DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin

Werts, Catherine; Charbit, Alain; Bachellier, Sophie; Hofnung, Maurice

doi:10.1007/bf00265433

Cited by 22 publications

(9 citation statements)

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“…A group of phenylalanine and tryptophan residues occurs in the N-terminal portion of OprB. Several investigations have consistently linked residues in the N-terminal third of porins to channel size and ion selectivity Misra and Benson, 1988;Tommassen et al, 1985) and formation, of a binding site in substrate-selective porins (Werts et al, 1992). In Rhodobacter capsulatus porin and OmpF and PhoE of E. coli, crystal-structure analysis has demonstrated that the third external loop in the N-terminal portion of these proteins folds into the p barrel (Cowan et al, 1992;Weiss and Schulz, 1992).…”

Section: Discussionmentioning

confidence: 99%

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

Wylie

Worobec

1994

European Journal of Biochemistry

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OprB is a glucose-selective porin known to be produced by Pseudomonas aeruginosa and Pseudomonas putida. We have cloned and sequenced the oprB gene of I? aeruginosa and obtained expression of OprB in Escherichia coli. The mature protein consists of 423 amino acid residues with a deduced molecular mass of 47597 Da. Several clusters of amino acid residues, potentially involved in the structure or function of the protein, were identified. An area of regional homology with E. coli LamB was also identified. Carbohydrate-inducible proteins, potentially homologous to OprB, were identified in several rRNA homology-group-I pseudomonads by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis analysis, Western immunoblotting and N-terminal amino acid sequencing. These species also contained DNA that hybridized to a P. aeruginosa oprB gene probe.Porin proteins form trans-membrane water-filled channels in the outer membrane of Gram-negative bacteria and allow the diffusion of substrates between the external environment and the periplasm of the cell (Nikaido and Vaara, 1985). Two types of porins have been recognized; nonspecific porins and substrate-selective porins. Nonspecific porins form channels which allow nonspecific diffusion of substrates based on solute mass, charge and polarity. Substrateselective porins show some substrate specificity and possess a binding site for a given substrate within the channel. In comparison to nonspecific porins, very few substrate-selective porins have been identified and characterized.The glucose-selective porin, OprB, of Pseudomonas aeruginosa was first identified by Hancock and Carey (1980) following SDSPAGE analysis of outer membranes of cells grown on glucose as the sole carbon source. Porin function was demonstrated by the efflux of several carbohydrates from vesicles reconstituted from purified OprB, lipopolysaccharide and phospholipid. Trias et al. (1988), using the liposome-swelling assay, characterized OprB as a substrate-selective porin by demonstrating selectivity of OprB for glucose and xylose. Subsequently, a binding site for glucose and several other carbohydrates was demonstrated in P. aeruginosa OprB and its homologue in Pseudomonas putida by Wylie et al. (1993) and Saravolac et al. (1991), respectively. f? aeruginosa OprB bound glucose with a K, of 380 mM. K, for glucose binding to I? putida OprB was within the same order of magnitude, with a value of 110 mM. P. putida OprB was also shown to bind maltose and maltotriose with a higher affinity than glucose, although neither l? aeruginosa nor P. putida is able to utilize maltose or maltotriose as a carbon source. Single-channel-conductance measurements indicated that both P. aeruginosa and P. putida OprB form small constricted channels [25 pS (picosiemens) for P. aeruginosa within the pore of these proteins. Surprisingly, although I? aeruginosa and I? putida OprB appear closely related based on immunological and biochemical evidence (Saravolac et al., 1991 ; Wylie et al., 1993), the two porins show opposite ion sel...

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Section: Discussionmentioning

confidence: 99%

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

Wylie

Worobec

1994

European Journal of Biochemistry

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“…Cloning of the lamB Gene of K. oxytoca M5a1-The lamB gene of K. oxytoca was cloned via PCR using phosphorylated oligonucleotides derived from the nucleotide sequence of the homologous gene from Klebsiella pneumoniae (13). Oligonucleotide LAMB3 is a 24-mer (5Ј-ATGAT-GATTACTCTGCGCAAACTT-3Ј) and starts with the ATG of the reading frame, whereas the 27-mer oligonucleotide LAMB4 (5Ј-TTAC-CACCACACTTCCATCTGGGCACC-3Ј) starts with the codon complementary to the stop codon of lamB.…”

