Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-DependentStreptococcus pneumoniaeCapsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall

Abstract

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of highmolecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.

Mutations in Pneumococcal cpsE Generated via In Vitro Serial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

“…This notion was further supported by similar observations made in three other pneumococcal serotypes (types 3, 37, and 8), which also arose due to spontaneous tandem sequence duplications in capsule biosynthesis genes, including a 223-bp duplication in cps8E (41,42). We suspect that mutations in cpsE occur at a higher frequency, since mutations in other serotype-specific genes are toxic, presumably due to the accumulation of intracellular capsule precursors, which are alleviated by a suppressor mutation in cpsE (68). In contrast to (3526) and acapsular (3105) serotype 19A conjunctival isolates were grown to midexponential phase in THY-Oxyrase, stained with India ink, and visualized by microscopy to verify their capsule status.…”

Discovery of Streptococcus pneumoniae Serotype 6 Variants with Glycosyltransferases Synthesizing Two Differing Repeating Units

“…An unencapsulated variant, named MBO163, was made by allelic exchange of the wchAwciN-wciO gene region with a Janus cassette (Cassette 1) (see Fig. 6) as described (17,27,28). Additional genetic constructs with desired mutations at wciN␣ were generated by overlap extension PCR.…”

Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

“…The experiment was done once. (35,47,77,86) and controlled at both the transcriptional and posttranscriptional levels (57,63,90). We thus decided to perform a second screen, this time using an acapsular serotype 4 TIGR4 strain.…”

“…The other five mutants were hyperbiofilm formers, and their transposon insertions were all mapped to different locations within cps4E (SP0350) (Fig. 3B), a gene in the capsule locus that encodes a glycosyl-phosphotransferase that transfers glucose-1-phosphate units to the growing undecaprenyl phosphate glycolipid (17); incidentally, cps4E is one of the few genes in the serotype 4 capsule locus that can be disrupted without compromising bacterial viability (37,90), which might explain why it was the only gene hit in the 15-kb capsule locus in our screen. All of the cps4E mutants isolated were acapsular, as determined by the Quellung reaction using an antibody against the serotype 4 capsule polysaccharide (not shown).…”