Effects of turbulence on diatoms of the genus pseudo-nitzschia spp. And associated bacteria.

Turbulence is one of the least investigated environmental factors impacting the ecophysiology of phytoplankton, both at the community and individual species level. Here, we investigated, for the first time, the effect of a turbulence gradient (${\rm{Reynolds\ number}}$, from ${\rm{R}}{{\rm{e}}}_{\rm{\lambda }} = 0$ to ${\rm{R}}{{\rm{e}}}_{\rm{\lambda }} = 360$) on two species of the marine diatom Pseudo-nitzschia and their associated bacterial communities under laboratory conditions. Cell abundance, domoic acid (DA) production, chain formation, and Chl a content of P. fraudulenta and P. multiseries were higher for intermediate turbulence (${\rm{R}}{{\rm{e}}}_{\rm{\lambda }} = 160$ or $240$). DA was detectable only in P. multiseries samples. These observations were supported by transcriptomic analyses results which suggested the turbulence related induction of the expression of the DA production locus, with a linkage to an increased photosynthetic activity of the total metatranscriptome. This study also highlighted a higher richness of the bacterial community associated with the non-toxic strain of P. fraudulenta in comparison to the toxic strain of P. multiseries. Bacillus was an important genus in P. multiseries cultures (relative abundance 15.5%) and its highest abundances coincided with the highest DA levels. However, associated bacterial communities of both Pseudo-nitzschia species did not show clear patterns relative to turbulence intensity.

Trajectories of nutrients concentrations and ratios in the French coastal ecosystems: 20 years of changes in relation with large-scale and local drivers.

Along with their important diversity, coastal ecosystems receive various amounts of nutrients, principally arising from the continent and from the related human activities (mainly industrial and agricultural activities). During the 20th century, nutrients loads have increased following the increase of both the global population and need of services. Alongside, climate change including temperature increase or atmospheric circulation change has occurred. These processes, Ecosystem state changes are hard to monitor and predict. To study the long-term changes of nutrients concentrations in coastal ecosystems, eleven French coastal ecosystems were studied over 20 years as they encompass large climatic and land pressures, representative of temperate ecosystems, over a rather small geographical area. Both univariate (time series decomposition) and multivariate (relationships between ecosystems and drivers) statistical analyses were used to determine ecosystem trajectories as well as typologies of ecosystem trajectories. It appeared that most of the French coastal ecosystems exhibited trajectories towards a decrease in nutrients concentrations. Differences in trajectories mainly depended on continental and human influences, as well as on climatic regimes. One single ecosystem exhibited very different trajectories, the Arcachon Bay with an increase in nutrients concentrations. Ecosystem trajectories based on ordination techniques were proven to be useful tools to monitor ecosystem changes. This study highlighted the importance of local environments and the need to couple uni- and multi-ecosystem studies. Although the studied ecosystems were influenced by both local and large-scale climate, by anthropogenic activities loads, and that their trajectories were mostly similar based on their continental influence, non-negligible variations resulted from their internal functioning.

Underwater light climate and wavelength dependence of microalgae photosynthetic parameters in a temperate sea.

Studying how natural phytoplankton adjust their photosynthetic properties to the quantity and quality of underwater light (i.e. light climate) is essential to understand primary production. A wavelength-dependent photoacclimation strategy was assessed using a multi-color pulse-amplitude-modulation chlorophyll fluorometer for phytoplankton samples collected in the spring at 19 locations across the English Channel. The functional absorption cross section of photosystem II, photosynthetic electron transport (PETλ) parameters and non-photochemical quenching were analyzed using an original approach with a sequence of three statistical analyses. Linear mixed-effects models using wavelength as a longitudinal variable were first applied to distinguish the fixed effect of the population from the random effect of individuals. Population and individual trends of wavelength-dependent PETλ parameters were consistent with photosynthesis and photoacclimation theories. The natural phytoplankton communities studied were in a photoprotective state for blue wavelengths (440 and 480 nm), but not for other wavelengths (green (540 nm), amber (590 nm) and light red (625 nm)). Population-detrended PETλ values were then used in multivariate analyses (partial triadic analysis and redundancy analysis) to study ecological implications of PETλ dynamics among water masses. Two wavelength ratios based on the microalgae saturation parameter Ek (in relative and absolute units), related to the hydrodynamic regime and underwater light climate, clearly confirmed the physiological state of microalgae. They also illustrate more accurately that natural phytoplankton communities can implement photoacclimation processes that are influenced by in situ light quality during the daylight cycle in temporarily and weakly stratified water. Ecological implications and consequences of PETλ are discussed in the context of turbulent coastal ecosystems.

Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily.

The MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the ß-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed.

Colicin M hydrolyses branched lipids II from Gram-positive bacteria.

Lipids II found in some Gram-positive bacteria were prepared in radioactive form from l-lysine-containing UDP-MurNAc-pentapeptide. The specific lateral chains of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus (di-L-alanine, D-isoasparagine, and pentaglycine, respectively) were introduced by chemical peptide synthesis using the Fmoc chemistry. The branched nucleotides obtained were converted into the corresponding lipids II by enzymatic synthesis using the MraY and MurG enzymes. All of the lipids were hydrolysed by Escherichia coli colicin M at approximately the same rate as the meso-diaminopimelate-containing lipid II found in Gram-negative bacteria, thereby opening the way to the use of this enzyme as a broad spectrum antibacterial agent.

Synthesis and biological evaluation of potential new inhibitors of the bacterial transferase MraY with a ß-ketophosphonate structure.

Stable analogs of bacterial transferase MraY substrate or product with a pyrophosphate surrogate in their structure are described. ß-ketophosphonates were designed as pyrophosphate bioisosteres and were investigated as UDP-GlcNAc mimics. The developed strategy allows introduction of structural diversity at a late stage of the synthesis. The biological activity of the synthesized compounds was evaluated on the MraY enzyme.

Synthesis and biological evaluation of a diazepanone-based library of liposidomycins analogs as MraY inhibitors.

New inhibitors of the bacterial tranferase MraY are described. A scaffold strategy based on the diazepanone central core of liposidomycins, natural inhibitors of MraY has been developed. It involves the introduction of key structural fragments required for biological activity on enantiopure diazepanones by reductive amination, esterification and glycosylation. Biological evaluation of these compounds on MraY enzyme revealed interesting inhibitory activity for compounds displaying three fragments on the scaffold: a palmitoyl chain, an aminoribose part and an alkyluracil moiety. The inhibitors were also evaluated on MurG enzyme. The best compounds resulted in inhibition with IC50 values in the 100 µM range for one or the other enzyme.

Bacterial transferase MraY inhibitors: synthesis and biological evaluation.

New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interesting inhibitory activity for both amino compounds.

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis.

To evaluate their role in the active site of the MurG enzyme from Escherichia coli, 13 residues conserved in the sequences of 73 MurG orthologues were submitted to site-directed mutagenesis. All these residues lay within, or close to, the active site of MurG as defined by its tridimensional structure [Ha et al., Prot. Sci. 9 (2000) 1045-1052, and Hu et al., Proc. Natl. Acad. Sci. USA 100 (2003) 845-849]. Thirteen mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms. The effects of the mutations on the activity were checked: (i) by functional complementation of an E. coli murG mutant strain by the mutated genes; and (ii) by the determination of the steady-state kinetic parameters of the purified proteins. Most mutations resulted in an important loss of activity and, in the case of N134A, in the production of a highly unstable protein. The results correlated with the assigned or putative functions of the residues based on the tridimensional structure.

The essential peptidoglycan glycosyltransferase MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli.

In Escherichia coli many enzymes including MurG are directly involved in the synthesis and assembly of peptidoglycan. MurG is an essential glycosyltransferase catalysing the last intracellular step of peptidoglycan synthesis. To elucidate its role during elongation and division events, localization of MurG using immunofluorescence microscopy was performed. MurG exhibited a random distribution in the cell envelope with a relatively higher intensity at the division site. This mid-cell localization was dependent on the presence of a mature divisome. Its localization in the lateral cell wall appeared to require the presence of MreCD. This could be indicative of a potential interaction between MurG and other proteins. Investigating this by immunoprecipitation revealed the association of MurG with MreB and MraY in the same protein complex. In view of this, the loss of rod shape of DeltamreBCD strain could be ascribed to the loss of MurG membrane localization. Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation. It is postulated that the involvement of MurG in the peptidoglycan synthesis concurs with two complexes, one implicated in cell elongation and the other in division. A model representing the first complex is proposed.

Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis.

The MraY translocase catalyzes the first membrane step of bacterial cell wall peptidoglycan synthesis (i.e. the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid), a reversible reaction yielding undecaprenylpyrophosphoryl-N-acetylmuramoyl-pentapeptide (lipid intermediate I). This essential integral membrane protein, which is considered as a very promising target for the search of new antibacterial compounds, has thus far been clearly underexploited due to its intrinsic refractory nature to overexpression and purification. We here report conditions for the high level overproduction and for the first time the purification to homogeneity of milligram quantities of MraY protein. The kinetic parameters and effects of pH, salts, cations, and detergents on enzyme activity are described, taking the Bacillus subtilis MraY translocase as a model.