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Mark Roberts - One of the best experts on this subject based on the ideXlab platform.
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The periplasmic chaperone Skp is required for successful Salmonella Typhimurium infection in a murine typhoid model
Microbiology, 2011Co-Authors: Gary Rowley, Claire Lewis, Andrew Stevenson, Bronislava Rezuchova, Dagmar Homerova, Jan Kormanec, Henrieta Skovierova, Aileen Sherry, Mark RobertsAbstract:The alternative sigma factor σ E (RpoE) is essential for survival in vivo of Salmonella Typhimurium but is dispensable during growth in the laboratory. We have been identifying σ E-regulated genes and studying their regulation and function to elucidate their potential role in the severe attenuation of S. Typhimurium RpoE mutants. In this study we identify five promoters that control the rseP, yaeT (bamA), skp region. A confirmed σ E-dependent promoter, yaeTp1, and a second downstream promoter, yaeTp2, are located within the upstream gene rseP and direct expression of the downstream genes. The only known function of RseP is σ E activation, and it is therefore not expected to be essential for S. Typhimurium in vitro. However, it proved impossible to delete the entire rseP gene due to the presence of internal promoters that regulate the essential gene yaeT. We could inactivate rseP by deleting the first third of the gene, leaving the yaeT promoters intact. Like the RpoE mutant, the rseP mutant exhibited severe attenuation in vivo. We were able to delete the entire coding sequence of skp, which encodes a periplasmic chaperone involved in targeting misfolded outer-membrane proteins to the β-barrel assembly machinery. The skp mutant was attenuated in mice after oral and parenteral infection. Virulence could be complemented by providing skp in trans but only by linking it to a heterologous σ E-regulated promoter. The reason the skp mutant is attenuated is currently enigmatic, but we know it is not through increased sensitivity to a variety of RpoE-activating host stresses, such as H2O2, polymyxin B and high temperature, or through altered secretion of effector proteins by either the Salmonella pathogenicity island (SPI)-1 or the SPI-2 type III secretion system.
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role of the alternative sigma factors sigmae and sigmas in survival of salmonella enterica serovar typhimurium during starvation refrigeration and osmotic shock
Microbiology, 2007Co-Authors: Alisdair Mcmeechan, Tristan A Cogan, Frieda Jorgensen, Gary Rowley, Claire Lewis, Andrew Stevenson, Mark Roberts, T J HumphreyAbstract:The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of σ E and σ S, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for σ E- and σ S-mediated survival, the ability of S. Typhimurium RpoE, rpoS and RpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 °C) was examined. Under these conditions, the RpoE mutant was significantly (P<0.05) compromised compared to the parent and to an rpoS mutant. The double mutant in RpoE and rpoS displayed a cumulative defect in survival. In hyperosmotic environments (low a w) containing 6 % NaCl and at refrigeration temperature, both sigma factors were important for maximum survival but σ S played the dominant role. Analysis of the metabolic activity of starved populations at 4.5 and 37 °C revealed significantly (P<0.001) elevated electron-transport system activity in mutants in RpoE and rpoS, indicating a role for σ E- and σ S-regulated genes in maintaining energy homeostasis. Together these data demonstrate that σ E and σ S are important for survival of S. Typhimurium in conditions encountered during food processing and that the relative contribution of σ E and σ S is critically dependent on the precise nature of the stress.
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Role of the alternative sigma factors sigmaE and sigmaS in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock.
