SS pu...