Interplay Between Capsule Expression and Uracil Metabolism in Streptococcus pneumoniae D39

Abstract

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen S. pneumoniae. In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the de novo synthesis of pyrimidines and in the -10 box of capsule operon promoter (Pcps). By directed mutagenesis we showed that the point mutation in Pcps was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased Pcps activity and capsule amounts. Importantly, Pcps expression is further decreased by mutating the first gene of the de novo synthesis of pyrimidines, carA. In contrast, the absence of uracil from the culture medium showed no effect on the spontaneous mutant strain. Co-cultivation of the wild-type and the mutant strain indicated a competitive advantage of the spontaneous mutant (non-producing capsule) in medium devoid of uracil. We propose a model in that uracil may act as a signal for the production of different capsule amounts in S. pneumoniae.

Keywords: Streptococcus pneumoniae; capsule biosynthesis; gene expression; spontaneous mutations; uracil metabolism.

Figure 1

Colony phenotype, growth profile, and capsule production in D39 and its derivatives. (A) Colony phenotype of D39, D39SM, and R6 strains in Glc-M17 blood agar plates. (B) Growth profiles of strains D39 (squares), D39SM (diamonds), D39Pcps_T→C (circles) and R6 (triangles) in Glc-CDM (closed symbols) and Glc-CDM without uracil (open symbols), at 37°C, without pH control (initial pH 6.5), in static rubber-stoppered bottles. The growth rates (h⁻¹) for each strain are indicated in the graph and the values shown are means from three biological replicates ± SD. (C) Estimation of capsule was performed based on the determination of its glucuronic acid content in D39 and its derivatives grown in Glc-CDM (black bars) and Glc-CDM lacking uracil (white bars), at mid-exponential phases of growth. Capsule measurements were performed in duplicate using samples from two independent cultures and the values represented are means ± SD. ^***p-value = 0.0002.

Figure 2

Expression of Pcps and Pcps_T→C in D39 and D39SM. (A) Schematic overview of the dexB-cps2A region and magnification of the cps promoter area of D39SM. Hooked arrow, cps promoter; lollipop, putative terminator; zoomed area (inset), promoter region of the cps gene cluster; −35 and −10 binding box, and the starting codon of the cps2A gene are indicated in bold; the number of bp of the cps2A gene next to the starting codon is subscripted after {n}; ^*, point mutation (T→C) in −10 binding box of D39SM strain. (B) Transcription of cps and cps_T→C promoters was estimated by measuring β-galactosidase activities in exponentially growing D39 and D39SM strains, carrying the pPP2 integrative lacZ reporter plasmid, in Glc-CDM (black bars) and Glc-CDM depleted of uracil (white bars). β-galactosidase activities are expressed as Miller units. (C) Colony phenotype of D39SM and D39Pcps_T→C strains in Glc-M17 blood agar plates. All the determinations were done in duplicate in two independent experiments and the values represented are means ± SD. ^*p-value < 0.05.

Figure 3

Capsule promoter expression in D39 carA mutant exposed to different amounts of uracil. Transcription of Pcps was estimated by measuring β-galactosidase activities in exponentially growing carA mutant and wild-type strains, carrying the pPP2 integrative lacZ reporter plasmid, in Glc-CDM with 0 (0U), 5 (5U), and 10 (10 U) mg L⁻¹ uracil. β-galactosidase activities are expressed as Miller units. The values represent the means of the Pcps expression ratios ± SD obtained from two biological independent samples assayed in duplicate. ^**p-value = 0.0025.

Figure 4

Growth profiles of strains D39 wild-type and D39SM in co-cultivation. D39 and D39SM strains were inoculated at a cell ratio of 1:1 (□), 1:4 (▴), and 4:1 (•) in (A) CDM or (B) CDM without uracil, containing 1% Glc, and grown at 37°C, without pH control (initial pH 6.5), in static rubber-stoppered bottles. The growth rates of the co-cultures are presented in the graphs. The values are averages from three independent cultures ± SD. For comparison the growth curves and growth rates of monocultures of D39 (■) and D39SM (♦) are also shown. +U, with uracil; -U, without uracil.

Figure 5

Proposed model for the effect of uracil on pyrimidine metabolism, biomass and capsule production in S. pneumoniae. The intracellular reactions depicted are catalyzed by the following enzymes (the genes that code for the enzymes are in parenthesis): carbamoyl-phosphate synthase, small and large subunit (carAB), aspartate carbamoyltransferase (pyrB), dihydroorotase (pyrC), dihydroorotate dehydrogenase A (pyrDa), dihydroorotate dehydrogenases (pyrDb-pyrK), orotate phosphoribosyltransferase (pyrE), orotidine 5-phosphate decarboxylase (pyrF), uridylate kinase (pyrH), pyrimidine-nucleoside phosphorylase (pdp), uridine kinase (udk), nucleoside-diphosphate kinase (ndk). Narrower red and green arrows indicate the pathways active when uracil is absent and present, respectively. Bold red and green arrows pointed up indicate an increase in biomass and capsule expression in the absence or presence of uracil, respectively, whereas pointed down arrows indicate a decrease. Asp, aspartate; CAA, carbamoyl-aspartate; CP, carbamoyl-phosphate; DHO, dihydroorotate; Gln, glutamine; Glu, glutamate; ${\text{HCO}}_{3^{-}}$, bicarbonate; OMP, orotate monophosphate; P_i, inorganic phosphate; U, uracil; UR, uridine. The nomenclature for the nucleotide abbreviations are provided in the list of abbreviations of this manuscript.