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  • 1.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Åhlund, Monika
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bröms, Jeanette
    Department of Medical Countermeasures, Swedish Defense Research Agency, Division of NBC12 Defense, Umeå, Sweden.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, s. 6683-6700Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

  • 2.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Beckstette, Michael
    Heroven, Ann Kathrin
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Dersch, Petra
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 20, s. 2876-2886Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and Delta tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26 degrees C and 37 degrees C. The most significant changes in the transcriptome of the Delta tatC mutant were seen at 26 degrees C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo. This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.

  • 3.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Beckstette, Michael
    Heroven, Ann Kathrin
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dersch, Petra
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 20, s. 2876-2886Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Twin-arginine translocation (Tat) system mediates secretion of folded proteins that in bacteria, plants and archaea are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26oC and 37oC. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26oC during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper conditions as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.

  • 4. Bao, Xiaofeng
    et al.
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sturdevant, Gail L.
    Gong, Zheng
    Xu, Shuang
    Caldwell, Harlan D.
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Fan, Huizhou
    Benzylidene acylhydrazides inhibit chlamydial growth in a type III secretion- and iron chelation-independent manner2014Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 196, nr 16, s. 2989-3001Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value.

  • 5.
    Berglin, Ewa
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Edlund, Maj-Britt K.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Nyberg, Göran K.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Carlsson, Jan
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli1982Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 152, nr 1, s. 81-88Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

  • 6.
    Björnfot, Ann-Catrin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins2009Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 13, s. 4259-4267Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca(2+) conditions) and calcium-depleted medium (-Ca(2+) conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca(2+)-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca(2+) conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under -Ca(2+) conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

  • 7.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra J
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Carlsson, Katrin E
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion2005Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, nr 22, s. 7738-7752Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.

  • 8.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    A conserved α-helix essential for a type VI secretion-like system of Francisella tularensis2009Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 8, s. 2431-2446Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These include iglA and iglB, the homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33-132, necessary for efficient binding to IglB as well as for IglAB protein stability and intra-macrophage growth. In particular, residues 103-122, overlapping with a highly conserved alpha-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in yeast, but markedly impaired intra-macrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to T6S, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhimurium and Escherichia coli were also shown to interact in yeast and the interaction was dependent on the preservation of the same alpha-helix. Heterologous interactions formed between non-native IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity and the same interaction is conserved in other human pathogens.

  • 9.
    Bylund, Göran O.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lövgren, J. Mattias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wikström, P. Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant2001Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, nr 20, s. 6095-6106Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits. The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA. Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a DeltarimM mutant by increasing the expression of rbfA. Nine of these mutations were in the nusA gene, which is located upstream from rbfA in the metY-nusA-infB operon; three mutations deleted the transcriptional terminator between infB and rbfA; one was an insertion of IS2 in infB, creating a new promoter for rbfA; and one was a duplication, placing a second copy of rbfA downstream from a promoter for the yhbM gene. Two of the nusA mutations were identical, while another mutation (nusA98) was identical to a previously isolated mutation, nusA11, shown to decrease termination of transcription. The different nusA mutations were found to increase the expression of rbfA by increasing the read-through of two internal transcriptional terminators located just downstream from the metY gene and that of the internal terminator preceding rbfA. Induced expression of the nusA(+) gene from a plasmid in a nusA(+) strain decreased the read-through of the two terminators just downstream from metY, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the metY-nusA-infB operon expression is these terminators. All of the nusA mutations produced temperature-sensitive phenotypes of rimM(+) strains. The nusA gene has previously been shown to be essential at 42 degrees C and below 32 degrees C. Here, we show that nusA is also essential at 37 degrees C.

