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  • 1. Alvarez, Francisco J.
    et al.
    Ryman, Kicki
    Hooijmaijers, Cornelis
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ljungdahl, Per O.
    Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 8, p. 2770-2780Article in journal (Refereed)
    Abstract [en]

    The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

  • 2.
    Ampomah, Osei Yaw
    et al.
    Swedish University of Agricultural Sciences.
    Huss-Danell, Kerstin
    Swedish University of Agricultural Sciences.
    Nodulation of Thermopsis lupinoides by a Mesorhizobium huakuii strain with a unique nodA gene in Kamtchatka, Russia2011In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 77, no 15, p. 5513-5516Article in journal (Refereed)
    Abstract [en]

    Very little is known about rhizobia that form nodules on Thermopsis spp. We report the isolation of a Mesorhizobium huakuii strain with a unique nodA gene that form nodules on Thermopsis lupinoides in Kamtchatka, Russia. The isolate did not form nodules on Thermopsis chinensis or Thermopsis caroliniana, which suggests it may be host specific.

  • 3.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hallström, Björn
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp Strain PCC 68032013In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 23, p. 7419-7427Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are emerging as promising hosts for production of advanced biofuels such as n-butanol and alkanes. However, cyanobacteria suffer from the same product inhibition problems as those that plague other microbial biofuel hosts. High concentrations of butanol severely reduce growth, and even small amounts can negatively affect metabolic processes. An understanding of how cyanobacteria are affected by their biofuel product can enable identification of engineering strategies for improving their tolerance. Here we used transcriptome sequencing (RNA-Seq) to assess the transcriptome response of Synechocystis sp. strain PCC 6803 to two concentrations of exogenous n-butanol. Approximately 80 transcripts were differentially expressed at 40 mg/liter butanol, and 280 transcripts were different at 1 g/liter butanol. Our results suggest a compromised cell membrane, impaired photosynthetic electron transport, and reduced biosynthesis. Accumulation of intracellular reactive oxygen species (ROS) scaled with butanol concentration. Using the physiology and transcriptomics data, we selected several genes for overexpression in an attempt to improve butanol tolerance. We found that overexpression of several proteins, notably, the small heat shock protein HspA, improved tolerance to butanol. Transcriptomics-guided engineering created more solvent-tolerant cyanobacteria strains that could be the foundation for a more productive biofuel host.

  • 4. Bracher, J. M.
    et al.
    de Hulster, E.
    Koster, C. C.
    van den Broek, M.
    Daran, J. -MG.
    van Maris, Antonius J.A.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Delft University of Technology, Netherlands.
    Pronk, J. T.
    Laboratory evolution of a biotin-requiring Saccharomyces cerevisiae strain for full biotin prototrophy and identification of causal mutations2017In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 83, no 16, article id e00892-17Article in journal (Refereed)
    Abstract [en]

    Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ= 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.

  • 5. Briolay, Anne
    et al.
    Bouzenzana, Jamel
    Guichardant, Michel
    Deshayes, Christian
    Sindt, Nicolas
    Bessueille, Laurence
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cell Wall Polysaccharide Synthases Are Located in Detergent-Resistant Membrane Microdomains in Oomycetes2009In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 75, no 7, p. 1938-1949Article in journal (Refereed)
    Abstract [en]

    The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1 -> 3)-beta-D-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1 -> 3)-beta-D-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

  • 6. Eriksson, M.
    et al.
    Sodersten, E.
    Yu, Z. T.
    Dalhammar, Gunnel
    KTH, Superseded Departments, Biotechnology.
    Mohn, W. W.
    Degradation of polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-reducing conditions in enrichment cultures from northern soils2003In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, no 1, p. 275-284Article in journal (Refereed)
    Abstract [en]

    The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs) at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 mug/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20degreesC removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7degreesC, 53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions, naphthalene, 2-methyl naphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7degreesC, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7degreesC, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations, including members of the genera Acidovorax, Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.

