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  • 51.
    Ny, Tor
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björk, G R
    Growth rate-dependent regulation of transfer ribonucleic acid (5-methyluridine) methyltransferase in Escherichia coli B/r.1980In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 141, no 1, p. 67-73Article in journal (Refereed)
    Abstract [en]

    Enzymes catalyzing the transfer of methyl groups from S-adenosyl-l-methionine to a precursor transfer ribonucleic acid (tRNA) and forming 5-methyluridine (m(5)U), 1-methylguanine (m(1)G), or 5-methylaminomethyl-2-thio-uridine (mam(5)s(2)U) are denoted tRNA(m(5)U)-(EC 2.1.1.35), tRNA(m(1)G)-(EC 2.1.1.31), and tRNA(mam(5)s(2)U)methyltransferase. We have studied the regulation of these tRNA biosynthetic enzymes in Escherichia coli under various physiological conditions and in bacterial mutants known to affect the regulation of components of the translational apparatus. Such studies have revealed that tRNA(m(5)U)-methyltransferase increases with the growth rate in the same fashion as stable RNA, whereas the activity of two other tRNA methyltransferases remains constant in relation to the growth rate. Thus, these tRNA biosynthetic enzymes were not coordinately regulated. Regulation of both tRNA(m(5)U)methyltransferase and stable RNA was similar during shift-up and shift-down experiments. This enzyme showed a stringent regulation in relA(+) strain (T. Ny and G. R. Björk, J. Bacteriol. 130:635-641, 1977) but also in two temperature-sensitive mutants, fusA and fusB, known to influence the accumulation of guanosine 5'-diphosphate 3'-diphosphate and RNA synthesis at nonpermissive temperatures. The tRNA(m(5)U)methyltransferase showed a gene dose effect when its structural gene, trmA, was carried on a plasmid or on lambda transducing phages. Although the regulation of tRNA-(m(5)U)methyltransferase was surprisingly coupled to that of stable RNA, this enzyme was expressed at a much lower level.

  • 52.
    Ny, Tor
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björk, G R
    Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli.1977In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 130, no 2, p. 635-41Article in journal (Refereed)
    Abstract [en]

    This paper describes the regulation of a transfer ribonucleic acid (tRNA) biosynthetic enzyme, the tRNA(m5U)methyltransferase (EC 2.1.1.35). This enzyme catalyzes the formation of 5-methyluridine (m5U, ribothymidine) in all tRNA chains of Escherichia coli. Partial deprivation of charged tRNAVal can be imposed by shifting strains carrying a temperature-sensitive valyl-tRNA ligase from a permissive to a semipermissive temperature. By using two such strains differing only in the allelic state of the relA gene, it was possible to show the tRNA(m5U)methyltransferase to be stringently regulated. Upon partial deprivation of charged tRNAVal, the differential rate of tRNA(m5U)methyltransferase synthesis was found to decrease in a strain with stringent RNA control (relA+), whereas it increased in the strain carrying the relA allele. This increase of accumulation of tRNA(m5U)methyltransferase activity required protein synthesis. Thus, when tRNA is partially uncharged in the cell, the relA gene product influences the expression of tRNA(m5U)methyltransferase gene.

  • 53.
    Olsson, Jan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 13, p. 4110-4123Article in journal (Refereed)
    Abstract [en]

    To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.

  • 54. Ormonde, P.
    et al.
    Hörstedt, P.
    O'Toole, R.
    Milton, Debra L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum2000In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 182, no 8, p. 2326-2328Article in journal (Refereed)
    Abstract [en]

    To understand further the role of the flagellum of Vibrio anguillarum in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by V. anguillarum, both in vivo and in vitro.

  • 55.
    Pavel, H
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsman, M
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols.1994In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, no 24Article in journal (Refereed)
    Abstract [en]

    The pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 carries the dmp system, which comprises the divergently transcribed dmpR gene and the dmp operon coding for the catabolic enzymes required for growth on (methyl)phenols. The constitutively expressed DmpR transcriptional activator positively controls the expression of the RpoN-dependent dmp operon promoter in the presence of the aromatic effector in the growth medium. However, the magnitude of the transcriptional response differs depending on the position of the methyl substituent on the aromatic ring. Experiments involving an elevated copy number of the dmp system demonstrate that growth on para-substituted methylphenols is limited by the level of the catabolic enzymes. An effector specificity mutant of DmpR, DmpR-E135K, that responded to the presence of 4-ethylphenol, a noneffector of the wild-type protein, was isolated by genetic selection. The single point mutation in DmpR-E135K, which results in a Glu-to-Lys change in residue 135, also results in a regulator with enhanced recognition of para-substituted methylphenols. The DmpR-E135K mutation, when introduced into the wild-type strain, confers enhanced utilization of the para-substituted methylphenols. These experiments demonstrate that the aromatic effector activation of wild-type DmpR by the para-substituted methylphenols is a major factor limiting the catabolism of these compounds.

