Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Müller, Norbert; Heine, Hans-Georg; Boos, Winfried

doi:10.1007/bf00334143

Cited by 14 publications

(7 citation statements)

References 36 publications

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“…DNA and amino acid sequences for the 38-kDa lipoprotein were analyzed further by using the GenBank sequence data base and the Lipman and Pearson algorithms (44, 64). Amino acid similarity with the MglB gene product (glucose/galactose-binding protein [GGBP]) (36,54,84) of E. coli was identified. Using the BESTFIT alignment program (55), the overall similarity between the mature 38-kDa lipoprotein and the GGBP was 58.9%, with 34.2% identity (Fig.…”

Section: Resultsmentioning

confidence: 99%

Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli

et al. 1994

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The recent discovery that abundant and immunogenic lipoproteins constitute the integral membrane proteins of Treponema pallidum has prompted efforts to investigate their importance in the physiology and ultrastructure of the organism and in immune responses during infection. Earlier studies identified a 38-kDa lipoprotein of T. pallidum believed to be specific to the pathogen. In the present study, monoclonal antibodies generated against the 38-kDa lipoprotein of T. pallidum reacted with cognate 37-kDa molecules in the nonpathogens Treponema phagedenis, Treponema denticola, and Treponema refringens. Cloning and expression of the 38-kDa-lipoprotein gene of T. pallidum in Escherichia coli revealed that the recombinant product displayed a slightly larger (39-kDa) apparent molecular mass but remained reactive with anti-38-kDa-protein monoclonal antibodies. The recombinant product was processed and acylated in E. coli. DNA and amino acid sequence analyses indicated an open reading frame encoding 403 amino acids, with the first 25 amino acids corresponding to a leader peptide terminated by a signal peptidase II processing site of Val-Val-Gly-Cys. The predicted mature protein is 378 amino acids in length with a deduced molecular weight of 40,422 (excluding acylation). Southern blotting failed to demonstrate in nonpathogenic treponemes genomic sequences homologous with the 38-kDa-lipoprotein gene of T. pallidum. Computer analysis revealed that the 38-kDa lipoprotein of T. pallidum had 34.2% identity and 58.9% similarity with the glucose/galactose-binding protein (MglB) of E. coli and Salmonella typhimurium. Furthermore, of the 19 amino acids of MglB involved in carbohydrate binding, the 38-kDa lipoprotein had identity with 11. These studies have allowed the first putative functional assignment (carbohydrate binding) to a T. pallidum integral membrane protein. Recognition of this potential physiological role for the 38-kDa lipoprotein underscores the possibility that the membrane biology of T. pallidum may more closely resemble that of gram-positive organisms, which also utilize lipoproteins as anchored transporters, than that of gram-negative bacteria to which T. pallidum often is analogized.

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Section: Resultsmentioning

confidence: 99%

Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli

et al. 1994

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“…Analysis of the mgl region. We previously cloned the mgl region as a 6.3-kilobase (kb) EcoRI fragment in pACYC184 to generate plasmid pHG4 starting from a library of EcoRI fragments in Xgt7 (29). Figure 1 shows the restriction endonuclease analysis of this EcoRI fragment after recloning into pBR322 to generate plasmid pHG30.…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, at least two gene products are very similar. The GBPs produced by both species are of nearly identical size, and both cross-react with antibodies against the E. coli protein (29). Furthermore, S. typhimurium GBP was fully active in transport and chemotaxis, when the remaining mgl gene products and the chemotactic signal transducer Trg (taxis to ribose and galactose) came fromn E. coli.…”

Section: Inmentioning

confidence: 99%

“…In addition to GBP (the mglB gene product) a membrane protein with an apparent molecular mass of 51,000 daltons has been identified as the mglA product by both studies, whereas the mglC gene product is claimed by Harayama et al (15) to be a membrane protein with a molecular mass of 38,000 daltons. We have cloned the corresponding region from S. typhimurium (29). In our present paper we analyzed this region by Tn5 mutagenesis, construction of lacZ fusions to mgl genes, and subcloning of restriction fragments.…”

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confidence: 99%

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Characterization of the Salmonella typhimurium mgl operon and its gene products

Müller¹,

Heine²,

Boos³

1985

J Bacteriol

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In Salmonella typhimurium and Escherichia coli the high-affinity galactose transport system, which contains a periplasmic galactose-binding protein as an essential component, is encoded by the mgl genes. The entire mgl region of S. typhimurium is contained on a 6.3-kilobase EcoRI restriction fragment, which has been cloned into plasmid vectors. We determined the extent of the mgl region on this fragment by TnS mutagenesis, examination of lacZ fusions to mgl genes, and subcloning smaller restriction fragments. Polyacrylamide gel electrophoresis of protein preparations derived from strains carrying different plasmids was used to identify the mgl gene products. We conclude that the mgl operon consists of four genes that form a single transcription unit: mglB, mglA, mglE, and mglC. The mglB gene codes for galactose-binding protein (33,000 daltons), mglA codes for a membrane-bound protein of 51,000 daltons, and mglC codes for a 29,000-dalton membrane protein. The mglE product was less well characterized. Its existence was inferred from a mglE-lacZ protein fusion located between mglA and mglC. In addition, the coupled transcription-translation in vitro system indicated that mglE codes for

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“…For labeling, the minicells were washed in MMA and suspended in 0.5 ml of MMA-0.4% lactate (optical density, 0.5). After a 45-min preincubation at 37°C, 25 ,ul of methionine assay medium (Difco Laboratories, Detroit, Mich.) and 5 ,uCi of L-[35S]methionine (Amersham International, England) per sample were added. After 60 min of incubation at 37°C, unlabeled methionine (0.25 mM) was added for a 10-min chase.…”

Section: Methodsmentioning

confidence: 99%

Defective secretion of maltose- and ribose-binding proteins caused by a truncated periplasmic protein in Escherichia coli

Hengge¹,

Boos²

1985

J Bacteriol

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The secretion in Escherichia coli of a C-terminally truncated periplasmic enzyme from Salmonella typhimurium, the glpQ-encoded glycerolphosphate phosphodiesterase, was studied. Plasmid pRH100, carrying the truncated glpQ gene, directs the synthesis of a 30,000-molecular-weight (30 K) protein that is processed to a mature 27.5 K protein. (The mature wild-type protein is a 38 K protein.) The truncated protein is not released into the periplasm but remains membrane associated, although it becomes protease sensitive after conversion of cells to spheroplasts. The presence of pRH100 strongly reduces the amount of some other proteins in the periplasm, including the maltose- and ribose-binding proteins. The reduction does not occur at the level of transcription or early translation, as shown by lacZ fusions to the gene coding for the structural gene of the maltose-binding protein. Outer membrane proteins are not affected. A hydroxylamine-induced mutation in the sequence of glpQ corresponding to the mature polypeptide overcomes the inhibitory effect of pRH100. The mutated gene no longer directs the synthesis of the 30/27.5 K protein but directs that of a new 19 K protein which is not membrane bound. We propose that sorting signals in the mature GIpQ protein are necessary for effective translocation to the periplasm and that the C-terminal third of the protein is essential for release into the periplasm.

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Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Cited by 14 publications

References 36 publications

Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli

Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli

Characterization of the Salmonella typhimurium mgl operon and its gene products

Defective secretion of maltose- and ribose-binding proteins caused by a truncated periplasmic protein in Escherichia coli

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