  • 7.
    Eriksson, Margareta
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Inorganic Chemistry (closed 20110630).
    Östensson, Rasmus
    Kloo, Lars
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Inorganic Chemistry (closed 20110630).
    Bacteriological study of an aqueous solution with ozone and SDS or SDSperoxide2005In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336Article in journal (Other academic)
  • 8.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Muraleedharan, Madhu Nair
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Surface Expression of omega-Transaminase in Escherichia coli2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 7, p. 2293-2298Article in journal (Refereed)
    Abstract [en]

    Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus omega-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

  • 9.
    Hugerth, Luisa W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Wefer, Hugo A.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jakobsson, Hedvig E.
    Lindberg, Mathilda
    Rodin, Sandra
    Engstrand, Lars
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 16, p. 5116-5123Article in journal (Refereed)
    Abstract [en]

    The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e. g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.

  • 10.
    Kostallas, George
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 22, p. 7500-7508Article in journal (Refereed)
    Abstract [en]

    We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1: 100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.

  • 11.
    Lindh, Jenny M.
    et al.
    Dept of genetics microbiology and toxicology, Stockholm University.
    Terenius, Olle
    Dept of genetics microbiology and toxicology, Stockholm University.
    Faye, Ingrid
    Dept of genetics microbiology and toxicology, Stockholm University.
    16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts2005In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 71, no 11, p. 7217-7223Article in journal (Refereed)
    Abstract [en]

    Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the gamma-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

  • 12. Liu, Zihe
    et al.
    Liu, Lifang
    Osterlund, Tobias
    Hou, Jin
    Huang, Mingtao
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Petranovic, Dina
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark .
    Nielsen, Jens
    Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 17, p. 5542-5550Article in journal (Refereed)
    Abstract [en]

    The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.

  • 13.
    Löfblom, John
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications2007In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, no 21, p. 6714-6721Article in journal (Refereed)
    Abstract [en]

    For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

  • 14.
    McKee, Lauren S.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Martinez-Abad, Antonio
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Ruthes, Andrea C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. AlbaNova Univ Ctr, KTH Royal Inst .
    Vilaplana, Francisco
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Brumer, Harry
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Focused Metabolism of beta-Glucans by the Soil Bacteroidetes Species Chitinophaga pinensis2019In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 85, no 2, article id UNSP e02231-18Article in journal (Refereed)
    Abstract [en]

    The genome and natural habitat of Chitinophaga pinensis suggest it has the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and on the fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of beta-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of beta-glucans isolated from different sources. Most noncrystalline beta-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, beta-glucan was the only type of polysaccharide that was effectively hydrolyzed by secreted enzymes. This contrasts with the secretion of enzymes with a broad range of activities observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass and suggest that the species may have two metabolic modes: a "scavenging mode," where multiple different types of glycan may be degraded, and a more "focused mode" of beta-glucan metabolism. The significant accumulation of some types of beta-gluco-oligosaccharides in growth media may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed polysaccharide utilization loci. We present a hypothetical model for beta-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community. IMPORTANCE It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis, a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source but is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota.

  • 15.
    Samuelson, Patrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Svedberg, M.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Staphylococcal surface display of metal-binding polyhistidyl peptides2000In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 66, no 3, p. 1243-1248Article in journal (Refereed)
    Abstract [en]

    Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni2+- and Cd2+-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

  • 16.
    Valk, Laura C.
    et al.
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    Frank, Jeroen
    Radboud Univ Nijmegen, Soehngen Inst Anaerob Microbiol, Nijmegen, Netherlands..
    de la Torre-Cortes, Pilar
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    van 't Hof, Max
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    Pronk, Jack T.
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    van Loosdrecht, Mark C. M.
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp nov "Candidatus Galacturonibacter soehngenii"2018In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 18, article id UNSP e01370-18Article in journal (Refereed)
    Abstract [en]

