| 51. |
- Aldick, Thomas, et al.
(författare)
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Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin
- 2009
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Ingår i: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 71:6, s. 1496-508
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Tidskriftsartikel (refereegranskat)abstract
- Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.
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| 52. |
- Andersson, Dan I., et al.
(författare)
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Biological roles of translesion synthesis DNA polymerases in eubacteria
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:3, s. 540-548
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Forskningsöversikt (refereegranskat)abstract
- Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.
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| 53. |
- Bagchi, Sonchita, et al.
(författare)
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Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces
- 2008
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:4, s. 1037-1050
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Tidskriftsartikel (refereegranskat)abstract
- Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.
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| 54. |
- 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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| 55. |
- Bernander, Rolf
(författare)
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The cell cycle of Sulfolobus
- 2007
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 66:3, s. 557-562
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Forskningsöversikt (refereegranskat)abstract
- Much of the current information about the archaeal cell cycle has been generated through studies of the genus Sulfolobus. The overall organization of the cell cycle in these species is well understood, and information about the regulatory principles that govern cell cycle progression is rapidly accumulating. Exciting progress regarding the control and molecular details of the chromosome replication process is evident, and the first insights into the elusive crenarchaeal mitosis and cytokinesis machineries are within reach.
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| 56. |
- Bernardo, Lisandro M D, et al.
(författare)
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The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription
- 2006
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 60:3, s. 749-764
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Tidskriftsartikel (refereegranskat)abstract
- The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.
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| 57. |
- Bilsland, Elizabeth, 1973, et al.
(författare)
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Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance
- 2004
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 53:6, s. 1743-56
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a role in oxidative and metal stress resistance for the MAPK-activated protein kinases Rck1 and Rck2 in Saccharomyces cerevisiae. We show that Hog1 is robustly phosphorylated in a Pbs2-dependent way during oxidative stress, and that Rck2 also is phosphorylated under these circumstances. Hog1 concentrates in the nucleus in oxidative stress. Hog1 localization is partially dependent on Rck2, as rck2 cells have more nuclear Hog1 than wild-type cells. We find several proteins with a role in oxidative stress resistance using Rck1 or Rck2 as baits in a two-hybrid screen. We identify the transcription factor Yap2 as a putative target for Rck1, and the Zn2+ transporter Zrc1 as a target for Rck2. Yap2 is normally cytoplasmic, but rapidly migrates to the nucleus upon exposure to oxidative stress agents. In a fraction of untreated pbs2 cells, Yap2 is nuclear. Zrc1 co-immunoprecipitates with Rck2, and ZRC1 is genetically downstream of RCK2. These data connect activation of the Hog1 MAPK cascade with effectors having a role in oxidative stress resistance.
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| 58. |
- Björkman, Johanna, et al.
(författare)
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Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium
- 1999
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Ingår i: Molecular Microbiology. - : BLACKWELL SCIENCE LTD. - 0950-382X .- 1365-2958. ; 31:1, s. 53-58
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.
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| 59. |
- Bouet, Jean-Yves, et al.
(författare)
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Plasmid partition and incompatibility : the focus shifts
- 2007
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 65:6, s. 1405-1414
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Forskningsöversikt (refereegranskat)abstract
- The mitotic apparatus that a plasmid uses to ensure its stable inheritance responds to the appearance of an additional copy of the plasmid's centromere by segregating it from the pre-existing copies: if the new copy arises by replication of the plasmid the result is partition, if it arrives on a different plasmid the result is incompatibility. Incompatibility thus serves as a probe of the partition mechanism. Coupling of distinct plasmids via their shared centromeres to form mixed pairs has been the favoured explanation for centromere-based incompatibility, because it supports a long-standing assumption that pairing of plasmid replicas is a prerequisite for their partition into daughter cells. Recent results from molecular genetic and fluorescence microscopy studies challenge this mixed pairing model. Partition incompatibility is seen to result from various processes, including titration, randomized positioning and a form of mixed pairing that is based on co-activation of the same partition event rather than direct contact between partition complexes. The perspectives thus opened onto the partition mechanism confirm the continuing utility of incompatibility as an approach to understanding bacterial mitosis. The results considered are compatible with the view that direct pairing of plasmids is not essential to plasmid partition.
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| 60. |
- Brandis, Gerrit, 1985-, et al.
(författare)
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Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
- 2012
-
Ingår i: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 85:1, s. 142-151
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant.
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