The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.

A polymerase chain reaction (PCR)-based procedure without any cloning step was developed for a rapid mutagenesis/deletion of chromosomal target genes in Yersinia. For this purpose, a PCR fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus is electroporated into a recipient strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-sacB. Two PCR procedures were tested to generate an amplification product formed of an antibiotic resistance gene flanked by short (55 bp) or long (500 bp) homology extensions. Using this method, three chromosomal loci were successfully disrupted in Yersinia pseudotuberculosis. The use of this technique allows rapid and efficient large-scale mutagenesis of Yersinia target chromosomal genes.

Enteroaggregative Escherichia coli (EAEC) is defined by a characteristic "stacked-brick" aggregative adherence (AA) pattern to cultured cells. In well-studied EAEC prototype strains (called typical EAEC strains), the AA phenotype requires aggregative adherence fimbriae (AAFs). However, previous studies suggest that known AAF alleles are not found in all EAEC strains. To define mechanisms contributing to adherence in an atypical strain, we studied EAEC strain C1096. An E. coli K12 derivative carrying two plasmids, designated pSERB1 and pSERB2, from C1096 adhered to cell lines and exhibited an AA pattern. Nucleotide sequence analysis of pSERB1 indicated that it is related to plasmids of the IncI1 incompatibility group. These plasmids encode genes involved in pilus-mediated conjugal transfer, as well as pilL-V, which encodes a second pilus of the type IV family. Insertional inactivation of the gene predicted to encode the major type IV pilin subunit (pilS) reduced conjugal transfer of the plasmid by 4 orders of magnitude. Adherence of the mutant strain to polystyrene and to HT29 cells was reduced by approximately 21% and 75%, respectively. In a continuous-flow microfermentor, the pilS inactivation reduced mature biofilm formation on a glass slide by approximately 50%. In addition, the simultaneous presence of both pSERB1 and pSERB2 plasmids promoted pilS-independent biofilm formation. We conclude that the IncI1 plasmid of EAEC C1096 encodes a type IV pilus that contributes to plasmid conjugation, epithelial cell adherence, and adherence to abiotic surfaces. We also observe that AA can be mediated by factors distinct from AAF adhesins.

The production of biofilms by bacteria is a lifestyle that is thought to require or involve a differential gene expression compared with that of planktonic bacteria. Recently, we have witnessed a change of focus from the simple hunt for hypothetical essential biofilm genes to the identification of late and more complex biofilm functions. However, finding common bacterial biofilm gene-expression patterns through global expression analysis remains difficult. Owing to the apparently minimal overlap between functions involved in biofilm formation by different bacteria, exploring the biofilm lifestyle could prove to be a case-by-case task for which global approaches show their limits.

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hIn recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies. More than 10 years ago, it was demonstrated that GGDEF domains can produce and EAL domains can degrade the bacterial second messenger cyclic di-GMP (c-di-GMP) (1-4). With these assignments, it also became clear that bacterial genomes-in particular, those of gammaproteobacteria-usually contain multiple genes encoding these diguanylate cyclases (DGC) and c-di-GMP phosphodiesterases (PDE) (5, 6). Crystal structures of GGDEF and EAL domains have been elucidated, and studies of structure-function relationships have identified the key amino acid residues required for substrate and cation binding and catalysis (7). This also allowed identification of a subset of GGDEF and EAL (GGDEF/EAL) domain proteins, in which these key amino acids are not conserved, as "degenerate" and enzymatically inactive. In a few cases, it could be demonstrated that these degenerate GGDEF/EAL domain proteins have alternative functions based on direct interactions with other macromolecules (8-11). A subset of proteins combine GGDEF and EAL domains in a single polypeptide, where one domain is usually enzymatically active and the other is degenerate and plays a regulatory role in these "composite" proteins (3). Most GGDEF/EAL domain proteins also contain N-terminal sensory input domains that control their output activities, and a majority are localized or anchored in the cytoplasmic membrane via their membrane-intrinsic or periplasmic sensory domains (12).In studies of the molecular principles and physiological functions of c-di-GMP signaling, Escherichia coli has served as one of a few model species (13,14). The commonly used E. coli K-12 laboratory strain has a total of 29 proteins with GGDEF and/or EAL domains, including 12 and 10 proteins featuring the GGDEF and EAL domains alone, respectively, and 7 composite proteins carrying both domains. Based on direct measurements of purified proteins and/or the presence of key conserved amino acids and their elimination by point mutations, DGC and PDE activities can ...

SummaryNumerous bacteria are able to use free and haemoprotein-bound haem as iron sources because of the action of small secreted proteins called haemophores. Haemophores have very high affinity for haem, and can therefore extract haem from the haemcarrier proteins and deliver it to the cells by means of specific cell surface receptors. Haem is then taken up and the empty haemophores are recycled. Here, we report a study of the regulation of the Serratia marcescens has operon which is involved in haemophoredependent haem acquisition. We characterized two genes encoding proteins homologous to specific ECF sigma and antisigma factors. We showed that they regulate the synthesis of the haemophore-specific outer membrane receptor, HasR, by a signal transduction mechanism similar to the siderophore surfacesignalling systems. We also showed the essential role of HasR itself in this process. Using haem-loaded and haem-free haemophore, we identified the stimulus for the HasR-mediated signal transduction as being the binding of the haem-loaded haemophore to HasR. Thus, unlike siderophore-uptake systems, in which the signalling molecule is the transported substrate itself, in the haemophore-dependent haem uptake system the inducer and the transported substrate are different compounds.

SummaryIn Gram-negative bacteria, the TonB -ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonB SM , and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonB SM and HasB are significantly similar and can replace each other for haem acquisition. However, TonB SM , but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonB SM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonB EC for haem acquisition. As the change affects a domain involved in specific TonB EC -ExbB EC interactions, HasB may be unable to interact with ExbB EC , and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonB EC -dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.