Microbiology, 2007Co-Authors: Alisdair Mcmeechan, Tristan A Cogan, Frieda Jorgensen, Gary Rowley, Claire Lewis, Andrew Stevenson, Mark Roberts, T J HumphreyAbstract:The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of sigmaE and sigmaS, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for sigmaE- and sigmaS-mediated survival, the ability of S. Typhimurium RpoE, rpoS and RpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 degrees C) was examined. Under these conditions, the RpoE mutant was significantly (P
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Effect of Inactivation of degS on Salmonella enterica Serovar Typhimurium In Vitro and In Vivo
Infection and immunity, 2005Co-Authors: Gary Rowley, Andrew Stevenson, Jan Kormanec, Mark RobertsAbstract:The alternative sigma factor (RpoE σE) enables Salmonella enterica serovar Typhimurium to adapt to stressful conditions, such as oxidative stress, nutrient deprivation, and growth in mammalian tissues. Infection of mice by Salmonella serovar Typhimurium also requires σE. In Escherichia coli, activation of the σE pathway is dependent on proteolysis of the anti-sigma factor RseA and is initiated by DegS. DegS is also important in order for E. coli to cause extraintestinal infection in mice. We constructed a degS mutant of the serovar Typhimurium strain SL1344 and compared its behavior in vitro and in vivo with those of its wild-type (WT) parent and an isogenic RpoE mutant. Unlike E. coli degS strains, the Salmonella serovar Typhimurium degS strain grew as well as the WT strain at 42°C. The degS mutant survived very poorly in murine macrophages in vitro and was highly attenuated compared with the WT strain for both the oral and parenteral routes of infection in mice. However, the degS mutant was not as attenuated as the serovar Typhimurium RpoE mutant: 100- to 1,000-fold more degS bacteria than RpoE bacteria were present in the livers and spleens of mice 24 h after intraperitoneal challenge. In most assays, the RpoE mutant was more severely affected than the degS mutant and a σE-dependent reporter gene was more active in the degS mutant than the RpoE strain. These findings indicate that degS is important for activation of the σE pathway in serovar Typhimurium but that alternative pathways for σE activation probably exist.
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Transcriptional analysis of the RpoE gene encoding extracytoplasmic stress response sigma factor σE in Salmonella enterica serovar Typhimurium
FEMS microbiology letters, 2003Co-Authors: Henrieta Miticka, Gary Rowley, Mark Roberts, Bronislava Rezuchova, Dagmar Homerova, Sue Humphreys, Jaci Farn, Jan KormanecAbstract:The RpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium), which encodes the extracytoplasmic stress response sigma factor σE, is critically important for the virulence of S. Typhimurium. We analysed expression of RpoE by wild-type and mutant bacteria grown in different conditions by S1-nuclease mapping using RNA, and using in vivo reporter gene fusions. Three promoters, RpoEp1, RpoEp2 and RpoEp3, were located upstream of the S. Typhimurium RpoE gene. The promoters were differentially expressed during growth and under several stress conditions including cold shock. Expression from the RpoEp3 promoter was absent in an S. Typhimurium RpoE mutant, demonstrating its dependence upon σE. The level of mRNA corresponding to RpoEp3 was also higher in a cpxR mutant, indicating a negative regulation of the promoter by the Cpx system. Using this RpoE-dependent promoter, we optimised a two-plasmid system for identification of promoters recognised by S. Typhimurium σE. The RpoEp3 promoter was active in the Escherichia coli two-plasmid system and has an identical transcription start point as in S. Typhimurium but only after induction of S. Typhimurium RpoE expression.
Jan Kormanec - One of the best experts on this subject based on the ideXlab platform.
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The periplasmic chaperone Skp is required for successful Salmonella Typhimurium infection in a murine typhoid model
Microbiology, 2011Co-Authors: Gary Rowley, Claire Lewis, Andrew Stevenson, Bronislava Rezuchova, Dagmar Homerova, Jan Kormanec, Henrieta Skovierova, Aileen Sherry, Mark RobertsAbstract:The alternative sigma factor σ E (RpoE) is essential for survival in vivo of Salmonella Typhimurium but is dispensable during growth in the laboratory. We have been identifying σ E-regulated genes and studying their regulation and function to elucidate their potential role in the severe attenuation of S. Typhimurium RpoE mutants. In this study we identify five promoters that control the rseP, yaeT (bamA), skp region. A confirmed σ E-dependent promoter, yaeTp1, and a second downstream promoter, yaeTp2, are located within the upstream gene rseP and direct expression of the downstream genes. The only known function of RseP is σ E activation, and it is therefore not expected to be essential for S. Typhimurium in vitro. However, it proved impossible to delete the entire rseP gene due to the presence of internal promoters that regulate the essential gene yaeT. We could inactivate rseP by deleting the first third of the gene, leaving the yaeT promoters intact. Like the RpoE mutant, the rseP mutant exhibited severe attenuation in vivo. We were able to delete the entire coding sequence of skp, which encodes a periplasmic chaperone involved in targeting misfolded outer-membrane proteins to the β-barrel assembly machinery. The skp mutant was attenuated in mice after oral and parenteral infection. Virulence could be complemented by providing skp in trans but only by linking it to a heterologous σ E-regulated promoter. The reason the skp mutant is attenuated is currently enigmatic, but we know it is not through increased sensitivity to a variety of RpoE-activating host stresses, such as H2O2, polymyxin B and high temperature, or through altered secretion of effector proteins by either the Salmonella pathogenicity island (SPI)-1 or the SPI-2 type III secretion system.
