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Uncovering novel cell wall chemistries in gram negative bacteria: from development or dedicated peptidoglycan chemometric tools to functional genomics
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Felipe Cava)ORCID iD: 0000-0003-0835-368X
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacteria are surrounded by an external cell wall whose main component is a polymeric net-like structure called the peptidoglycan (PG) or murein sacculus. PG plays crucial roles in bacterial physiology (eg morphogenesis, growth fitness and regulation of innate immunity). Based on the characteristics of this macromolecule, bacteria are grouped as gram negative and positive. Gram negatives present a thin PG layer in the periplasmic space, while Gram positive bacteria contain one thick multi-layered sacculus covering the cytoplasmic membrane. Although the PG sacculus is widely conserved between bacteria, variations in its chemical structure (ie sugars and peptide components) have been reported as a coping mechanism to stress. For example, V. choleraeis able to downregulate PG biosynthesis through non-canonical D-amino acids (NCDAAs) cell wall editing when entering stationary phase. NCDAAs production relies on Bsr enzymes, broad spectrum racemases which are expressed in V. cholerae under the control of stress sigma factor RpoS. In this thesis, we present a comprehensive study that allows us to determine the basic structural and biochemical features required for prominent D-amino acid production by Bsr enzymes.

V. cholerae’s PG editing by NCDAAs revealed the existence of previously unappreciated  chemical modification in the cell wall of bacteria. Such an observation made us question whether the latest technology could reveal, otherwise undetectable, novel PG traits and furthermore, revisit the existence of murein in bacteria which were previously defined as PG-less. Finally, these studies would promote a global assessment of the degree of PG-chemical variability at a Kingdom scale.

On the search for novel functional chemistries and associated mechanisms of cell wall regulation, we analysed the cell wall of hundreds of different species. Here, I present two proof of concept studies: i) investigation of the existence of PG in the Plantomycetes Kuenenia stuttgartiensis, a species previously classified as PG-less; and ii) PG chemical diversity within Class Alphaproteobacteria. To do so, we developed and experimentally validated an innovative chemometric pipeline to rapidly analyse large PG datasets. Chemometric analyses revealed 3 PG clusters within Alphaproteobacteria, which included unprecedented PG modifications widely conserved in family Acetobacteria: amidation at the α-(L)-carboxyl of meso-diaminopimelic acid and the presence of (1–3) cross-linked muropeptides between L-Ala and D-(meso)-diaminopimelate residues from adjacent moieties. Fluctuations of the relative abundance of these PG traits were growth phase and media composition dependent. Functional studies demonstrated that Acetobacteria atypical muropeptides enabled cellular protection against Type VI secreted endopeptidases and negatively affected innate immune system recognition suggesting relevant functional roles in the environmental adaptability of these bacteria.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2019. , p. 63
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2027
Keywords [en]
Bacteria, cell wall, peptidoglycan, peptidoglycan variations, D-amino acids
National Category
Microbiology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-157645ISBN: 978-91-7855-045-6 (print)OAI: oai:DiVA.org:umu-157645DiVA, id: diva2:1300554
Public defence
2019-04-26, Hörsal d Unod T 9, Umeå University Hospital, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2019-04-05 Created: 2019-03-28 Last updated: 2019-04-05Bibliographically approved
List of papers
1. Structural basis for the broad specificity of a new family of amino-acid racemases
Open this publication in new window or tab >>Structural basis for the broad specificity of a new family of amino-acid racemases
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2014 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 70, p. 79-90Article in journal (Refereed) Published
Abstract [en]

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2014
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-86070 (URN)10.1107/S1399004713024838 (DOI)000329942900010 ()
Funder
Knut and Alice Wallenberg Foundation
Available from: 2014-02-17 Created: 2014-02-17 Last updated: 2019-03-28Bibliographically approved
2. Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus
Open this publication in new window or tab >>Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus
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2016 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 352, no 6289, p. 1105-1109Article in journal (Refereed) Published
Abstract [en]

Metal acquisition is a vital microbial process in metal-scarce environments, such as inside a host. Using metabolomic exploration, targeted mutagenesis, and biochemical analysis, we discovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking of a broad-spectrum metallophore related to plant nicotianamine (here called staphylopine). The biosynthesis of staphylopine reveals the association of three enzyme activities: a histidine racemase, an enzyme distantly related to nicotianamine synthase, and a staphylopine dehydrogenase belonging to the DUF2338 family. Staphylopine is involved in nickel, cobalt, zinc, copper, and iron acquisition, depending on the growth conditions. This biosynthetic pathway is conserved across other pathogens, thus underscoring the importance of this metal acquisition strategy in infection.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-123368 (URN)10.1126/science.aaf1018 (DOI)000376480800040 ()27230378 (PubMedID)
Available from: 2016-07-05 Created: 2016-07-01 Last updated: 2019-03-28Bibliographically approved
3. Chemometric Analysis of Bacterial Peptidoglycan Reveals Atypical Modifications That Empower the Cell Wall against Predatory Enzymes and Fly Innate Immunity
Open this publication in new window or tab >>Chemometric Analysis of Bacterial Peptidoglycan Reveals Atypical Modifications That Empower the Cell Wall against Predatory Enzymes and Fly Innate Immunity
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2016 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 29, p. 9193-9204Article in journal (Refereed) Published
Abstract [en]

Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology and provides cell wall integrity against environmental insults. While several studies have reported a significant degree of variability in the chemical composition and organization of peptidoglycan in the domain Bacteria, the real diversity of this polymer is far from fully explored. This work exploits rapid ultraperformance liquid chromatography and multivariate data analysis to uncover peptidoglycan chemical diversity in the Class Alphaproteobacteria, a group of Gram negative bacteria that are highly heterogeneous in terms of metabolism, morphology and life-styles. Indeed, chemometric analyses revealed novel peptidoglycan structures conserved in Acetobacteria: amidation at the alpha-(L)-carboxyl of meso-diaminopimelic acid and the presence of muropeptides cross-linked by (1-3) L-Ala-D-(meso)diaminopimelate cross-links. Both structures are growth-controlled modifications that influence sensitivity to Type VI secretion system peptidoglycan endopeptidases and recognition by the Drosophila innate immune system, suggesting relevant roles in the environmental adaptability of these bacteria. Collectively our findings demonstrate the discriminative power of chemometric tools on large cell wall-chromatographic data sets to discover novel peptidoglycan structural properties in bacteria.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Chemical Sciences Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-125551 (URN)10.1021/jacs.6b04430 (DOI)000380730000039 ()27337563 (PubMedID)
Available from: 2016-09-16 Created: 2016-09-13 Last updated: 2019-03-28Bibliographically approved
4. PG-metrics: a chemometric-based approach for classifying bacterial peptidoglycan data sets and uncovering their subjacent chemical variability
Open this publication in new window or tab >>PG-metrics: a chemometric-based approach for classifying bacterial peptidoglycan data sets and uncovering their subjacent chemical variability
2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 10, article id e0186197Article in journal (Refereed) Published
Abstract [en]

Bacteria cells are protected from osmotic and environmental stresses by an exoskeleton-like polymeric structure called peptidoglycan ( PG) or murein sacculus. This structure is fundamental for bacteria's viability and thus, the mechanisms underlying cell wall assembly and how it is modulated serve as targets for many of our most successful antibiotics. Therefore, it is now more important than ever to understand the genetics and structural chemistry of the bacterial cell walls in order to find new and effective methods of blocking it for the treatment of disease. In the last decades, liquid chromatography and mass spectrometry have been demonstrated to provide the required resolution and sensitivity to characterize the fine chemical structure of PG. However, the large volume of data sets that can be produced by these instruments today are difficult to handle without a proper data analysis work-flow. Here, we present PG-metrics, a chemometric based pipeline that allows fast and easy classification of bacteria according to their muropeptide chromatographic profiles and identification of the subjacent PG chemical variability between e.g. bacterial species, growth conditions and, mutant libraries. The pipeline is successfully validated here using PG samples from different bacterial species and mutants in cell wall proteins. The obtained results clearly demonstrated that PG-metrics pipeline is a valuable bioanalytical tool that can lead us to cell wall classification and biomarker discovery.

Place, publisher, year, edition, pages
Public library science, 2017
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-141817 (URN)10.1371/journal.pone.0186197 (DOI)000413167500024 ()29040278 (PubMedID)
Available from: 2017-11-27 Created: 2017-11-27 Last updated: 2019-03-28Bibliographically approved
5. Anammox Planctomycetes have a peptidoglycan cell wall
Open this publication in new window or tab >>Anammox Planctomycetes have a peptidoglycan cell wall
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 6878Article in journal (Refereed) Published
Abstract [en]

Planctomycetes are intriguing microorganisms that apparently lack peptidoglycan, a structure that controls the shape and integrity of almost all bacterial cells. Therefore, the planctomycetal cell envelope is considered exceptional and their cell plan uniquely compartmentalized. Anaerobic ammonium-oxidizing (anammox) Planctomycetes play a key role in the global nitrogen cycle by releasing fixed nitrogen back to the atmosphere as N-2. Here using a complementary array of state-of-the-art techniques including continuous culturing, cryo-transmission electron microscopy, peptidoglycan-specific probes and muropeptide analysis, we show that the anammox bacterium Kuenenia stuttgartiensis contains peptidoglycan. On the basis of the thickness, composition and location of peptidoglycan in K. stuttgartiensis, we propose to redefine Planctomycetes as Gram-negative bacteria. Our results demonstrate that Planctomycetes are not an exception to the universal presence of peptidoglycan in bacteria.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-106512 (URN)10.1038/ncomms7878 (DOI)000355526200001 ()25962786 (PubMedID)
Available from: 2015-07-15 Created: 2015-07-14 Last updated: 2019-03-28Bibliographically approved

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