Annick Jacq - Academia.edu (original) (raw)

Papers by Annick Jacq

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion... more Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (priA) were characterized. The prlU1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39°C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23°C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15°C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 3rC, at which about 20% sequestration was attained. At 15°C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.

PLOS Genetics, 2009

The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that d... more The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signalinducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.

PLOS One, 2011

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affec... more The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600 T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.

Applied and Environmental Microbiology, 2008

DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be in... more DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924 Vt , which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlA Vt but not duf924 Vt . These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model.

PLOS Genetics, 2009

The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that d... more The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signalinducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.

Biochimica Et Biophysica Acta-biomembranes, 2008

The results here show for the first time that pH and monovalent cations can regulate cytosolic fr... more The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca 2+ in E. coli through Ca 2+ influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca 2+ was 0.2-0.5 µM. In the presence of external Ca 2+ (1 mM) at alkaline pH this rose to 4 µM, being reduced to 0.9 µM at acid pH. Removal of external Ca 2+ caused an immediate decrease in cytosolic free Ca 2+ at 50-100nM s − 1 . Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca 2+ /H + exchanger, appeared not to be a major Ca 2+ -efflux pathway. In the absence of added Na + , but with 1 mM external Ca 2+ , cytosolic free Ca 2+ rose to approximately 10 µM. The addition of Na + (half maximum 60 mM) largely blocked this increase and immediately stimulated Ca 2+ efflux. However, this effect was not specific, since K + also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca 2+ efflux. The results were consistent with H + and monovalent cations competing with Ca 2+ for a non-selective ion influx channel. Ca 2+ entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca 2+ in Escherichia coli. The number of Ca 2+ ions calculated to move per cell per second ranged from b 1 to 100, depending on conditions. Yet a single eukaryote Ca 2+ channel, conductance 100 pS, should conduct N 6 million ions per second. This raises fundamental questions about the nature and regulation of Ca 2+ transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

Journal of Bacteriology, 2002

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia ... more The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

Biochimica Et Biophysica Acta-biomembranes, 2006

Verapamil is used clinically as a Ca 2+ channel inhibitor for the treatment of various disorders ... more Verapamil is used clinically as a Ca 2+ channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia.

Molecular Genetics and Genomics, 2003

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escher... more DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

Molecular Microbiology, 1997

DjlA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the... more DjlA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N-terminus. The overproduction of DjlA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two-component signal transduction pathway. We now show that both the TMD and the J-region are essential for the induction of cps expression observed with the overproduction of DjlA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjlA. These mutations were shown not to affect the localization, stability or topology of the mutant DjlA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjlA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

Molecular and General Genetics, 1998

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants... more In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required. the full-length, wild-type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Biochimie, 1999

Please cite this article in press as: Campbell, A.K., et al., Bacterial metabolic 'toxins': A new... more Please cite this article in press as: Campbell, A.K., et al., Bacterial metabolic 'toxins': A new mechanism for lactose and food intolerance, and irritable bowel syndrome. Toxicology (2010), a b s t r a c t Lactose and food intolerance cause a wide range of gut and systemic symptoms, including gas, gut pain, diarrhoea or constipation, severe headaches, severe fatigue, loss of cognitive functions such as concentration, memory and reasoning, muscle and joint pain, heart palpitations, and a variety of allergies . These can be explained by the production of toxic metabolites from gut bacteria, as a result of anaerobic digestion of carbohydrates and other foods, not absorbed in the small intestine. These metabolites include alcohols, diols such as butan 2,3 diol, ketones, acids, and aldehydes such as methylglyoxal ). These 'toxins' induce calcium signals in bacteria and affect their growth, thereby acting to modify the balance of microflora in the gut . These bacterial 'toxins' also affect signalling mechanisms in cells around the body, thereby explaining the wide range of symptoms in people with food intolerance. This new mechanism also explains the most common referral to gastroenterologists, irritable bowel syndrome (IBS), and the illness that afflicted Charles Darwin for 50 years (Campbell and Matthews, 2005a,b). We propose it will lead to a new understanding of the molecular mechanism of type 2 diabetes and some cancers.

Molecular and General Genetics, 1998

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants... more In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required, the full-length, wild- type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Molecular Microbiology, 1994

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors vera... more Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross-reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca2+-binding protein from Saccharo-polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.

Molecular Microbiology, 1996

We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J-region motif,... more We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J-region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N-out, C-in). Thus, DjlA appears to present an extremely short N-terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C-terminal J-region. Analysis of the TMD of DjIA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four-residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10-fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell-envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two-component signal-transduction systems.

Molecular and General Genetics, 1983

The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sit... more The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is locatd in the minimum oriC (35–270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3′OH-5′P in the direction of the E. coli genetic map is recognized, at the second site the 5′P-3′OH strand.

European Journal of Biochemistry, 1980

After extensive dialysis of Escherichia coli membranes treated with Triton X-100, three membrane ... more After extensive dialysis of Escherichia coli membranes treated with Triton X-100, three membrane proteins (A, B and B') with an affinity for DNA have been isolated and purified. They bind to either double-stranded or single-stranded DNA. A and B' proteins preferentially attach to DNA even in the presence of poly(uridy1ic acid). Only protein B' can recognize some base sequence of DNA because pulse-labelled DNA made at the initiation of replication in a synchronized dnaC mutant has been selectively retained by the protein.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion... more Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (priA) were characterized. The prlU1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39°C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23°C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15°C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 3rC, at which about 20% sequestration was attained. At 15°C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.