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The expression of the RpoE operon is fine-tuned by the internal rseAp promoter in Salmonella enterica serovar Typhimurium
Biologia, 2010Co-Authors: Dagmar Homerova, Bronislava Rezuchova, Henrieta Skovierova, Jan KormanecAbstract:Three promoters, located upstream of the RpoE gene encoding an extracytoplasmic sigma factor σ ^E, direct expression of the RpoE operon ( RpoE, rseA, rseB, rseC ) in Salmonella enterica serovar Typhimurium ( S. Typhimurium). One of them, RpoEp3 , has been found to be directly recognized by RNA polymerase containing sigma factor σ ^E. Using the Northern blot analysis we detected a complex pattern of transcripts indicating an internal promoter in the coding region of the RpoE gene. The promoter, rseAp , has been located by S1 mapping analysis. Its proposed −10 and −35 elements with 15 intervening nucleotides exhibited high similarity with the consensus sequence of σ ^E promoters, suggesting the direct dependence of rseAp upon σ ^E. Activity of rseAp increased towards stationary phase, after heat shock, cold shock, and in the presence of artificially induced RpoE expression, the conditions previously shown to activate σ ^E-dependent promoters. In vivo experiments revealed increase of the rseAp activity during growth and confirmed its clear dependence upon σ ^E. The proposed role of the internal rseAp promoter is to facilitate a feedback control of σ ^E level after the envelope stress is removed.
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Effect of Inactivation of degS on Salmonella enterica Serovar Typhimurium In Vitro and In Vivo
Infection and immunity, 2005Co-Authors: Gary Rowley, Andrew Stevenson, Jan Kormanec, Mark RobertsAbstract:The alternative sigma factor (RpoE σE) enables Salmonella enterica serovar Typhimurium to adapt to stressful conditions, such as oxidative stress, nutrient deprivation, and growth in mammalian tissues. Infection of mice by Salmonella serovar Typhimurium also requires σE. In Escherichia coli, activation of the σE pathway is dependent on proteolysis of the anti-sigma factor RseA and is initiated by DegS. DegS is also important in order for E. coli to cause extraintestinal infection in mice. We constructed a degS mutant of the serovar Typhimurium strain SL1344 and compared its behavior in vitro and in vivo with those of its wild-type (WT) parent and an isogenic RpoE mutant. Unlike E. coli degS strains, the Salmonella serovar Typhimurium degS strain grew as well as the WT strain at 42°C. The degS mutant survived very poorly in murine macrophages in vitro and was highly attenuated compared with the WT strain for both the oral and parenteral routes of infection in mice. However, the degS mutant was not as attenuated as the serovar Typhimurium RpoE mutant: 100- to 1,000-fold more degS bacteria than RpoE bacteria were present in the livers and spleens of mice 24 h after intraperitoneal challenge. In most assays, the RpoE mutant was more severely affected than the degS mutant and a σE-dependent reporter gene was more active in the degS mutant than the RpoE strain. These findings indicate that degS is important for activation of the σE pathway in serovar Typhimurium but that alternative pathways for σE activation probably exist.
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Transcriptional analysis of the RpoE gene encoding extracytoplasmic stress response sigma factor σE in Salmonella enterica serovar Typhimurium
FEMS microbiology letters, 2003Co-Authors: Henrieta Miticka, Gary Rowley, Mark Roberts, Bronislava Rezuchova, Dagmar Homerova, Sue Humphreys, Jaci Farn, Jan KormanecAbstract:The RpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium), which encodes the extracytoplasmic stress response sigma factor σE, is critically important for the virulence of S. Typhimurium. We analysed expression of RpoE by wild-type and mutant bacteria grown in different conditions by S1-nuclease mapping using RNA, and using in vivo reporter gene fusions. Three promoters, RpoEp1, RpoEp2 and RpoEp3, were located upstream of the S. Typhimurium RpoE gene. The promoters were differentially expressed during growth and under several stress conditions including cold shock. Expression from the RpoEp3 promoter was absent in an S. Typhimurium RpoE mutant, demonstrating its dependence upon σE. The level of mRNA corresponding to RpoEp3 was also higher in a cpxR mutant, indicating a negative regulation of the promoter by the Cpx system. Using this RpoE-dependent promoter, we optimised a two-plasmid system for identification of promoters recognised by S. Typhimurium σE. The RpoEp3 promoter was active in the Escherichia coli two-plasmid system and has an identical transcription start point as in S. Typhimurium but only after induction of S. Typhimurium RpoE expression.
