Expression, Two-Dimensional Crystallization, and Three-Dimensional Reconstruction of the β8 Outer Membrane Protein Omp21 from Comamonas acidovorans

Baldermann, C.; Engelhardt, Harald

doi:10.1006/jsbi.2000.4261

Cited by 10 publications

(4 citation statements)

References 44 publications

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“…An 18-␤-strand porin has also been suggested (10,33). While ␤ barrel proteins with 8 ␤-strands, such as E. coli OmpX (29), Neisseria meningitidis NspA (28), and Comamonas acidovorans Omp21 (2), and a 10-␤-strand protein, OpcA, of N. meningitidis (19), have been suggested to serve as outer membrane anchors rather than forming pores (2,12), the proteoliposome swelling data presented here suggest that the 12 ␤-strands of P28 and OMP-1F form pores. Confirmation of the secondary structure of P28 and OMP-1F awaits crystal structure analysis.…”

Section: Discussionmentioning

confidence: 99%

Expression and Porin Activity of P28 and OMP-1F during Intracellular Ehrlichia chaffeensis Development

2008

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Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gramnegative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel ␤-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a ␤-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37°C and the late-exponential growth phase at 28°C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid-and late-exponential growth phases at 28°C and 37°C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.

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Section: Discussionmentioning

confidence: 99%

Expression and Porin Activity of P28 and OMP-1F during Intracellular Ehrlichia chaffeensis Development

2008

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“…That OmpW is likely to be devoid of porin activity is also supported by the observation that the protein does not exist in the oligomeric or tightly peptidoglycan-associated forms that are usually the characteristics of porins. It is pertinent to mention that the Omp21 of C. acidovorans was also found to be devoid of any porin activity by ion conductance assay (Baldermann & Engelhardt, 2000).…”

Section: Discussionmentioning

confidence: 99%

Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

et al. 2005

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The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0?1 % or 2 % SDS at 25 6C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His 6 -OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of b-structures (~40 %) with minor amounts (8-15 %) of a-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66?6 % and 33?3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR " mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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“…One example of the opacity protein family protein was actually shown by X-ray crystallography to have a 10-strand ␤-barrel structure (511), and another example from this family, NspA, was recently characterized to be an 8-strand ␤-barrel protein (691). As Baldermann and Engelhardt argue (31), what is important in these proteins are the external loops that may be needed for interaction with the external structure, such as the surface of the host cell membrane or the substrates of enzyme reaction, and the 8-or 10-strand ␤-barrel may simply serve as the OM anchor. None of these proteins has been shown to form channels, as far as I am aware.…”

Section: Other Porinsmentioning

confidence: 99%

“…Q51498), which is part of the cluster coding for naphthalene utilization and is also likely to code for an OM channel. This AlkL-OmpW group also includes Omp21 of Comamonas acidovorans, which was studied as a two-dimensional crystal (31) and showed patterns similar to other eight-strand ␤-barrel proteins. With slow porins like OmpA and OprF, there was a possibility of alternative folding to produce large channels.…”

Section: Other Porinsmentioning

confidence: 99%

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Nikaido

2003

Microbiol Mol Biol Rev

3,776

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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Expression, Two-Dimensional Crystallization, and Three-Dimensional Reconstruction of the β8 Outer Membrane Protein Omp21 from Comamonas acidovorans

Cited by 10 publications

References 44 publications

Expression and Porin Activity of P28 and OMP-1F during Intracellular Ehrlichia chaffeensis Development

Expression and Porin Activity of P28 and OMP-1F during Intracellular Ehrlichia chaffeensis Development

Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

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