Evolution des Régulations Endocriniennes

We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled oligonucleotides). All sequences were capable of forming stable quadruplexes, with T(m) above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and T(m) was found in K(+): each added base leads to a 2°C drop in T(m) or ∼0.3 kcal/mol loss in ΔG°. The trend was less clear in Na(+), with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases).

Microcins are gene-encoded antibacterial peptides, with molecular masses below 10 kDa, produced by enterobacteria. They are secreted under conditions of nutrient depletion and exert potent antibacterial activity against closely related species. Typical gene clusters encoding the microcin precursor, the self-immunity factor, the secretion proteins and frequently the post-translational modification enzymes are located either on plasmids or on the chromosome. In contrast to most of the antibiotics of microbial origin, which are non-ribosomally synthesized by multimodular enzymes termed peptide synthetases, microcins are ribosomally synthesized as precursors, which are further modified enzymatically. They form a restricted class of potent antibacterial peptides. Fourteen microcins have been reported so far, among which only seven have been isolated and characterized. Despite the low number of known representatives, microcins exhibit a diversity of structures and antibacterial mechanisms. This review provides an updated overview of microcin structures, antibacterial activities, genetic systems and biosyntheses, as well as of their mechanisms of action.

OBJECTIVE: To define the role that repeat expansions of a GGGGCC hexanucleotide sequence of the C9orf72 gene play in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A genetic model for ALS was developed to determine whether loss of function of the zebrafish orthologue of C9orf72 (zC9orf72) leads to abnormalities in neuronal development. METHODS: C9orf72 mRNA levels were quantified in brain and lymphoblasts derived from FTLD and ALS/FTLD patients and in zebrafish. Knockdown of the zC9orf72 was performed using 2 specific antisense morpholino oligonucleotides to block transcription. Quantifications of spontaneous swimming and tactile escape response, as well as measurements of axonal projections from the spinal cord, were performed. RESULTS: Significantly decreased expression of C9orf72 transcripts in brain and lymphoblasts was found in sporadic FTLD and ALS/FTLD patients with normal-size or expanded hexanucleotide repeats. The zC9orf72 is selectively expressed in the developing nervous system at developmental stages. Loss of function of the zC9orf72 transcripts causes both behavioral and cellular deficits related to locomotion without major morphological abnormalities. These deficits were rescued upon overexpression of human C9orf72 mRNA transcripts. INTERPRETATION: Our results indicate C9orf72 haploinsufficiency could be a contributing factor in the spectrum of ALS/FTLD neurodegenerative disorders. Loss of function of the zebrafish orthologue of zC9orf72 expression in zebrafish is associated with axonal degeneration of motor neurons that can be rescued by expressing human C9orf72 mRNA, highlighting the specificity of the induced phenotype. These results reveal a pathogenic consequence of decreased C9orf72 levels, supporting a loss of function mechanism of disease.

Amyloid deposits within the cerebral tissue constitute a characteristic lesion associated with Alzheimer disease. They mainly consist of the amyloid peptide Abeta and display an abnormal content in Zn(2+) ions, together with many truncated, isomerized, and racemized forms of Abeta. The region 1-16 of Abeta can be considered the minimal zinc-binding domain and contains two aspartates subject to protein aging. The influence of zinc binding and protein aging related modifications on the conformation of this region of Abeta is of importance given the potentiality of this domain to constitute a therapeutic target, especially for immunization approaches. In this study, we determined from NMR data the solution structure of the Abeta-(1-16)-Zn(2+) complex in aqueous solution at pH 6.5. The residues His(6), His(13), and His(14) and the Glu(11) carboxylate were identified as ligands that tetrahedrally coordinate the Zn(II) cation. In vitro aging experiments on Abeta-(1-16) led to the formation of truncated and isomerized species. The major isomer generated, Abeta-(1-16)-l-iso-Asp(7), displayed a local conformational change in the His(6)-Ser(8) region but kept a zinc binding propensity via a coordination mode involving l-iso-Asp(7). These results are discussed here with regard to Abeta fibrillogenesis and the potentiality of the region 1-16 of Abeta to be used as a therapeutic target.