  • 56.
    Pinne, Marija
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Denker, Katrin
    Nilsson, Elin
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Bergström, Sven
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13.2006In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 188, no 12, p. 4207-4217Article in journal (Refereed)
    Abstract [en]

    The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13. Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.

  • 57.
    Powlowski, J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sahlman, L
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600.1993In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 175, no 2Article in journal (Refereed)
    Abstract [en]

    The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.

  • 58.
    Powlowski, J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.1990In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 172, no 12Article in journal (Refereed)
    Abstract [en]

    An in vitro study of the multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600 was performed. Phenol-stimulated oxygen uptake from crude extracts was strictly dependent on the addition of NAD(P)H and Fe2+ to assay mixtures. Five of six polypeptides required for growth on phenol were necessary for in vitro activity. One of the polypeptides was purified to homogeneity and found to be a flavin adenine dinucleotide containing iron-sulfur protein with significant sequence homology, at the amino terminus, to plant-type ferredoxins. This component, as in other oxygenase systems, probably functions to transfer electrons from NAD(P)H to the iron-requiring oxygenase component. Phenol hydroxylase from this organism is thus markedly different from bacterial flavoprotein monooxygenases commonly used for hydroxylation of other phenolic compounds, but bears a number of similarities to multicomponent oxygenase systems for unactivated compounds.

  • 59. Seele, Jana
    et al.
    Singpiel, Alena
    Spoerry, Christian
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Valentin-Weigand, Peter
    Baums, Christoph G.
    Identification of a Novel Host-Specific IgM Protease in Streptococcus suis2013In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 195, no 5, p. 930-940Article in journal (Refereed)
    Abstract [en]

    Streptococcus suis serotype 2 is a highly invasive, extracellular pathogen in pigs with the capacity to cause severe infections in humans. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis. The objective of this work was to identify the bacterial factor responsible for IgM degradation. The results of this study showed that a member of the IdeS family, designated Ide(Ssuis) (Immunoglobulin M-degrading enzyme of S. suis), is responsible and sufficient for IgM cleavage. Recombinant Ide(Ssuis) was found to degrade only IgM but neither IgG nor IgA. Interestingly, Western blot analysis revealed that Ide(Ssuis) is host specific, as it exclusively cleaves porcine IgM but not IgM from six other species, including a closely related member of the Suidae family. As demonstrated by flow cytometry and immunofluorescence microscopy, Ide(Ssuis) modulates binding of IgM to the bacterial surface. Ide(Ssuis) is the first prokaryotic IgM-specific protease described, indicating that this enzyme is involved in a so-far-unknown mechanism of host-pathogen interaction at an early stage of the host immune response. Furthermore, cleavage of porcine IgM by Ide(Ssuis) is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host.

  • 60.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bartilson, M
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moore, T
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators.1993In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 175, no 6Article in journal (Refereed)
    Abstract [en]

    The catabolic plasmid pVI150 of Pseudomonas sp. strain CF600 encodes all the genetic information required for the regulated metabolism of phenol and some of its methyl-substituted derivatives. The structural dmp genes of the pathway are clustered in a single operon that lies just downstream of a -24 TGGC, -12 TTGC nif/ntr-like promoter sequence. Promoters of this class are recognized by a minor form of RNA polymerase utilizing sigma 54 (NtrA, RpoN). Primer extension analysis demonstrated that the dmp operon transcript initiates downstream of the -24, -12 promoter. Transposon insertion mutants, specifically defective in the regulation of the dmp operon, were isolated, and complementation of a phenol-utilization regulatory mutant was used to identify the regulatory locus, dmpR. The 67-kDa dmpR gene product alone was shown to be sufficient for activation of transcription from the dmp operon promoter. Nucleotide sequence determination revealed that DmpR belongs to the NtrC family of transcriptional activators that regulate transcription from -24, -12 promoters. The deduced amino acid sequence of DmpR has high homology (40 to 67% identity) with the central and carboxy-terminal regions of these activators, which are believed to be involved in the interaction with the sigma 54 RNA polymerase and in DNA binding, respectively. The amino-terminal region of DmpR was found to share 64% identity with the amino-terminal region of XylR, which is also a member of this family of activators. This region has been implicated in effector recognition of aromatic compounds that is required for the regulatory activity of XylR.