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New members of the Escherichia coli σE regulon identified by a two-plasmid system
FEMS microbiology letters, 2003Co-Authors: Bronislava Rezuchova, Mark Roberts, Henrieta Miticka, Dagmar Homerova, Jan KormanecAbstract:A previously established method, based on a two-plasmid system, was used to identify promoters recognized by RNA polymerase containing the extracytoplasmic stress response sigma factor σE in Escherichia coli. In addition to previously identified RpoE-dependent promoters, 11 new promoters potentially directing the expression of 15 genes were identified that were active only after over-expression of RpoE. The promoters were confirmed and transcriptional start points of the promoters were determined by primer extension analysis and S1-nuclease mapping. All the promoters contained sequences similar to the consensus sequence of RpoE-dependent promoters. The new RpoE-dependent promoters governed expression of genes encoding proteins involved in primary metabolism (fusA, tufA, recR), phospholipid and lipopolysaccharide biosynthesis (psd, lpxP), signal transduction (sixA), proposed inner or outer membrane proteins (bacA, sbmA, smpA, yeaY), and proteins with unknown function (ybaB, yaiW, yiiS, yiiT, yfeY).
Shwu-jen Liaw - One of the best experts on this subject based on the ideXlab platform.
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New Aspects of RpoE in Uropathogenic Proteus mirabilis
Infection and immunity, 2014Co-Authors: Ming Che Liu, Kuan-ting Kuo, Hsiung-fei Chien, Yi-lin Tsai, Shwu-jen LiawAbstract:Proteus mirabilis is a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms for P. mirabilis to establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulating mrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted from RpoE mutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness of P. mirabilis in the host, including the avoidance of immune attacks. Accordingly, RpoE mutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and the RpoE mutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression of RpoE by the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness of P. mirabilis, to build up a UTI.
Gary Rowley - One of the best experts on this subject based on the ideXlab platform.
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The periplasmic chaperone Skp is required for successful Salmonella Typhimurium infection in a murine typhoid model
Microbiology, 2011Co-Authors: Gary Rowley, Claire Lewis, Andrew Stevenson, Bronislava Rezuchova, Dagmar Homerova, Jan Kormanec, Henrieta Skovierova, Aileen Sherry, Mark RobertsAbstract:The alternative sigma factor σ E (RpoE) is essential for survival in vivo of Salmonella Typhimurium but is dispensable during growth in the laboratory. We have been identifying σ E-regulated genes and studying their regulation and function to elucidate their potential role in the severe attenuation of S. Typhimurium RpoE mutants. In this study we identify five promoters that control the rseP, yaeT (bamA), skp region. A confirmed σ E-dependent promoter, yaeTp1, and a second downstream promoter, yaeTp2, are located within the upstream gene rseP and direct expression of the downstream genes. The only known function of RseP is σ E activation, and it is therefore not expected to be essential for S. Typhimurium in vitro. However, it proved impossible to delete the entire rseP gene due to the presence of internal promoters that regulate the essential gene yaeT. We could inactivate rseP by deleting the first third of the gene, leaving the yaeT promoters intact. Like the RpoE mutant, the rseP mutant exhibited severe attenuation in vivo. We were able to delete the entire coding sequence of skp, which encodes a periplasmic chaperone involved in targeting misfolded outer-membrane proteins to the β-barrel assembly machinery. The skp mutant was attenuated in mice after oral and parenteral infection. Virulence could be complemented by providing skp in trans but only by linking it to a heterologous σ E-regulated promoter. The reason the skp mutant is attenuated is currently enigmatic, but we know it is not through increased sensitivity to a variety of RpoE-activating host stresses, such as H2O2, polymyxin B and high temperature, or through altered secretion of effector proteins by either the Salmonella pathogenicity island (SPI)-1 or the SPI-2 type III secretion system.
