A signal sequence mutant defective in export of ribose-binding protein and a corresponding pseudorevertant isolated without imposed selection.

Iida, Akihiro; Groarke, James M.; Park, S.; Thom, J R; Zabicky, J.H.; Hazelbauer, Gerald L.; Randall, Linda L.

doi:10.1002/j.1460-2075.1985.tb03863.x

Cited by 28 publications

(10 citation statements)

References 28 publications

(14 reference statements)

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“…Determination of the site of TnlO insertion in the E. coli chromosome was carried out by the method of Yonetani (44). Chromosomal DNA from strain AI80 was prepared as described previously (15). After digestion of the chromosomal DNA with appropriate restriction enzymes, the DNA fragments formed were separated by agarose gel electrophoresis and transferred onto nitrocellulose filters (BA85; Schleicher & Schuell) as described previously (36).…”

Section: Materiauls and Methodsmentioning

confidence: 99%

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

1993

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We isolated a transposon TnlO insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.Transketolase (EC 2.2.1.1) catalyzes transfer of the glycol aldehyde moiety from a ketose or its phosphate to an aldose or its phosphate. The enzyme is found in animals, plants, and bacteria and is involved in the pentose phosphate pathway responsible for production of NADPH and several sugar phosphate intermediates such as ribose 5-phosphate, erythrose 4-phosphate, and sedoheptulose 7-phosphate. Ribose 5-phosphate is utilized for the biosynthesis of purine and pyrimidine nucleotides and histidine, erythrose 4-phosphate is used for the biosynthesis of aromatic amino acids, and sedoheptulose 7-phosphate is used for the biosynthesis of cell wall components in gram-negative bacteria. The reaction is also important in CO2 fixation in photosynthetic organisms.In Escherichia coli Josephson and Fraenkel (18,19) first isolated mutants defective in transketolase activity. These mutants were unable to use L-arabinose or D-xylose as a sole carbon source and required shikimic acid or aromatic amino acids for growth on a minimal medium. The requirement for aromatic amino acids was shown to be slightly leaky, and the existence of low residual transketolase activity was suggested by the authors. The mutations were mapped around 62 min on the E. coli chromosome. A similar mutant was also isolated from Salmonella typhimurium, and the role of transketolase in supplying sedoheptulose 7-phosphate was examined (7). In this report, we describe isolation of a transposon TnlO insertion mutant of E. coli defective in transketolase as a ribose-sensitive mutant on MacConkey plates containing D-ri...

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Section: Materiauls and Methodsmentioning

confidence: 99%

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

1993

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“…Strain AI287 or H020 carrying plasmid pAI27, which harbors the rbsB103 mutation, was used to isolate revertants that could suppress the rbsBl03 signal sequence mutation defect (10,32). rbsB201, rbsB202 (32), and rbsB212 revertants were isolated from strain A1287, whereas rbsB298 and rbsB299 are from H020 carrying pAI27 (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…The 5OVE mutation in the mature region was isolated by its ability to suppress the signal sequence defect -17LP (rbsB103 [10]) and was shown to reduce the rate of protein folding (32). Since no processing of precursor RBP was observed with the signal sequence mutation alone in either the presence or absence of SecB, the suppressor change in the mature protein makes it possible for SecB to improve export.…”

Section: Methodsmentioning

confidence: 99%

Involvement of SecB, a chaperone, in the export of ribose-binding protein

et al. 1992

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Ribose-binding protein (RBP) is an exported protein of Escherichia coli that functions in the periplasm. The export of RBP involves the secretion machinery of the cell, consisting of a cytoplasmic protein, SecA, and the integral membrane translocation complex, including SecE and SecY. SecB protein, a chaperone known to mediate the export of some periplasmic and outer membrane proteins, was previously reported not to be involved in RBP translocation even though small amounts of in vitro complexes between SecB and RBP have been detected. In our investigation, it was shown that a dependence on SecB could be demonstrated under conditions in which export was compromised. Species of RBP which carry two mutations, one in the leader that blocks export and a second in the mature protein which partially suppresses the export defect, were shown to be affected by SecB for efficient translocation. Five different changes which suppress the effect of the signal sequence mutation -17LP are all located in the N domain of the tertiary structure of RBP. All species of RBP show similar interaction with SecB. Furthermore, a leaky mutation, -14AE, generated by site-specific mutagenesis causes reduced export in the absence of SecB. These results indicate that SecB can interact with RBP during secretion, although it is not absolutely required under normal circumstances.The ribose-binding protein (RBP) of Escherichia coli functions in the periplasm as a component of a high-affinity transport system for ribose (38) and as a primary receptor for bacterial chemotaxis toward ribose (1). RBP is exported by components involved in the translocation of other secreted proteins, such as maltose-binding protein (MBP), bacteriophage lambda receptor (LamB), and other periplasmic and outer membrane proteins. However, it has been shown that the translocation of RBP synthesized in the cytoplasm is independent of translocation of the export component SecB (5, 13, 15), although some interaction between SecB and RBP has been demonstrated in vitro (8,12,14).SecB function was originally identified by a genetic defect in protein secretion. The protein exists as an oligomer with a monomeric subunit of 16.6 kDa (36), which appears to be required for the export of a subset of proteins that are secreted via a SecA-and SecY (PrlA)-dependent pathway (9). The periplasmic protein MBP and outer membrane proteins LamB, OmpA, and OmpF are SecB dependent, whereas ,-lactamase, lipoprotein, phage M13 coat protein, and RBP are SecB independent (13). SecB has been proposed to function as a molecular chaperone that maintains protein folding intermediates, perhaps structurally similar to the molten globule (21), by preventing the protein from attaining its final conformation (4). Other chaperones, for example, GroEL and Hsp7O, are also known to be involved in the export of ,B-lactamase (3) and in the import of protein into the mitochondria (7), respectively. However, the crossspecificity of these proteins for their target substrates with respect to protein translocation appea...

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“…Both genetic (3,19) and biophysical (5,34) evidence suggests that the export competence of certain E. coli signal peptides depends on the ability of their h regions to adopt this secondary structure. Although introduction of helix-breaking proline residues into the h region of MBP (3,13), RBP (13,25) Under certain circumstances, the C region (20) and even the first residues of the mature region (2,53) (45).…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis

Collier

1994

J Bacteriol

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A signal sequence mutant defective in export of ribose-binding protein and a corresponding pseudorevertant isolated without imposed selection.

Cited by 28 publications

References 28 publications

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12

Involvement of SecB, a chaperone, in the export of ribose-binding protein

Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis

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