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  • 1
    Online Resource
    Online Resource
    Proceedings of the National Academy of Sciences ; 2020
    In:  Proceedings of the National Academy of Sciences Vol. 117, No. 11 ( 2020-03-17), p. 6129-6138
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 117, No. 11 ( 2020-03-17), p. 6129-6138
    Abstract: In oval-shaped Streptococcus pneumoniae , septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    RVK:
    RVK:
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2020
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  • 2
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 81, No. 20 ( 2015-10-15), p. 7244-7252
    Abstract: During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae . Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
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    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
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  • 3
    In: mBio, American Society for Microbiology, Vol. 6, No. 4 ( 2015-09)
    Abstract: Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for more than a million deaths each year. Like all other organisms, S. pneumoniae must be able to segregate its chromosomes properly. Not only is understanding the molecular mechanisms underlying chromosome segregation in S. pneumoniae therefore of fundamental importance, but also, this knowledge might offer new leads for ways to target this pathogen. Here, we identified a link between the pneumococcal chromosome segregation system and the competence-developmental system. Competence allows S. pneumoniae to take up and integrate exogenous DNA in its chromosome. This process plays a crucial role in successful adaptation to—and escape from—host defenses, antibiotic treatments, and vaccination strategies. We show that the chromosome segregation protein ParB acts as a repressor of competence. To the best of our knowledge, this is the first example of a ParB protein controlling bacterial competence.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 2557172-2
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  • 4
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2019-03-06)
    Abstract: The RNA binding proteins EloR and KhpA are important components of the regulatory network that controls and coordinates cell elongation and division in S . pneumoniae . Loss of either protein reduces cell length, and makes the essential elongasome proteins PBP2b and RodA dispensable. It has been shown previously in formaldehyde crosslinking experiments that EloR co-precipitates with KhpA, indicating that they form a complex in vivo . In the present study, we used 3D modeling and site directed mutagenesis in combination with protein crosslinking to further study the relationship between EloR and KhpA. Protein-protein interaction studies demonstrated that KhpA forms homodimers and that KhpA in addition binds to the KH-II domain of EloR. Site directed mutagenesis identified isoleucine 61 (I61) as crucial for KhpA homodimerization. When substituting I61 with phenylalanine, KhpA lost the ability to homodimerize, while it still interacted clearly with EloR. In contrast, both homo- and heterodimerization were lost when I61 was substituted with tyrosine. By expressing these KhpA versions in S . pneumoniae , we were able to show that disruption of EloR/KhpA heterodimerization makes the elongasome redundant in S . pneumoniae . Of note, loss of KhpA homodimerization did not give rise to this phenotype, demonstrating that the EloR/KhpA complex is crucial for regulating the activity of the elongasome. In support of this conclusion, we found that localization of KhpA to the pneumococcal mid-cell region depends on its interaction with EloR. Furthermore, we found that the EloR/KhpA complex co-localizes with FtsZ throughout the cell cycle.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2019
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  • 5
    In: BMC Musculoskeletal Disorders, Springer Science and Business Media LLC, Vol. 21, No. 1 ( 2020-12)
    Abstract: Modic Changes (MCs, magnetic resonance imaging (MRI) signal changes in the vertebral bone marrow extending from the vertebral endplate) may represent a subgroup of nonspecific chronic low back pain that could benefit from a specific management. The primary aim was to compare clinical characteristics between patients with type 1 versus type 2 MCs. The secondary aim was to explore associations between clinical characteristics and MC related short tau inversion recovery (STIR) signals. Methods This cross-sectional study used baseline data prospectively collected between 2015 and 2017 on the 180 patients included in the AIM-study (Antibiotics In Modic changes), a randomized controlled trial in a Norwegian hospital out-patient setting of patients with chronic low back pain, a lumbar disc herniation within the last 2 years, low back pain intensity