Biosynthesis of Cell Wall Lipopolysaccharide in Mutants of Salmonella V. A Mutant of Salmonella typhimurium Defective in the Synthesis of Cytidine Diphosphoabequose

Abstract

A mutant of Salmonella typhimurium LT2 was found to be unable to convert cytidine diphospho4-keto-6-deoxy-D-glucose into cytidine diphosphoabequose. The mutation maps in the rfb gene cluster, which is known to be involved in the biosynthesis of the peripheral, "O side-chain" portion of cell wall lipopolysaccharide. In spite of the fact that, in the 0 side chains, abequose is not a part of the main chain but occurs as short branches, the mutant appears to be unable to polymerize oligosaccharide "repeat units" into long 0 side chains. The following evidence indicates that this failure is the result of the absence of cytidine diphosphoabequose rather than that of a superimposed second mutation in other genes of the rfb cluster. (i) The mutant does not behave like a multisite mutant in genetic crosses, and it gives rise, at a high frequency, to "revertants" where the ability to synthesize cytidine diphosphoabequose and the ability to synthesize normal lipopolysaccharide with 0 side chains are both restored. (ii) The mutant strain has normal levels of activity of all of the other enzymes known to be involved in 0 side-chain synthesis, except that the levels of several enzymes were lowered by about 30% owing to the polarity effect of the mutation. That the lowering of these enzymes is not responsible for the failure of the mutant to synthesize 0 side chains is clear from the fact that there were revertants which had regained some ability to synthesize abequose but still had lowered levels of these other enzymes, and that this type of revertant produced lipopolysaccharide with considerable amounts of 0 side chains. 433 on July 31, 2020 by guest GlcNAc P, P-ethanolamine |Man-Rha-Gal-n-Man-Rha-GaltGlc-Gal-Glc-Hep-Hep-(KDO).-(Lipid A) 8 ml of 0.025 M tris(hydroxymethyl)aminomethane(Tris)-hydrochloride buffer (pH 7.5) containing 0.2 mg of pancreatic ribonuclease, and the suspension was incubated at 55 C for 2 hr. This preparation was then purified by repeated ultracentrifugations at 105,000 X g for 90 min, until absorption of the supernatant fluid at 260 nm became negligible.

Dead‐end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate

“…Growth and morphological defects are also observed in mutants that accumulate Und-PP-linked O-antigen intermediates. Such mutants are genetically unstable (Yuasa et al, 1969) and cause significant growth defects (Klena and Schnaitman, 1993), which suggested to Tatar et al that these unprocessed intermediates were toxic (Tatar et al, 2007). However, given our current results, it seems just as likely that these dead-end intermediates sequestered part of the pool of Und-P, thereby affecting PG synthesis and producing the phenotypes.…”

“…The deleterious effects triggered by the accumulation of various Und-PP-linked metabolic intermediates have been known for some time (Yuasa et al, 1969;Rick et al, 1988;Danese et al, 1998;Marolda et al, 2006;Tatar et al, 2007;Castelli and Vescovi, 2011), but the mechanism of this toxicity has remained elusive. In theory, there are three possible explanations.…”

“…Given the central role of Und-P in the biosynthesis of various glycan polymers, it should come as little surprise that the sequestration hypothesis has been suggested to account for several phenotypes observed in a diverse set of organisms including: E. coli (Paradis-Bleau et al, 2014), Salmonella enterica (Yuasa et al, 1969;Liu et al, 2015), Shigella dysenteriae (Klena and Schnaitman, 1993), Pseudomonas aeruginosa (Burrows and Lam, 1999), Bacillus subtilis (D'Elia et al, 2009) and Streptococcus pneumoniae (Xayarath and Yother, 2007). Thus, the sequestration of Und-P into non-productive intermediate compounds may explain many previous results obtained across a fairly wide range of bacteria.…”

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

“…The RfbF, RbfJ and RfbI proteins catalyze steps in the synthesis of the abequose precursor (Rubenstein and Strominger, 1974;Wyk and Reeves, 1989;Lindqvist et al, 1994). Yuasa et al (Yuasa et al, 1969) and Hong et al (Hong et al, 2012) have reported that inactivation of rfbJ causes failure to assemble O-antigen chains, and we found that P22 fails to infect rfbF and rfbI SGD mutants (DSTM14_2586; DSTM14_2587; Table 1 and Supporting Information Table S1). Our SGD panel does not include mutants of the other abequose pathway genes, so they could not be tested individually.…”

Periplasmic protein EipA determines envelope stress resistance and virulence inBrucella abortus

“…One possibility is that EipA influences synthesis, recycling or degradation of bactoprenol. Bactoprenol is a carrier molecule for intermediates in the synthesis of O-chain and peptidoglycan and mutations preventing its recycling or synthesis result in a variety of deleterious or toxic effects such as antibiotic sensitivity, morphological and growth defects, LPS O-chain defects, and reduced virulence (Jorgenson & Young, 2016, Tatar et al, 2007, Yuasa et al, 1969, Bhavsar et al, 2001, Rick et al, 2003, Manat et al, 2014. EipA may also directly interact with enzymes involved in synthesis of peptidoglycan and influence the integrity of the cell wall.…”