  • 10.
    Bylund, Göran O
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nord, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Lövgren, J Mattias
    Wikström, P Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Alterations in the β flap and β' dock domains of the RNA polymerase abolish NusA-mediated feedback regulation of the metY-nusA-infB operon2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 16, s. 4113-4122Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The RimM protein in Escherichia coli is important for the in vivo maturation of 30S ribosomal subunits and a ΔrimM mutant grows poorly due to assembly and translational defects. These deficiencies are suppressed partially by mutations that increase the synthesis of another assembly protein, RbfA, encoded by the metY-nusA-infB operon. Among these suppressors are mutations in nusA that impair the NusA-mediated negative-feedback regulation at internal intrinsic transcriptional terminators of the metY-nusA-infB operon. We describe here the isolation of two new mutations, one in rpoB and one in rpoC (encoding the β and β' subunits of the RNA polymerase, respectively), that increase the synthesis of RbfA by preventing NusA from stimulating termination at the internal intrinsic transcriptional terminators of the metY-nusA-infB operon. The rpoB2063 mutation changed the isoleucine in position 905 of the β flap-tip helix to a serine, while the rpoC2064 mutation duplicated positions 415 to 416 (valine-isoleucine) at the base of the β' dock domain. These findings support previously published in vitro results, which have suggested that the β flap-tip helix and β' dock domain at either side of the RNA exit tunnel mediate the binding to NusA during transcriptional pausing and termination.

  • 11. Charpentier, Emmanuelle
    et al.
    Gerbaud, G
    Courvalin, P
    Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.1999Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 181, nr 11, s. 3368-3374Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.

  • 12.
    Croxatto, Antony
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Chalker, Victoria J
    Lauritz, Johan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Jass, Jana
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hardman, Andrea
    Williams, Paul
    Cámara, Miguel
    Milton, Debra L
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    VanT, a Homologue of Vibrio harveyi LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in Vibrio anguillarum2002Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, nr 6, s. 1617-1629Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZhpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

  • 13. Delgado, Camila
    et al.
    Florez, Laura
    Lollett, Ivonne
    Lopez, Christine
    Kangeyan, Shiva
    Kumari, Hansi
    Stylianou, Marios
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Smiddy, Robert J.
    Schneper, Lisa
    Sautter, Robert T.
    Smith, David
    Szatmari, George
    Mathee, Kalai
    Pseudomonas aeruginosa Regulated Intramembrane Proteolysis: Protease MucP Can Overcome Mutations in the AlgO Periplasmic Protease To Restore Alginate Production in Nonmucoid Revertants2018Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 200, nr 16, artikkel-id e00215-18Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen Pseudomonas aeruginosa from a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the algT (algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes, algO, also known as prc or PA3257, and mucP. Interestingly, our algO mutations were complemented by both mucP and algO, leading us to delete, clone, and overexpress mucP, algO, mucE, and mucD in both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Pseudomonas aeruginosa. IMPORTANCE Infection by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

  • 14. Dunny, Gary
    et al.
    Berntsson, Ronnie Per-Arne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Enterococcal sex pheromones: evolutionary pathways to complex, two-signal systems2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 11, s. 1556-1562Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Gram-positive bacteria carry out intercellular communication using secreted peptides. Important examples of this type of communication are the enterococcal sex pheromone systems, in which the transfer of conjugative plasmids is controlled by intercellular signaling among populations of donors and recipients. This review focuses on the pheromone response system of the conjugative plasmid pCF10. The peptide pheromones regulating pCF10 transfer act by modulating the ability of the PrgX transcription factor to repress the transcription of an operon encoding conjugation functions. Many Gram-positive bacteria regulate important processes, including the production of virulence factors, biofilm formation, sporulation, and genetic exchange using peptide-mediated signaling systems. The key master regulators of these systems comprise the RRNPP (RggRap/NprR/PlcR/PrgX) family of intracellular peptide receptors; these regulators show conserved structures. While many RRNPP systems include a core module of two linked genes encoding the regulatory protein and its cognate signaling peptide, the enterococcal sex pheromone plasmids have evolved to a complex system that also recognizes a second host-encoded signaling peptide. Additional regulatory genes not found in most RRNPP systems also modulate signal production and signal import in the enterococcal pheromone plasmids. This review summarizes several structural studies that cumulatively demonstrate that the ability of three pCF10 regulatory proteins to recognize the same 7-amino-acid pheromone peptide arose by convergent evolution of unrelated proteins from different families. We also focus on the selective pressures and structure/function constraints that have driven the evolution of pCF10 from a simple, single-peptide system resembling current RRNPPs in other bacteria to the current complex inducible plasmid transfer system.

  • 15.
    Edqvist, Petra
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olsson, Jan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lavander, Moa
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Sundberg, Lena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lloyd, Scott
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System2003Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 185, nr 7, s. 2259-2266Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

  • 16. Egge-Jacobsen, Wolfgang
    et al.
    Salomonsson, Emelie Naslund
    Aas, Finn Erik
    Forslund, Anna-Lena
    Winther-Larsen, Hanne C.
    Maier, Josef
    Macellaro, Anna
    Kuoppa, Kerstin
    Oyston, Petra C. F.
    Titball, Richard W.
    Thomas, Rebecca M.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Prior, Joann L.
    Koomey, Michael
    O-Linked glycosylation of the PilA pilin protein of francisella tularensis: identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 19, s. 5487-5497Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-HexHex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.

  • 17. El Mouali, Youssef
    et al.
    Kim, Hyunhee
    Ahmad, Irfan
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Brauner, Annelie
    Liu, Ying
    Skurnik, Mikael
    Galperin, Michael Y.
    Romling, Ute
    Stand-Alone EAL Domain Proteins Form a Distinct Subclass of EAL Proteins Involved in Regulation of Cell Motility and Biofilm Formation in Enterobacteria2017Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, nr 18, artikkel-id e00179-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta-and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC. IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/CdgR-like proteins that regulate bacterial motility by binding to the flagellar master regulator, FlhDC, and are found primarily in enterobacteria. The presence of apparently inactive EAL-only proteins in the bacteria that do not express FlhD suggests the existence of additional EAL interaction partners.

  • 18. Elbir, Haitham
    et al.
    Gimenez, Gregory
    Robert, Catherine
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Cutler, Sally
    Raoult, Didier
    Drancourt, Michel
    Complete genome sequence of Borrelia crocidurae2012Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 194, nr 14, s. 3723-3724Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We announce the draft genome sequence of Borrelia crocidurae (strain Achema). The 1,557,560-bp genome (27% GC content) comprises one 919,477-bp linear chromosome and 638,083-bp plasmids that together carry 1,472 open reading frames, 32 tRNAs, and three complete rRNAs, with almost complete colinearity between B. crocidurae and Borrelia duttonii chromosomes.

  • 19.
    Engström, Patrik
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nguyen, Bidong D.
    Normark, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Ingela
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bastidas, Robert J.
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Elofsson, Mikael
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Fields, Kenneth A.
    Valdivia, Raphael H.
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity2013Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 195, nr 18, s. 4221-4230Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.

  • 20. Fernández, S
    et al.
    Shingler, V
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    De Lorenzo, V
    Cross-regulation by XylR and DmpR activators of Pseudomonas putida suggests that transcriptional control of biodegradative operons evolves independently of catabolic genes.1994Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, nr 16Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Pu promoter of the toluene degradation plasmid pWW0 of Pseudomonas putida drives expression of an operon involved in the sequential oxidation of toluene and m- and p-xylenes to benzoate and toluates, respectively. Similarly, the Po promoter of plasmid pVI150 controls expression of an operon of Pseudomonas sp. strain CF600 which is required for the complete catabolism of phenol and cresols. These promoters, which both belong to the sigma 54-dependent class, are regulated by their cognate activators, XylR and DmpR, respectively. XylR and DmpR are homologous proteins, and both require aromatic compounds as effector molecules for activity. However, these two proteins respond to different profiles of aromatic compounds. The activity of each promoter in the presence of the heterologous regulator was monitored using lacZ and luxAB reporter systems. Genetic evidence is presented that the two activators can functionally substitute each other in the regulation of their corresponding promoters by binding the same upstream DNA segment. Furthermore, when coexpressed, the two proteins appear to act simultaneously on each of the promoters, expanding the responsiveness of these systems to the presence of effectors of both proteins. Potential mechanisms for the occurrence of evolutionary divergence between XylR and DmpR are discussed in view of the DNA sequence similarities among Pu, Po, and a third XylR-responsive promoter, Ps.

  • 21. Forns, Núria
    et al.
    Baños, Rosa C
    Balsalobre, Carlos
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Juárez, Antonio
    Madrid, Cristina
    Temperature-dependent conjugative transfer of R27: Role of chromosome- and plasmid-encoded Hha and H-NS proteins2005Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, nr 12, s. 3950-3959Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 22.
    Gaca, Anthony O.
    et al.
    Rochester, New York, USA .
    Kudrin, Pavel
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Colomer-Winter, Cristina
    Rochester, New York, USA .
    Beljantseva, Jelena
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Liu, Kuanqing
    Madison, Wisconsin, USA .
    Anderson, Brent
    Madison, Wisconsin, USA .
    Wang, Jue D.
    Madison, Wisconsin, USA .
    Rejman, Dominik
    Prague, Czech Republic.
    Potrykus, Katarzyna
    Gdańsk, Poland; Bethesda, Maryland, USA.
    Cashel, Michael
    Bethesda, Maryland, USA.
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Lemos, Jose A.
    Rochester, New York, USA .
    From (p)ppGpp to (pp)pGpp: characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis2015Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 197, nr 18, s. 2908-2919Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p) ppGpp. In Enterococcus faecalis, (p) ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel(Ef)) and the small alarmone synthetase (SAS) RelQ(Ef). Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ(Ef) synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p) ppGpp synthesis from GDP and GTP, RelQ(Ef) also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p) ppGpp. We found that pGpp, like (p) ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ(Ef) was activated only by ppGpp. Furthermore, enzymatic activity of RelQ(Ef) is insensitive to relacin, a (p) ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p) ppGpp. IMPORTANCE Accumulation of the nucleotide second messengers (p) ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p) ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p) ppGpp synthetase RelQ of Enterococcus faecalis (RelQ(Ef)), we found that, in addition to (p) ppGpp, RelQ(Ef) is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p) ppGpp. These findings provide a new regulatory feature of RelQ(Ef) and suggest that pGpp may represent a new member of the (pp) pGpp family of alarmones.

  • 23. Howell, Matthew
    et al.
    Aliashkevich, Alena
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Salisbury, Anne K.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Bowman, Grant R.
    Brown, Pamela J. B.
    Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens2017Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, nr 17, artikkel-id UNSP e00101-17Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Agrobacterium tumefaciens is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The A. tumefaciens homolog of the Caulobacter crescentus polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of A. tumefaciens. Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division. IMPORTANCE A. tumefaciens is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of A. tumefaciens growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in A. tumefaciens may be broadly applicable for devising antimicrobial strategies.

  • 24.
    Johansson, Anders
    et al.
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Farlow, Jason
    Larsson, Pär
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Dukerich, Meghan
    Chambers, Elias
    Byström, Mona
    Fox, James
    Chu, May
    Forsman, Mats
    FOI, Umeå.
    Sjöstedt, Anders
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Keim, Paul
    Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis.2004Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, nr 17, s. 5808-5818Artikkel i tidsskrift (Fagfellevurdert)
  • 25.
    Johansson, J
    et al.
    Umeå universitet, Medicinska fakulteten, Mikrobiologi.
    Eriksson, S
    Umeå universitet, Medicinska fakulteten, Mikrobiologi.
    Sondén, B
    Umeå universitet, Medicinska fakulteten, Mikrobiologi.
    Wai, S N
    Umeå universitet, Medicinska fakulteten, Mikrobiologi.
    Uhlin, B E
    Umeå universitet, Medicinska fakulteten, Mikrobiologi.
    Heteromeric interactions among nucleoid-associated bacterial proteins: localization of StpA-stabilizing regions in H-NS of Escherichia coli.2001Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, nr 7, s. 2343-2347Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nucleoid-associated proteins H-NS and StpA in Escherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS-StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpA(F21C), forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.

  • 26.
    Jäger, Gunilla
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Leipuviene, Ramune
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Pollard, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Qian, Qiang
    Björk, Glenn
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium.2004Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, nr 3, s. 750-757Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and BACTERIA: In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.

