Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli

Abstract

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon ؉ cells, increased 38-fold in lon ؉ cells carrying a Tn10::⌬kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::⌬kan insertion. Sequencing of both sides of the Tn10::⌬kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.Regulation of colanic acid capsular polysaccharide gene (cps) expression in Escherichia coli is multilayered, as evidenced by the numerous direct and indirect regulatory mechanisms that have been identified to date (for reviews, see references 6 and 8). The current model proposes two pathways for activating cps expression (6). One pathway involves a twocomponent regulatory system (24). RcsC (regulator of capsule synthesis), which has been described as a membrane-bound sensor protein based on homology with the sensor component of two-component sensor regulator pairs (24), appears to be activated by environmental stimuli, such as desiccation (18) or osmotic shock (22). Presumably, activated RcsC either directly or indirectly modifies RcsB, the proposed effector of the twocomponent system, which in turn activates cps expression (6, 7). In the alternate pathway, the other positive effector of cps expression, RcsA, presumably forms a complex with RcsB, resulting in the activation of cps expression (2, 6, 25). RcsA is highly unstable and appears to be degraded in a Lon-dependent fashion (26; for a review, see reference 6). In lon ϩ cells, RcsA levels are low, and these cells produce little colanic acid (26; for a review, see reference 6). Conversely, in ⌬lon cells, RcsA levels are high, leading to increased colanic acid production and mucoid colonies (26; for a review, see reference 6). In this model, both pathways require RcsB for high-level expression of cps (6).Current evidence suggests that additional regulators of cps expression exist: mutations in hns (6 min) (21, 29), capS (22.5 min) (14), opsX (62 min) (30), or capT (unknown location) (14) lead to an increase in colanic acid production. Furthermore, mutations within the rfa locus (82 min) both alter lipopolysaccharide structure and synthesis and increase colanic acid production (19). Given the complexity of the cps system, identification of additional cps regulators seems probable. (SG20780 [2]) strains carrying the zce-23::⌬kan insertion were assayed for ␤-galactosidase activity (Table 1). A 38-fold increase in the level of cps expression was observed in either Luria-Bertani (LB) or minimal (M63 salts, 0.4% glucose, 0.1% Casamino Acids) medium with the introduction of the zce-23:: ⌬kan insertion into a lon ϩ strain. The zce-23::⌬kan insertion had no effect on cps expression in ⌬lon cells.The increase in cpsB10::lacZ expression is abolished in lon ؉ s...

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

“…For this reason, the physiological roles of the Rcs system have often been inferred from the ability of null mutations in genes of the phosphorelay to suppress mutations in other loci. For instance, in E. coli, null mutations in mdoH lead to an increase in colanic acid production that is suppressed by null mutations in rcsA, rcsB or rcsC (Ebel et al, 1997). In a similar fashion, viable igaA alleles of Salmonella enterica serovar Typhimurium result in induction of wca genes, repression of flhDC and avirulence, and all these phenotypes are suppressed by null mutations in rcsB or rcsC (Cano et al, 2002;Costa & Antó n, 2001;Domínguez-Bernal et al, 2004).…”

“…Furthermore, several genetic conditions that result in alterations in envelope composition or integrity are known to activate the Rcs system. For example, a mutation in the mdoH gene involved in the biosynthesis of membrane-derived oligosaccharides (Ebel et al, 1997) Here, we describe the isolation of Salmonella rcsC mutants with constitutive activation of the Rcs system, and the characterization of amino acid substitutions that lead to different degrees of activation of the system. Virulence trials with constitutively activated rcsC mutants provide a direct demonstration of the link between activation of the Rcs system and avirulence in mice.…”

“…Furthermore, several genetic conditions that result in alterations in envelope composition or integrity are known to activate the Rcs system. For example, a mutation in the mdoH gene involved in the biosynthesis of membrane-derived oligosaccharides (Ebel et al, 1997) or overproduction of the DnaJ-like transmembrane protein DjlA (Kelley & Georgopoulos, 1997) has been shown to stimulate the expression of genes regulated by the E. coli Rcs system. In Salmonella enterica serovar Typhimurium, mutations in the essential gene igaA (Cano et al, 2001) [also known as yrfF or mucM (Costa & Antó n, 2001)] result in mucoidy and reduced motility (Cano et al, 2002).…”

Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli

“…These include stresses caused by mutations in: (i) the pgsA gene, encoding phosphatidylglycerophosphate synthase (Shiba et al, 2004); (ii) the mdoH gene for glucosyltransferase, responsible for the synthesis of periplasmic membrane-derived oligosaccharides (Ebel et al, 1997;Shiba et al, 2004); (iii) the tolB gene for a periplasmic component of the Tol system, involved in maintenance of cell envelope integrity (Mouslim & Groisman, 2003); and (iv) the rfaP and rfaG genes, encoding phosphoryl-and glucosyltransferase, respectively, responsible for the assembly of the lipopolysaccharide core (Parker et al, 1992;.…”

Escherichia coli tol and rcs genes participate in the complex network affecting curli synthesis

“…However, many reports suggest that the Rcs system might sense alterations of the cell envelope. The Rcs system has been reported to be activated by environmental signals, such as desiccation, osmotic shock and Zn 2+ concentration (Hagiwara et al, 2003;Ophir & Gutnick, 1994;Sledjeski & Gottesman, 1996), in the presence of mutations affecting cell envelope components, such as tol (Clavel et al, 1996;Mouslim & Groisman, 2003), rfa (Parker et al, 1992), mdoH (Ebel et al, 1997), dsbA and dsbB (El-Kazzaz et al, 2004), or after overproduction of envelope proteins, such as TolB/Pal (Majdalani et al, 2002), RcsF (Gervais & Drapeau, 1992), DjlA (Toutain et al, 2003), LolA and OmpG (Chen et al, 2001). …”