When translation elongation is impaired, the mRNA is uniformly destabilized by the RNA degradosome, while the concentration of mRNA is altered along the molecule.

mRNA sits at the crossroads of transcription, translation and mRNA degradation. Many questions remain about the coupling of these three processes in Escherichia coli and, in particular, how translation may have an effect on mRNA degradation and transcription. To characterize the interplay between mRNA degradation and translation while accounting for transcription, we altered the translation initiation or elongation and measured the effects on mRNA stability and concentration. Using a mapping method, we analysed mRNA concentration and stability at the local scale all along the transcript. We showed that a decrease in translation initiation efficiency destabilizes the mRNA and leads to a uniform decrease in mRNA concentration throughout the molecule. Prematurely terminating translation elongation by inserting a stop codon is associated with a drop in local mRNA concentration downstream of the stop codon, due to the uncoupling of transcription and translation. In contrast, this translation alteration uniformly destabilizes the coding and ribosome-free regions, in a process triggered by RNase E activity, and its ability to form the RNA degradome. These results demonstrate how ribosomes protect mRNA molecules and highlight how translation, mRNA degradation and transcription are deeply interconnected in the quality control process that avoids unproductive gene expression in cells.

Adhesion of the genome-sequenced Lactococcus lactis subsp. cremoris IBB477 strain is mediated by specific molecular determinants.

Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow.

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.

Dynamics of detachment of Escherichia coli from plasma-mediated coatings under shear flow.

A series of plasma-mediated coatings, containing silver nanoparticles embedded in an organosilicon or silica-like matrix, were deposited onto stainless steel and chemically characterized. Their anti-adhesive properties were evaluated in vitro towards Escherichia coli by performing shear-flow induced detachment experiments. Increasing the wall shear stress facilitated E. coli cell detachment, irrespective of the coating characteristics. When nanosilver was incorporated, cell detachment was lower, probably due to the affinity of the embedded silver for biological components of the cell wall. The presence of methyl groups in the matrix network could also promote enhanced hydrophobic interactions. Within the population fraction remaining attached to the coating under increasing shear flow, different association phenotypes were observed, viz. progressively lying flat, moving laterally, remaining tethered, or rotating by a single anchoring point, until alignment with the flow direction. This re-orientation phenotype and its relation with detachment were dependent of the coating. The effects of such heterogeneities should be more deeply explored.

Synergistic Regulation of Transcription and Translation in Escherichia coli Revealed by Codirectional Increases in mRNA Concentration and Translation Efficiency.

Translational regulation was investigated at the genome-scale in Escherichia coli cells. Using the polysome profiling method, the ribosome occupancy (RO) and ribosome density (RD) of different mRNA copies were determined for several hundred mRNAs during the exponential- and stationary-phases, providing the most complete characterization of such regulation in E. coli. Although for most genes, nearly all mRNAs (>90%) were undergoing translation, they were loaded with far fewer than the theoretical maximum number of ribosomes, suggesting translation limitation at the initiation step. Multiple linear regression was used to identify key intrinsic factors involved in the genome-wide regulation of RO and RD (i.e., open reading frame GC%, protein function, and localization). Unexpectedly, mRNA concentration, a factor that depends on cell physiology, was predicted to positively regulate RO and RD during the exponential- and stationary-phases. Using a set of selected genes controlled by an inducible promoter, we confirmed that increasing the mRNA concentration upon transcription induction led to increases in both RO and ribosome load. The fact that this relationship between mRNA concentration and translation parameters was also effective when E. coli cells naturally adapted to carbon source changes demonstrates its physiological relevance. This work demonstrated that translation regulation is positively controlled by transcript availability. This new mechanism contributed to the codirectional regulation of transcription and translation with synergistic effects on gene expression and provided a systemic understanding of E. coli cell function. IMPORTANCE The process of gene expression is divided into translation and transcription. Considerable efforts have been made in bacteria to characterize the mechanisms underlying translational regulation and identify the regulatory factors for particular mRNAs. However, to understand bacterial physiology and adaptation, it is important to elucidate genome-wide translational regulation and examine its coordination with transcriptional regulation. Here, we provided a genome-wide picture of translational regulation in Escherichia coli. For most genes, nearly all mRNA copies were found to undergo translation but were loaded with a low number of ribosomes. We showed that mRNA concentration had a positive effect on translation regulation, linking translational regulation to transcriptional regulation as well as to cell physiology and growth conditions. The codirectional regulation of transcription and translation had synergistic effects on gene expression, contributing to E. coli cell function optimization. This finding could be used in biotechnology to optimize strategies for recombinant protein synthesis.

Multiplexing polysome profiling experiments to study translation in Escherichia coli.

Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome fractionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.

Characterization of Mucus-Related Properties of Streptococcus thermophilus: From Adhesion to Induction.

Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut. Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis. The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilusvia transit as a sub-dominant bacterium (103 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract.

Purification and characterization of cassiicolin, the toxin produced by Corynespora cassiicola, causal agent of the leaf fall disease of rubber tree.

Cassiicolin, a phytotoxin produced by the necrotrophic fungus Corynespora cassiicola, was purified to homogeneity from a rubber tree isolate. The optimized protocol involves reverse phase chromatography followed by size exclusion chromatography, with monitoring of the toxicity on detached rubber tree leaves. Cassiicolin appeared to be a peptide composed of 27 amino acids, glycosylated on the second residue, with a N-terminal pyroglutamic acid and 6 cysteines involved in disulfide bonds. Its molecular mass was estimated to be 2885 Da. No significant sequence homology with other proteins could be found. The availability of pure toxin in sufficient amount is a prerequisite for its structure determination, which is a key step in the understanding of the aggression mechanism.

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin.

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0-200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.

Unraveling the role of surface mucus-binding protein and pili in muco-adhesion of Lactococcus lactis.

Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100-200 nm) and long distances (up to 600-800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants.