Positive allosteric feedback regulation of the stringent response enzyme RelA by its product.

During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.

Antimicrobial effect of probiotic bacteriocins on Streptococcus mutans biofilm in a dynamic oral flow chamber model - an in vitro study.

Aim: To determine the antimicrobial activity of the bacteriocin-producing probiotic strains Streptococcus salivarius K12 and Streptococcus salivarius M18 alone or in combination against caries-associated Streptococcus mutans. Methods: Antimicrobial activity of S. salivarius K12 and/or S. salivarius M18 against S. mutans ATCC 25175 growth and biofilm formation on hydroxyapatite (HA) discs was determined in a flow chamber model by recording the colony forming units (CFU/ml) after 48 h of co-cultivation. The biofilm was analyzed by scanning electron microscopy (SEM) and by confocal laser scanning microscopy (CLSM). Additionally, the simultaneous antagonism assay was used to assess the inhibitory effect of S. salivarius K12 and/or S. salivarius M18 against S. mutans ATCC 25175 and 21 clinical isolates of S. mutans. Results: Co-cultivation of S. mutans and S. salivarius K12 and/or S. salivarius M18 led to the inhibition of S. mutans viability, thereby, preventing its biofilm formation on HA discs. Furthermore, S. salivarius K12 and S. salivarius M18 exhibited antimicrobial activity against most clinical isolates of S. mutans. Conclusion: The in vitro flow chamber system used in this study allows the simulation of time-dependent administration of S. salivarius probiotic strains, either alone or in combination, to investigate the prevention of S. mutans biofilm formation in a standardized model.

Inhibition of biofilm formation and virulence factors of cariogenic oral pathogen Streptococcus mutans by natural flavonoid phloretin.

Objectives: To evaluate the effect and mechanism of action of the flavonoid phloretin on the growth and sucrose-dependent biofilm formation of Streptococcus mutans. Methods: Minimum inhibitory concentration, viability, and biofilm susceptibility assays were conducted to assess antimicrobial and antibiofilm effect of phloretin. Biofilm composition and structure were analysed with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Water-soluble (WSG) and water-insoluble glucan (WIG) were determined using anthrone method. Lactic acid measurements and acid tolerance assay were performed to assess acidogenicity and aciduricity. Reverse transcription quantitative PCR (RT-qPCR) was used to measure the expression of virulence genes essential for surface attachment, biofilm formation, and quorum sensing. Results: Phloretin inhibited S. mutans growth and viability in a dose-dependent manner. Furthermore, it reduced gtfB and gtfC gene expression, correlating with the reduction of extracellular polysaccharides (EPS)/bacteria and WIG/WSG ratio. Inhibition of comED and luxS gene expression, involved in stress tolerance, was associated with compromised acidogenicity and aciduricity of S. mutans. Conclusions: Phloretin exhibits antibacterial properties against S. mutans, modulates acid production and tolerance, and reduces biofilm formation. Clinical significance: Phloretin is a promising natural compound with pronounced inhibitory effect on key virulence factors of the cariogenic pathogen, S. mutans.

Mutant structure of metabolic switch protein in complex with monomeric c-di-GMP reveals a potential mechanism of protein-mediated ligand dimerization.

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of c-di-GMP inducing a conformational change that involves loop 7 of the protein that leads to downstream signaling. Here, we report a crystal structure of a partial loop 7 deletion mutant, SmbA∆loop in complex with c-di-GMP determined at 1.4 Å resolution. SmbA∆loop binds monomeric c-di-GMP indicating that loop 7 is required for c-di-GMP dimerization. Thus the complex probably represents the first step of consecutive c-di-GMP binding to form an intercalated dimer as has been observed in wild-type SmbA. Considering the prevalence of intercalated c-di-GMP molecules observed bound to proteins, the proposed mechanism may be generally applicable to protein-mediated c-di-GMP dimerization. Notably, in the crystal, SmbA∆loop forms a 2-fold symmetric dimer via isologous interactions with the two symmetric halves of c-di-GMP. Structural comparisons of SmbA∆loop with wild-type SmbA in complex with dimeric c-di-GMP or ppGpp support the idea that loop 7 is critical for SmbA function by interacting with downstream partners. Our results also underscore the flexibility of c-di-GMP, to allow binding to the symmetric SmbA∆loop dimer interface. It is envisaged that such isologous interactions of c-di-GMP could be observed in hitherto unrecognized targets.

Inhibitory effect of natural flavone luteolin on Streptococcus mutans biofilm formation.

