Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.
Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed.IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5'-monophosphate from guanine and inosine-5'-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the ΔhgprtM. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.
- MeSH
- antituberkulotika chemie farmakologie MeSH
- hypoxanthinfosforibosyltransferasa antagonisté a inhibitory chemie genetika metabolismus MeSH
- inhibitory enzymů chemie farmakologie MeSH
- katalýza MeSH
- metabolické sítě a dráhy MeSH
- mikrobiální viabilita MeSH
- Mycobacterium smegmatis genetika růst a vývoj metabolismus MeSH
- plazmidy genetika MeSH
- puriny metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.
- MeSH
- buněčná membrána metabolismus MeSH
- chlorid draselný farmakologie MeSH
- gating iontového kanálu MeSH
- koliciny metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lipidové dvojvrstvy metabolismus MeSH
- permeabilita MeSH
- Shigella boydii metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP.IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.
The σI sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI Further analysis revealed that the majority of these genes were affected indirectly by σI The σI regulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σI in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilisIMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σI regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σI affects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.
- MeSH
- Bacillus subtilis genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika MeSH
- genetická transkripce * MeSH
- operon MeSH
- promotorové oblasti (genetika) * MeSH
- regulace genové exprese u bakterií * MeSH
- regulon MeSH
- sigma faktor genetika MeSH
- transkriptom MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome. IMPORTANCE: Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- delece genu MeSH
- molekulární sekvence - údaje MeSH
- posttranslační úpravy proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- Streptomyces coelicolor genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- deoxyguanosin analogy a deriváty biosyntéza chemie MeSH
- dipeptidy biosyntéza chemie MeSH
- Enterococcus faecalis účinky léků enzymologie genetika metabolismus MeSH
- fyziologický stres MeSH
- guanosindifosfát metabolismus MeSH
- guanosinpentafosfát metabolismus MeSH
- guanosintetrafosfát biosyntéza MeSH
- guanosintrifosfát metabolismus MeSH
- hořčík MeSH
- ligasy genetika metabolismus MeSH
- molekulární struktura MeSH
- regulace genové exprese enzymů MeSH
- regulace genové exprese u bakterií MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
RNA polymerase in bacteria is a multisubunit protein complex that is essential for gene expression. We have identified a new subunit of RNA polymerase present in the high-A+T Firmicutes phylum of Gram-positive bacteria and have named it ε. Previously ε had been identified as a small protein (ω1) that copurified with RNA polymerase. We have solved the structure of ε by X-ray crystallography and show that it is not an ω subunit. Rather, ε bears remarkable similarity to the Gp2 family of phage proteins involved in the inhibition of host cell transcription following infection. Deletion of ε shows no phenotype and has no effect on the transcriptional profile of the cell. Determination of the location of ε within the assembly of RNA polymerase core by single-particle analysis suggests that it binds toward the downstream side of the DNA binding cleft. Due to the structural similarity of ε with Gp2 and the fact they bind similar regions of RNA polymerase, we hypothesize that ε may serve a role in protection from phage infection.
- MeSH
- Bacillus subtilis enzymologie MeSH
- DNA řízené RNA-polymerasy chemie genetika metabolismus MeSH
- fylogeneze MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- podjednotky proteinů MeSH
- regulace genové exprese enzymů MeSH
- regulace genové exprese u bakterií MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the δ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of δ from Bacillus subtilis. We demonstrate that δ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that δ is essential for cell survival when facing a competing strain in a changing environment. Hence, although δ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis.
- MeSH
- Bacillus subtilis enzymologie genetika fyziologie MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- DNA řízené RNA-polymerasy chemie genetika metabolismus MeSH
- exprese genu MeSH
- fyziologická adaptace MeSH
- genetická transkripce MeSH
- genový knockout MeSH
- mikrobiální viabilita MeSH
- podjednotky proteinů MeSH
- promotorové oblasti (genetika) genetika MeSH
- regulace genové exprese u bakterií genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chlorosomes are large light-harvesting complexes found in three phyla of anoxygenic photosynthetic bacteria. Chlorosomes are primarily composed of self-assembling pigment aggregates. In addition to the main pigment, bacteriochlorophyll c, d, or e, chlorosomes also contain variable amounts of carotenoids. Here, we use X-ray scattering and electron cryomicroscopy, complemented with absorption spectroscopy and pigment analysis, to compare the morphologies, structures, and pigment compositions of chlorosomes from Chloroflexus aurantiacus grown under two different light conditions and Chlorobaculum tepidum. High-purity chlorosomes from C. aurantiacus contain about 20% more carotenoid per bacteriochlorophyll c molecule when grown under low light than when grown under high light. This accentuates the light-harvesting function of carotenoids, in addition to their photoprotective role. The low-light chlorosomes are thicker due to the overall greater content of pigments and contain domains of lamellar aggregates. Experiments where carotenoids were selectively extracted from intact chlorosomes using hexane proved that they are located in the interlamellar space, as observed previously for species belonging to the phylum Chlorobi. A fraction of the carotenoids are localized in the baseplate, where they are bound differently and cannot be removed by hexane. In C. tepidum, carotenoids cannot be extracted by hexane even from the chlorosome interior. The chemical structure of the pigments in C. tepidum may lead to π-π interactions between carotenoids and bacteriochlorophylls, preventing carotenoid extraction. The results provide information about the nature of interactions between bacteriochlorophylls and carotenoids in the protein-free environment of the chlorosome interior.
- MeSH
- bakteriální chromatofory MeSH
- biologické pigmenty MeSH
- Chloroflexus cytologie metabolismus MeSH
- difrakce rentgenového záření MeSH
- fykobiliproteiny chemie fyziologie MeSH
- karotenoidy chemie metabolismus MeSH
- molekulární struktura MeSH
- organely fyziologie MeSH
- světlo * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