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role of the alternative sigma factors sigmae and sigmas in survival of salmonella enterica serovar typhimurium during starvation refrigeration and osmotic shock
Microbiology, 2007Co-Authors: Alisdair Mcmeechan, Tristan A Cogan, Frieda Jorgensen, Gary Rowley, Claire Lewis, Andrew Stevenson, Mark Roberts, T J HumphreyAbstract:The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of σ E and σ S, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for σ E- and σ S-mediated survival, the ability of S. Typhimurium RpoE, rpoS and RpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 °C) was examined. Under these conditions, the RpoE mutant was significantly (P<0.05) compromised compared to the parent and to an rpoS mutant. The double mutant in RpoE and rpoS displayed a cumulative defect in survival. In hyperosmotic environments (low a w) containing 6 % NaCl and at refrigeration temperature, both sigma factors were important for maximum survival but σ S played the dominant role. Analysis of the metabolic activity of starved populations at 4.5 and 37 °C revealed significantly (P<0.001) elevated electron-transport system activity in mutants in RpoE and rpoS, indicating a role for σ E- and σ S-regulated genes in maintaining energy homeostasis. Together these data demonstrate that σ E and σ S are important for survival of S. Typhimurium in conditions encountered during food processing and that the relative contribution of σ E and σ S is critically dependent on the precise nature of the stress.
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Role of the alternative sigma factors sigmaE and sigmaS in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock.
Microbiology, 2007Co-Authors: Alisdair Mcmeechan, Tristan A Cogan, Frieda Jorgensen, Gary Rowley, Claire Lewis, Andrew Stevenson, Mark Roberts, T J HumphreyAbstract:The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of sigmaE and sigmaS, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for sigmaE- and sigmaS-mediated survival, the ability of S. Typhimurium RpoE, rpoS and RpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 degrees C) was examined. Under these conditions, the RpoE mutant was significantly (P
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Effect of Inactivation of degS on Salmonella enterica Serovar Typhimurium In Vitro and In Vivo
Infection and immunity, 2005Co-Authors: Gary Rowley, Andrew Stevenson, Jan Kormanec, Mark RobertsAbstract:The alternative sigma factor (RpoE σE) enables Salmonella enterica serovar Typhimurium to adapt to stressful conditions, such as oxidative stress, nutrient deprivation, and growth in mammalian tissues. Infection of mice by Salmonella serovar Typhimurium also requires σE. In Escherichia coli, activation of the σE pathway is dependent on proteolysis of the anti-sigma factor RseA and is initiated by DegS. DegS is also important in order for E. coli to cause extraintestinal infection in mice. We constructed a degS mutant of the serovar Typhimurium strain SL1344 and compared its behavior in vitro and in vivo with those of its wild-type (WT) parent and an isogenic RpoE mutant. Unlike E. coli degS strains, the Salmonella serovar Typhimurium degS strain grew as well as the WT strain at 42°C. The degS mutant survived very poorly in murine macrophages in vitro and was highly attenuated compared with the WT strain for both the oral and parenteral routes of infection in mice. However, the degS mutant was not as attenuated as the serovar Typhimurium RpoE mutant: 100- to 1,000-fold more degS bacteria than RpoE bacteria were present in the livers and spleens of mice 24 h after intraperitoneal challenge. In most assays, the RpoE mutant was more severely affected than the degS mutant and a σE-dependent reporter gene was more active in the degS mutant than the RpoE strain. These findings indicate that degS is important for activation of the σE pathway in serovar Typhimurium but that alternative pathways for σE activation probably exist.
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Characterisation of the S. Typhimurium σE regulon
2005Co-Authors: Gary RowleyAbstract:The extracytoplasmic stress response (ESR) of the Enterobacteriacea consists mainly of two partially overlapping pathways, one regulated by the alternative sigma factor, sigmaE encoded for by RpoE and the other by the two component regulator cpxRA. This thesis mainly focuses on the regulated arm of the ESR but does discuss potential crosstalk between these two systems. Characterisation of the RpoE regulon in E. coli has been performed using a number of methods, each of which identified different genes (Dartigalongue, Missiakas, and Raina, 2001;Rezuchova et al., 2003). RpoE has been described as essential for survival of E. coli laboratory strains (De Las, Connolly, and Gross, 1997a), but deletion of the RpoE structural gene in S. Typhimurium is not lethal (Humphreys et al, 1999). However the S. Typhimurium RpoE mutation is critical for survival in vivo, as it appears to be for a number of pathogenic bacteria (Humphreys et al, 1999;Hild et al., 2000;Craig, Nobbs, and High, 2002;Heungens, Cowles, and Goodrich-Blair, 2002;Kovacikova and Skorupski, 2002;Testerman et al, 2002;Heusipp, Schmidt, and Miller, 2003). Considering the different locations and microenvironments encountered by these different pathogens it is likely that each of the RpoE regulons may contain different genes. To establish which are important for S. Typhimurium our collaborators and I have utilised a number of techniques to identify RpoE regulated genes. In collaboration with the group of Dr. Jan Kormanec, Slovakia, a two plasmid system has been utilised. We have also been given access to the S. Typhimurium microarrays at the Institute of Food Research, and used a promoter consensus search derived from known sigmaE regulated genes to search the S. Typhimurium genome for putatively sigmaE regulated genes. With a combination of these techniques we have identified a number of putatively sigmaE regulated genes, and have selected a portion of these to be mutated with lambda red mutagenesis and characterised both in vitro and in vivo.