Human biomonitoring (HBM) studies have demonstrated widespread and daily exposure to bisphenol A (BPA). Moreover, BPA structural analogues (e.g. BPS, BPF, BPAF), used as BPA replacements, are being increasingly detected in human biological matrices. BPA and some of its analogues are classified as endocrine disruptors suspected of contributing to adverse health outcomes such as altered reproduction and neurodevelopment, obesity, and metabolic disorders among other developmental and chronic impairments. One of the aims of the H2020 European Human Biomonitoring Initiative (HBM4EU) is the implementation of effect biomarkers at large scales in future HBM studies in a systematic and standardized way, in order to complement exposure data with mechanistically-based biomarkers of early adverse effects. This review aimed to identify and prioritize existing biomarkers of effect for BPA, as well as to provide relevant mechanistic and adverse outcome pathway (AOP) information in order to cover knowledge gaps and better interpret effect biomarker data. A comprehensive literature search was performed in PubMed to identify all the epidemiologic studies published in the last 10 years addressing the potential relationship between bisphenols exposure and alterations in biological parameters. A total of 5716 references were screened, out of which, 119 full-text articles were analyzed and tabulated in detail. This work provides first an overview of all epigenetics, gene transcription, oxidative stress, reproductive, glucocorticoid and thyroid hormones, metabolic and allergy/immune biomarkers previously studied. Then, promising effect biomarkers related to altered neurodevelopmental and reproductive outcomes including brain-derived neurotrophic factor (BDNF), kisspeptin (KiSS), and gene expression of nuclear receptors are prioritized, providing mechanistic insights based on in vitro, animal studies and AOP information. Finally, the potential of omics technologies for biomarker discovery and its implications for risk assessment are discussed. To the best of our knowledge, this is the first effort to comprehensively identify bisphenol-related biomarkers of effect for HBM purposes.

Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily.

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.

During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.

Steroid hormones regulate many physiological processes in vertebrates, nematodes, and arthropods through binding to nuclear receptors (NR), a metazoan-specific family of ligand-activated transcription factors. The main steps controlling the diversification of this family are now well-understood. In contrast, the origin and evolution of steroid ligands remain mysterious, although this is crucial for understanding the emergence of modern endocrine systems. Using a comparative genomic approach, we analyzed complete metazoan genomes to provide a comprehensive view of the evolution of major enzymatic players implicated in steroidogenesis at the whole metazoan scale. Our analysis reveals that steroidogenesis has been independently elaborated in the 3 main bilaterian lineages, and that steroidogenic cytochrome P450 enzymes descended from those that detoxify xenobiotics.

Considered during the past decades as cell dust, microparticles are now deemed true biomarkers and vectors of biological information between cells. Depending on their origin, the composition of microparticles varies and the subsequent message transported by them, such as proteins, mRNA, or miRNA, can differ. Recent studies have described microparticles as "cargos" of deleterious information in blood vessel wall under pathological situations such as hypertension, myocardial infarction, and metabolic syndrome. In addition, it has been reported that depending on their origin, microparticles also possess a therapeutic potential regarding angiogenesis. Microparticles can act directly through the interaction ligand/receptor or indirectly on angiogenesis by modulating soluble factor production involved in endothelial cell differentiation, proliferation, migration, and adhesion; by reprogramming endothelial mature cells; and by inducing changes in levels, phenotype, and function of endothelial progenitor cells. This results in an increase in formation of in vitro capillary-like tubes and the generation of new vessels in vivo under ischemic conditions, for instance. Taking into consideration these properties of microparticles, recent evidence provides new basis to expand the possibility that microparticles might be used as therapeutic tools in pathologies associated with an alteration of angiogenesis.

