<i>rfaP</i> mutants of <i>Salmonella typhimurium</i>

Helander, Ilkka M.; Vaara, Martti; Sukupolvi, Soila; Rhen, Mikael; Saarela, Sirkku; Zähringer, Ulrich; Mäkelä, P. Helena

doi:10.1111/j.1432-1033.1989.tb15147.x

Cited by 44 publications

(44 citation statements)

References 30 publications

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“…It should be noted that the rfaP mutant is sensitive to 0 antigen-specific phage P22, indicating that some 0 antigen is still produced. This is consistent with other observations that loss of rfaP results in incomplete termination of the core (5,8). Complementation of the rfaG mutant by both the SG3.4 and UG1.5 fragments indicates that the second gene in the contiguous block is rfaG, and complementation of the rfaP mutant by fragments XA2.1 and SA4.9 but not by SG3.4 indicates that the third gene in the contiguous block is rfaP.…”

Section: Resultssupporting

confidence: 82%

Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12

1992

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The rfa locus of Escherichia coli K-12 includes a block of about 10 closely spaced genes transcribed in the same direction which are involved in synthesis and modification of the hexose region of the llpopolysaccharide core. We have sequenced the first three genes in this block. The function of the first of these genes is unknown, but we have designated it rfaQ on the basis of its location and similarity to other rfa genes. Complementation of SalmoneUa typhimurium rfa mutants with E. coli rfa restriction fragments indicated that the second and third genes in the block were rfaG and rfaP. The deduced sizes of the RfaQ, RfaG, and RfaP proteins are 36,298, 42,284, and 30,872 Da, respectively, and the proteins are basic and lack extensive hydrophobic domins. RfaQ shares regions of homology with proteins RfaC and RfaF, which are involved in synthesis of the heptose region of the core. Proteins RfaB, RfaG, and RfaK share a region of homology, which suggests that they belong to a second family of Rfa proteins which are thought to be hexose transferases.This study deals with rfaG, the gene for addition of the first glucose residue to the lipopolysaccharide (LPS) core, and with rfaP, a gene involved in attachment of phosphatecontaining substituents to the inner core. Figure 1 shows a simplified structure of the inner core region of Salmonella typhimurium and what are thought to be the primary sites of action of the relevant rfa genes (8). It can be assumed that this part of the core structure is very similar or identical in Escherichia coli K-12, since cloned DNA fragments from E. coli K-12 efficiently complement mutations in rfaC, rfaF, and rfaG in S. typhimurium (3,12).Two E. coli K-12 recombinant plasmids from the ClarkeCarbon collection, pLC10-7 and pLC17-24, were identified by Creeger and Rothfield as containing inserts which would complement rfaG mutations in S. typhimurium (3). The inserts of these plasmids overlap by about 4 kb, and the region of overlap includes two HindIII sites near the pyrE end of the rfa locus at 81 min on the E. coli map (1). In vitro transcription and translation of restriction fragments derived from the insert of pLC10-7 showed that these Hindlll sites lie at the beginning of a series of contiguous genes which are transcribed counterclockwise from pyrE. The in vitro products of the first three of these genes, reading away from pyrE, had apparent molecular masses of 42, 39, and 35 kDa. TnJO insertions in the second gene resulted in a glucosedeficient LPS (chemotype Rd1) phenotype, suggesting that this is the rfaG gene, while an insertion in the third gene resulted in a deep rough phenotype and LPS of the RcPchemotype, which includes the first glucose residue. These properties suggest that this gene is rfaP (1).In this report, we describe the sequence and properties of these three genes and show, by complementation of Salmonella mutations, that two of them are indeed rfaG and rfaP. The other is a novel gene, which we designated rfaQ. The relationship of these genes to the physical map of ...

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Section: Resultssupporting

confidence: 82%

Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12

1992

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“…10A) used to be thought to result in the truncated core and therefore to lead to a deep rough phenotype (265), disruption of the waaP gene, responsible for this phosphorylation, did not produce a complete truncation of the core (758). Therefore, the mutant used in the early study, isolated by chemical mutagenesis followed by selection for core-truncated mutants (265), must have contained multiple mutations. Even more importantly, the "clean" waaP mutant did not show decreased levels of OM proteins yet was hypersensitive to hydrophobic agents (759,760).…”

Section: General Structure Of Lpsmentioning

confidence: 99%

“…Although a defect in the phosphorylation of the distal core heptose (Hep II in Fig. 10A) used to be thought to result in the truncated core and therefore to lead to a deep rough phenotype (265), disruption of the waaP gene, responsible for this phosphorylation, did not produce a complete truncation of the core (758). Therefore, the mutant used in the early study, isolated by chemical mutagenesis followed by selection for core-truncated mutants (265), must have contained multiple mutations.…”

Section: General Structure Of Lpsmentioning

confidence: 99%

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Nikaido

2003

Microbiol Mol Biol Rev

3,838

3,800

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Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions

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“…Re LPS was extracted by the phenol-chloroform-petroleum ether method (4). Ra LPS was extracted by the phenol-water method (11) and purified by treatment with RNase and ultracentrifugation at 100,000 possesses the full length of R-core, whereas Re LPS possesses the shortest R-core consisting of two molecules of 3-deoxy-D-manno-octulosonic acid (1,5,6,10,12). Experimental conditions to form crystals of LPS.…”

Section: Methodsmentioning

confidence: 99%

Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

Kato

Ohta

Kido

et al. 1993

Microbiology and Immunology

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Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three-dimensional crystals when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516Bacteriol. 172: -1528Bacteriol. 172: (1990), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 A. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the { 1011} planes of solid hexagonal column crystals.

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rfaP mutants of Salmonella typhimurium

Cited by 44 publications

References 30 publications

Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12

Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

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