Streptococcus mutans is one of the key pathogens responsible for dental caries, which is known to be one of the most prevalent biofilm-associated diseases worldwide. S. mutans virulence strongly depends on its biofilm formation and enamel demineralization abilities due to the production of surface adhesins, exopolysaccharides, and acid in the presence of sugar. Luteolin is an abundant natural flavone with a prominent anti-bacterial function. However, it remains unclear how luteolin affects S. mutans pathogenicity including its acidogenicity and biofilm formation. In this study, the effect of luteolin on S. mutans growth, acid production, and its early and late biofilm formation and biofilm disruption was tested. Luteolin shows strong anti-biofilm activity, while it remains non-toxic for bacterial cell viability. In the biofilm, luteolin reduces the expression of S. mutans virulence genes such as gbpC, spaP, gtfBCD, and ftf encoding for surface adhesins and extracellular polysaccharides (EPS)-producing enzymes, which reflects in the strong reduction of bacteria and EPS. Further, it reduces water-insoluble glucan production in the biofilm, potentially, via direct interference with glucosyltransfereases (Gtfs). Moreover, at biofilm inhibitory concentrations, luteolin significantly reduces acid production by S. mutans. Finally, luteolin could target S. mutans amyloid proteins to disrupt the biofilm based on the observation that it inhibits the uptake of the amyloid dye, thioflavin T, by S. mutans extracellular proteins and failed to inhibit biofilm formation by the mutant strain lacking three main amyloid proteins. In conclusion, luteolin appears to be a potent natural compound with pleiotropic anti-biofilm properties against one of the main cariogenic human pathogens, S. mutans. IMPORTANCE Flavonoids are natural compounds with proven anti-bacterial and anti-biofilm properties. Here, we describe the anti-biofilm properties of natural flavone luteolin against the main cariogenic bacteria, S. mutans. Luteolin inhibited gene expression of cell surface adhesins, fructosyltransferases, and glucosyltransferases, which promotes a significant reduction of bacterial and EPS biomass in early and late biofilms. Moreover, luteolin could directly target S. mutans Gtfs and functional amyloids to modulate pathogenic biofilms. These observations provide important insights into the anti-biofilm properties of luteolin while laying out a framework for future therapeutic strategies targeting biofilm-associated virulence factors of oral pathogens.

Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus.

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA. While c-di-GMP binding inhibits SmbA, (p)ppGpp competes for the same binding site to sustain SmbA activity. We demonstrate that (p)ppGpp specifically promotes Caulobacter growth on glucose, whereas c-di-GMP inhibits glucose consumption. We find that SmbA contributes to this metabolic switch and promotes growth on glucose by quenching the associated redox stress. The identification of an effector protein that acts as a central regulatory hub for two global second messengers opens up future studies on specific crosstalk between small-molecule-based regulatory networks.

GTPases IF2 and EF-G bind GDP and the SRL RNA in a mutually exclusive manner.

Translational GTPases (trGTPases) are involved in all four stages of protein biosynthesis: initiation, elongation, termination and ribosome recycling. The trGTPases Initiation Factor 2 (IF2) and Elongation Factor G (EF-G) respectively orchestrate initiation complex formation and translocation of the peptidyl-tRNA:mRNA complex through the bacterial ribosome. The ribosome regulates the GTPase cycle and efficiently discriminates between the GDP- and GTP-bound forms of these proteins. Using Isothermal Titration Calorimetry, we have investigated interactions of IF2 and EF-G with the sarcin-ricin loop of the 23S rRNA, a crucial element of the GTPase-associated center of the ribosome. We show that binding of IF2 and EF-G to a 27 nucleotide RNA fragment mimicking the sarcin-ricin loop is mutually exclusive with that of GDP, but not of GTP, providing a mechanism for destabilization of the ribosome-bound GDP forms of translational GTPases.

Structure of the Dom34-Hbs1 complex and implications for no-go decay.

No-go decay (NGD) targets mRNAs with stalls in translation elongation for endonucleolytic cleavage in a process involving the Dom34 and Hbs1 proteins. The crystal structure of a Schizosaccharomyces pombe Dom34-Hbs1 complex reveals an overall shape similar to that of eRF1-eRF3-GTP and EF-Tu-tRNA-GDPNP. Similarly to eRF1 and GTP binding to eRF3, Dom34 and GTP bind to Hbs1 with strong cooperativity, and Dom34 acts as a GTP-dissociation inhibitor (GDI). A marked conformational change in Dom34 occurs upon binding to Hbs1, leading Dom34 to resemble a portion of a tRNA and to position a conserved basic region in a position expected to be near the peptidyl transferase center. These results support the idea that the Dom34-Hbs1 complex functions to terminate translation and thereby commit mRNAs to NGD. Consistent with this role, NGD at runs of arginine codons, which cause a strong block to elongation, is independent of the Dom34-Hbs1 complex.

Thermodynamic characterization of ppGpp binding to EF-G or IF2 and of initiator tRNA binding to free IF2 in the presence of GDP, GTP, or ppGpp.

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 µM K(d)versus 9.1-13.9 µM K(d) at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA(fMet) demonstrating that IF2 binds to initiator tRNA with submicromolar K(d) and that affinity is altered by the G nucleotides only slightly. This--in conjunction with earlier reports on IF2 interactions with fMet-tRNA(fMet) in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA(fMet) binding and GTP was suggested to strongly promote fMet-tRNA(fMet) binding--sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA(fMet) selection.

Thermodynamics of GTP and GDP binding to bacterial initiation factor 2 suggests two types of structural transitions.

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 degrees C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 degrees C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (-868+/-25 and -577+/-23 cal mol(-1) K(-1), respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the gamma-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.