  • 27. KALLA, SR
    et al.
    LIND, LK
    LIDHOLM, J
    Gustafsson, Petter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    TRANSCRIPTIONAL ORGANIZATION OF THE PHYCOCYANIN SUBUNIT GENE CLUSTERS OF THE CYANOBACTERIUM ANACYSTIS-NIDULANS UTEX-6251988Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 170, nr 7, s. 2961-2970Artikkel i tidsskrift (Fagfellevurdert)
  • 28. Kumru, Ozan S.
    et al.
    Bunikis, Ignas
    Sorokina, Irina
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Zueckert, Wolfram R.
    Specificity and role of the borrelia burgdorferi CtpA protease in outer membrane protein processing2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 20, s. 5759-5765Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used site-directed mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.

  • 29.
    Lavander, Moa
    et al.
    Department of Medical Countermeasures, Division of NBC-Defense, Swedish Defense Research Agency.
    Sundberg, Lena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Medical Countermeasures, Division of NBC-Defense, Swedish Defense Research Agency.
    Edqvist, Petra
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lloyd, Scott
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Proteolytic Cleavage of the FlhB Homologue YscU of Yersinia pseudotuberculosis Is Essential for Bacterial Survival2002Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, nr 16, s. 4500-4509Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pathogenic Yersinia species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells. The secretion machinery is constituted of ca. 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS. A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK. The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression. Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part. Expression of the latter in a wild-type strain resulted in elevated Yop secretion. The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH. A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion. Variants of YscU with single substitutions in the conserved NPTH sequence--i.e., N263A, P264A, or T265A--were not cleaved but retained function in Yop secretion. Elevated expression of these YscU variants did, however, result in severe growth inhibition. From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold.

  • 30.
    Lee, Je Hee
    et al.
    Chunlab, Inc., Seoul National University.
    Valeriano, Valerie Diane
    Dankook University.
    Shin, Yu-ri
    Dankook University, Republic of Korea.
    Chae, Jong Pyo
    Dankook University, Republic of Korea.
    Kim, Geun-Bae
    Chung-Ang University, Republic of Korea.
    Ham, Jun-Sang
    Rural Development Administration, Suwon, Republic of Korea.
    Chun, Jongsik
    Chunlab, Inc., Seoul National University, Republic of Korea.
    Kang, Dae-Kyung
    Dankook University, Republic of Korea.
    Genome Sequence of Lactobacillus mucosae LM1, Isolated from Piglet Feces2012Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 194, nr §7, s. 4766-Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Lactobacillus mucosae LM1,isolated from stool samples of a healthy piglet, displays good in vitro mucin adhesion and antimicrobial activity against pathogenic bacteria.To elucidate its antimicrobial effects and to find its epithelial cell and mucin adhesion genes, the genomic sequence of L. mucosae LM1 was investigated.

  • 31.
    Leipuviene, Ramune
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Qian, Qiang
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways.2004Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, nr 3, s. 758-766Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s(2)C), 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms(2)io(6)A (10-fold) and s(2)C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms(2)io(6)A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s(4)U and the s(2)U moiety of (c)mnm(5)s(2)U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s(2)C and MiaB in the synthesis of ms(2)io(6)A) in the transfer of sulfur to the tRNA.

  • 32.
    Liljeqvist, Maria
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Valdes, Jorge
    Holmes, David S
    Dopson, Mark
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Draft genome of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS32011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 16, s. 4304-4305Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5° to 30°C (optimum, ≈25°C). It gains energy from the oxidation of ferrous iron and inorganic sulfur compounds and obtains organic carbon from carbon dioxide. Here, we present the draft genome sequence of A. ferrivorans SS3 that will permit investigation of genes involved in growth in acidic environments at low temperatures.

  • 33.
    Lundgren, Hans K
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn R
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Structural alterations of the cysteine desulfurase IscS of Salmonella enterica serovar Typhimurium reveal substrate specificity of IscS in tRNA thiolation.2006Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 188, nr 8, s. 3052-3062Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s(4)U) and 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s(2)C) and N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm(5)s(2)U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms(2)io(6)A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms(2)io(6)A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.