score ≥ 5 (on a 0–10 scale) and current type 1 or type 2 MCs at the previously herniated lumbar disc level. We used prespecified clinical characteristics including self-report measures, physiologic measures and functional measures from clinical history and examination. The diagnostic accuracy of various clinical characteristics to discriminate between patients with type 1 MCs (with or without additional type 2 MCs) and patents with type 2 MCs only (not type 1) were assessed by calculating the area under the receiver-operating curve. We assessed the correlations of clinical characteristics with details of MC related STIR signal increase. Results No clinical characteristic differed between patients with type 1 ( n  = 118) versus type 2 (but not type 1) ( n  = 62) MCs. The clinical characteristics showed no/minor differences or no/weak correlations with MC related STIR signal increase. Patients with a positive Springing test (at any lumbar level) had slightly less volume of STIR signal increase than those with a negative test (mean difference 1.3 on a 0–48 scale, 95% CI 0.3 to 2.3). Conclusion Clinical characteristics were similar for patients with type 1 MCs and patients with type 2 MCs, and showed no clinically relevant correlations with MC related STIR signal increase. Trial registration ClinicalTrials.gov NCT02323412 , First registered 23 December 2014
    Type of Medium: Online Resource
    ISSN: 1471-2474
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
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  • 6
    Online Resource
    Online Resource
    Wiley ; 2021
    In:  Molecular Microbiology Vol. 116, No. 1 ( 2021-07), p. 41-52
    In: Molecular Microbiology, Wiley, Vol. 116, No. 1 ( 2021-07), p. 41-52
    Abstract: Until recently, class A penicillin‐binding proteins (aPBPs) were the only enzymes known to catalyze glycan chain polymerization from lipid II in bacteria. Hence, the discovery of two novel lipid II polymerases, FtsW and RodA, raises new questions and has consequently received a lot of attention from the research community. FtsW and RodA are essential and highly conserved members of the divisome and elongasome, respectively, and work in conjunction with their cognate class B PBPs (bPBPs) to synthesize the division septum and insert new peptidoglycan into the lateral cell wall. The identification of FtsW and RodA as peptidoglycan glycosyltransferases has raised questions regarding the role of aPBPs in peptidoglycan synthesis and fundamentally changed our understanding of the process. Despite their dethronement, aPBPs are essential in most bacteria. So, what is their function? In this review, we discuss recent progress in answering this question and present our own views on the topic.
    Type of Medium: Online Resource
    ISSN: 0950-382X , 1365-2958
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2021
    detail.hit.zdb_id: 1501537-3
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  • 7
    Online Resource
    Online Resource
    American Society for Microbiology ; 2011
    In:  Applied and Environmental Microbiology Vol. 77, No. 10 ( 2011-05-15), p. 3335-3342
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 77, No. 10 ( 2011-05-15), p. 3335-3342
    Abstract: The membrane proteins IIC and IID of the mannose phosphotransferase system (Man-PTS) together form a membrane-located complex that serves as a receptor for several different bacteriocins, including the pediocin-like class IIa bacteriocins and the class IIc bacteriocin lactococcin A. Bacterial strains sensitive to class IIa bacteriocins readily give rise to resistant mutants upon bacteriocin exposure. In the present study, we have therefore investigated lactococcin A-resistant mutants of Lactococcus lactis as well as natural food isolates of Listeria monocytogenes with different susceptibilities to class IIa bacteriocins. We found two major mechanisms of resistance. The first involves downregulation of Man-PTS gene expression, which takes place both in spontaneous resistant mutants and in natural resistant isolates. The second involves normal expression of the Man-PTS system, but the underlying mechanism of resistance for these cells is unknown. In some cases, the resistant phenotype was linked to a shift in the metabolism; i.e., reduced growth on glucose due to reduction in Man-PTS expression was accompanied by enhanced growth on another sugar, such as galactose. The implications of these findings in terms of metabolic heterogeneity are discussed.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 8
    Online Resource
    Online Resource
    American Society for Microbiology ; 2021
    In:  Journal of Bacteriology Vol. 203, No. 9 ( 2021-04-08)
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 203, No. 9 ( 2021-04-08)