    Agricultural residues such as sugar beet pulp and citrus peel are rich in pectin, which contains galacturonic acid as a main monomer. Pectin-rich residues are underexploited as feedstocks for production of bulk chemicals or biofuels. The anaerobic, fermentative conversion of D-galacturonate in anaerobic chemostat enrichment cultures provides valuable information toward valorization of these pectin-rich feedstocks. Replicate anaerobic chemostat enrichments, with D-galacturonate as the sole limiting carbon source and inoculum from cow rumen content and rotting orange peels, yielded stable microbial communities, which were dominated by a novel Lachnospiraceae species, for which the name "Candidatus Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H-2 as coproducts. The observed molar ratio of acetate and the combined amounts of H-2 and formate deviated significantly from 1, which suggested that some of the hydrogen and CO2 formed during D-galacturonate fermentation was converted into acetate via the Wood-Ljungdahl acetogenesis pathway. Indeed, metagenomic analysis of the enrichment cultures indicated that the genome of "Candidatus G. soehngenii" encoded enzymes of the adapted Entner-Doudoroff pathway for D-galacturonate metabolism as well as enzymes of the Wood-Ljungdahl pathway. The simultaneous operation of these pathways may provide a selective advantage under D-galacturonate-limited conditions by enabling a higher specific ATP production rate and lower residual D-galacturonate concentration than would be possible with a strictly fermentative metabolism of this carbon and energy source. IMPORTANCE This study on D-galacturonate metabolism by open, mixed-culture enrichments under anaerobic, D-galacturonate-limited chemostat conditions shows a stable and efficient fermentation of D-galacturonate into acetate as the dominant organic fermentation product. This fermentation stoichiometry and population analyses provide a valuable baseline for interpretation of the conversion of pectin-rich agricultural feedstocks by mixed microbial cultures. Moreover, the results of this study provide a reference for studies on the microbial metabolism of D-galacturonate under different cultivation regimes.

  • 17. Vanden Wymelenberg, A.
    et al.
    Denman, S.
    Dietrich, D.
    Bassett, J.
    Yu, X. C.
    Atalla, R.
    Predki, P.
    Rudsander, U.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Cullen, D.
    Transcript analysis of genes encoding a family 61 endoglucanase and a putative membrane-anchored family 9 glycosyl hydrolase from Phanerochaete chrysosporium2002In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 68, no 11, p. 5765-5768Article in journal (Refereed)
    Abstract [en]

    Phanerochaete chrysosporium cellulase genes were cloned and characterized. The cel61A product was structurally similar to fungal endoglucanases of glycoside hydrolase family 61, whereas the cel9A product revealed similarities to Thermobifida fusca Cel9A (E4), an enzyme with both endo- and exocellulase characteristics. The fungal Cel9A is apparently a membrane-bound protein, which is very unusual for microbial cellulases. Transcript levels of both genes were substantially higher in cellulose-grown cultures than in glucose-grown cultures. These results show that P. chrysosporium possesses a wide array of conventional and unconventional cellulase genes.

  • 18.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lehtio, J.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains2001In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 67, no 10, p. 4678-4684Article in journal (Refereed)
    Abstract [en]

    Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase CeI7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

  • 19.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Fluorescence-activated cell sorting of specific affibody-displaying staphylococci2003In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, no 9, p. 5328-5335Article in journal (Refereed)
    Abstract [en]

    Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.

  • 20.
    Ytterberg, A. Jimmy
    et al.
    Karolinska Inst, Dept Med Biochem & Biophys, Chem Div 1, Stockholm, Sweden.;Karolinska Inst, Dept Med, Rheumatol Unit, Stockholm, Sweden.;Swedish Orphan Biovitrum AB Publ, Stockholm, Sweden..
    Zubarev, Roman A.
    Karolinska Inst, Dept Med Biochem & Biophys, Chem Div 1, Stockholm, Sweden..
    Baumgarten, Thomas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Posttranslational Targeting of a Recombinant Protein Promotes Its Efficient Secretion into the Escherichia coli Periplasm2019In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 85, no 13, article id UNSP e00671Article in journal (Refereed)
    Abstract [en]

    Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins. IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.

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