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Direct involvement of DprA, the transformation-dedicated RecA loader, in the shut-off of pneumococcal competence

Significance This article concerns the control of competence for bacterial genetic transformation. Competence is transient in the human pathogen Streptococcus pneumoniae, involving the specific expression of ∼100 genes that are turned ON suddenly and OFF almost as abruptly. Although the mechanism rendering all cells in a culture simultaneously competent is well understood, how competence stops has remained unknown. Here, we unravel the mechanism of shut-off, describing the discovery that a key recombination protein, DprA, exerts a negative control on competence through direct, physical interaction with the master regulator of competence, the response regulator ComE, to abolish transcription from ComE-activated promoters. Natural bacterial transformation is a genetically programmed process allowing genotype alterations that involves the internalization of DNA and its chromosomal integration catalyzed by the universal recombinase RecA, assisted by its transformation-dedicated loader, DNA processing protein A (DprA). In Streptococcus pneumoniae, the ability to internalize DNA, known as competence, is transient, developing suddenly and stopping as quickly. Competence is induced by the comC-encoded peptide, competence stimulating peptide (CSP), via a classic two-component regulatory system ComDE. Upon CSP binding, ComD phosphorylates the ComE response-regulator, which then activates transcription of comCDE and the competence-specific σX, leading to a sudden rise in CSP levels and rendering all cells in a culture competent. However, how competence stops has remained unknown. We report that DprA, under σX control, interacts with ComE∼P to block ComE-driven transcription, chiefly impacting σX production. Mutations of dprA specifically disrupting interaction with ComE were isolated and shown to map mainly to the N-terminal domain of DprA. Wild-type DprA but not ComE interaction mutants affected in vitro binding of ComE to its promoter targets. Once introduced at the dprA chromosomal locus, mutations disrupting DprA interaction with ComE altered competence shut-off. The absence of DprA was found to negatively impact growth following competence induction, highlighting the importance of DprA for pneumococcal physiology. DprA has thus two key roles: ensuring production of transformants via interaction with RecA and competence shut-off via interaction with ComE, avoiding physiologically detrimental consequences of prolonged competence. Finally, phylogenetic analyses revealed that the acquisition of a new function by DprA impacted its evolution in streptococci relying on ComE to regulate comX expression.

Diversity of β-lactam resistance mechanisms in cystic fibrosis isolates of Pseudomonas aeruginosa: a French multicentre study

OBJECTIVESnTo investigate the resistance mechanisms of β-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France.nnnMETHODSnTwo-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to β-lactams were investigated. Their β-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic β-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations.nnnRESULTSnMLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to β-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to β-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary β-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene.nnnCONCLUSIONSnβ-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of β-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpAIS200/IS605). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpAREP) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpAREP is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpAREP was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpAREP activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpAREP shared the same general organization and similar catalytic characteristics with TnpAIS200/IS605 transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpAREP may therefore be one of the first examples of transposase domestication in prokaryotes.

Recognition of cell surface acceptors by two human α-2,6-sialyltransferases produced in CHO cells ☆

The action of sialyltransferases (STs) on cell surface glycoconjugates is a key process in shaping cell phenotype in a variety of cells mostly involved in migratory and adhesive pathways. The factors determining cell-specific pattern of glycosylation are so far poorly understood. Most STs are resident proteins of the Golgi apparatus, where acceptors are sialylated while they are in transit to the cell surface. To identify putative structural features that may account for their acceptor preference, we analyzed 53 cloned animal and human STs. We could identify conserved regions and peptide motifs representative of ST subfamilies, located at the C-terminal end of the hypervariable region upstream from the L-sialyl motif. Residues 93-100 in human ST6Gal I (hST6Gal I) were shown to be crucial for enzymatic activity when deleted and expressed in CHO cells. The Delta100 hST6Gal I mutant protein was fully recognized by polyclonal anti-hST6Gal I antibodies and followed the intracellular secretory pathway. This indicated that the conserved QVWxKDS sequence is essential for the whole catalytic domain to acquire a biologically active conformation. When full-length epitope-tagged hST6Gal I and hST6GalNAc I constructs were transfected in CHO cells, the alpha-2,6 sialylated glycotope was found to be largely restricted to intracellular resident acceptors and enzymatic activity based on fluorescent lectin staining. In contrast, both enzymes deprived of their membrane anchor and part of the hypervariable region but still possessing the conserved domains exhibited a very efficient transfer of sialic acid to cell surface glycoconjugates. Colocalization of the ST6Gal I mutant proteins with early and late Golgi markers such as giantin or rab6 proteins confirmed that soluble STs migrate forward in these subcompartments where they can act upon newly synthesized acceptors and follow the secretory pathway. It is thus concluded that downstream from the transmembrane domain, native STs possess peptide sequences that allow them to sialylate glycoprotein acceptors selectively along their transit within Golgi stacks.

