Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

Characterization of Pseudomonas aeruginosa l,d-Transpeptidases and Evaluation of Their Role in Peptidoglycan Adaptation to Biofilm Growth.

Peptidoglycan is an essential component of the bacterial cell envelope that sustains the turgor pressure of the cytoplasm, determines cell shape, and acts as a scaffold for the anchoring of envelope polymers such as lipoproteins. The final cross-linking step of peptidoglycan polymerization is performed by classical d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family and by l,d-transpeptidases (LDTs), which are dispensable for growth in most bacterial species and whose physiological functions remain elusive. In this study, we investigated the contribution of LDTs to cell envelope synthesis in Pseudomonas aeruginosa grown in planktonic and biofilm conditions. We first assigned a function to each of the three P. aeruginosa LDTs by gene inactivation in P. aeruginosa, heterospecific gene expression in Escherichia coli, and, for one of them, direct determination of its enzymatic activity. We found that the three P. aeruginosa LDTs catalyze peptidoglycan cross-linking (LdtPae1), the anchoring of lipoprotein OprI to the peptidoglycan (LdtPae2), and the hydrolysis of the resulting peptidoglycan-OprI amide bond (LdtPae3). Construction of a phylogram revealed that LDTs performing each of these three functions in various species cannot be assigned to distinct evolutionary lineages, in contrast to what has been observed with PBPs. We showed that biofilm, but not planktonic bacteria, displayed an increase proportion of peptidoglycan cross-links formed by LdtPae1 and a greater extent of OprI anchoring to peptidoglycan, which is controlled by LdtPae2 and LdtPae3. Consistently, deletion of each of the ldt genes impaired biofilm formation and potentiated the bactericidal activity of EDTA. These results indicate that LDTs contribute to the stabilization of the bacterial cell envelope and to the adaptation of peptidoglycan metabolism to growth in biofilm. IMPORTANCE Active-site cysteine LDTs form a functionally heterologous family of enzymes that contribute to the biogenesis of the bacterial cell envelope through formation of peptidoglycan cross-links and through the dynamic anchoring of lipoproteins to peptidoglycan. Here, we report the role of three P. aeruginosa LDTs that had not been previously characterized. We show that these enzymes contribute to resistance to the bactericidal activity of EDTA and to the adaptation of cell envelope polymers to conditions that prevail in biofilms. These results indicate that LDTs should be considered putative targets in the development of drug-EDTA associations for the control of biofilm-related infections.

Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance.

Bacterial antibiotic resistance is a global health concern of increasing importance and intensive study. Although biofilms are a common source of infections in clinical settings, little is known about the development of antibiotic resistance within biofilms. Here, we use experimental evolution to compare selection of resistance mutations in planktonic and biofilm Escherichia coli populations exposed to clinically relevant cycles of lethal treatment with the aminoglycoside amikacin. Consistently, mutations in sbmA, encoding an inner membrane peptide transporter, and fusA, encoding the essential elongation factor G, are rapidly selected in biofilms, but not in planktonic cells. This is due to a combination of enhanced mutation rate, increased adhesion capacity and protective biofilm-associated tolerance. These results show that the biofilm environment favors rapid evolution of resistance and provide new insights into the dynamic evolution of antibiotic resistance in biofilms.

Selection for nonspecific adhesion is a driver of FimH evolution increasing Escherichia coli biofilm capacity.

Bacterial interactions with surfaces rely on the coordinated expression of a vast repertoire of surface-exposed adhesins. However, how bacteria dynamically modulate their adhesion potential to achieve successful surface colonization is not yet well understood. Here, we investigated changes in adhesion capacity of an initially poorly adherent Escherichia coli strain using experimental evolution and positive selection for mutations improving adhesion and biofilm formation on abiotic surfaces. We showed that all identified evolved populations and clones acquired mutations located almost exclusively in the lectin domain of fimH, the gene coding for the α-d-mannose-specific tip adhesin of type 1 fimbriae, a key E. coli virulence factor. While most of these fimH mutants showed reduced mannose-binding ability, they all displayed enhanced binding to abiotic surfaces, indicating a trade-off between FimH-mediated specific and nonspecific adhesion properties. Several of the identified mutations were already reported in the FimH lectin domain of pathogenic and environmental E. coli, suggesting that, beyond pathoadaptation, FimH microevolution favoring nonspecific surface adhesion could constitute a selective advantage for natural E. coli isolates. Consistently, although E. coli deleted for the fim operon still evolves an increased adhesion capacity, mutants selected in the ∆fim background are outcompeted by fimH mutants revealing clonal interference for adhesion. Our study therefore provides insights into the plasticity of E. coli adhesion potential and shows that evolution of type 1 fimbriae is a major driver of the adaptation of natural E. coli to colonization.

Long-term acclimation to cadmium exposure reveals extensive phenotypic plasticity in Chlamydomonas.

