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Journal of Bacteriology Jun 2023Flavobacterium johnsoniae is a free-living member of the phylum that is found in soil and water. It is frequently used as a model species for studying a type of gliding...
Flavobacterium johnsoniae is a free-living member of the phylum that is found in soil and water. It is frequently used as a model species for studying a type of gliding motility dependent on the type IX secretion system (T9SS). -Glycosylation has been reported in several species, and the -glycosylation of S-layer proteins in Tannerella forsythia was shown to be important for certain virulence features. In this study, we characterized the -glycoproteome of F. johnsoniae and identified 325 -glycosylation sites within 226 glycoproteins. The structure of the major glycan was found to be a hexasaccharide with the sequence Hex-(Me-dHex)-Me-HexA-Pent-HexA-Me-HexNAcA. Bioinformatic localization of the glycoproteins predicted 68 inner membrane proteins, 60 periplasmic proteins, 26 outer membrane proteins, 57 lipoproteins, and 9 proteins secreted by the T9SS. The glycosylated sites were predominantly located in the periplasm, where they are postulated to be beneficial for protein folding/stability. Six proteins associated with gliding motility or the T9SS were demonstrated to be -glycosylated. Flavobacterium johnsoniae is a Gram-negative bacterium that is found in soil and water. It is frequently used as a model species for studying gliding motility and the T9SS. In this study, we characterized the -glycoproteome of F. johnsoniae and identified 325 -glycosylation sites within 226 glycoproteins. The glycosylated domains were mainly localized to the periplasm. The function of -glycosylation is likely related to protein folding and stability; therefore, the finding of the glycosylation sites has relevance for studies involving expression of the proteins. Six proteins associated with gliding motility or the T9SS were demonstrated to be -glycosylated, which may impact the structure and function of these components.
Topics: Bacterial Proteins; Flavobacterium; Polysaccharides; Glycosylation; Proteome
PubMed: 37162352
DOI: 10.1128/jb.00093-23 -
Nature Communications May 2024Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions...
Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.
Topics: ATP-Binding Cassette Transporters; Light; Bacterial Proteins; Protein Binding; Rhodopsins, Microbial; Optogenetics
PubMed: 38773114
DOI: 10.1038/s41467-024-48650-1 -
Nature Communications Aug 2023Proteins from the signal transduction ATPases with numerous domains (STAND) family are known to play an important role in innate immunity. However, it remains less well...
Proteins from the signal transduction ATPases with numerous domains (STAND) family are known to play an important role in innate immunity. However, it remains less well understood how they function in transcriptional regulation. MalT is a bacterial STAND that controls the Escherichia coli maltose system. Inactive MalT is sequestered by different inhibitory proteins such as MalY. Here, we show that MalY interacts with one oligomerization interface of MalT to form a 2:2 complex. MalY represses MalT activity by blocking its oligomerization and strengthening ADP-mediated MalT autoinhibition. A loop region N-terminal to the nucleotide-binding domain (NBD) of MalT has a dual role in mediating MalT autoinhibition and activation. Structural comparison shows that ligand-binding induced oligomerization is required for stabilizing the C-terminal domains and conferring DNA-binding activity. Together, our study reveals the mechanism whereby a prokaryotic STAND is inhibited by a repressor protein and offers insights into signaling by STAND transcription activators.
Topics: Escherichia coli; Maltose; DNA-Binding Proteins; Escherichia coli Proteins; Transcription Factors; Bacterial Proteins
PubMed: 37582800
DOI: 10.1038/s41467-023-40447-y -
Current Opinion in Microbiology Aug 2023The Lyme disease spirochete persists in nature through cycles between ticks and vertebrates. Although the spirochete interacts with numerous, distinct tissues and... (Review)
Review
The Lyme disease spirochete persists in nature through cycles between ticks and vertebrates. Although the spirochete interacts with numerous, distinct tissues and environmental conditions during its infectious cycle, Borrelia burgdorferi appears to possess a limited ability to sense its external environment. This apparent paradox is being resolved through detailed investigations of the molecular mechanisms through which B. burgdorferi controls production of virulence-associated factors such as the Erp outer surface proteins. The results have led to development of a model for how B. burgdorferi controls expression of its diverse proteins, wherein physiological and metabolic states that are unique to specific points in the infectious cycle trigger changes in gene and protein expression levels.
Topics: Animals; Borrelia burgdorferi; Bacterial Proteins; Ticks; Lyme Disease
PubMed: 37279610
DOI: 10.1016/j.mib.2023.102332 -
Molecules (Basel, Switzerland) Apr 2024Antibiotic resistance in Gram-negative bacteria remains one of the most pressing challenges to global public health. Blocking the transportation of lipopolysaccharides...
Antibiotic resistance in Gram-negative bacteria remains one of the most pressing challenges to global public health. Blocking the transportation of lipopolysaccharides (LPS), a crucial component of the outer membrane of Gram-negative bacteria, is considered a promising strategy for drug discovery. In the transportation process of LPS, two components of the LPS transport (Lpt) complex, LptA and LptC, are responsible for shuttling LPS across the periplasm to the outer membrane, highlighting their potential as targets for antibacterial drug development. In the current study, a protein-protein interaction (PPI) model of LptA and LptC was constructed, and a molecular screening strategy was employed to search a protein-protein interaction compound library. The screening results indicated that compound 18593 exhibits favorable binding free energy with LptA and LptC. In comparison with the molecular dynamics (MD) simulations on currently known inhibitors, compound 18593 shows more stable target binding ability at the same level. The current study suggests that compound 18593 may exhibit an inhibitory effect on the LPS transport process, making it a promising hit compound for further research.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Discovery; Gram-Negative Bacteria; Lipopolysaccharides; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Carrier Proteins
PubMed: 38675646
DOI: 10.3390/molecules29081827 -
Microbiology Spectrum Jun 2023Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are...
Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG dd-carboxypeptidases (DD-CPases) DacC and DacA in cell growth under alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline DD-CPase, the enzyme activity and protein stability of which are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for growth under salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all of these roles of DacC and DacA were independent of ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs)-dd-transpeptidases-mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of DD-CPases in bacterial growth and shape maintenance under stress conditions and provide novel insights into the cellular functions of DD-CPases associated with PBPs. Most bacteria have a peptidoglycan architecture for cell shape maintenance and protection against osmotic challenges. Peptidoglycan dd-carboxypeptidases control the amount of pentapeptide substrates, which are used in the formation of 4-3 cross-links by the peptidoglycan synthetic dd-transpeptidases, penicillin-binding proteins (PBPs). Seven dd-carboxypeptidases exist in Escherichia coli, but the physiological significance of their redundancy and their roles in peptidoglycan synthesis are poorly understood. Here, we showed that DacC is an alkaline dd-carboxypeptidase for which both protein stability and enzyme activity are significantly enhanced at high pH. Strikingly, dd-carboxypeptidases DacC and DacA physically interacted with PBPs, and these interactions were necessary for cell shape maintenance as well as growth under alkaline and salt stresses. Thus, cooperation between dd-carboxypeptidases and PBPs may allow E. coli to overcome various stresses and to maintain cell shape.
Topics: Penicillin-Binding Proteins; Peptidoglycan; Peptidyl Transferases; Escherichia coli; Carboxypeptidases; Bacterial Proteins
PubMed: 37098975
DOI: 10.1128/spectrum.00014-23 -
Virulence Dec 2023Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. (group A , GAS) is one of the most...
Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. (group A , GAS) is one of the most common pathogens of monomicrobial necrotizing fasciitis. Spontaneous inactivating mutation in the CovR/CovS two-component regulatory system is related to the increase of EVs production via an unknown mechanism. This study aimed to investigate whether the CovR/CovS-regulated RopB, the transcriptional regulator of GAS exoproteins, would participate in regulating EVs production. Results showed that the size, morphology, and number of EVs released from the wild-type strain and the mutant were similar, suggesting RopB is not involved in controlling EVs production. Nonetheless, RopB-regulated SpeB protease degrades streptolysin O and bacterial proteins in EVs. Although SpeB has crucial roles in modulating protein composition in EVs, the SpeB-positive EVs failed to trigger HaCaT keratinocytes pyroptosis, suggesting that EVs did not deliver SpeB into keratinocytes or the amount of SpeB in EVs was not sufficient to trigger cell pyroptosis. Finally, we identified that EV-associated enolase was resistant to SpeB degradation, and therefore could be utilized as the internal control protein for verifying SLO degradation. This study revealed that RopB would participate in modulating protein composition in EVs via SpeB-dependent protein degradation and suggested that enolase is a potential internal marker for studying GAS EVs.
