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A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anoxic activated sludge from a municipal wastewater treatment plant. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by polymerase chain reaction using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 132 and 249 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was done using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota (93.8% of the operational taxonomic units [OTUs]) and Crenarchaeota (6.2% of the OTUs). Within the bacterial library, 84.8% of the OTUs represent novel putative phylotypes never described before and affiliated with ten divisions. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 60.4% of the OTUs, followed by Bacteroidetes (22.1%) and gram-positives (6.1%). Interestingly, we detected a novel Proteobacteria monophyletic group distinct from the five known subclasses, which we named New Lineage of Proteobacteria (NLP) lineage, and it is composed of eight clones representing 4.6% of the Proteobacteria. A new 16S rRNA-targeted hybridization probe was designed and fluorescent in situ hybridization analyses shows representatives of NLP as cocci-shaped microorganisms. The Chloroflexi, Acidobacterium, and Nitrospira phyla and TM7 candidate division are each represented by ≤3% of clone sequences. A comprehensive set of eight 16S and 23S rRNA-targeted oligonucleotide probes was used to quantify these major groups by dot blot hybridization within 12 samples. The Proteobacteria accounted for 82.5 ± 4.9%, representing the most abundant phyla. The Bacteroidetes and Planctomycetales groups accounted for 4.9 ± 1.3% and 4 ± 1.7%, respectively. Firmicutes and Actinobacteria together accounted for only 1.9 ± 0.5%. The set of probes covers 93.4 ± 14% of the total bacterial population rRNA within the anoxic basin.

Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations ( approximately 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions. P1 transductions from K-12 into the two B lineages produced 317 single-base-pair differences and 9 insertions or deletions, reflecting differences between B DNA in BL21(DE3) and integrated restriction fragments of K-12 DNA inherited by REL606. Two sites showed selective enrichment of spontaneous mutations. No unselected spontaneous single-base-pair mutations were evident. The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences. Limited sequencing of other B strains defined characteristic properties of B and allowed assembly of the inferred genome of the ancestral B of Delbrück and Luria. Comparison of the B and K-12 genomes shows that more than half of the 3793 proteins of their basic genomes are predicted to be identical, although approximately 310 appear to be functional in either B or K-12 but not in both. The ancestral basic genome appears to have had approximately 4039 coding sequences occupying approximately 4.0 Mbp. Repeated horizontal transfer from diverged Escherichia coli genomes and homologous recombination may explain the observed variable distribution of single-base-pair differences. Fifteen sites are occupied by phage-related elements, but only six by comparable elements at the same site. More than 50 sites are occupied by IS elements in both B and K, 16 in common, and likely founding IS elements are identified. A signature of widespread cryptic phage P4-type mobile elements was identified. Complex deletions (dense clusters of small deletions and substitutions) apparently removed nonessential genes from approximately 30 sites in the basic genomes.

Antecedents of Escherichia coli B have been traced through publications, inferences, and personal communication to a strain from the Institut Pasteur in Paris used by d'Herelle in his studies of bacteriophages as early as 1918 (a strain not in the current collection). This strain appears to have passed from d'Herelle to Bordet in 1920, and from Bordet to at least three other laboratories by 1925. The strain that Gratia received from Bordet was apparently passed to Bronfenbrenner by 1924 and from him to Luria around 1941. Delbrück and Luria published the first paper calling this strain B in 1942. Its choice as the common host for phages T1-T7 by the phage group that developed around Delbrück, Luria, and Hershey in the 1940s led to widespread use of B along with E. coli K-12, chosen about the same time for biochemical and genetic studies by Tatum and Lederberg. Not all currently available strains related to B are descended from the B of Delbrück and Luria; at least three strains with somewhat different characteristics were derived independently by Hershey directly from the Bronfenbrenner strain, and a strain that appears to have passed from Bordet to Wollman is in the current Collection of the Institut Pasteur. The succession of manipulations and strains that led from the B of Delbrück and Luria to REL606 and BL21(DE3) is given, established in part through evidence from their recently determined complete genome sequences.

In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69: 7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.

We show some thermodynamic results concerning chemical kinetics, either from statistical physics, under the strong assumption (EXS) of the statistical exponential distribution of the physical states, or from kinetics. In that frame, the reaction rates are modeled according to the stochastic activation collision theory, under the weaker assumption (EXW) of the statistical exponential distribution not on the whole space but only within each structure; moreover a symmetry condition (SY) concerning the local properties of the rate constants is assumed. The Boltzmann distribution and an extension of the more general result of the thermodynamic law of mass action are proved. Moreover, we show that the assumptions (EXS) and (EXW) are equivalent, under (SY). At last, we deal with the stochastic example of the RNA structure for which we calculate the temporal probabilities of the structures by means of a jump process.

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the lambda attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have >99% sequence identity over approximately 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.

[Departement_IRSTEA]MA [TR1_IRSTEA]TED / SOWASTE; International audience; We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA-DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as “ Candidatus Cloacamonas acidaminovorans.” “ Candidatus Cloacamonas acidaminovorans” is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomycetes . Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.

