A Functional Genomics Approach to Establish the Complement of Carbohydrate Transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium∶solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection

NERAZLIKOVNOST RAZLIKA

Analiziraju se leksikološko-leksikografska polazišta i metodološki postupci u izradi Razlikovnog rječnika srpskog i hrvatskog jezika Vladimira Brodnjaka

The factor H-binding fragment of PspC as a vaccine antigen for the induction of protective humoral immunity against experimental pneumococcal sepsis

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Carbon source utilisation profiles of EI mutants.

<p>The relative metabolic activity of EI mutants of G54 (panel A) and DP1004 (panel B) was determined by phenotype microarray. Relative metabolic activity is expressed as percent of the mean metabolic activity of the mutants with respect to the wild type using triplicate experiments (Biolog Omnilog-PM software parameter average height). Shading refers to carbon sources whose uptake is not influenced (white bars), influenced in part (grey bars) or abolished (black bars) by mutation of <i>ptsI</i>.</p

Pneumococcal carbohydrate uptake systems.

(a)<p>conservation as defined by analysis of the published pneumococcal genomes;</p>(b)<p>substrates as from literature and genomic context.</p>(c)<p>indel insertion/deletion.</p>(d)<p>in the text and on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033320#pone-0033320-g001" target="_blank">Figure 1</a> the operon of Hungary 19 is described, since the one of G54 is not complete.</p

Growth profiles of sugar uptake system mutants on different carbon sources (continued from <b>Fig. 2</b>).

<p>As for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033320#pone-0033320-g002" target="_blank">Figure 2</a> pneumococci were grown in CAT medium containing 0.3% of the sugar indicated above every single panel and OD590 nm values were recorded at 10 minute intervals automatically in a thermostatic 96 well microplate reader. In all panels growth of the wild type strain on the respective sugar is shown in blue while its growth in CAT without added sugar is shown in green. Growth on lactose is shown in panel A, on growth sucrose (panel B), trehalose (panel C), amygdalin (panel D), ascorbate (panel E), N-acetylmannosamine (ManNAc) (panel F), sialic acid (NeuNAc) (panel G), the alpha-galactosides raffinose, melibiose and stachyose (panes H–J) and glycerol (panel K). All mutants shown are generated in the D39 derivative DP1004, except for panels F and G where strain G54 and mutants in G54 were used for evaluation of growth in ManNAc and NeuNAc.</p

Growth profiles of sugar uptake system mutants on different carbon sources.

<p>Pneumococci were grown in CAT medium containing 0.3% of the sugar indicated above every single panel and OD590 nm values were recorded at 10 minute intervals automatically in a thermostatic 96 well microplate reader. In all panels growth of the wild type strain DP1004 on the respective sugar is shown in blue while its growth in CAT without added sugar is shown in green. Growth on N-acetylglucosamine (GlcNAc) is shown in panel A, mannose (panel B), galactose (panel C), glucose (panle D), glucosamine (GlcN) (panel D), the beta-glucosides cellobiose, gentiobiose, amygdalin, esculin, 1-O-methyl-betaglucoside in panel F-J, hyaluronic acid (panel K, galactose (panel L9, maltose (panel M), maltotriose (panel N), maltodextrin (panel O), glycogen (panel P), fructose (panel Q) and lactulose (R). All mutants shown are generated in the D39 derivative DP1004.</p

Fermentation assays.

<p>Acid generation from sugar substrates is indicated by colour change (yellow) of the pH indicator phenol red after incubation for 24 hours. (A) Negative control, ΔSP1895-6-7, ΔSP2108-9-10 , ΔSP1580 and wild type in presence of 0.3% of glucose, glycogen, maltodextrin, melibiose, stachyose and raffinose; (B) wild type and ΔSP0321-3-4-5 in serial dilutions of hyaluronic acid; (C) wild type and ΔSP0394-6 in serial dilutions of mannitol.</p

Genomic distribution of pneumococcal carbohydrate uptake systems.

*<p>strains are described in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033320#pone.0033320-Donati1" target="_blank">[32]</a>;</p>**<p>Transport systems are numbered as in TIGR4 according to the EIIC gene in case of PTS and to the substrate binding protein for ABC transporters.</p><p>+, presence of the operon; −, absence of the operon; fs, frame shift in a gene of the operon (no re-sequencing of genome data done); a, allelic variant of the transporter with respect to TIGR4.</p

Schematic representation of all the genomic regions containing the pneumococcal sugar transporters.

<p>The genomic regions for the described 21 described PTS systems (PTS ORFs in light blue), the seven ABC transporters (black), the single sodium∶solute symporter (green) with downstream sodium ATPase (light green), the glycerol permease (red) of strain TIGR4 are shown. In addition to the substrate specific parts of the transporters of TIGR4 also the locus for the central PTS enzymes EI (SP1176 <i>ptsI</i>; light blue) and HpR (SP1177 <i>ptsH</i>; light blue) is shown, as also the monocystronic <i>msmK</i> gene encoding the ATP binding cassette protein energising some of the CUT1 permeases. The PTS transporter SPH1925-6-7, not present in TIGR4, is shown as in the genome of strain Hungary 19F.</p