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D N Collier

Publications and source records attributed to D N Collier.

11 recordsLinked to original sources
PubMed2001

Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele.

The amiE gene encodes an aliphatic amidase capable of converting fluoroacetamide to the toxic compound fluoroacetate and is one of many genes whose expression is subject to catabolite repression control in Pseudomonas aeruginosa. The protein product of the crc gene, Crc, is required for repression of amiE and most other genes subject to catabolite repression control in this bacterium. When grown in a carbon source such as succinate, wild-type P. aeruginosa is insensitive to fluoroacetamide (due to repression of amiE expression). In contrast, mutants harboring the crc-10 null allele cannot grow in the presence of fluoroacetamide (due to lack of repression of amiE). Selection for succinate-dependent, fluoroacetamide-resistant derivatives of the crc-10 mutant yielded three independent pseudorevertants containing suppressors that restored a degree of catabolite repression control. Synthesis of Crc protein was not reestablished in these pseudorevertants. All three suppressors of crc-10 were extragenic, and all three also suppressed a Delta crc::tetA allele. In each of the three pseudorevertants, catabolite repression control of amidase expression was restored. Catabolite repression control of mannitol dehydrogenase production was also restored in two of the three isolates. None of the suppressors restored repression of glucose-6-phosphate dehydrogenase or pyocyanin production.

Amidohydrolases↗
PubMed1994

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.

Signal peptides of gram-positive exoproteins generally carry a higher net positive charge at their amino termini (N regions) and have longer hydrophobic cores (h regions) and carboxy termini (C regions) than do signal peptides of Escherichia coli envelope proteins. To determine if these differences are functionally significant, the ability of Bacillus subtilis to secrete four different E. coli envelope proteins was tested. A pulse-chase analysis demonstrated that the periplasmic maltose-binding protein (MBP), ribose-binding protein (RBP), alkaline phosphatase (PhoA), and outer membrane protein OmpA were only inefficiently secreted. Inefficient secretion could be ascribed largely to properties of the homologous signal peptides, since replacing them with the B. amyloliquefaciens alkaline protease signal peptide resulted in significant increases in both the rate and extent of export. The relative efficiency with which the native precursors were secreted (OmpA >> RBP > MBP > PhoA) was most closely correlated with the overall hydrophobicity of their h regions. This correlation was strengthened by the observation that the B. amyloliquefaciens levansucrase signal peptide, whose h region has an overall hydrophobicity similar to that of E. coli signal peptides, was able to direct secretion of only modest levels of MBP and OmpA. These results imply that there are differences between the secretion machineries of B. subtilis and E. coli and demonstrate that the outer membrane protein OmpA can be translocated across the cytoplasmic membrane of B. subtilis.

ATP-Binding Cassette Transporters↗
PubMed1994

Expression of Escherichia coli SecB in Bacillus subtilis facilitates secretion of the SecB-dependent maltose-binding protein of E. coli.

Less than 20% of the Escherichia coli maltose-binding protein (MBP) synthesized in Bacillus subtilis is exported. However, a portion of the secreted MBP was processed cotranslationally. Coexpression of SecB, a secretion-related chaperone of E. coli, stimulated posttranslational export of MBP in B. subtilis but inhibited its cotranslational processing. Export of a SecB-independent MBP-ribose-binding protein hybrid precursor was not enhanced by SecB. A slowly folding MBP derivative (MBP-Y283D) was more efficiently secreted than wild-type MBP, suggesting that the antifolding activity of SecB promotes posttranslational secretion of MBP in B. subtilis.

ATP-Binding Cassette Transporters↗
PubMed1990

SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins.

The efficient export of the Escherichia coli maltose-binding protein (MBP) is known to be SecB dependent, whereas ribose-binding protein (RBP) export is SecB independent. When the MBP and RBP signal peptides were exchanged precisely at the signal peptidase processing sites, the resultant RBP-MBP and MBP-RBP hybrid proteins both were efficiently exported in SecB+ cells. However, only MBP-RBP was efficiently exported in SecB- cells; RBP-MBP exhibited a significant export defect, a finding that was consistent with previous proposals that SecB specifically interacts with the mature moiety of precursor MBP to promote export. The relatively slow, totally posttranslational export mode exhibited by certain mutant RBP and MBP-RBP species in SecB+ cells was not affected by the loss of SecB. In contrast, MBP and RBP-MBP species with similarly altered signal peptides were totally export defective in SecB- cells. Both export-defective MBP and RBP-MBP interfered with SecB-mediated protein export by depleting cells of functional SecB. In contrast, neither export-defective RBP nor MBP-RBP elicited such an interference effect. These and other data indicated that SecB is unable to interact with precursor RBP or that any interaction between these two proteins is considerably weaker than that of SecB with precursor MBP. In addition, no correlation could be established between a SecB requirement for export and PrlA-mediated suppression of signal peptide export defects. Finally, previous studies have established that wild-type MBP export can be accomplished cotranslationally, whereas wild-type RBP export is strictly a posttranslational process. In this study, cotranslational export was not detected for either MBP-RBP or RBP-MBP. This indicates that the export mode exhibited by a given precursor protein (cotranslational versus posttranslational) is determined by properties of both the signal peptide and the mature moiety.

ATP-Binding Cassette Transporters↗
PubMed1989

Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein.

An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.

ATP-Binding Cassette Transporters↗
PubMed1989

Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli.

It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export.

ATP-Binding Cassette Transporters↗
PubMed1988

The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein.

Evidence is presented that the E. coli secB gene encodes a soluble protein that interacts with the mature region of the precursor maltose-binding protein (MBP), and promotes MBP export by preventing premature folding of the newly synthesized polypeptide into an export-incompetent form. The interaction of SecB with MBP was indicated by the finding that synthesis of various export-defective MBP species interfered with normal protein export by limiting SecB availability. The antifolding activity of SecB was demonstrated by the following: the defect in MBP export in SecB- cells was suppressed by mutational alterations affecting MBP folding; export of a mutant MBP that is accomplished in a strictly posttranslational mode was totally blocked in SecB- cells; and the rate of folding of wild-type MBP synthesized in vitro was found to be accelerated when SecB was absent and greatly retarded when excess SecB was present.

ATP-Binding Cassette Transporters↗
PubMed1983

Effect of dietary regimen on rotavirus-Escherichia coli weanling diarrhea of piglets.

Previously, we induced weanling diarrhea in piglets by infecting them with rotavirus followed by hemolytic enteropathogenic Escherichia coli. We postulated that rotavirus, by damaging the epithelium of the small intestines, produced an enteroenvironment which favored the selection and growth of enteropathogenic E. coli. Furthermore, diet might affect the enteroenvironment and influence the course of the disease. To test this, newly weaned 3-week-old piglets were assigned to one of four dietary regimens and infected with rotavirus followed 24 h later with enteropathogenic E. coli. The course of the disease was followed by monitoring the severity of diarrhea and the fecal shedding of rotavirus and enteropathogenic E. coli in these dually infected piglets. The dietary regimen designed to tax the digestive and absorptive capacities of the piglets (high nutrient intake fed three times a day) produced the most prolonged diarrhea, colonization of the gut by hemolytic enteropathogenic E. coli, and persistent shedding of rotavirus (P less than 0.01). The same nutrient intake divided into 24 equal increments and fed hourly produced a less severe response (P less than 0.01). The least severe response was seen in piglets fed one-third the nutrient intake either hourly or three times a day (P less than 0.01). We conclude that dietary regimen plays an important role in rotavirus-E. coli-induced weanling diarrhea.

Animals↗