We propose a common rigorous foundation to the classical collision theory and that of the classical activated complex. Based on the notion of activated complex, this foundation relies on a stochastic approach showing up the different influence factors of a chemical reaction. The thermodynamic formulation is obtained here by assuming the exponential statistical distribution within each stable chemical state. The general model we obtain yields two stochastic formulations called the stochastic transition state theory (denoted STST) and the stochastic activated collision theory (denoted SACT) respectively, depending on whether the rate of the reaction is of the same scale as, either the rate of passing over the potential energy barrier, for the STST, or the rate of reaching the activated complex state (which is a generalization of the collision rate), for the SACT. The modeling is first done in the case of a closed small system undergoing stochastic changes of chemical state and then, by extension, in the fra...

International audience; A rigorous mathematical modeling of the RNA sequential folding process during transcription is proposed. It is based, at each transcription step, on a homogeneous markovian jump process, the state space of which is the set of structures constructible on the part of the RNA already transcribed. A theoretical formula permitting the computation of the structures probabilities at the end of the RNA transcription is derived. Successive approximations, aimed at reducing the size of the state space, permit the design of a prediction algorithm. The algorithm is tested on some structural RNAs (tRNA, 5S, 16S, hammerhead, ...), results are discussed and possible improvements are proposed.

We have determined the DNA sequence of the rIIA gene and have discovered a small open reading frame, rIIA.1, between genes 60 and rIIA. The predicted molecular weights of these proteins are 82,840 for rIIA and 8,124 for rIIA.1. The rIIA protein has a repeated motif which suggests that the gene has evolved by duplication. It also has a motif which suggests that it belongs to a group of ompR-like proteins that control regulation of gene expression in response to changes in the external environment. We have sequenced three different missense mutants whose mutations lie in the Ala segment of the rIIA genetic map. All three changes are found within the first 35 bp of the rIIA coding sequence. The region of control of protein synthesis is identical in the rIIA gene and in gene 44 of T4. We relate this finding to the high sensitivity of both RNAs to translational repression by the T4 regA gene product.

The effect of the E. coli termination factor rho on middle mode and anti-late T4 transcription was examined. RNA from CAM-treated rho ts-infected cells contains only about 25% of the normal T4 gene 43 RNA levels, despite the fact that the DE content of this RNA is qualitatively normal. Gene 43 RNA is not found during the early period of infection of rho+ bacteria by mot− bacteriophage, but about 20–25% normal levels are found when mot− phage infect rho ts hosts. Thus gene 43 is transcribed in two modes: a major middle mode which uses mot as an initiation cofactor and a minor early mode which seems to need mot expression to help overcome rho action. Apparently, the middle mode RNA from gene 43 is translated much more efficiently than is the early RNA transcribed from this gene. CAM RNA from rho ts infections contains nearly as much anti-late RNA as does normal RNA (without antibiotic) from these cells. Thus anti-late synthesis, which is ordinarily mot dependent, becomes mot independent if there is no rho activity in the hosts cells. These data along with experiments on an in vitro system for middle mode expression ( C. Thermes, P. Daegelen, V. de Franciscis, and E. Brody (1976) Proc. Nat. Acad. Sci. USA 73, 2569–2573) are consistent with a model in which the mot product binds to DNA where it acts either as a melting protein or as a gyrase. This DNA change leads to changed interactions both with rho and RNA polymerase.

We have constructed a bacteriophage T4 isar which is deleted in the rII and D2 regions and simultaneously allows cells to be superinfected after a primary infection. After infecting with this phage, we add chloramphenicol to the cells, and after a short period we introduce the rII and D2 regions into the cell by superinfection with a phage carrying these regions. Transcription of the rII and D2 regions takes place very rapidly after superinfection. We conclude that there is a stable, diffusible T4 effect which overcomes the restriction of T4 transcription seen when infection is done in the presence of chloramphenicol. The kinetics of rIIA and rIIB synthesis are much more rapid when these genes are first introduced into a T4 isar infected cell than in a normal infection. We think that a promoter close to rIIA is used in superinfection of T4isar infected cells; this promoter is different from the promoter usually used for the synthesis of rIIA RNA.

We have used ts lethal mutants in Escherichia coli termination factor rho to examine its role in T4 development. The rho ts mutation has been transduced into E. coli B from E. coli K12. This mutation has a number of different properties in the E. coli B genetic background, but the overall effect on T4 regulation seems to be the same. Rho-defective bacteria produce T4 after infection but the burst size is lower than in rho + host cells. We have found that two protein bands appear after T4 + infections of rho ts , but not rho + , bacteria. One of these is gp alt and the other is gp 7. These proteins seem to be expressed in a different mode than the late mode in rho + cells. Their appearance is sensitive to a mutation in the T4 mot gene. When T4 + infects E. coli rho + bacteria in the presence of chloramphenicol (CAM), only a subfraction of early RNA is found. This RNA comes from promoter proximal portions of early polycistronic transcription units and is called immediate early RNA (IE). CAM-sensitive RNA is defined as delayed early (DE) RNA. CAM RNA from rho ts bacteria has almost normal amounts of DE RNA. Thus, CAM-induced polarity, which limits T4 transcription to IE RNA, is mediated by rho factor.