  • 61.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moore, T
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600.1994In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, no 6Article in journal (Refereed)
    Abstract [en]

    The dmp operon of the pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 encodes the enzymes involved in the catabolism of phenol and methylphenols. The regulator of this dmp pathway, DmpR, is a member of the NtrC family of transcriptional activators and controls transcription of the dmp operon in response to aromatic effector compounds (V. Shingler, M. Bartilson, and T. Moore, J. Bacteriol. 175:1596-1604, 1993). Using a lux gene fusion reporter system, in which the DmpR-regulated operon promoter controls the expression of luciferase activity, we have shown in the study reported here that DmpR is activated by, but responds differentially to, the presence of a wide range of aromatic compounds. In many microbial regulatory systems, including some members of the NtrC family, the response to environmental fluctuations involves information transfer from surface sensory proteins to transcriptional regulators. However, DmpR-mediated activation of phenol metabolism in response to aromatic compounds occurs in the absence of a specific sensory protein. We used hybrids between DmpR and XylR, a structurally related regulator of toluene and xylene metabolism, to demonstrate that it is the amino-terminal domains of these regulators that determine the specificity of transcriptional activation. The results suggest that it is the direct interaction of aromatic compounds with the DmpR and XylR proteins that regulates their transcriptional promoting activity.

  • 62.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Powlowski, J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Marklund, U
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.1992In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 174, no 3Article in journal (Refereed)
    Abstract [en]

    The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.

  • 63.
    Singh, Bhupender
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Mortezaei, Narges
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Savarino, Stephen J.
    Enteric Diseases Department, Naval Medical Research Center, Silver Spring, MD, 20910, USA.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bullitt, Esther
    Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Antibodies damage the resilience of fimbriae, causing them to be stiff and tangled2017In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, no 1, article id e00665-16Article in journal (Refereed)
    Abstract [en]

    As adhesion fimbriae are a major virulence factor for many pathogenic Gram-negative bacteria, they are also potential targets for antibodies. Fimbriae are commonly required for initiating the colonization that leads to disease, and their success as adhesion organelles lies in their ability to both initiate and sustain bacte- rial attachment to epithelial cells. The ability of fimbriae to unwind and rewind their helical filaments presumably reduces their detachment from tissue surfaces with the shear forces that accompany significant fluid flow. Therefore, the disruption of func- tional fimbriae by inhibiting this resilience should have high potential for use as a vaccine to prevent disease. In this study, we show that two characteristic biome- chanical features of fimbrial resilience, namely, the extension force and the exten- sion length, are significantly altered by the binding of antibodies to fimbriae. The fimbriae that were studied are normally expressed on enterotoxigenic Escherichia coli, which are a major cause of diarrheal disease. This alteration in biomechanical properties was observed with bivalent polyclonal antifimbrial antibodies that recog- nize major pilin subunits but not with the Fab fragments of these antibodies. Thus, we propose that the mechanism by which bound antibodies disrupt the uncoiling of natural fimbria under force is by clamping together layers of the helical filament, thereby increasing their stiffness and reducing their resilience during fluid flow. In addition, we propose that antibodies tangle fimbriae via bivalent binding, i.e., by binding to two individual fimbriae and linking them together. Use of antibodies to disrupt physical properties of fimbriae may be generally applicable to the large number of Gram-negative bacteria that rely on these surface-adhesion molecules as an essential virulence factor.

    I M P O R T A N C E Our study shows that the resiliency of colonization factor antigen I (CFA/I) and coli surface antigen 2 (CS2) fimbriae, which are current targets for vac- cine development, can be compromised significantly in the presence of antifimbrial antibodies. It is unclear how the humoral immune system specifically interrupts in- fection after the attachment of enterotoxigenic Escherichia coli (ETEC) to the epithe- lial surface. Our study indicates that immunoglobulins, in addition to their well- documented role in adaptive immunity, can mechanically damage the resilience of fimbriae of surface-attached ETEC, thereby revealing a new mode of action. Our data suggest a mechanism whereby antibodies coat adherent and free-floating bacteria to impede fimbrial resilience. Further elucidation of this possible mechanism is likely to inform the development and refinement of preventive vaccines against ETEC diar- rhea. 

  • 64.
    Skärfstad, E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    O'Neill, E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Garmendia, J
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.2000In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 182, no 11Article in journal (Refereed)
    Abstract [en]

    The Pseudomonas derived sigma(54)-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.