Background: Plant composition changes with topography and edaphic gradients that correlate with soil-water and nutrient availability. Data on soil water for the Amazon Basin are scarce, limiting the possibility of distinguishing between soil and soil-water influences on plant composition. Aim: We tested a new proxy for water table depth, the terrain height above nearest drainage (HAND), as a predictor of composition in trees, lianas, palms, shrubs, and herbs and compared HAND to conventional measures of height above sea level (HASL) and horizontal distances from nearest drainage (HDND). Methods: Plant-species composition in 72 plots distributed across 64 km2 of lowland evergreen terra firme forest was summarised using non-metric multidimensional scaling (NMDS). NMDS scores were regressed against estimates of HAND, HASL and HDND. Results: Plant composition was highly correlated with the vertical distance from water table, capturing up to 82% of variation. All life forms showed highest turnover rates in the zone with seasonally water-saturated soils, which can extend 350 m from stream margins. Conclusions: Floristic composition is closely related to water table depth, and HAND appears to be the most robust available topographical metric of soil-water gradients. Brazilian conservation laws protecting 30-m-wide riparian buffers are likely to be too narrow to encompass the full zone of highest floristic turnover and may be ineffective in safeguarding riparian plant diversity.

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Three pyrrolo[1,2-a]quinoxalines, 15 bispyrrolo[1,2-a]quinoxalines, bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines, and bispyrrolo[1,2-a]thieno[3,2-e]pyrazines were synthesized from various substituted nitroanilines or nitropyridines and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. Bispyrrolo[1,2-a]quinoxalines showed superior antimalarial activity with respect to monopyrrolo[1,2-a]quinoxalines. The best activity was observed with bispyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that the presence of a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus increased the pharmacological activity. Drug effects upon beta-hematin formation were assayed and showed similar or higher inhibitory activities than CQ. A possible mechanism of interaction implicating binding of pyrroloquinoxalines to beta-hematin was supported by molecular modeling.

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).

Bivalves have evolved a range of complex shell forming mechanisms that are reflected by their incredible diversity in shell mineralogy and microstructures. A suite of proteins exported to the shell matrix space plays a significant role in controlling these features, in addition to underpinning some of the physical properties of the shell itself. Although, there is a general consensus that a minimum basic protein tool kit is required for shell construction, to date, this remains undefined. In this study, the shell matrix proteins (SMPs) of four highly divergent bivalves (The Pacific oyster, Crassostrea gigas; the blue mussel, Mytilus edulis; the clam, Mya truncata, and the king scallop, Pecten maximus) were analyzed in an identical fashion using proteomics pipeline. This enabled us to identify the critical elements of a "basic tool kit" for calcification processes, which were conserved across the taxa irrespective of the shell morphology and arrangement of the crystal surfaces. In addition, protein domains controlling the crystal layers specific to aragonite and calcite were also identified. Intriguingly, a significant number of the identified SMPs contained domains related to immune functions. These were often are unique to each species implying their involvement not only in immunity, but also environmental adaptation. This suggests that the SMPs are selectively exported in a complex mix to endow the shell with both mechanical protection and biochemical defense.

Magnoliales, consisting of six families of tropical to warm-temperate woody angiosperms, were long considered the most archaic order of flowering plants, but molecular analyses nest them among other eumagnoliids. Based on separate and combined analyses of a morphological matrix (115 characters) and multiple molecular data sets (seven variable chloroplast loci and five more conserved genes; 14 536 aligned nucleotides), phylogenetic relationships were investigated simultaneously within Magnoliales and Myristicaceae, using Laurales, Winterales, and Piperales as outgroups. Despite apparent conflicts among data sets, parsimony and maximum likelihood analyses of combined data converged towards a fully resolved and well-supported topology, consistent with higher-level molecular analyses except for the position of Magnoliaceae: Myristicaceae + (Magnoliaceae + ((Degeneria+Galbulimima) + (Eupomatia+ Annonaceae))). Based on these results, we discuss morphological evolution in Magnoliales and show that several supposedly plesiomorphic traits are synapomorphies of Magnoliineae, the sister group of Myristicaceae (e.g. laminar stamens). Relationships within Annonaceae are also resolved with strong support (Anaxagorea basal, then ambavioids). In contrast, resolution of relationships within Myristicaceae is difficult and still incomplete, due to a very low level of molecular divergence within the family and a long stem lineage. However, our data provide good evidence that Mauloutchia is nested among other Afro-Malagasy genera, contradicting the view that its androecium and pollen are plesiomorphic © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 142, 125–186.

