The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli

The cAMP-CRP regulon coordinates transcription regulation of several energy-related genes, the lac operon among them. Lactose, or IPTG, induces the lac operon expression by binding to the LacI repressor, and releasing it from the promoter sequence. At the same time, the expression of the lac operon requires the presence of the CRP-cAMP complex, which promotes the binding of the RNA polymerase to the promoter region. The modified nucleotide cAMP accumulates in the absence of glucose and binds to the CRP protein, but its ability to bind to DNA can be impaired by lysine-acetylation of CRP. Here we add another layer of control, as acetylation of CRP seems to be modified by ppGpp. In cells grown in glycerol minimal media, ppGpp seems to repress the expression of lacZ, where ΔrelA mutants show higher expression of lacZ than in WT. These differences between the WT and ΔrelA strains seem to depend on the levels of acetylated CRP. During the growth in minimal media supplemented with glycerol, ppGpp promotes the acetylation of CRP by the Nε-lysine acetyltransferases YfiQ. Moreover, the expression of the different genes involved in the production and degradation of Acetyl-phosphate (ackA-pta) and the enzymatic acetylation of proteins (yfiQ) are stimulated by the presence of ppGpp, depending on the growth conditions.

Three ParA Dimers Cooperatively Assemble on Type Ia Partition Promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by partition systems composed of two proteins, ParA and ParB, and a centromere site. Auto-regulation of Par operon expression is important for efficient partitioning and is primarily mediated by ParA for type Ia plasmid partition systems. For the F-plasmid, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface-plasmon-resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. Molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter. We propose that three ParAF dimers bind cooperatively to overlapping motifs, thus covering the promoter region. A similar organization is found on closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

High vitamin B2 production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

Manufacturing of probiotics and functional foods using lactic acid bacteria that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analyzed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for lactic acid bacteria, exceeding by ~25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~6-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavine, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

Three ParA dimers cooperatively assemble on type Ia partition promoters

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by a partition system (Par) composed of two proteins, ParA and ParB, and a centromere site. The auto-regulation of Par operon expression is important for efficient partitioning, and is primarily mediated by ParA for type Ia plasmid partition systems. For the plasmid F, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface plasmon resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. In addition, molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter region. We propose that three ParAF dimers bind cooperatively to overlapping motifs thus covering the promoter region. A similar organization is found on both closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

Different csrA Expression Levels in C versus K-12 E. coli Strains Affect Biofilm Formation and Impact the Regulatory Mechanism Presided by the CsrB and CsrC Small RNAs

Escherichia coli C is a strong biofilm producer in comparison to E. coli K-12 laboratory strains due to higher expression of the pgaABCD operon encoding the enzymes for the biosynthesis of the extracellular polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). The pgaABCD operon is negatively regulated at the post-transcriptional level by two factors, namely CsrA, a conserved RNA-binding protein controlling multiple pathways, and the RNA exonuclease polynucleotide phosphorylase (PNPase). In this work, we investigated the molecular bases of different PNAG production in C-1a and MG1655 strains taken as representative of E. coli C and K-12 strains, respectively. We found that pgaABCD operon expression is significantly lower in MG1655 than in C-1a; consistently, CsrA protein levels were much higher in MG1655. In contrast, we show that the negative effect exerted by PNPase on pgaABCD expression is much stronger in C-1a than in MG1655. The amount of CsrA and of the small RNAs CsrB, CsrC, and McaS sRNAs regulating CsrA activity is dramatically different in the two strains, whereas PNPase level is similar. Finally, the compensatory regulation acting between CsrB and CsrC in MG1655 does not occur in E. coli C. Our results suggest that PNPase preserves CsrA-dependent regulation by indirectly modulating csrA expression.

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

ABSTRACTFree L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

Escherichia coli S2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of remnant signal peptides. However, whether RseP has additional cellular functions is unclear. In this study, we attempted to identify new RseP substrates to explore still unknown physiological roles of this protease. Our mass spectrometry-based quantitative proteomic analysis revealed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with the fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. Ferric citrate signal-dependent generation of this cleavage product is the essential and sufficient role of RseP in the transcriptional activation of the fec operon. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

The Route of Sucrose Utilization by Streptococcus mutans Affects Intracellular Polysaccharide Metabolism

Streptococcus mutans converts extracellular sucrose (Suc) into exopolysaccharides (EPS) by glucosyl-transferase and fructosyl-transferase enzymes and internalizes Suc for fermentation through the phosphotransferase system (PTS). Here, we examined how altering the routes for sucrose utilization impacts intracellular polysaccharide [IPS; glycogen, (glg)] metabolism during carbohydrate starvation. Strain UA159 (WT), a mutant lacking all exo-enzymes for sucrose utilization (MMZ952), and a CcpA-deficient mutant (∆ccpA) were cultured with sucrose or a combination of glucose and fructose, followed by carbohydrate starvation. At baseline (0h), and after 4 and 24h of starvation, cells were evaluated for mRNA levels of the glg operon, IPS storage, glucose-1-phosphate (G1P) concentrations, viability, and PTS activities. A pH drop assay was performed in the absence of carbohydrates at the baseline to measure acid production. We observed glg operon activation in response to starvation (p<0.05) in all strains, however, such activation was significantly delayed and reduced in magnitude when EPS synthesis was involved (p<0.05). Enhanced acidification and greater G1P concentrations were observed in the sucrose-treated group, but mostly in strains capable of producing EPS (p<0.05). Importantly, only the WT exposed to sucrose was able to synthesize IPS during starvation. Contrary to CcpA-proficient strains, IPS was progressively degraded during starvation in ∆ccpA, which also showed increased glg operon expression and greater PTS activities at baseline. Therefore, sucrose metabolism by secreted enzymes affects the capacity of S. mutans in synthesizing IPS and converting it into organic acids, without necessarily inducing greater expression of the glg operon.

Deletion of SarX decreases biofilm formation of Staphylococcus aureus in a polysaccharide intercellular adhesin (PIA) dependent manner

Background: Biofilm formation by Staphylococcus aureus is an important virulence determinant mediated by the polysaccharide intercellular adhere (PIA) encoded by ica operon or by surface and extracellular proteins. Previous studies have shown that S. epidermidis SarX protein regulated the transcriptional activity of the agr and ica loci and controled the biofilm phenotype, primarily by regulating icaADBC transcription and PIA production.Results: In this study, our results indicated that biofilm formation and detachment of S. aureus were significantly decreased in the sarX mutant strain. sarX mutant in S. aureus biofilm formation was related to the production of PIA and not to that of eDNA. RT-PCR results showed that deletion of sarX was associated with a 1.8-fold reduction in spa transcription, which was complemented by sarX. Expression of Spa protein was decreased in sarX mutant strain.Conclusions: sarX promoted biofilm production of S. aureus may mediated primarily through increasing ica operon expression and PIA production. Deletion of sarX was associated with reduction in spa transcription.