Topics: Streptococcus pyogenes; Cysteine Proteases; Bacterial Proteins; Extracellular Vesicles; Phosphopyruvate Hydratase
PubMed: 37621107
DOI: 10.1080/21505594.2023.2249784 -
Science Advances Feb 2024Subcellular compartments often serve to store nutrients or sequester labile or toxic compounds. As bacteria mostly do not possess membrane-bound organelles, they often...
Subcellular compartments often serve to store nutrients or sequester labile or toxic compounds. As bacteria mostly do not possess membrane-bound organelles, they often have to rely on protein-based compartments. Encapsulins are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes. Here, we show that desulfurase encapsulins can sequester and store large amounts of crystalline elemental sulfur. We determine the 1.78-angstrom cryo-EM structure of a 24-nanometer desulfurase-loaded encapsulin. Elemental sulfur crystals can be formed inside the encapsulin shell in a desulfurase-dependent manner with l-cysteine as the sulfur donor. Sulfur accumulation can be influenced by the concentration and type of sulfur source in growth medium. The selectively permeable protein shell allows the storage of redox-labile elemental sulfur by excluding cellular reducing agents, while encapsulation substantially improves desulfurase activity and stability. These findings represent an example of a protein compartment able to accumulate and store elemental sulfur.
Topics: Bacterial Proteins; Bacteria; Prokaryotic Cells; Oxidation-Reduction; Sulfur
PubMed: 38306423
DOI: 10.1126/sciadv.adk9345 -
Frontiers in Cellular and Infection... 2023Many pathogens use Type III and Type IV protein secretion systems to secrete virulence factors from the bacterial cytosol into host cells. These systems operate through... (Review)
Review
Many pathogens use Type III and Type IV protein secretion systems to secrete virulence factors from the bacterial cytosol into host cells. These systems operate through a one-step mechanism. The secreted substrates (protein or nucleo-protein complexes in the case of Type IV conjugative systems) are guided to the base of the secretion channel, where they are directly delivered into the host cell in an ATP-dependent unfolded state. Despite the numerous disparities between these secretion systems, here we have focused on the structural and functional similarities between both systems. In particular, on the structural similarity shared by one of the main ATPases (EscN and VirD4 in Type III and Type IV secretion systems, respectively). Interestingly, these ATPases also exhibit a structural resemblance to F-ATPases, which suggests a common mechanism for substrate secretion. The correlation between structure and function of essential components in both systems can provide significant insights into the molecular mechanisms involved. This approach is of great interest in the pursuit of identifying inhibitors that can effectively target these systems.
Topics: Type IV Secretion Systems; Bacterial Proteins; Bacteria; Protein Transport; Adenosine Triphosphatases; Type III Secretion Systems
PubMed: 38089815
DOI: 10.3389/fcimb.2023.1255852 -
Applied Microbiology and Biotechnology Aug 2023Despite the advances in understanding the regulatory networks for secondary metabolite production in Streptomyces, the participation of the two-component systems (TCS)... (Review)
Review
Despite the advances in understanding the regulatory networks for secondary metabolite production in Streptomyces, the participation of the two-component systems (TCS) in this process still requires better characterization. These sensing systems and their responses to environmental stimuli have been described by evaluating mutant strains with techniques that allow in-depth regulatory responses. However, defining the stimulus that triggers their activation is still a task. The transmembrane nature of the sensor kinases and the high content of GC in the streptomycetes represent significant challenges in their study. In some examples, adding elements to the assay medium has determined the respective ligand. However, a complete TCS description and characterization requires specific amounts of the involved proteins that are most difficult to obtain. The availability of enough sensor histidine kinase concentrations could facilitate the identification of the ligand-protein interaction, and besides would allow the establishment of its phosphorylation mechanisms and determine their tridimensional structure. Similarly, the advances in the development of bioinformatics tools and novel experimental techniques also promise to accelerate the TCSs description and provide knowledge on their participation in the regulation processes of secondary metabolite formation. This review aims to summarize the recent advances in the study of TCSs involved in antibiotic biosynthesis and to discuss alternatives to continue their characterization. KEY POINTS: • TCSs are the environmental signal transducers more abundant in nature. • The Streptomyces have some of the highest number of TCSs found in bacteria. • The study of signal transduction between SHKs and RRs domains is a big challenge.
Topics: Streptomyces; Anti-Bacterial Agents; Ligands; Histidine Kinase; Signal Transduction; Bacterial Proteins; Gene Expression Regulation, Bacterial
PubMed: 37341754
DOI: 10.1007/s00253-023-12623-z