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. 2010 Sep;192(17):4413-24.
doi: 10.1128/JB.00494-10. Epub 2010 Jul 9.

Functional identification of the Proteus mirabilis core lipopolysaccharide biosynthesis genes

Eleonora Aquilini  1 , Joana Azevedo, Natalia Jimenez, Lamiaa Bouamama, Juan M Tomás, Miguel Regué
Affiliations

Functional identification of the Proteus mirabilis core lipopolysaccharide biosynthesis genes

Eleonora Aquilini et al. J Bacteriol. 2010 Sep.

Abstract

In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyltransferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster.

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Figures

FIG. 1.
FIG. 1.
Chemical structures of the core LPSs of P. mirabilis strains R110 and 51/57 (51), K. pneumoniae types 1 (50) and 2 (41), and S. marcescens N28b (11).
FIG. 2.
FIG. 2.
Genetic organization of the chromosomal region (waa gene cluster) containing the core LPS biosynthesis genes of P. mirabilis strains R110, 51/57, and HI4320 (34), K. pneumoniae strains 52145 (41) and C3 (39), S. marcescens N28b (11), and E. coli K-12 (20). Common inner (black arrows)- and outer (gray arrows)-core genes, specific outer-core genes (light gray arrows), O-PS ligase genes (stripped arrows), genes with unknown functions (white arrows), and genes unrelated to core LPS (dotted arrows) are illustrated. The horizontal bars indicate regions with a significantly lower C+G percentage than the whole wa gene cluster. Genes common to the seven P. mirabilis strains studied (R110, 51/57, 14/57, 50/57, TG83, OXK, and CECT170) are underlined.
FIG. 3.
FIG. 3.
SDS-Tricine-PAGE analysis of LPS samples from K. pneumoniae 52145, 52145ΔwaaC, 52145ΔwaaC(pGEMT-WaaCR110), 52145ΔwaaF, 52145ΔwaaF(pGEMT-WaaFR110), 52145ΔwaaE, 52145ΔwaaE(pGEMT-WaaER110), 52145ΔwaaQ, and 52145ΔwaaQ(pGEMT-WaaQR110).
FIG. 4.
FIG. 4.
(A) PCR-amplified DNA products obtained using oligonucleotides BOf and BOr and genomic DNAs from P. mirabilis R110 (lane 1) and 51/57 (lane 2). Lane 0, molecular mass marker. SDS-PAGE (B) and SDS-Tricine-PAGE (C) analyses of LPSs from K. pneumoniae 52145, 52145ΔwabO, and 52145ΔwabO(pGEMT- WabOR110) are also shown.
FIG. 5.
FIG. 5.
SDS-PAGE (A) and SDS-Tricine-PAGE (B) analyses of LPSs from K. pneumoniae 52145, 52145ΔwabG, 52145ΔwabG(pGEMT-WabGR110), 52145ΔwabH, 52145ΔwabH(pGEMT-WabHR110), 52145ΔwabN, and 52145ΔwabN(pGEMT-WabNR110).
FIG. 6.
FIG. 6.
(A) SDS-Tricine-PAGE analysis of LPS samples from K. pneumoniae 52145, 52145ΔwabH, 52145ΔwabH(pGEMT-WamAR110), 52145ΔwabH(pGEMT-WamAR110)(pBAD18-Cm-WamB), 52145ΔwabH(pGEMT-WamBR110), and 52145ΔwabH(pGEMT-WamCR110). Positive-ion MALDI-TOF analyses of acid-released core OSs from the LPSs of K. pneumoniae 52145ΔwabH (B), 52145ΔwabH(pGEMT-WamAR110) (C), and 52145ΔwabH(pGEMT-WamAR110)(pBAD-WamB) (D) are also shown.
FIG. 7.
FIG. 7.
(A) SDS-Tricine-PAGE analysis of LPS samples from K. pneumoniae 52145, 52145ΔwabK, 52145ΔwabK(pGEMT-WamAR110), 52145ΔwabK(pGEMT-WamCR110), 52145ΔwabK(pGEMT-WamBR110), and 52145ΔwabK(pGEMT-WamD51/57. Positive-ion MALDI-TOF analyses of acid-released core OSs from the LPSs of K. pneumoniae 52145ΔwabK (B), 52145ΔwabK(pGEMT-WamCR110) (C), and 52145ΔwabK(pGEMT-WamD51/57 (D) are also shown.
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