Interaction of the <i>lamB</i> Protein with the Peptidoglycan Layer in <i>Escherichia coli</i> K12

Gabay, Joëlle E.; Yasunaka, Kakuko

doi:10.1111/j.1432-1033.1980.tb04393.x

Cited by 46 publications

(29 citation statements)

References 33 publications

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“…Two of these antibodies (E177 and E302) had been shown to recognize determinants located within 70 residues of the COOH terminus of LamB (18). In addition, the sequence changes due to mutations yielding resistance to killing by complement in the presence of three of these antibodies (E302, E72, and E347) were located in loops around residues 333 and 390 (14).…”

Section: Methodsmentioning

confidence: 99%

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Permissive sites and topology of an outer membrane protein with a reporter epitope

et al. 1991

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We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12 In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.Genetic fusions between proteins have proved to be useful tools in the study of a large number of biological problems. P-Galactosidase, alkaline phosphatase, and 1-lactamase have been used as reporter enzymes to study the location or the folding of proteins and for various biotechnological applications (for recent discussions, see references 7, 27, and 28). For topological studies, such fusions present at least two kinds of limitations. First, they usually delete the COOH-terminal end of the vehicle protein; this end can be critical for the normal biogenesis of the protein. Second, properties of the passenger enzyme (size, sequence, etc.) may alter the location or folding of the vehicle protein (see, for example, references 31 and 42). These two problems occur in the case of the outer membrane protein LamB (3, 4), which is why the topological information on this protein comes from other approaches (10, 21).LamB is representative of a class of integral membrane proteins, the porins (35), which are peculiar for at least two reasons. They are mostly hydrophilic, with no apparent hydrophobic transmembrane segment, and the structured regions are essentially composed of beta-strand segments. LamB plays a role in the penetration of maltose and maltodextrins through the outer membrane and is the cell surface receptor for a number of bacteriophages, including phage lambda; the active form is a trimer (reference 10 and references therein). In the absence of precise crystallo-* Corresponding author. graphic data, most of our knowledge of LamB organization relies on genetic, immunological, and biochemical data. On these bases, we have proposed a two-dimensional folding model that we would like to test and refine (9, 10).To circumvent the problems encountered with reporter enzymes, we started a new and complementary approach that consists of using as a reporter a small peptide corresponding to a foreign antigenic determinant (i.e., a reporter epitope) which we insert genetically into permissive sites of LamB (4,8; reviewed in reference 22). Such sites tolerate insertions without loss of most biological properties of the protein and therefore without major changes in its cellular location and structure. Permissive sites can be identified by a genetic method (8), and the hybrid proteins generated are then probed with a monoclonal antibody (MAb) against the reporter epitope. This approach led us to show that upon insertion after residues 153...

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Section: Methodsmentioning

confidence: 99%

“…Four MAbs recognize conformational determinants of the cell surfaceexposed regions of LamB located after residue 330 (14,18). They could therefore be used to examine changes in the conformation or the exposure of these determinants.…”

Section: Methodsmentioning

confidence: 99%

Permissive sites and topology of an outer membrane protein with a reporter epitope

et al. 1991

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“…For the purpose of our study, a dnaA(Ts) mutation was P1 transduced into the mutD5 strain so that mismatch repair efficiency could be determined under conditions in which E. coli DNA replication was allowed or prevented. P1 transductions were performed as described elsewhere (20 (Lambda receptor was extracted as described elsewhere [12] from bacterial strain 6371, which produces high amounts of the LamB protein [27].) Infective centers were scored after being streaked on indicator bacteria at 32°C.…”

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Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli

1989

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The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III. The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired. We show that the mismatch repair system of mutDS strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication. This recovery depends on de novo protein synthesis.The mechanisms ensuring accuracy of DNA replication have been elucidated by study of mutator mutants affecting specific accuracy functions (for a review, see reference 4). In Escherichia coli, mutator mutations mapping in the dnaE gene presumably affect nucleotide selection by DNA polymerase III (28). Mutations mapping in the dnaQ gene (also called mutD) affect the proofreading 3'->5' exonuclease function of the DNA polymerase III holoenzyme (6, 8), whereas mutH, mutL, mutS, and mutU mutations cause defects in methyl-directed postreplicative mismatch correction (for reviews, see references 21 and 24). mutD5 and dnaQ49 mutants are the most potent mutator strains known. All classes of transition, transversion, and frameshift mutations are increased up to 105-fold when mutD5 strains are grown in rich medium (5, 10, 11). The mutD5 gene of E. coli encodes the epsilon subunit, which carries out the 3'-*5' proofreading exonuclease function of the DNA polymerase III holoenzyme (6,8). However, the high-mutation-rate effect of mutD5 strains cannot be explained only by the defect in exonuclease activity, as this mutation rate is comparable to the error rate of in vitro replication with a polymerase devoid of proofreading activity (9, 16). The strong mutator phenotype of mutD5 suggested that mismatch repair functions may be impaired as well. This prediction was supported by results from DNA heteroduplex transfection experiments (23,25,26). One hypothesis is that the high error rate in DNA replication saturates mismatch repair in mutD5 mutants (23). Therefore, we have tested the mismatch repair capacity of mutD5 strains under conditions in which chromosomal DNA replication was or was not allowed. Mismatch repair activity was determined by the extent of pure infective centers derived from infection with packaged hemimethylated heteroduplexes of lambda DNA. Under these conditions, bacteria proficient in mismatch repair produced essentially pure infective centers, whereas bacteria deficient in mismatch repair produced mostly mixed infective centers.To evaluate mismatch repair activity, pure hemimethy-* Corresponding author. t Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. lated heteroduplexes of lambda DNA containing an A. C or a G T mismatch were artificially constructed and introduced into mutD5 and other appropriate strains. Hemimethylated heteroduplexes of lambda DNA with defined mismatches were prepared as previously described (19) by reannealing the separated DNA strands of a lambda mutant carry...

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ABSTRACTAnalysis of amino acid sequences reported for the major outer membrane proteins of Escherichia coli, including the porins (OmpF, OmpC, and PhoE), the phage A receptor (LamB), and another protein (OmpA), revealed several regions of local homology that is statistically significant.The implications of this observation are discussed in relation to the evolutionary origins of these proteins, as well as to the mechanism of export of these proteins to the outer membrane.All Gram-negative (4,10,11). Third, because all of these proteins are located in the outer membrane, there may be a common "signal" for their translocation.

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Amino acid sequence homology among the major outer membrane proteins of Escherichia coli.

Nikaido¹,

Wu²

1984

Proc. Natl. Acad. Sci. U.S.A.

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Analysis of amino acid sequences reported for the major outer membrane proteins of Escherichia coli, including the porins (OmpF, OmpC, and PhoE), the phage A receptor (LamB), and another protein (OmpA), revealed several regions of local homology that is statistically significant.The implications of this observation are discussed in relation to the evolutionary origins of these proteins, as well as to the mechanism of export of these proteins to the outer membrane.All Gram-negative (4,10,11). Third, because all of these proteins are located in the outer membrane, there may be a common "signal" for their translocation. Various observations suggest that if such a signal were present, it likely would be located within the sequence of the mature proteins. For example, it is known that the amino terminal "leader" sequence of the LamB precursor by itself was unable to direct the translocation to the outer membrane of another polypeptide fused to it (12). Fourth, translocation of proteins into the outer membrane could require a complex transport mechanism. It is unlikely that such a mechanism was "reinvented" for each new outer membrane protein as it evolved; therefore, many outer membrane proteins may have been derived from a common ancestral protein.Sequence homologies between E. coli outer membrane proteins have been examined before. Although strong homologies were found among three porins (OmpF, OmpC, and PhoE) (13,14), no significant homology was reported between OmpA and OmpF (15) Searches for Potentially Homologous Regions. All computer programs were written for a Tandy model I personal computer; to speed up the execution, most of the routines were written in Z80 assembly language.t Potentially homologous regions between proteins were identified by a two-step process. (i) Initially, regions of potential homology were searched for by comparisons of short segments without the creation of gaps. Thus, the program compared all possible short segments (e.g., containing seven amino acid residues) from one protein with all or a limited subset of segments of the same length from the second protein. If a comparison revealed a set number (e.g., four) or more of identical amino acids at corresponding positions, the matched region was analyzed further as described below.We emphasize that, for a number of reasons, the matches detected here can serve only as the starting points for the search for homology: homology of short sequences is statistically very unreliable (22); gaps may be needed for optimum alignment, but they were not introduced; and, finally, the search was performed purposefully at low degrees of stringency so as not to miss the real regions of homology. (ii) We then optimized the alignment of the more extended segments containing 11-30 amino acid residues, centered on the short segments of potential match identified above. This procedure permitted the insertion of gaps, if necessary, with a set amount of penalty ("gap penalty factor" of 2) given to the creation of each gap and also another penalty ...

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Interaction of the lamB Protein with the Peptidoglycan Layer in Escherichia coli K12

Cited by 46 publications

References 33 publications

Permissive sites and topology of an outer membrane protein with a reporter epitope

Permissive sites and topology of an outer membrane protein with a reporter epitope

Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli

Amino acid sequence homology among the major outer membrane proteins of Escherichia coli.

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