  • 65. Svensson, Kerstin
    et al.
    Larsson, Pär
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Johansson, Daniel
    Byström, Mona
    Forsman, Mats
    Johansson, Anders
    Evolution of subspecies of Francisella tularensis2005In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, no 11, p. 3903-3908Article in journal (Refereed)
    Abstract [en]

    Analysis of unidirectional genomic deletion events and single nucleotide variations suggested that the four subspecies of Francisella tularensis have evolved by vertical descent. The analysis indicated an evolutionary scenario where the highly virulent F. tularensis subsp. tularensis (type A) appeared before the less virulent F. tularensis subsp. holarctica (type B). Compared to their virulent progenitors, attenuated strains of F. tularensis exhibited specific unidirectional gene losses.

  • 66.
    Sze, C C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Laurie, A D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    In vivo and in vitro effects of integration host factor at the DmpR-regulated sigma(54)-dependent Po promoter.2001In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 9Article in journal (Refereed)
    Abstract [en]

    Transcription from the Pseudomonas CF600-derived sigma(54)-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many sigma(54)-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at sigma(54) promoters such as aiding binding of the regulator or recruitment of sigma(54)-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.

  • 67.
    Sze, C C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moore, T
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Growth phase-dependent transcription of the sigma(54)-dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150.1996In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 178, no 13Article in journal (Refereed)
    Abstract [en]

    Transcription from Pseudomonas-derived -24, -12 Po promoter of the pVI150-encoded dmp operon is mediated by the sigma 54-dependent DmpR activator in response to the presence of aromatic pathway substrates in the medium. However, global regulatory mechanisms are superimposed on this regulatory system so that the specific response to aromatic effectors is absent in cultures until the stationary phase is reached. Here we genetically dissect the system to show that the growth phase response is faithfully mimicked by a minimal system composed of the dmpR regulatory gene and the Po promoter regulatory region and can be reproduced in heterologous Escherichia coli. Using this system, we show that the growth phase-dependent DmpR-mediated response to aromatic compounds is limited to fast-growing cultures. Thus, during exponential growth of cultures in minimal media containing different carbon sources, the response to aromatics is immediate, while the response is suppressed in cultures grown on rich media until the exponential-to-stationary phase transition. Elements known to be involved in the DmpR-mediated transcription from Po were analyzed for the ability to influence the growth phase response. Most dramatically, overexpression of DmpR was shown to completely abolish the growth phase response, suggesting that a negatively acting factor may mediate this level of regulation. The possible mechanism of action and integration (of the specific regulation of the dmp operon-encoded catabolic enzymes with the physiological status of the bacteria are discussed.

  • 68. Sze, Chun Chau
    et al.
    Bernardo, Lisandro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 3, p. 760-770Article in journal (Refereed)
    Abstract [en]

    Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.

  • 69. Thein, Marcus
    et al.
    Bunikis, Ignas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Denker, Katrin
    Larsson, Christer
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cutler, Sally
    Drancourt, Michel
    Schwan, Tom G
    Mentele, Reinhard
    Lottspeich, Friedrich
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes2008In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 21, p. 7035-7042Article in journal (Refereed)
    Abstract [en]

    Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia. During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii, B. hermsii, B. recurrentis, and B. turicatae. The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic beta-sheets.

  • 70. Valdes, Jorge
    et al.
    Quatrini, Raquel
    Hallberg, Kevin
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Valenzuela, Pablo DT
    Holmes, David S
    Draft genome sequence of the extremely acidophilic bacterium acidithiobacillus caldus ATCC 51756 reveals metabolic versatility in the genus acidithiobacillus2009In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 18, p. 5877-5878Article in journal (Refereed)
    Abstract [en]

    Acidithiobacillus caldus is an extremely acidophilic, moderately thermophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and reduced inorganic sulfur compounds. Here we present the draft genome sequence of Acidithiobacillus caldus ATCC 51756 ( the type strain of the species), which has permitted the prediction of genes for survival in extremely acidic environments, including genes for sulfur oxidation and nutrient assimilation.

  • 71. Vanderlinde, Elizabeth M.
    et al.
    Strozen, Timothy G.
    Hernandez, Sara B.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Howard, S. Peter
    Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System2017In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, no 8, article id UNSP e00617-16Article in journal (Refereed)
    Abstract [en]

    In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila, outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the D, D-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG crosslinking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila. In a heterologous assembly model in E. coli, expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.