Sunee Korbsrisate - One of the best experts on this subject based on the ideXlab platform.
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The RpoE operon regulates heat stress response in Burkholderia pseudomallei.
FEMS microbiology letters, 2008Co-Authors: Muthita Vanaporn, Visith Thongboonkerd, Paiboon Vattanaviboon, Sunee KorbsrisateAbstract:Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei RpoE locus in the response to heat stress. The RpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter- lacZ fusion revealed that heat stress induction of rpoH , which encodes heat shock sigma factor (σH), was abolished in the B. pseudomallei RpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of σE-dependent promoters. Moreover, the putative heat-induced σH-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the RpoE operon mutant. Altogether, our data suggest that the RpoE operon regulates B. pseudomallei heat stress response through the function of rpoH .
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Altered proteome in Burkholderia pseudomallei RpoE operon knockout mutant: insights into mechanisms of RpoE operon in stress tolerance, survival, and virulence.
Journal of proteome research, 2007Co-Authors: Visith Thongboonkerd, Muthita Vanaporn, Napat Songtawee, Rattiyaporn Kanlaya, Supachok Sinchaikul, Shui-tein Chen, Anna Easton, Karen Chu, Gregory J. Bancroft, Sunee KorbsrisateAbstract:We have previously shown that the alternative sigma factor U E (RpoE), encoded by RpoE, is involved in stress tolerance and survival of Burkholderia pseudomallei. However, its molecular and pathogenic mechanisms remain unclear. In the present study, we applied gel-based, differential proteomics to compare the cellular proteome of an RpoE operon knockout mutant (RpoE Mut) to that of wild-type (K96243 WT) B. pseudomallei. Quantitative intensity analysis (n ) 5 gels from 5 individual culture flasks in each group) revealed significantly differential expression of 52 proteins, which were subsequently identified by Q-TOF MS/MS. These included oxidative, osmotic, and other stress response proteins; chaperones; transcriptional/translational regulators; metabolic enzymes; proteins involved in cell wall synthesis, fatty synthesis, glycogen synthesis, and storage; exported proteins; secreted proteins; adhesion molecule; protease/peptidase; protease inhibitor; signaling proteins; and other miscellaneous proteins. The down-regulation of several stress response proteins, chaperones, transcriptional/ translational regulators, and proteins involved in cell wall synthesis in RpoE Mut provided some new insights into the mechanisms of the RpoE operon for the stress tolerance and survival of B. pseudomallei. In addition, the proteomic data and in vivo study indicated that the RpoE operon is also involved in the virulence of B. pseudomallei. Our findings underscore the usefulness of proteomics for unraveling pathogenic mechanisms of diseases at the molecular level.
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The Burkholderia pseudomallei RpoE (AlgU) operon is involved in environmental stress tolerance and biofilm formation.
FEMS microbiology letters, 2005Co-Authors: Sunee Korbsrisate, Muthita Vanaporn, Paiboon Vattanaviboon, Phansupa Kerdsuk, Wannapa Kespichayawattana, Pornpimon Kiatpapan, Ganjana LertmemongkolchaiAbstract:Burkholderia pseudomallei, the causative agent of melioidosis, can be isolated from soil and water. To persist, adapt and survive within and outside their human host, bacteria rely on regulatory mechanisms that allow them to respond rapidly to stressful situations. We have examined the possible role of B. pseudomallei alternative sigma factor σE (RpoE) in the stress response and found that RpoE and its putative regulators (bprE-rseB-mucD) are transcribed in a single transcriptional unit. Inactivation of the RpoE operon changed the B. pseudomallei phenotype. Changes included increased susceptibility to killing by menadione and H2O2, susceptibility to high osmolarity, reduced ability to form biofilms, and reduced survival in macrophage J774A.1. Therefore, we conclude that RpoE controls gene expression that contributes, at least in part, to B. pseudomallei adaptation to adverse environmental conditions.