PLOS Genetics, 2009

The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that d... more The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signalinducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.

PLOS One, 2011

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affec... more The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600 T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.

Applied and Environmental Microbiology, 2008

DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be in... more DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924 Vt , which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlA Vt but not duf924 Vt . These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model.

PLOS Genetics, 2009

The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that d... more The Bae, Cpx, Psp, Rcs, and s E pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signalinducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response.

Biochimica Et Biophysica Acta-biomembranes, 2008

The results here show for the first time that pH and monovalent cations can regulate cytosolic fr... more The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca 2+ in E. coli through Ca 2+ influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca 2+ was 0.2-0.5 µM. In the presence of external Ca 2+ (1 mM) at alkaline pH this rose to 4 µM, being reduced to 0.9 µM at acid pH. Removal of external Ca 2+ caused an immediate decrease in cytosolic free Ca 2+ at 50-100nM s − 1 . Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca 2+ /H + exchanger, appeared not to be a major Ca 2+ -efflux pathway. In the absence of added Na + , but with 1 mM external Ca 2+ , cytosolic free Ca 2+ rose to approximately 10 µM. The addition of Na + (half maximum 60 mM) largely blocked this increase and immediately stimulated Ca 2+ efflux. However, this effect was not specific, since K + also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca 2+ efflux. The results were consistent with H + and monovalent cations competing with Ca 2+ for a non-selective ion influx channel. Ca 2+ entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca 2+ in Escherichia coli. The number of Ca 2+ ions calculated to move per cell per second ranged from b 1 to 100, depending on conditions. Yet a single eukaryote Ca 2+ channel, conductance 100 pS, should conduct N 6 million ions per second. This raises fundamental questions about the nature and regulation of Ca 2+ transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

Journal of Bacteriology, 2002

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia ... more The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

Biochimica Et Biophysica Acta-biomembranes, 2006

Verapamil is used clinically as a Ca 2+ channel inhibitor for the treatment of various disorders ... more Verapamil is used clinically as a Ca 2+ channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia.

Molecular Genetics and Genomics, 2003

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escher... more DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

Molecular Microbiology, 1997

DjlA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the... more DjlA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N-terminus. The overproduction of DjlA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two-component signal transduction pathway. We now show that both the TMD and the J-region are essential for the induction of cps expression observed with the overproduction of DjlA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjlA. These mutations were shown not to affect the localization, stability or topology of the mutant DjlA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjlA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

Molecular and General Genetics, 1998

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants... more In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required. the full-length, wild-type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Biochimie, 1999

Please cite this article in press as: Campbell, A.K., et al., Bacterial metabolic 'toxins': A new... more Please cite this article in press as: Campbell, A.K., et al., Bacterial metabolic 'toxins': A new mechanism for lactose and food intolerance, and irritable bowel syndrome. Toxicology (2010), a b s t r a c t Lactose and food intolerance cause a wide range of gut and systemic symptoms, including gas, gut pain, diarrhoea or constipation, severe headaches, severe fatigue, loss of cognitive functions such as concentration, memory and reasoning, muscle and joint pain, heart palpitations, and a variety of allergies . These can be explained by the production of toxic metabolites from gut bacteria, as a result of anaerobic digestion of carbohydrates and other foods, not absorbed in the small intestine. These metabolites include alcohols, diols such as butan 2,3 diol, ketones, acids, and aldehydes such as methylglyoxal ). These 'toxins' induce calcium signals in bacteria and affect their growth, thereby acting to modify the balance of microflora in the gut . These bacterial 'toxins' also affect signalling mechanisms in cells around the body, thereby explaining the wide range of symptoms in people with food intolerance. This new mechanism also explains the most common referral to gastroenterologists, irritable bowel syndrome (IBS), and the illness that afflicted Charles Darwin for 50 years (Campbell and Matthews, 2005a,b). We propose it will lead to a new understanding of the molecular mechanism of type 2 diabetes and some cancers.

Molecular and General Genetics, 1998

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants... more In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required, the full-length, wild- type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Molecular Microbiology, 1994

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors vera... more Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross-reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca2+-binding protein from Saccharo-polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.

Molecular Microbiology, 1996

We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J-region motif,... more We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J-region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N-out, C-in). Thus, DjlA appears to present an extremely short N-terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C-terminal J-region. Analysis of the TMD of DjIA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four-residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10-fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell-envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two-component signal-transduction systems.

Molecular and General Genetics, 1983

The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sit... more The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is locatd in the minimum oriC (35–270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3′OH-5′P in the direction of the E. coli genetic map is recognized, at the second site the 5′P-3′OH strand.

European Journal of Biochemistry, 1980

After extensive dialysis of Escherichia coli membranes treated with Triton X-100, three membrane ... more After extensive dialysis of Escherichia coli membranes treated with Triton X-100, three membrane proteins (A, B and B') with an affinity for DNA have been isolated and purified. They bind to either double-stranded or single-stranded DNA. A and B' proteins preferentially attach to DNA even in the presence of poly(uridy1ic acid). Only protein B' can recognize some base sequence of DNA because pulse-labelled DNA made at the initiation of replication in a synchronized dnaC mutant has been selectively retained by the protein.