  • 34.
    Lövgren, J. Mattias
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wikström, P. Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins2001Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, nr 18, s. 5352-5357Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimM mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion between rpsP, encoding the ribosomal protein S16, and rimM on the chromosome as a result of mutations in the rpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.

  • 35.
    Lövgren, J. Mattias
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wikström, P. Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The rlmB Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in Escherichia coli2001Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, nr 23, s. 6957-6960Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In Saccharomyces cerevisiae, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity. Here we show that the proposed Escherichia coli ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA. However, a DeltarlmB mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings. Thus, RlmB has no important role in ribosome assembly or function in E. coli.

  • 36. Matern, Andreas
    et al.
    Pedrolli, Danielle
    Großhennig, Stephanie
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Mack, Matthias
    Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 23, s. 3233-3243Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B2), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF.

  • 37.
    McGee, Karen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hörstedt, Per
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Milton, Debra L.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Identification and characterization of additional flagellin genes from Vibrio anguillarum1996Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 178, nr 17, s. 5188-5198Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made. Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament. However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.

  • 38.
    Milton, Debra L.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Chalker, V. J.
    Kirke, D.
    Hardman, A.
    Cámara, M.
    Williams, P.
    The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone: 2001Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, nr 12, s. 3537-3547Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

  • 39.
    Milton, Debra L.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hardman, A.
    Camara, M.
    Chhabra, S. R.
    Bycroft, B. W.
    Stewart, G. S.
    Williams, P.
    Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone1997Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 179, nr 9, s. 3004-3012Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers. This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators. Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout. In this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V. anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis. The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases. Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators. Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed. However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E. coli and C. violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.

  • 40.
    Milton, Debra L.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Norqvist, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum1992Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 174, nr 22, s. 7235-7244Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.

  • 41.
    Milton, Debra L.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    O'Toole, Ronan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hörstedt, Per
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Flagellin A is essential for the virulence of Vibrio anguillarum1996Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 178, nr 5, s. 1310-1319Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.

  • 42.
    Moell, Andrea
    et al.
    Boston, Massachusetts, USA .
    Doerr, Tobias
    Boston, Massachusetts, USA .
    Alvarez, Laura
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Chao, Michael C.
    Boston, Massachusetts, USA .
    Davis, Brigid M.
    Boston, Massachusetts, USA .
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Waldor, Matthew K.
    Boston, Massachusetts, USA .
    Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators2014Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 196, nr 22, s. 3937-3948Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.

  • 43.
    Moreno, Renata
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Hidalgo, Aurelio
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus de Cantoblanco, Madrid, Spain.
    Cava, Felipe
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Fernández-Lafuente, Roberto
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus de Cantoblanco, Madrid, Spain.
    Guisán, José Manuel
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus de Cantoblanco, Madrid, Spain.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Use of an antisense RNA strategy to investigate the functional significance of Mn-catalase in the extreme thermophile Thermus thermophilus2004Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, nr 22, s. 7804-7806Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a "bicistronic" transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target.

  • 44.
    Myrtennäs, Kerstin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar. FOI.
    Sjödin, Andreas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Byström, Mona
    Granberg, Malin
    Brittnacher, Mitchell J.
    Rohmer, Laurence
    Jacobs, Michael A.
    Sims-Day, Elizabeth H.
    Levy, Ruth
    Zhou, Yang
    Hayden, Hillary S.
    Lim, Regina
    Chang, Jean
    Guenthener, Donald
    Kang, Allison
    Haugen, Eric
    Gillett, Will
    Kaul, Rajinder
    Forsman, Mats
    Larsson, Par
    FOI.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Genome sequence of Francisella Tularensis subspecies holarctica Strain FSC200, isolated from a child with Tularemia2012Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 194, nr 24, s. 6965-6966Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here we report the complete, accurate 1.89-Mb genome sequence of Francisella tularensis subsp. holarctica strain FSC200, isolated in 1998 in the Swedish municipality Ljusdal, which is in an area where tularemia is highly endemic. This genome is important because strain FSC200 has been extensively used for functional and genetic studies of Francisella and is well-characterized.

