Proteomics-Based Identification of Outer-Membrane Proteins Responsible for Import of Macromolecules inSphingomonassp. A1:  Alginate-Binding Flagellin on the Cell Surface†,‡

ABSTRACT During the initial steps of biofilm formation, bacteria have to adapt to a major change in their environment. The adhesion-induced phenotypic changes in a type 1 fimbriated Escherichia coli strain included reductions in the levels of several outer membrane proteins, one of which was identified as OmpX. Here, the phenotypes of mutant strains that differ at the ompX locus were studied with regard to adhesion, cell surface properties, and resistance to stress and antimicrobial compounds. The kinetics of adhesion were measured online by an extended quartz crystal microbalance technique for wild-type and mutant strains with a fimbriated or nonfimbriated background. Deletion of ompX led to significantly increased cell-surface contact in fimbriated strains but to decreased cell-surface contact in a nonfimbriated strain. Phenotypic characterization of the ompX mutant demonstrated that ompX interferes with proper regulation of cell surface structures that play a key role in mediating firm contact of the cell with a surface (i.e., type 1 fimbriae, flagellae, and exopolysaccharides). These phenotypic changes were accompanied by increased tolerance to several antibiotic compounds and sodium dodecyl sulfate. Based on these results, we propose that changes in the composition of outer membrane proteins during fimbria-mediated adhesion may be part of a coordinated adaptive response to the attached mode of growth.

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Outer membrane proteins and cell surface structure of Selenomonas ruminantium.

Journal of Bacteriology ◽

10.1128/jb.141.2.899-907.1980 ◽

1980 ◽

Vol 141 (2) ◽

pp. 899-907 ◽

Cited By ~ 18

Author(s):

Y Kamio ◽

H Takahashi

Keyword(s):

Membrane Proteins ◽

Cell Surface ◽

Surface Structure ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Selenomonas Ruminantium ◽

Cell Surface Structure

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Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface ofBacteroides thetaiotaomicron

Journal of Bacteriology ◽

10.1128/jb.182.19.5365-5372.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5365-5372 ◽

Cited By ~ 142

Author(s):

Joseph A. Shipman ◽

James E. Berleman ◽

Abigail A. Salyers

Keyword(s):

Membrane Proteins ◽

Cell Surface ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Binding Activity ◽

Obligate Anaerobe ◽

Binding Assays ◽

Starch Utilization

ABSTRACT Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of 14C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.

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Outer Membrane Changes in a Toluene-Sensitive Mutant of Toluene-Tolerant Pseudomonas putidaIH-2000

Journal of Bacteriology ◽

10.1128/jb.181.15.4493-4498.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4493-4498 ◽

Cited By ~ 35

Author(s):

Hideki Kobayashi ◽

Hideto Takami ◽

Hisako Hirayama ◽

Kuniko Kobata ◽

Ron Usami ◽

...

Keyword(s):

Fatty Acid ◽

Membrane Proteins ◽

Cell Surface ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Dodecanoic Acid ◽

Sensitive Mutant ◽

Membrane Changes

ABSTRACT We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerantPseudomonas putida IH-2000 by transposon mutagenesis with Tn5. The gene disrupted by insertion of Tn5 was identified as cyoC, which is one of the subunits of cytochrome o. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of cyoC caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.

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Bacterial Phage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface

Journal of Bacteriology ◽

10.1128/jb.183.23.6924-6935.2001 ◽

2001 ◽

Vol 183 (23) ◽

pp. 6924-6935 ◽

Cited By ~ 25

Author(s):

Hildegard Etz ◽

Duc Bui Minh ◽

Carola Schellack ◽

Eszter Nagy ◽

Andreas Meinke

Keyword(s):

Amino Acids ◽

Membrane Proteins ◽

Cell Surface ◽

Outer Membrane ◽

Cell Sorting ◽

Fusion Proteins ◽

Outer Membrane Proteins ◽

Wild Type ◽

E Coli ◽

Multiple Copies

ABSTRACT Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B12 and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and λ, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.

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