Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1

ABSTRACT Gram-negative outer membrane (OM) integrity is maintained in part by Mg2+ cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated i...

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Autores principales:	Angela M. DeLucia, David A. Six, Ruth E. Caughlan, Patricia Gee, Ian Hunt, Joseph S. Lam, Charles R. Dean
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2011
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9c68738d0918443f84488cc00f0aab62
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spelling	oai:doaj.org-article:9c68738d0918443f84488cc00f0aab622021-11-15T15:38:45ZLipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO110.1128/mBio.00142-112150-7511https://doaj.org/article/9c68738d0918443f84488cc00f0aab622011-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00142-11https://doaj.org/toc/2150-7511ABSTRACT Gram-negative outer membrane (OM) integrity is maintained in part by Mg2+ cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM. IMPORTANCE Gram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase gene waaP can be deleted in several Gram-negative bacteria but not in Pseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly in P. aeruginosa cells, which halted growth. WaaP depletion also caused gross changes in cell morphology and led to the accumulation of an aberrant LPS lacking several core sugars and all core phosphates. The aberrant LPS failed to reach the OM, suggesting that WaaP is essential in P. aeruginosa because it is required to produce the full-length LPS that is recognized by the OM transport/assembly machinery in this organism. Therefore, WaaP may constitute a good target for the development of novel antipseudomonal agents.Angela M. DeLuciaDavid A. SixRuth E. CaughlanPatricia GeeIan HuntJoseph S. LamCharles R. DeanAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 2, Iss 4 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Angela M. DeLucia David A. Six Ruth E. Caughlan Patricia Gee Ian Hunt Joseph S. Lam Charles R. Dean Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1
description	ABSTRACT Gram-negative outer membrane (OM) integrity is maintained in part by Mg2+ cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM. IMPORTANCE Gram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase gene waaP can be deleted in several Gram-negative bacteria but not in Pseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly in P. aeruginosa cells, which halted growth. WaaP depletion also caused gross changes in cell morphology and led to the accumulation of an aberrant LPS lacking several core sugars and all core phosphates. The aberrant LPS failed to reach the OM, suggesting that WaaP is essential in P. aeruginosa because it is required to produce the full-length LPS that is recognized by the OM transport/assembly machinery in this organism. Therefore, WaaP may constitute a good target for the development of novel antipseudomonal agents.
format	article
author	Angela M. DeLucia David A. Six Ruth E. Caughlan Patricia Gee Ian Hunt Joseph S. Lam Charles R. Dean
author_facet	Angela M. DeLucia David A. Six Ruth E. Caughlan Patricia Gee Ian Hunt Joseph S. Lam Charles R. Dean
author_sort	Angela M. DeLucia
title	Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1
title_short	Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1
title_full	Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1
title_fullStr	Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1
title_full_unstemmed	Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1
title_sort	lipopolysaccharide (lps) inner-core phosphates are required for complete lps synthesis and transport to the outer membrane in <named-content content-type="genus-species">pseudomonas aeruginosa</named-content> pao1
publisher	American Society for Microbiology
publishDate	2011
url	https://doaj.org/article/9c68738d0918443f84488cc00f0aab62
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Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> PAO1

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