  • 45.
    Netterling, Sakura
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Vaitkevicius, Karolis
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nord, Stefan
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    A listeria monocytogenes RNA-helicase essential for growth and ribosomal maturation at low temperatures, uses its C-terminus for appropriate interaction with the ribosome2012Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 194, nr 16, s. 4377-4385Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food-industry. In this work, we have investigated the physiological role of one DExD-box RNA-helicase in Listeria monocytogenes. The RNA-helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to a down-regulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low temperature growth, motility, 23S rRNA processing and appropriate ribosomal maturation. However, Lmo1722 lacking the C-terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C-terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, ribosomal RNA processing and is important for ribosomal maturation being associated mainly with the 50S subunit of the ribosome.

  • 46.
    Ng, L C
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Poh, C L
    Shingler, V
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Aromatic effector activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls.1995Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 177, nr 6Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pseudomonas putida P35X (NCIB 9869) metabolizes phenol and monomethylphenols via a chromosomally encoded meta-cleavage pathway. We have recently described a 13.4-kb fragment of the chromosome that codes for the first eight genes of the catabolic pathway and a divergently transcribed positive regulator, phhR. The eight structural genes lie in an operon, the phh operon, downstream of a -24 TGGC, -12 TTGC promoter sequence. Promoters of this class are recognized by RNA polymerase that utilizes the alternative sigma 54 factor encoded by rpoN (ntrA) and are positively regulated by activators of the NtrC family. In this study, we have identified the coding region for the 63-kDa PhhR gene product by nucleotide sequencing of a 2,040-bp region and polypeptide analysis. PhhR was found to have homology with the NtrC family of transcriptional activators, in particular with DmpR, the pVI150-encoded regulator of (methyl)phenol catabolism by Pseudomonas sp. strain CF600. By using a luciferase reporter system, PhhR alone was shown to be sufficient to activate transcription from the phh operon promoter in an RpoN+ background but not an RpoN- background. Luciferase reporter systems were also used to directly compare the aromatic effector profiles of PhhR and DmpR. Evidence that the difference in the growth substrate ranges of strains P35X and CF600 is due to the effector activation specificities of the regulators of these systems rather than the substrate specificities of the catabolic enzymes is presented.

  • 47.
    Nilsson, Kristina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lundgren, Hans K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hagervall, Tord G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn R
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium2002Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, nr 24, s. 6830-6835Artikkel i tidsskrift (Fagfellevurdert)
  • 48.
    Nordlund, I
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Powlowski, J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, V
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.1990Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 172, nr 12Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.

  • 49. Novak, R
    et al.
    Cauwels, A
    Charpentier, E
    Tuomanen, E
    Identification of a Streptococcus pneumoniae gene locus encoding proteins of an ABC phosphate transporter and a two-component regulatory system.1999Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 181, nr 4, s. 1126-1133Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.

  • 50.
    Ny, Tor
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björk, G R
    Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12.1980Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 142, nr 2, s. 371-9Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A hybrid plasmid from the Clarke and Carbon collection has been isolated. This plasmid carries the trmA gene of E. coli, which is necessary for the formation of 5-methyluridine (m5U,ribothymidine) present in all transfer ribonucleic acid (tRNA) chains of the organism so far sequenced. A restriction map of the argCBH-trmA regions is presented. By using cloning in vitro, the trmA gene was located on a 2.9-kilobase pair deoxyribonucleic acid (DNA) fragment. These results and comparison with lambda dargECBH transducing phages established the gene order: argECBH trmA bfe in the 88-min region of the E. coli chromosomal map. Plasmids carrying this 2.9-kilobase pair DNA fragment overproduce the enzyme tRNA(m5U)methyltransferase (EC 2.1.1.35) 20 to 40 times. When this 2.9-kilobase pair chromosomal DNA fragment was expressed in a minicell system, a polypeptide of a molecular weight of 42,000 was synthesized. This polypeptide was tentatively identified as the tRNA(m5U)methyltransferase. These results support the earlier suggestion that the trmA gene is the structural gene for the tRNA(m5U)methyltransferase.

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