| 1. |
- Balsalobre, Carlos, et al.
(författare)
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Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli.
- 2006
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Ingår i: J Bacteriol. - 0021-9193. ; 188:16, s. 5935-44
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Tidskriftsartikel (refereegranskat)abstract
- We have found that the cyclic AMP (cAMP) receptor protein (CRP)-cAMP regulatory complex in Escherichia coli is subject to osmoregulation at the level of crp gene expression. This osmoregulation was lost in a cya mutant strain but could be restored by external addition of cAMP, suggesting that the intracellular level of cAMP is a key factor in the osmoregulation of CRP. The ability of the cell to maintain optimal CRP activity was essential for the growth and survival of the bacteria under low-osmolarity conditions as shown by studies with different crp mutant alleles. A suppressor mutant with a novel amino acid substitution (L124R) in CRP showed restored growth at low osmolarity. CRP(L124R) was not activated by cAMP and was shown to be dominant negative over the wild type. Our findings suggest that the fine-tuning of the CRP activity may be critical for bacterial viability and adaptability to changing osmotic conditions.
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| 2. |
- Balsalobre, Carlos, et al.
(författare)
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Release of the type I secreted α-haemolysin via outer membrane vesicles from Escherichia coli
- 2006
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Ingår i: Molecular Microbiology. - Hoboken, NJ, United States : Wiley-Blackwell. - 0950-382X .- 1365-2958. ; 59:1, s. 99-112
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Tidskriftsartikel (refereegranskat)abstract
- The α-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the α-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The α-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The α-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the α-haemolysin were distinct from the OMVs not containing α-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of α-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted α-haemolysin from the bacteria to the host cells during bacterial infections.
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| 3. |
- Forns, Núria, et al.
(författare)
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Temperature-dependent conjugative transfer of R27 : Role of chromosome- and plasmid-encoded Hha and H-NS proteins
- 2005
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Ingår i: Journal of Bacteriology. - Washington : American society of microbiology. - 0021-9193 .- 1098-5530. ; 187:12, s. 3950-3959
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Tidskriftsartikel (refereegranskat)abstract
- IncHI plasmids encode multiple-antibiotic resistance in Salmonella enterica serovar Typhi. These plasmids have been considered to play a relevant role in the persistence and reemergence of this microorganism. The IncHII plasmid R27, which can be considered the prototype of IncHI plasmids, is thermosensitive for transfer. Conjugation frequency is highest at low temperature (25 to 30 degrees C), decreasing when temperature increases. R27 codifies an H-NS-like protein (open reading frame 164 [ORF164]) and an Hha-like protein (ORF182). The H-NS and Hha proteins participate in the thermoregulation of gene expression in Escherichia coli. Here we investigated the hypothetical role of such proteins in thermoregulation of R27 conjugation. At a nonpermissive temperature (33 degrees C), transcription of several ORFs in both transfer region I (Tra1) and Tra2 from R27 is upregulated in cells depleted of Hha-like and H-NS-like proteins. Both chromosome- and plasmid-encoded Hha and H-NS proteins appear to potentially modulate R27 transfer. The function of R27-encoded Hha-like and H-NS proteins is not restricted to modulation of R27 transfer. Different mutant phenotypes associated with both chromosomal hha and hns mutations are compensated in cells harboring R27.
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| 4. |
- Müller, Claudia M, et al.
(författare)
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Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP.
- 2009
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Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 5:2, s. e1000303-
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Tidskriftsartikel (refereegranskat)abstract
- Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.
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| 5. |
- Sjöström, Annika E., 1963-, et al.
(författare)
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The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression
- 2009
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Ingår i: Microbial Pathogenesis. - : Elsevier Ltd. - 0882-4010 .- 1096-1208. ; 46:5, s. 243-252
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Tidskriftsartikel (refereegranskat)abstract
- The genomes of pathogenic E. coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaXII gene associated to the SfaII fimbrial determinant in the E. coli isolate IHE3034. sfaXII belongs to a subfamily of genes, the 17kDa genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaXII gene had an inhibitory effect on type 1 fimbriae expression. Down regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaXII on expression of the recombinase FimB that catalyzes OFF to ON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaXII gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaXII gene, and presumably other members in the 17kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli.
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| 6. |
- Åberg, Anna, 1977-
(författare)
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New insights into the role of ppGpp and DksA through their effect on transcriptional regulation of housekeeping and colonization related genes of Escherichia coli
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria have the ability to sense different environmental signals. When an environmental stress is detected, bacteria rapidly adjust their gene expression profile to be able to survive and thrive. The transduction of such environmental signals often requires the coordinated involvement of several factors that constitute complex regulatory networks. Hence, depending on the combination of signals, a unique gene expression profile required to adapt to the specific stress conditions is generated. Proteins are the best-known regulatory factors. However, non-proteinaceous molecules are also important in signal-responsive control of bacterial gene expression. Alarmones are low molecular weight non-proteinaceous regulatory factors which can characteristically be rapidly turned-over to mediate instant changes in gene expression. One such alarmone is the modified nucleotide ppGpp, which directly binds to RNA polymerase to alter its activity. The levels of this alarmone are expected to rapidly increase in response to any environmental stress that result in slow proliferation. DksA, a putative ppGpp co-regulator that likewise directly targets RNA polymerase, has been suggested to be required for both the positive and negative regulation mediated by ppGpp in Escherichia coli. This thesis describes dissection of the role of ppGpp and DksA on transcriptional regulation, primarily using the fim genetic determinant that encodes for the type 1 fimbriae. Type 1 fimbriae are involved in adhesion to abiotic surface and initial adhesion/invasion of bladder cells, as well as in biofilm formation. We found that ppGpp regulates phase variation by increasing the sub-population of cells that express the fimbriae. The effect of ppGpp was ultimately traced to its role in transcription of the fimB gene that encodes a recombinase involved in the phase variation process (paper 1). In contrast, we unexpectedly found that lack of DksA causes an increase, rather than a decrease, in transcription from the fimB P2 promoter in vivo. However, in vitro transcription studies demonstrated that ppGpp and DksA, both independently and co-dependently, stimulate transcription from the fimB P2 promoter. These seemingly contradictory results from the in vivo and in vitro transcriptional studies were shown to be, at least in part, a consequence of the increased association of Gre-factors with RNA polymerase that can occur in the absence of DksA in vivo (paper 2). The results outlined above have implications for the role of ppGpp and/or DksA in global gene expression. Using gene expression profile (microarray analysis) during the transition from logarithmic to stationary phase of E. coli, we found that while most of the genes regulated by ppGpp and DksA are regulated in the same direction by the two factors, many were not. In addition to the fim genes, genes involved in flagella functioning, taxis responses, and a few genes encoding different transport systems are also differentially regulated in ppGpp- and DksA-deficient strains in vivo. Our results clearly indicate that the effect of deficiencies in ppGpp and DksA is far more complex than phenotypic similarity of the corresponding mutants anticipated by the proposed concerted action of ppGpp and DksA on gene expression (paper 2 & 3).
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| 7. |
- Åberg, Anna, et al.
(författare)
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(p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB.
- 2006
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 60:6, s. 1520-1533
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Tidskriftsartikel (refereegranskat)abstract
- In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp-deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp(0) suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp(0) strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp-stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.
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| 8. |
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| 9. |
- Åberg, Anna, et al.
(författare)
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Similar and divergent effects of ppGpp and DksA deficiencies on transcription in Escherichia coli
- 2009
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 191:10, s. 3226-3236
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Tidskriftsartikel (refereegranskat)abstract
- The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp(0)) and/or DksA (DeltadksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp(0) DeltadksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.
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