Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp− Escherichia coli

Lin, Bo; Short, Steven A.; Eskildsen, Manuel; Klempner, Mark S.; Hu, Linden T.

doi:10.1016/s0167-4889(00)00121-x

Cited by 41 publications

(46 citation statements)

References 12 publications

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“…While the B. burgdorferi opp operon is chromosomally encoded, at least two other OppA orthologues have been identified on borrelial plasmids (oppA-IV on cp26 and oppA-V on lp-54). In previous studies using a complementation system with the E. coli integral membrane proteins OppB and OppC, the ability of the borrelial OppA orthologues to bind and facilitate transport of peptides was confirmed (10).…”

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confidence: 87%

“…Whereas E. coli DppA appears to have fairly narrow substrate specificity, OppA in other bacteria has been shown to have very broad substrate specificity (6, 14-16, 20, 21). In comparison to E. coli OppA, the B. burgdorferi OppA proteins appear to have overlapping but narrower specificities, allowing for the potential of separate roles for each OppA protein (10). We were interested in the mechanisms of regulation and expression of the B. burgdorferi OppA proteins and whether they could be differentially expressed under changing environmental conditions.…”

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confidence: 99%

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Effects of Environmental Changes on Expression of the Oligopeptide Permease ( opp ) Genes of Borrelia burgdorferi

Wang

Lin²,

Kidder

et al. 2002

J Bacteriol

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We analyzed expression of a putative oligopeptide permease (Opp) of Borrelia burgdorferi. Unlike the opp operons of other bacteria for which there is a single substrate binding protein, B. burgdorferi codes for three substrate binding proteins (OppA-I to -III) in its opp operon and an additional two homologs on plasmids (OppA-IV and -V). Instead of a single promoter region regulating transcription of the entire operon, as seen in other bacterial opp operons, it appears that among oppA-I, -II, and -III, as well as oppA-IV and -V, each has a potential upstream promoter region. We tested the function of these putative promoter sequences by fusion to a promoterless ␤-galactosidase reporter gene in pCB182. Each of the promoter regions was found to be active. The level of activity in the reporter constructs closely paralleled the level of expression of each gene in in vitro-grown B. burgdorferi. Changes in carbon and nitrogen availability differentially affected individual promoters, but no changes in promoter activity were seen when Escherichia coli bacteria (with the promoter constructs) were grown in various concentrations of phosphate and leucine and changes in pH. Expression of specific oppA genes with B. burgdorferi varied significantly between its mouse and fed and unfed tick hosts. Differences in regulation of opp gene expression suggest a potential role in environmental response by the organism.

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confidence: 87%

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confidence: 99%

Effects of Environmental Changes on Expression of the Oligopeptide Permease ( opp ) Genes of Borrelia burgdorferi

Wang

Lin²,

Kidder

et al. 2002

J Bacteriol

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“…Western blotting was performed as described previously (18) to confirm the identities of the purified proteins. Antibodies to the OppA proteins were kind gifts from James Bono and Patricia Rosa (Rocky Mountain Laboratories, Hamilton, Mont.…”

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confidence: 99%

“…Two other OppA orthologues (OppA-4 and OppA-5) have been identified from borrelial plasmids (cp26 and lp-54, respectively). Lin et al confirmed the ability of the B. burgdorferi OppA orthologues to transport tripeptides by using a complementation system in E. coli (18).…”

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confidence: 95%

Analysis of Differences in the Functional Properties of the Substrate Binding Proteins of the Borrelia burgdorferi Oligopeptide Permease ( opp ) Operon

Wang¹,

Kidder²,

Scagliotti³

et al. 2004

J Bacteriol

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The Borrelia burgdorferi genome encodes five orthologues of the substrate binding protein oligopeptide permease A (OppA). It was previously shown that these genes are under the control of separate promoters and are differentially expressed under various environmental conditions. We were interested in determining whether there are also differences in substrate specificities among the proteins. The substrate specificities of recombinant proteins were determined by screening for high-affinity peptides by use of a combinatorial phage display heptapeptide library. Different heptapeptides with high affinities for OppA-1, OppA-2, and OppA-3 were identified. No heptapeptide binding OppA-4 or OppA-5 could be identified. Competitive binding assays were performed under various conditions to determine the substrate preferences of the OppA proteins. OppA-1 retained maximal activity over a broad range of pHs (5.5 to 7.5), whereas OppA-2 and OppA-3 showed peak activities at pHs below 5.5. OppA-1 and OppA-2 showed preferences for tripeptides over dipeptides and longer-chain peptides. Although a wide variety of amino acyl side chains were tolerated by all three OppA proteins, OppA-1 showed the broadest substrate specificity and was able to accommodate peptides composed of bulky hydrophobic residues; OppA-2 and OppA-3 showed preferences for peptides composed of small nonpolar amino acids. All three OppA proteins showed preferences for peptides composed of L-rather than D-amino acids. OppA-3 showed the greatest tolerance for changes in stereochemistry. Substantial differences in the substrate specificities of the OppA proteins of B. burgdorferi suggest that they may have distinct functions in the organism.

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“…A number of bacterial species cannot synthesize amino acids, fatty acids, and certain other essential elements (Matthews, 1975;Lin et al, 2001), and uptake essential nutrients from their environment. The ABC transporters mediate a large fraction of the nutrient uptake capacity in bacteria.…”

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confidence: 99%

Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein

et al. 2015

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ABSTRACT. In this study, the in vivo interaction system of oligopeptide permease (Opp) proteins was analyzed, and a high expression system of inner membrane protein OppC was constructed by flexible usage of the green fluorescent protein (GFP). The Escherichia coli OppC gene, which encodes a transmembrane component of oligopeptide transporter, was cloned into different vectors. Recombinant plasmids were transformed into different E. coli strains, and the expression conditions were optimized. The effect of plasmids and expression strains on OppC production was evaluated by in-gel and western blot analyses. OppC produced by the pWaldo-GFPe vector, harboring the GFP reporter gene, transformed into E. coli C43(DE3) provided sufficient functional protein for biochemical and biophysical studies. In vivo protein-protein interactions were detected among oligopeptide permease proteins using a GFP fragment reassembly 17835 OppC expression and interaction analysis using GFP ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 17834-17846 (2015) protocol. The substrate binding protein OppA showed no interaction with the other components, while the ATP-binding component OppD did not interact with OppF. OppD and OppF interacted with the transmembrane components OppB and OppC. OppB also showed direct interaction with OppC. In vivo OppC functionality was determined by constructing an OppC gene deletion strain. OppC was shown to be essential for peptide uptake, and non-essential for cell viability. These results could help in elucidating the oligopeptide transport mechanism in bacteria.

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Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp− Escherichia coli

Cited by 41 publications

References 12 publications

Effects of Environmental Changes on Expression of the Oligopeptide Permease ( opp ) Genes of Borrelia burgdorferi

Effects of Environmental Changes on Expression of the Oligopeptide Permease ( opp ) Genes of Borrelia burgdorferi

Analysis of Differences in the Functional Properties of the Substrate Binding Proteins of the Borrelia burgdorferi Oligopeptide Permease ( opp ) Operon

Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein

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