Section: Methodsmentioning

confidence: 99%

Properties of a Cyclodextrin-specific, Unusual Porin from Klebsiella oxytoca

Pajatsch¹,

Andersen²,

Mathes³

et al. 1999

Journal of Biological Chemistry

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The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized. CymA is essential for growth on cyclodextrins, but it can also complement the deficiency of a lamB (maltoporin) mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates. CymA was overproduced in E. coli and purified to apparent homogeneity. CymA is a component of the outer membrane, is processed from a signal peptidecontaining precursor, and possesses a high content of antiparallel ␤-sheet. Incorporation of CymA into lipid bilayers and conductance measurements revealed that it forms ion-permeable channels, which exhibit a substantial current noise. CymA-induced membrane conductance decreased considerably upon addition of ␣-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K S , of 28 M for its binding to CymA. CymA assembled in vitro to two-dimensionally crystalline tubular membranes, which, on electron microscopy, are characterized by a p1-related two-sided plane group. The crystallographic unit cell contains four monomeric CymA molecules showing a central pore. The lattice parameters are a ‫؍‬ 16.1 nm, b ‫؍‬ 3.8 nm, ␥ ‫؍‬ 93°. CymA does not form trimeric complexes in lipid membranes and shows no tendency to trimerize in solution. CymA thus is an atypical porin with novel properties specialized to transfer cyclodextrins across the outer membrane.

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“…Residues 40 through 60 of LamB in Escherichia coli, Salmonella typhimurium, Shigella spp., and Klebsiella * Corresponding author. pneumoniae are identical except for one substitution (29). Residues 40 to 49 also constitute part of a conserved region identified by Nikaido and Wu (22) (Fig.…”

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confidence: 66%

“…Short in-frame deletions overlapping residues 39 to 49 of mature LamB resulted in the formation of unstable protein that was rapidly degraded (24), possibly as a result of incorrect outer membrane routing. Apart from its potential role in localization, the N-terminal third of the sequence has also been postulated to be important for sugar selectivity and channel formation (14,29) (Fig. 1) in outer membrane proteins such as LamB, OmpA, and OmpF.…”

mentioning

confidence: 99%

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function

Chan

Ferenci

1993

J Bacteriol

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A new strategy for combinatorial mutagenesis was developed and applied to residues 40 through 60 of LamB protein (maltoporin), with the aim of identifying amino acids important for LamB structure and function. The strategy involved a template containing a stop codon in the target sequence and a pool of random degenerate oligonucleotides covering the region. In vitro mutagenesis followed by selection for function (Dex+, ability to utilize dextrins) corrected the nonsense mutation and simultaneously forced incorporation of a random mutation(s) within the region. The relative importance of each residue within the target was indicated by the frequency and nature of neutral and deleterious mutations recovered at each position. Residues 41 through 43 in LamB accepted few neutral substitutions, whereas residues 55 through 57 were highly flexible in this regard.Consistent with this finding was that the majority of defective mutants were altered at residues 41 to 43.Characterization of these mutants indicated that the nature of residues 41 to 43 influenced the amount of stable protein in the outer membrane. These results, as well as the conserved nature of this stretch of residues among outer membrane proteins, suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.Combinatorial mutagenesis studies are becoming important in the identification of important residues of proteins, including transport proteins (3,15,23,25,26). In this study, we developed a novel strategy for introducing random mutations into a defined region of maltoporin (LamB protein), with the aim of identifying residues significant in contributing to structure, assembly, and sugar transport. The strategy used with LamB can be applied to the mutagenesis of any gene coding for a selectable phenotype and has considerable advantages over cassette mutagenesis methods.Maltoporin in the outer membrane of Escherichia coli has been favored as a model in studies of phage (lambda) binding (4) and sugar channel selectivity (1) as well as protein export and assembly (9,20). Amino acid residues which play an important role in phage lambda and sugar binding have been identified (4,14). However, the events leading to the localization and assembly of LamB trimers into the outer membrane are still not clear. In addition to the signal sequence, specific regions in the mature protein may also play an important role in the biogenesis process (9, 20) but remain to be convincingly identified.There are several lines of evidence to suggest that residues 40 through 60 of mature LamB are of structural importance. Short in-frame deletions overlapping residues 39 to 49 of mature LamB resulted in the formation of unstable protein that was rapidly degraded (24), possibly as a result of incorrect outer membrane routing. Apart from its potential role in localization, the N-terminal third of the sequence has also been postulated to be important for sugar selectivity and channel formation (14,29) (Fig. 1) in outer membrane proteins such ...

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DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin

Cited by 22 publications

References 36 publications

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae

Properties of a Cyclodextrin-specific, Unusual Porin from Klebsiella oxytoca

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function

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