Increasing industrial and anthropogenic activities are producing and releasing more and more pollutants in the environment. Among them, toxic metals are one of the major threats for human health and natural ecosystems. Because photosynthetic organisms play a critical role in primary productivity and pollution management, investigating their response to metal toxicity is of major interest. Here, the green microalga Chlamydomonas (Chlamydomonas reinhardtii) was subjected to short (3 d) or chronic (6 months) exposure to 50 µM cadmium (Cd), and the recovery from chronic exposure was also examined. An extensive phenotypic characterization and transcriptomic analysis showed that the impact of Cd on biomass production of short-term (ST) exposed cells was almost entirely abolished by long-term (LT) acclimation. The underlying mechanisms were initiated at ST and further amplified after LT exposure resulting in a reversible equilibrium allowing biomass production similar to control condition. This included modification of cell wall-related gene expression and biofilm-like structure formation, dynamics of metal ion uptake and homeostasis, photosynthesis efficiency recovery and Cd acclimation through metal homeostasis adjustment. The contribution of the identified coordination of phosphorus and iron homeostasis (partly) mediated by the main phosphorus homeostasis regulator, Phosphate Starvation Response 1, and a basic Helix-Loop-Helix transcription factor (Cre05.g241636) was further investigated. The study reveals the highly dynamic physiological plasticity enabling algal cell growth in an extreme environment.

The Transition Toward Nitrogen Deprivation in Diatoms Requires Chloroplast Stand-By and Deep Metabolic Reshuffling.

Microalgae have adapted to face abiotic stresses by accumulating energy storage molecules such as lipids, which are also of interest to industries. Unfortunately, the impairment in cell division during the accumulation of these molecules constitutes a major bottleneck for the development of efficient microalgae-based biotechnology processes. To address the bottleneck, a multidisciplinary approach was used to study the mechanisms involved in the transition from nitrogen repletion to nitrogen starvation conditions in the marine diatom Phaeodactylum tricornutum that was cultured in a turbidostat. Combining data demonstrate that the different steps of nitrogen deficiency clustered together in a single state in which cells are in equilibrium with their environment. The switch between the nitrogen-replete and the nitrogen-deficient equilibrium is driven by intracellular nitrogen availability. The switch induces a major gene expression change, which is reflected in the reorientation of the carbon metabolism toward an energy storage mode while still operating as a metabolic flywheel. Although the photosynthetic activity is reduced, the chloroplast is kept in a stand-by mode allowing a fast resuming upon nitrogen repletion. Altogether, these results contribute to the understanding of the intricate response of diatoms under stress.

Transcription factors in microalgae: genome-wide prediction and comparative analysis.

BACKGROUND: Studying transcription factors, which are some of the key players in gene expression, is of outstanding interest for the investigation of the evolutionary history of organisms through lineage-specific features. In this study we performed the first genome-wide TF identification and comparison between haptophytes and other algal lineages. RESULTS: For TF identification and classification, we created a comprehensive pipeline using a combination of BLAST, HMMER and InterProScan software. The accuracy evaluation of the pipeline shows its applicability for every alga, plant and cyanobacterium, with very good PPV and sensitivity. This pipeline allowed us to identify and classified the transcription factor complement of the three haptophytes Tisochrysis lutea, Emiliania huxleyi and Pavlova sp.; the two stramenopiles Phaeodactylum tricornutum and Nannochloropsis gaditana; the chlorophyte Chlamydomonas reinhardtii and the rhodophyte Porphyridium purpureum. By using T. lutea and Porphyridium purpureum, this work extends the variety of species included in such comparative studies, allowing the detection and detailed study of lineage-specific features, such as the presence of TF families specific to the green lineage in Porphyridium purpureum, haptophytes and stramenopiles. Our comprehensive pipeline also allowed us to identify fungal and cyanobacterial TF families in the algal nuclear genomes. CONCLUSIONS: This study provides examples illustrating the complex evolutionary history of algae, some of which support the involvement of a green alga in haptophyte and stramenopile evolution.

Transcriptional response of stress-regulated genes to cadmium exposure in the cockle Cerastoderma glaucum from the gulf of Gabès area (Tunisia).

This study investigates cadmium effects on key messenger RNA (mRNA) expression (MT, MnSOD, CuZnSOD, CAT, ABCB1, HSP70, and CO1) by qPCR in the cockle Cerastoderma glaucum after chronic exposure to two high but environmentally relevant concentrations of CdCl2 (50 µg/L and 5 mg/L) for 12 h to 18 days. Cd accumulation measured in cockles' tissues is significantly higher in both treatment conditions compared to controls and in a dose-dependent manner. Stress on stress tests performed at different times of the experiment clearly demonstrated that exposure to both concentrations of Cd significantly affects cockle survival time in air. Important changes in gene transcription were also highlighted. In particular, MT, HSP70, CAT, and CuZnSOD seem to be relevant biomarkers of Cd exposure because (1) their mRNA levels increase upon exposure and (2) they are highly correlated to Cd accumulation in tissues. Results may be useful for control strategies and for the use of cockles as sentinel organisms.

First evidence of mariner-like transposons in the genome of the marine microalga Amphora acutiuscula (Bacillariophyta).

Mariner-like elements (MLEs) are transposable elements able to move in the host genomes by a "cut and paste" mechanism. They have been found in numerous organisms. We succeeded in amplifying complete and truncated MLEs in the marine diatom Amphora acutiuscula. Full-length MLEs of 2,100bp delimited by imperfect Terminal Inverted Repeats revealed an intact Open Reading Frame, suggesting that the MLEs could be active. The DNA binding domain of the corresponding putative transposase could have two Helix-Turn-Helix and a Nuclear Location Site motifs, and its catalytic domain includes a particular triad of aspartic acids DD43D not previously reported. The number of copies was estimated to be 38, including approximately 20 full-length elements. Phylogenetic analysis shows that these peculiar MLEs differ from plant and other stramenopile MLEs and that they could constitute a new sub-family of Tc1-mariner elements.