Articulated jaws are highly conserved structures characteristic of gnathostome evolution. Epithelial-mesenchymal interactions within the first pharyngeal arch (PA1) instruct cephalic neural crest cells (CNCCs) to form the different skeletal elements of the jaws. The endothelin-1 (Edn1)/endothelin receptor type-A (Ednra)-->Dlx5/6-->Hand2 signaling pathway is necessary for lower jaw formation. Here, we show that the Edn1 signaling is sufficient for the conversion of the maxillary arch to mandibular identity. Constitutive activation of Ednra induced the transformation of upper jaw, maxillary, structures into lower jaw, mandibular, structures with duplicated Meckel's cartilage and dermatocranial jaws constituted by 4 dentary bones. Misexpression of Hand2 in the Ednra domain caused a similar transformation. Skeletal transformations are accompanied by neuromuscular remodeling. Ednra is expressed by most CNCCs, but its constitutive activation affects predominantly PA1. We conclude that after migration CNCCs are not all equivalent, suggesting that their specification occurs in sequential steps. Also, we show that, within PA1, CNCCs are competent to form both mandibular and maxillary structures and that an Edn1 switch is responsible for the choice of either morphogenetic program.

Trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (trans-ACPD), a specific agonist of the glutamate phosphoinositide-coupled receptor (Qp receptor), increased the amplitude of the outward K+ current recorded in the whole-cell configuration of the patch-clamp technique in mouse cultured cerebellar granule cells. This effect was abolished by buffering internal Ca2+ with BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. Activation of a large-conductance K+ channel was observed when trans-ACPD or quisqualic acid (QA), another Qp receptor agonist, was applied outside the cell-attached patch pipettes. No activation was observed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a specific agonist of ionotropic non-N-methyl-d-aspartate (non-NMDA) receptors. The effects of trans-ACPD or QA were potentiated in the presence of external Ca2+. The channel was also directly activated by both micromolar concentrations of internal Ca2+ and membrane depolarization. Its unitary conductance was 100 - 115 pS under asymmetrical K+ and 195 - 235 pS under high symmetrical K+ conditions. In the absence of agonist, the channel was blocked by 1 mM external tetraethylammonium. This is the first description of a large conductance Ca2+-activated K+ channel in cultured cerebellar granule cells. It possesses properties similar to those of the so-called 'big K+ channel' described in other preparations. Our cell-attached experiments demonstrated an indirect coupling between Qp receptors and this channel. The most likely hypothesis is that the second messenger system inositol 1,4,5-triphosphate (IP3)-Ca2+ was involved in the coupling process. This hypothesis was further strengthened by our whole-cell experiments. On the basis of the voltage- and Ca2+-sensitivities of the studied channel, we estimated an increase of 350 to 570 nM in internal Ca2+ concentration when Qp receptors were stimulated by 100 microM trans-ACPD. Under physiological conditions, stimulation of Qp receptors by the endogenous neurotransmitter should lead to similar K+ channel activation and therefore would tend to reduce the efficacy of ionotropic glutamate synaptic receptor stimulation responsible for cell excitation.

The lower jaw skeleton is derived from cephalic neural crest (CNC) cells that reside in the mandibular region of the first pharyngeal arch. Endothelin-A receptor (Ednra) signaling in crest cells is crucial for their development, as Ednra(-/-) mice are born with severe craniofacial defects resulting in neonatal lethality. In this study, we undertook a more detailed analysis of mandibular arch development in Ednra(-/-) embryos to better understand the cellular and molecular basis for these defects. We show that most lower jaw structures in Ednra(-/-) embryos undergo a homeotic transformation into maxillary-like structures similar to those observed in Dlx5/Dlx6(-/-) embryos, though lower incisors are still present in both mutant embryos. These structural changes are preceded by aberrant expansion of proximal first arch gene expression into the distal arch, in addition to the previously described loss of a Dlx6/Hand2 expression network. However, a small distal Hand2 expression domain remains. Although this distal expression is not dependent on either Ednra or Dlx5/Dlx6 function, it may require one or more GATA factors. Using fate analysis, we show that these distal Hand2-positive cells probably contribute to lower incisor formation. Together, our results suggest that the establishment of a 'mandibular identity' during lower jaw development requires both Ednra-dependent and -independent signaling pathways.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.