  • 72. Veenendaal, Andreas K J
    et al.
    Sundin, Charlotta
    Blocker, Ariel J
    Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton.2009In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 2, p. 563-70Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

  • 73.
    Vogler, Amy J
    et al.
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Birdsell, Dawn
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Price, Lance B
    Translational Genomics Research Institute, Phoenix, Arizona 85004 .
    Bowers, Jolene R
    Translational Genomics Research Institute, Phoenix, Arizona 85004 .
    Beckstrom-Sternberg, Stephen M
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Auerbach, Raymond K
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Beckstrom-Sternberg, James S
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Clare, Ashley
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Buchhagen, Jordan L
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Petersen, Jeannine M
    Centers for Disease Control and Prevention, Fort Collins, Colorado 80521 .
    Pearson, Talima
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Vaissaire, Josée
    Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches en Pathologie Animale et Zoonoses, 94700 Maison-Alfort, France .
    Dempsey, Michael P
    Division of Microbiology, Armed Forces Institute of Pathology, Washington, D.C. 20306 .
    Foxall, Paul
    Affymetrix, Inc., Santa Clara, California 95051 USA.
    Engelthaler, David M
    Translational Genomics Research Institute, Phoenix, Arizona 85004 .
    Wagner, David M
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Keim, Paul
    Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-4073 .
    Phylogeography of Francisella tularensis: global expansion of a highly fit clone2009In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 8, p. 2474-2484Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

  • 74.
    Wang, Su-Yan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lauritz, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Jass, Jana
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Milton, Debra L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 6, p. 1630-1639Article in journal (Refereed)
    Abstract [en]

    ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.

  • 75.
    Zafra, Olga
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Cava, Felipe
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Blasco, Francis
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS 31, Marseille, France.
    Magalon, Axel
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS 31, Marseille, France.
    Berenguer, Jose
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus2005In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, no 12, p. 3990-3996Article in journal (Refereed)
    Abstract [en]

    The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.

  • 76.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fernández-Vázquez, Jorge
    Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Cabrer-Panes, Juan David
    Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Sánchez, Alex
    Departament d'Estadística, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Balsalobre, Carlos
    Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Similar and divergent effects of ppGpp and DksA deficiencies on transcription in Escherichia coli2009In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, no 10, p. 3226-3236Article in journal (Refereed)
    Abstract [en]

    The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp(0)) and/or DksA (DeltadksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp(0) DeltadksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

  • 77.
    Östberg, Yngve
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bunikis, Ignas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The etiological agent of Lyme disease, Borrelia burgdorferi, appears to contain2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 24, p. 8472-8477Article in journal (Refereed)
    Abstract [en]

    Small regulatory RNAs (sRNAs) have recently been shown to be the main controllers of several regulatory pathways. The function of sRNAs depends in many cases on the RNA-binding protein Hfq, especially for sRNAs with an antisense function. In this study, the genome of Borrelia burgdorferi was subjected to different searches for sRNAs, including direct homology and comparative genomics searches and ortholog- and annotation-based search strategies. Two new sRNAs were found, one of which showed complementarity to the rpoS region, which it possibly controls by an antisense mechanism. The role of the other sRNA is unknown, although observed complementarities against particular mRNA sequences suggest an antisense mechanism. We suggest that the low level of sRNAs observed in B. burgdorferi is at least partly due to the presumed lack of both functional Hfq protein and RNase E activity.

  • 78.
    Östberg, Yngve
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Carroll, James A
    Pinne, Marija
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Krum, Jonathan G
    Rosa, Patricia
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 7, p. 2074-2084Article in journal (Refereed)
    Abstract [en]

    The aetiological agent of Lyme disease, Borrelia burgdorferi cycles between its tick vector and mammalian hosts, implying that it can sense different environments and consequently change the expression of genes encoding several surface-associated proteins. The genome of the type strain B. burgdorferi B31 has revealed 175 different gene families. The p13 gene, situated on the chromosome, encodes a channel-forming protein that belongs to the gene family 48 consisting of eight additional paralogous genes. The heterogeneity of the P13 protein from different Lyme disease Borrelia strains was investigated. The predicted surface-exposed domains are the most heterogeneous regions and contain probable epitopes of P13. The membrane-spanning architecture of P13 was determined and a model for the location of this protein in the outer membrane is presented. The transcription of the paralogues of gene family 48 during in vitro culturing and in a mouse infection model was also analysed. The bba01 gene is the only p13 paralogue present in all three Lyme-disease-causing genospecies; it is stable during cultivation in vitro and the BBA01 protein was expressed in all Borrelia strains investigated. Conversely, paralogues bbi31, bbq06 and bbh41 were only detected in B. burgdorferi and the corresponding plasmids harbouring bbi31 and bbh41 were lost during in vitro passage. Finally, p13 and bbi31 are the only members of gene family 48 that are transcribed in mice, suggesting their importance during mammalian infection.

  • 79.
    Östberg, Yngve
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pinne, Marija
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Rosa, Patricia
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 24, p. 6811-6819Article in journal (Refereed)
    Abstract [en]

    P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.

12 51 - 79 of 79
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