Cationic antimicrobial polypeptides (CAP) play an important role in host defense against microbial infection and are key effectors of the host innate immune response (24). Microbial pathogens have evolved distinct mechanisms to resist killing by CAP, including expelling CAP through pumps and cleaving CAP with proteases (47). One of the important mechanisms of resistance to CAP in Gram-negative bacteria involves modification of lipopolysaccharide (LPS) with positively charged substituents, which leads to the repulsion of CAP (47). In a large number of bacterial species, the genes conferring resistance to CAP, including polymyxin B (PB), are regulated by bacterial two-component systems (31, 37, 39, 41, 42, 46). In Salmonella enterica serovar Typhimurium, evasion of CAP killing is regulated in part by the PmrA-PmrB two-component regulatory system which upregulates genes involved in covalent modifications of LPS (21, 22). The LPS modifications reduce the negative charge of LPS and consequently decrease attraction and binding of CAP to the outer membrane. The PhoP-PhoQ two-component system, a master regulator of S. enterica serovar Typhimurium virulence functions, also has been shown to be involved in regulating resistance to CAP (18). The activation of PhoP-PhoQ increases the expression of PmrD (31), which in turn leads to the activation of PmrA, resulting in modification of LPS. The PhoP-PhoQ system is activated by micromolar concentration of magnesium (18, 19), and transcription of PhoP-activated genes is upregulated by sublethal concentration of CAP (4, 8). Modulation of resistance to CAP by the PhoP-PhoQ and PmrA-PmrB two-component systems has also been observed with Pseudomonas aeruginosa (37, 41). Proteus mirabilis exhibits a form of multicellular behavior known as swarming migration (35, 36). It is believed that the ability of P. mirabilis to colonize the urinary tract is associated with its swarming motility. The swarming behavior of P. mirabilis is under the control of a complex regulatory network that may include bacterial two-component systems (34, 36, 49, 58, 59). In this respect, we have identified a gene, rsbA, which may encode a histidine-containing phosphotransmitter of the bacterial two-component system and act as a repressor of swarming and virulence factor expression in P. mirabilis (7, 34, 36). That swarming and virulence factor expression can be coregulated has been reported previously (2, 3, 35). It has been demonstrated that swarming and CAP resistance may be coregulated (1, 30, 40). For example, activation of the PhoP-PhoQ two-component system, which is known to enhance CAP resistance, can lead to inhibition of swarming through repressing the expression of flagellin in S. enterica serovar Typhimurium (1). Moreover, in P. mirabilis, LPS has been shown to play a critical role in swarming (6, 40), and LPS modification can affect both swarming and PB resistance (40). UDP-glucose pyrophosphorylase (GalU) is the enzyme for the biosynthesis of UDP-glucose from UTP and glucose-1-phosphate (45). UDP-glucose is the precursor for synthesis of different surface structures, LPS, and extracellular polysaccharides (EPS). In many Gram-negative pathogens, mutation in galU leads to attenuated virulence, mainly because of changes in LPS or capsular structures (16, 45, 57). UDP-glucose dehydrogenase (Ugd) is an enzyme that converts UDP-glucose into UDP-glucuronic acid (10). UDP-glucuronic acid is also necessary for the synthesis of EPS and LPS in many pathogenic bacteria (10, 21, 43, 53). Formation of these polysaccharides is critical to bacterial virulence (10, 28) because it enables the bacteria to evade attacks by host immune systems. Recent studies demonstrate that ugd mutation in Cryptococcus neoformans alters cell integrity and the mutant cells also become temperature sensitive and fail to grow in an animal model (17). Transcription of Salmonella ugd is controlled by three regulatory systems that respond to different signals (43, 44). The participation of multiple regulatory systems in the control of ugd expression suggests a role for the ugd gene product in a broad spectrum of environments. Till now, nothing has been known about the roles of galU and ugd in P. mirabilis. P. mirabilis is known to be highly resistant to the action of CAP, such as PB (40, 52). Although the detailed mechanisms underlying P. mirabilis resistance to PB are not clear, studies have shown that modification of LPS plays an important role in modulating CAP resistance in P. mirabilis (40, 52). Previously, we reported that RppA, a putative response regulator of the two-component system, can regulate PB susceptibility through modulating LPS modification in P. mirabilis (58). How RppA regulates LPS modification is not known. In this study, we used a Tn5 transposon mutagenesis approach to identify genes that may affect PB susceptibility in P. mirabilis. Two genes, ugd and galU, whose products may be involved in LPS synthesis and modification were identified. Knockout mutants of these genes were found to be extremely sensitive to PB, presumably because of changes in LPS. These mutants also had lower ability to swarm and express virulence factors. More importantly, we found that the expression of these genes was under the control of RppA. To our knowledge, this is the first report describing the roles and regulation of Ugd and GalU in P. mirabilis.