Strategies for the identification, the assembly and the classification of integrated biological systems in completely sequenced genomes

The proteins involved in a single biological process may form a stable supra-molecular assembly or be transiently in interaction. Although, the first annotation steps of a complete genome may allow the identification of the different partners, their assembly in a functional system, referred to as an integrated system, is a domain where methodological effort has to be done. Indeed, the knowledge required to assemble partners of such systems should be explicitly included in annotation software. The availability of a complete genome, and therefore of all the proteins encoded by that genome, motivated the development of automated approaches through the coordinated combination of different bio-informatic methods allowing the identification of the different partners, their assembly and the classification of the reconstructed systems in functional categories. In this data flux, the identification of the sequence partners represents the principal bottleneck. Here, we describe and compare the results obtained with different classes of methods (BLASTP2, PSI-BLAST, MAST and META-MEME) applied to the identification in complete genomes of a given family of integrated systems: the ABC transporters. PSI-BLAST appears to significantly outperform motif-based methods, and the results are discussed according to the nature of the proteins and the structure of the sub-families.

Toxin-Antitoxin Loci in Mycobacterium tuberculosis

Chromosomally encoded type II toxin–antitoxin (TA) systems generally consist of two adjacent genes in an operon encoding a stable toxin and a less stable, protease-sensitive cognate antitoxin. While the toxin and the antitoxin form a stable complex under normal growth conditions, the degradation of the antitoxin by stress-proteases under certain conditions leads to activation of the toxin and subsequent growth inhibition. Such stress-responsive TA systems have been associated with various cellular processes, including stabilization of genomic regions, protection against foreign DNA, biofilm formation, persistence, and control of the stress response. Yet, the contribution of chromosomal TA to bacterial virulence is presently unknown. Herein, we investigate the potential role of multiple chromosomally encoded TA systems in virulence, focusing on the tuberculosis agent Mycobacterium tuberculosis, which contains more than 70 TA loci in its genome. We describe what is currently known about the multiple TA families present in this bacterium, with emphasis on the recently discovered atypical stress-responsive toxin–antitoxin-chaperone (TAC) system, a TA system controlled by a SecB-like chaperone.

Protein-coding region discovery in organisms under-represented in databases

The prediction of coding sequences has received a lot of attention during the last decade. We can distinguish two kinds of methods, those that rely on training with sets of example and counter-example sequences, and those that exploit the intrinsic properties of the DNA sequences to be analyzed. The former are generally more powerful but their domains of application are limited by the availability of a training set. The latter avoid this drawback but can only be applied to sequences that are long enough to allow computation of the statistics. Here, we present a method that fills the gap between the two approaches. A learning step is applied using a set of sequences that are assumed to contain coding and non-coding regions, but with the boundaries of these regions unknown. A test step then uses the discriminant function obtained during the learning to predict coding regions in sequences from the same organism. The learning relies upon a correspondence analysis and prediction is presented on a graphical display. The method has been evaluated on a sample of yeast sequences, and the analysis of a set of expressed sequence tags from the Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza illustrates the relevance of the approach in its biological context.

Single-strand DNA processing: phylogenomics and sequence diversity of a superfamily of potential prokaryotic HuH endonucleases

BackgroundSome mobile genetic elements target the lagging strand template during DNA replication. Bacterial examples are insertion sequences IS608 and ISDra2 (IS200/IS605 family members). They use obligatory single-stranded circular DNA intermediates for excision and insertion and encode a transposase, TnpAIS200, which recognizes subterminal secondary structures at the insertion sequence ends. Similar secondary structures, Repeated Extragenic Palindromes (REP), are present in many bacterial genomes. TnpAIS200-related proteins, TnpAREP, have been identified and could be responsible for REP sequence proliferation. These proteins share a conserved HuH/Tyrosine core domain responsible for catalysis and are involved in processes of ssDNA cleavage and ligation. Our goal is to characterize the diversity of these proteins collectively referred as the TnpAY1 family.ResultsA genome-wide analysis of sequences similar to TnpAIS200 and TnpAREP in prokaryotes revealed a large number of family members with a wide taxonomic distribution. These can be arranged into three distinct classes and 12 subclasses based on sequence similarity. One subclass includes sequences similar to TnpAIS200. Proteins from other subclasses are not associated with typical insertion sequence features. These are characterized by specific additional domains possibly involved in protein/DNA or protein/protein interactions. Their genes are found in more than 25% of species analyzed. They exhibit a patchy taxonomic distribution consistent with dissemination by horizontal gene transfers followed by loss. The tnpAREP genes of five subclasses are flanked by typical REP sequences in a REPtron-like arrangement. Four distinct REP types were characterized with a subclass specific distribution. Other subclasses are not associated with REP sequences but have a large conserved domain located in C-terminal end of their sequence. This unexpected diversity suggests that, while most likely involved in processing single-strand DNA, proteins from different subfamilies may play a number of different roles.ConclusionsWe established a detailed classification of TnpAY1 proteins, consolidated by the analysis of the conserved core domains and the characterization of additional domains. The data obtained illustrate the unexpected diversity of the TnpAY1 family and provide a strong framework for future evolutionary and functional studies. By their potential function in ssDNA editing, they may confer adaptive responses to host cell physiology and metabolism.