In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or b... more In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for P. aeruginosa pathogenicity and M. tuberculosis survival, respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide-bond sensitive β-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development. This article is protected by copyright. All rights reserved.
Disulfide bonds influence the stability and activity of many proteins. In , the DsbA and DsbB enz... more Disulfide bonds influence the stability and activity of many proteins. In , the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the , use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a -like gene. and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The gene complements an deletion strain, restoring the oxidation of DsbA. While we have suggested that the gene linked to the gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. ...
Disulfide bonds are critical to the stability and function of many bacterial proteins. In the per... more Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli , intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restor...
Disulfide bonds contribute to protein stability, activity and folding in a variety of proteins in... more Disulfide bonds contribute to protein stability, activity and folding in a variety of proteins including many involved in bacterial virulence such as, toxins, adhesins, flagella and pili among others. Therefore, inhibitors of disulfide bond formation enzymes could have profound effects on pathogen virulence. In the Escherichia coli disulfide bond formation pathway, the periplasmic protein DsbA introduces disulfide bonds into substrates and then the cytoplasmic membrane protein DsbB reoxidizes DsbAs cysteines regenerating its activity. Thus, DsbB generates a protein disulfide bond de novo by transferring electrons to the quinone pool. We previously identified an effective pyridazinone-related inhibitor of DsbB enzymes from several gram-negative bacteria. In order to map the protein residues that are important for the interaction with this inhibitor, we randomly mutagenized by error-prone PCR the E. coli dsbB gene and selected dsbB mutants that confer resistance to this drug using two...
Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2015
Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is... more Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by...
Introduction Integral membrane proteins play essential roles in numerous cellular functions, incl... more Introduction Integral membrane proteins play essential roles in numerous cellular functions, including cell division, intra and inter-cellular signaling, and transport of macromolecules. 14 Mutations affecting structure and assembly of membrane proteins often lead to cell toxicity in microbes and pathologies such as cystic brosis and Alzheimer's disease in humans. 5,6 With the advent of modern genomics, integral membrane proteins have gained much attention. For example, they are often identi ed from genome-wide viability screens for new drug targets. Yet compared to soluble proteins, sufcient tools generally do not exist to map the interactions that are required for tertiary structure and oligomeric assembly of integral membrane proteins. In depth structural studies of several model integral membrane proteins such as the human erythrocyte protein glycophorin A (GpA) and phospholamban have dened some basic rules of membrane protein structure and assembly. Integral membrane protein
Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2011
In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide red... more In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide reductase (RNR), are maintained in the reduced state by the thioredoxin and glutathione/glutaredoxin pathways. An Escherichia coli mutant lacking both glutathione reductase and thioredoxin reductase cannot grow because RNR is disulfide bonded and nonfunctional. Here we report that suppressor mutations in the lpdA gene, which encodes the oxidative enzyme lipoamide dehydrogenase required for tricarboxylic acid (TCA) cycle functioning, restore growth to this redox-defective mutant. The suppressor mutations reduce LpdA activity, causing the accumulation of dihydrolipoamide, the reduced protein-bound form of lipoic acid. Dihydrolipoamide can then provide electrons for the reactivation of RNR through reduction of glutaredoxins. Dihydrolipoamide is oxidized in the process, restoring function to the TCA cycle. Thus, two electron transfer pathways are rewired to meet both oxidative and reductive nee...
A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of ... more A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.
Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothe... more Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematical...
... PC). [7] Disulfide Bond Formation in Periplasm Introduction of Escherichia coli ByFEDERICO KA... more ... PC). [7] Disulfide Bond Formation in Periplasm Introduction of Escherichia coli ByFEDERICO KATZEN and JON BECKWITH The formation of stable disulfide bonds in proteins takes place in specific compartments in the cell. In ...
Assembly of the division septum in bacteria is mediated by several proteins that localize to the ... more Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor...
In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-compon... more In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.
Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants res... more Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.
In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or b... more In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for P. aeruginosa pathogenicity and M. tuberculosis survival, respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide-bond sensitive β-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development. This article is protected by copyright. All rights reserved.
Disulfide bonds influence the stability and activity of many proteins. In , the DsbA and DsbB enz... more Disulfide bonds influence the stability and activity of many proteins. In , the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the , use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a -like gene. and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The gene complements an deletion strain, restoring the oxidation of DsbA. While we have suggested that the gene linked to the gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. ...
Disulfide bonds are critical to the stability and function of many bacterial proteins. In the per... more Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli , intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restor...
Disulfide bonds contribute to protein stability, activity and folding in a variety of proteins in... more Disulfide bonds contribute to protein stability, activity and folding in a variety of proteins including many involved in bacterial virulence such as, toxins, adhesins, flagella and pili among others. Therefore, inhibitors of disulfide bond formation enzymes could have profound effects on pathogen virulence. In the Escherichia coli disulfide bond formation pathway, the periplasmic protein DsbA introduces disulfide bonds into substrates and then the cytoplasmic membrane protein DsbB reoxidizes DsbAs cysteines regenerating its activity. Thus, DsbB generates a protein disulfide bond de novo by transferring electrons to the quinone pool. We previously identified an effective pyridazinone-related inhibitor of DsbB enzymes from several gram-negative bacteria. In order to map the protein residues that are important for the interaction with this inhibitor, we randomly mutagenized by error-prone PCR the E. coli dsbB gene and selected dsbB mutants that confer resistance to this drug using two...
Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2015
Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is... more Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by...
Introduction Integral membrane proteins play essential roles in numerous cellular functions, incl... more Introduction Integral membrane proteins play essential roles in numerous cellular functions, including cell division, intra and inter-cellular signaling, and transport of macromolecules. 14 Mutations affecting structure and assembly of membrane proteins often lead to cell toxicity in microbes and pathologies such as cystic brosis and Alzheimer's disease in humans. 5,6 With the advent of modern genomics, integral membrane proteins have gained much attention. For example, they are often identi ed from genome-wide viability screens for new drug targets. Yet compared to soluble proteins, sufcient tools generally do not exist to map the interactions that are required for tertiary structure and oligomeric assembly of integral membrane proteins. In depth structural studies of several model integral membrane proteins such as the human erythrocyte protein glycophorin A (GpA) and phospholamban have dened some basic rules of membrane protein structure and assembly. Integral membrane protein
Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2011
In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide red... more In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide reductase (RNR), are maintained in the reduced state by the thioredoxin and glutathione/glutaredoxin pathways. An Escherichia coli mutant lacking both glutathione reductase and thioredoxin reductase cannot grow because RNR is disulfide bonded and nonfunctional. Here we report that suppressor mutations in the lpdA gene, which encodes the oxidative enzyme lipoamide dehydrogenase required for tricarboxylic acid (TCA) cycle functioning, restore growth to this redox-defective mutant. The suppressor mutations reduce LpdA activity, causing the accumulation of dihydrolipoamide, the reduced protein-bound form of lipoic acid. Dihydrolipoamide can then provide electrons for the reactivation of RNR through reduction of glutaredoxins. Dihydrolipoamide is oxidized in the process, restoring function to the TCA cycle. Thus, two electron transfer pathways are rewired to meet both oxidative and reductive nee...
A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of ... more A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.
Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothe... more Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematical...
... PC). [7] Disulfide Bond Formation in Periplasm Introduction of Escherichia coli ByFEDERICO KA... more ... PC). [7] Disulfide Bond Formation in Periplasm Introduction of Escherichia coli ByFEDERICO KATZEN and JON BECKWITH The formation of stable disulfide bonds in proteins takes place in specific compartments in the cell. In ...
Assembly of the division septum in bacteria is mediated by several proteins that localize to the ... more Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor...
In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-compon... more In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.
Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants res... more Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.
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