-
Frontiers in Bioengineering and... 2021Main reasons to produce recombinant proteins in the periplasm of rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein... (Review)
Review
Main reasons to produce recombinant proteins in the periplasm of rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.
PubMed: 34970535
DOI: 10.3389/fbioe.2021.797334 -
Proceedings of the National Academy of... May 2022Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species,...
Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.
Topics: Biofilms; Cell Membrane; Cell Wall; Periplasm; Pseudomonas aeruginosa
PubMed: 35476526
DOI: 10.1073/pnas.2117633119 -
International Journal of Molecular... Nov 2021Bacteria must maintain the ability to modify and repair the peptidoglycan layer without jeopardising its essential functions in cell shape, cellular integrity and... (Review)
Review
Bacteria must maintain the ability to modify and repair the peptidoglycan layer without jeopardising its essential functions in cell shape, cellular integrity and intermolecular interactions. A range of new experimental techniques is bringing an advanced understanding of how bacteria regulate and achieve peptidoglycan synthesis, particularly in respect of the central role played by complexes of Sporulation, Elongation or Division (SEDs) and class B penicillin-binding proteins required for cell division, growth and shape. In this review we highlight relationships implicated by a bioinformatic approach between the outer membrane, cytoskeletal components, periplasmic control proteins, and cell elongation/division proteins to provide further perspective on the interactions of these cell division, growth and shape complexes. We detail the network of protein interactions that assist in the formation of peptidoglycan and highlight the increasingly dynamic and connected set of protein machinery and macrostructures that assist in creating the cell envelope layers in Gram-negative bacteria.
Topics: Bacterial Proteins; Cell Membrane; Cell Wall; Gram-Negative Bacteria; Penicillin-Binding Proteins; Peptidoglycan; Periplasmic Proteins
PubMed: 34884635
DOI: 10.3390/ijms222312831 -
Molekuliarnaia Biologiia 2019This review summarizes the main achievements of recent years in molecular organization research of yeast cell surface, i.e., the compartment that consists of the... (Review)
Review
This review summarizes the main achievements of recent years in molecular organization research of yeast cell surface, i.e., the compartment that consists of the coordinately functioning plasma membrane, periplasmic space, and cell wall. There are data on vesicular transport to the external environment through the cell wall and the formation of channels in the wall, which indicate the possibility of dynamic rearrangements of the molecular structure of the yeast cell wall. There is an idea about the mosaic arrangement of the compartments of the plasma membrane. The hypothesis has been suggested on the heterogeneity of the molecular structure of the cell wall, which is usually considered as uniform except for the budding zones. The groups of proteins that form the molecular assembly of the yeast cell surface have been described. Special attention has been paid for proteins with amyloid properties, including Bgl2p glucanosyltransglycosylase, which is important for virulence in pathogenic yeast, and Gas1p, the first of the studied proteins of the cell surface, which is involved in the regulation of ribosomal DNA transcriptional silencing. The data on the structure of receptors localized on the cell surface and the "moonlight" proteins, involved in the cell stress response of yeasts, have been given.
Topics: Amyloid; Cell Membrane; Cell Wall; Periplasm; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31876276
DOI: 10.1134/S0026898419060065 -
Journal of Bacteriology Jul 2022Three distinct genetic architectures (GAs) of Type VI secretion systems (T6SSs) have been described in gut Bacteroidales species, each with unique genes and...
Three distinct genetic architectures (GAs) of Type VI secretion systems (T6SSs) have been described in gut Bacteroidales species, each with unique genes and characteristics. Unlike the GA3 T6SSs, potent antagonism has not yet been demonstrated for the GA1 or GA2 T6SSs. We previously showed that the GA2 T6SS loci are contained on integrative and conjugative elements and that there are five subtypes. Collectively, GA2 are the most prevalent Bacteroidales T6SSs in the human populations analyzed. In this study, we provide a comprehensive bioinformatic analysis of the three variable regions of GA2 T6SS loci, which encode toxic effector and immunity proteins. In total, we identified 63 distinct effectors encoded within 31 nonredundant GA2 loci, 18 of which do not have described motifs or predicted functions. We provide experimental evidence for toxin activity for four different GA2 effectors, showing that each functions only when present in the periplasm, and experimentally confirm their cognate immunity proteins. Our data demonstrate that each GA2 locus encodes at least three distinct effectors with targets in both the cytoplasm and the periplasm. The data also suggest that the effectors of a given locus are loaded onto the tube by different mechanisms, which may allow all three effectors encoded within a single GA2 locus with distinct antibacterial activity to be loaded onto a single T6 tube, increasing the antagonistic effect. Humans are colonized with many gut Bacteroidales species at high density, allowing for extensive opportunities for contact-dependent antagonism. To begin to understand the antagonistic potential of the GA2 T6SSs of the gut Bacteroidales, we performed bioinformatic and experimental analyses of the three divergent regions containing the toxin effector and immunity genes. We show that each GA2 T6SS locus encodes at least three distinct toxic effectors including toxins linked to Rhs and Hcp with cytoplasmic targets, and unlinked effectors with targets in the periplasm. The diversity and modality of effectors exceeds that of the GA1 or GA3 T6SS loci (M. J. Coyne, K. G. Roelofs, and L. E. Comstock, BMC Genomics 17:58, 2016, https://doi.org/10.1186/s12864-016-2377-z) and suggests that these T6SSs have the potential to be potent antibacterial weapons in the human gut.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Bacteroidetes; Humans; Type VI Secretion Systems
PubMed: 35735993
DOI: 10.1128/jb.00122-22 -
Proceedings of the National Academy of... Jun 2021Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as experience dramatic changes in nutrient availability, pH, and...
Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of 's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.
Topics: Carbon; Cytoplasm; DNA Replication; Down-Regulation; Escherichia coli; Escherichia coli Proteins; Ion Channels; Mechanotransduction, Cellular; Nitrogen; Phosphorus
PubMed: 34117124
DOI: 10.1073/pnas.2104686118 -
Brazilian Journal of Microbiology :... Sep 2022Vibrio parahaemolyticus can degrade insoluble chitin with the help of chitinase enzymes that generate soluble N-acetyl glucosamine oligosaccharides (GlcNAc) to induce a...
Vibrio parahaemolyticus can degrade insoluble chitin with the help of chitinase enzymes that generate soluble N-acetyl glucosamine oligosaccharides (GlcNAc) to induce a state of natural competence for the uptake of extracellular DNA. In this study, we had evaluated the role of various regulatory factors such as TfoX, CytR, OpaR, and RpoS during natural transformation of V. parahaemolyticus. The results suggest that TfoX regulates natural competence via CytR in a chitin-dependent manner. CytR controls the release of GlcNAc from insoluble chitin and conversion of GlcNAc into smaller GlcNAc residues inside the periplasm by modulating the expression of endochitinase and periplasmic chitinases. In addition, CytR was also responsible for GlcNAc-mediated upregulation of competence-related genes such as pilA, pilB, comEA, and qstR. Next, we found that the quorum sensing regulator OpaR affects the natural transformation through its regulation of extracellular nuclease Dns. The ΔopaR mutant showed increased expression of Dns, which might degrade the eDNA. As a consequence, the transformation efficiency was decreased and eDNA-dependent growth was hugely enhanced. However, when Dns-containing DASW was substituted with fresh DASW, the transformation was detectable in ΔopaR mutant and eDNA-dependent growth was less. These results suggest that the occurrence of natural transformation and eDNA-dependent growth were inversely related to each other. Lastly, the general stress regulator RpoS was required for neither quorum-sensing dependent nor chitin-dependent regulation of natural competence in V. parahaemolyticus.
Topics: Bacterial Proteins; Chitin; Chitinases; Gene Expression Regulation, Bacterial; Vibrio cholerae; Vibrio parahaemolyticus
PubMed: 35761009
DOI: 10.1007/s42770-022-00788-0 -
Biomolecules Jun 2023Efflux pumps are a relevant factor in antimicrobial resistance. In , the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the...
Efflux pumps are a relevant factor in antimicrobial resistance. In , the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the bacterium. Therefore, small molecules interfering with efflux pump function are considered adjuvants for improving antimicrobial therapies. Several compounds targeting the periplasmic adapter protein AcrA and the efflux pump AcrB have been identified to act synergistically with different antibiotics. Among those, several 4(3-aminocyclobutyl)pyrimidin-2-amines have been shown to bind to both proteins. In this study, we intended to identify analogs of these substances with improved binding affinity to AcrA using virtual screening followed by experimental validation. While we succeeded in identifying several compounds showing a synergistic effect with erythromycin on , biophysical studies suggested that 4(3-aminocyclobutyl)pyrimidin-2-amines form colloidal aggregates that do not bind specifically to AcrA. Therefore, these substances are not suited for further development. Our study emphasizes the importance of implementing additional control experiments to identify aggregators among bioactive compounds.
Topics: Membrane Transport Proteins; Escherichia coli; Escherichia coli Proteins; Periplasm; Anti-Bacterial Agents; Multidrug Resistance-Associated Proteins
PubMed: 37371580
DOI: 10.3390/biom13061000 -
FEBS Letters Nov 2019The di-copper center Cu is an essential metal cofactor in cytochrome oxidase (Cox) of mitochondria and many prokaryotes, mediating one-electron transfer from cytochrome... (Review)
Review
The di-copper center Cu is an essential metal cofactor in cytochrome oxidase (Cox) of mitochondria and many prokaryotes, mediating one-electron transfer from cytochrome c to the site for oxygen reduction. Cu is located in subunit II (CoxB) of Cox and protrudes into the periplasm of Gram-negative bacteria or the mitochondrial intermembrane space. How the two copper ions are brought together to build CoxB·Cu is the subject of this review. It had been known that the reductase TlpA and the metallochaperones ScoI and PcuC are required for Cu formation in bacteria, but the mechanism of copper transfer has emerged only recently for the Bradyrhizobium diazoefficiens system. It consists of the following steps: (a) TlpA keeps the active site cysteine pair of CoxB in its dithiol state as a prerequisite for metal insertion; (b) ScoI·Cu rapidly forms a transient complex with apo-CoxB; (c) PcuC, loaded with Cu and Cu , dissociates this complex to CoxB·Cu , and a second PcuC·Cu ·Cu transfers Cu to CoxB·Cu , yielding mature CoxB·Cu . Variants of this pathway might exist in other bacteria or mitochondria.
Topics: Bacteria; Bacterial Proteins; Catalytic Domain; Copper; Electron Transport; Electron Transport Complex IV; Metabolic Networks and Pathways
PubMed: 31449676
DOI: 10.1002/1873-3468.13587 -
The Journal of Biological Chemistry Mar 2022Understanding the evolution of metallo-β-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and... (Review)
Review
Understanding the evolution of metallo-β-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies.
Topics: Anti-Bacterial Agents; Bacteria; Carbapenems; Periplasm; beta-Lactamases
PubMed: 35120928
DOI: 10.1016/j.jbc.2022.101665