    Abstract: The ellipsoid shape of Streptococcus pneumoniae is determined by the synchronized actions of the elongasome and the divisome, which have the task of creating a protective layer of peptidoglycan (PG) enveloping the cell membrane. The elongasome is necessary for expanding PG in the longitudinal direction, whereas the divisome synthesizes the PG that divides one cell into two. Although there is still little knowledge about how these two modes of PG synthesis are coordinated, it was recently discovered that two RNA-binding proteins called EloR and KhpA are part of a novel regulatory pathway controlling elongation in S. pneumoniae . EloR and KhpA form a complex that works closely with the Ser/Thr kinase StkP to regulate cell elongation. Here, we have further explored how this regulation occurs. EloR/KhpA is found at midcell, a localization fully dependent on EloR. Using a bacterial two-hybrid assay, we probed EloR against several elongasome proteins and found an interaction with the lytic transglycosylase homolog MltG. By using EloR as bait in immunoprecipitation assays, MltG was pulled down, confirming that they are part of the same protein complex. Fluorescence microscopy demonstrated that the Jag domain of EloR is essential for EloR’s midcell localization and its interaction with MltG. Since MltG is found at midcell independent of EloR, our results suggest that MltG is responsible for the recruitment of the EloR/KhpA complex to the division zone to regulate cell elongation. IMPORTANCE Bacterial cell division has been a successful target for antimicrobial agents for decades. How different pathogens regulate cell division is, however, poorly understood. To fully exploit the potential for future antibiotics targeting cell division, we need to understand the details of how the bacteria regulate and construct the cell wall during this process. Here, we have revealed that the newly identified EloR/KhpA complex, regulating cell elongation in S. pneumoniae , forms a complex with the essential peptidoglycan transglycosylase MltG at midcell. EloR, KhpA, and MltG are conserved among many bacterial species, and the EloR/KhpA/MltG regulatory pathway is most likely a common mechanism employed by many Gram-positive bacteria to coordinate cell elongation and septation.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 1481988-0
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  • 9
    Online Resource
    Online Resource
    American Society for Microbiology ; 2010
    In:  Journal of Bacteriology Vol. 192, No. 22 ( 2010-11-15), p. 5906-5913
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 192, No. 22 ( 2010-11-15), p. 5906-5913
    Abstract: Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive Listeria monocytogenes ( mpt ) and the nonsensitive Lactococcus lactis ( ptn ) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    Microbiology Society ; 2009
    In:  Microbiology Vol. 155, No. 9 ( 2009-09-01), p. 2949-2961
    In: Microbiology, Microbiology Society, Vol. 155, No. 9 ( 2009-09-01), p. 2949-2961
    Abstract: Membrane-located proteins (IIC and IID) of the mannose-phosphotransferase system (man-PTS) have previously been shown to serve as target receptors for several bacteriocins. Although many bacteria contain at least one such man-PTS in their genome, most bacteriocins display a narrow inhibitory spectrum, targeting predominantly bacteria closely related to the producers. In the present study we have analysed the receptor spectrum of one-peptide bacteriocins of class II. A phylogenetic analysis of 86 man-PTSs from a wide range of bacterial genera grouped the man-PTSs into three main clusters (groups I–III). Fourteen man-PTSs distributed across the phylogenetic tree were selected for experimental analysis in a heterologous host. Only members of group I could serve as receptors for class IIa bacteriocins, and the receptor efficiencies varied in a pattern directly related to their phylogenetic position. A multiple sequence alignment of IIC and IID proteins revealed three sequence regions (two in IIC and one in IID) that distinguish members of the bacteriocin-susceptible group from those of the other groups, suggesting that these amino acid regions confer the specific bacteriocin receptor function. Moreover, we demonstrated that variation in sensitivity might also exist within the same species due to differential expression levels of the receptor, since three strains of Lactobacillus sakei harbouring identical man-PTSs were shown to display different expression levels of a man-PTS gene that corresponded to the variation in bacteriocin sensitivity. Together, the results of our study show that the level of bacteriocin susceptibility for a bacterial cell is primarily determined by differences in its man-PTS proteins, although the expression levels of the corresponding genes also play an important role.
    Type of Medium: Online Resource
    ISSN: 1350-0872 , 1465-2080
    Language: English
    Publisher: Microbiology Society
    Publication Date: 2009
    detail.hit.zdb_id: 2008736-6
    SSG: 12
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