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the cell envelope 5 . The type III secretion pathway requires a complex apparatus of proteins, which forms a tightly regulated oligomeric structure spanning the inner and outer membranes 6,7 . Type IV secretion is perhaps the least understood of the Gramnegative bacterial secretion apparatuses 8 . The type IV export process involves the coordinate action of at least nine proteins, which are variously associated with the inner and outer membrane and which are localized within the periplasm and cytoplasm. Autotransporters

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.

We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae ). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis.

The autotransporters, a family of secreted proteins from Gram-negative bacteria, possess an overall unifying structure comprising three functional domains: the amino-terminal leader sequence, the secreted mature protein (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein. Members of this family have been implicated as important or putative virulence factors in many Gram-negative pathogens.

Antigen 43 (Ag43) is a prominent hetero-oligomeric protein complex in the outer membrane of Escherichia coli. It is composed of two subunits. α43 (M r 60, 000) and β43 (M r 53, 000) in 1:1 stoichiometry. α43 is surface expressed, extends beyond the O-side chains of smooth lipopolysaccharide and is bound to the cell surface through an interaction with β43 itself an integral outer membrane protein. α43 shows limited sequence homology with some enterobacterial adhesins. Expression of Ag43 is subject to reversible phase variation, the rates of variation from the Ag43+ve to Ag43-ve states in liquid minimal medium being ~2.2 × 10−3, the corresponding rates from Ag43-ve to Ag43f+ve states being ~1 × 10−3. Phase switching of genes encoding Ag43 are transcriptionally regulated by DNA methylation (deoxyadenosine methylase [dam] mutants being “locked OFF”) and by OxyR (oxyR mutants being “locked ON”). It is proposed that OxyR acts as a repressor of Ag43 transcription by binding to unmethylated GATC sites in the regulatory region of the gene. Sequencing and mapping has identified Ag43 as the likely product of the metastable flu gene first described in 1980 by Diderichsen and responsible for colony form variation in E. coli.

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.

Enteroaggregative Escherichia coli (EAEC) is a significant cause of diarrhoeal illness in both children and adults. Genetic heterogeneity and recovery of EAEC strains from both healthy and diseased individuals complicates our understanding of EAEC pathogenesis. We wished to establish if genetic or phenotypic attributes could be used to distinguish between strains asymptomatically colonising healthy individuals and those which cause disease. Genotypic screening of a collection of twenty four EAEC isolates from children with and without diarrhoea revealed no significant differences in the repertoire of putative virulence factors present in either group of strains. In contrast, EAEC strains from phylogroup A were more strongly associated with asymptomatic groups whereas strains from phylogroup D were more associated with cases of diarrhoea. Phenotypic screening revealed no differences in the ability of strains from either cohort of children to form biofilms, to adhere to and invade cells in tissue culture or to cause disease in the Caenorhabditis elegans model of infection. However, the latter assay did reveal significant reduction in nematode killing rates when specific virulence factors were deleted from human pathogenic strains. Our results suggest that current models of infection are not useful for distinguishing avirulent from pathogenic strains of EAEC but can be useful in studying the effect of specific virulence factors.

Introduction We have previously identified patients colonised with Pseudomonas aeruginosa (PA), in whom strains isolated from their sputum cannot be killed by their serum, yet are fully sensitive to healthy control serum (HCS). Addition of inhibitory patient serum to HCS also impaired killing of PA. Fractionation of patient serum identified that IgG was responsible for the inhibition. FACS demonstrated excessive binding of IgG2 from patient serum to autologous strains. Visualisation of lipopolysaccharide (LPS) isolated from bacterial strains by silver staining, showed that strains from patients with inhibitory serum demonstrated detectable O antigen expression – a component of the LPS cell wall of PA. We aimed to confirm this relationship between inhibitory IgG2 and LPS expression by PA. Methods Anti-LPS antibodies were removed from inhibitory serum (S4) by binding LPS from PA isolated from patient sputum, to polymyxin-B agarose overnight. Inhibitory serum was then passed over the LPS bound column. Antibodies specific for LPS bound to the column, and the flow through fractions of serum were collected. Bound antibody was subsequently buffer exchanged into PBS. LPS was isolated from a PA strain resistant to patient sera (B4) and attached to a 96 well plate. ELISA was performed by adding dilutions of patient or HCS to the plate followed by anti-human IgG2 conjugated to alkaline phosphotase. Results were derived 30 minutes after addition of developer. Results LPS removal of IgG from the patient’s serum restored bacterial killing. Adding the eluted antibody to HCS impaired killing. This confirmed that inhibitory IgG2 is LPS specific. The patients’ serum that blocked bacterial killing had high titres of IgG2 to LPS compared to those from patients with bactericidal serum (fig. 1) Conclusion We have established that PA strains deficient in LPS O antigen are generally sensitive to killing by patients’ serum, whilst the presence of O antigen leads to serum resistance mediated by IgG2. Results indicate that PA LPS O antigen repeats are central to serum resistance of autologous strains but not normal HCS. Current data indicates that bacterial killing is impaired in subjects colonised with PA expressing O antigen due to overproduction of LPS specific IgG2.

T-zone localized monocyte-derived dendritic cells promote Th1 priming to Salmonella Adriana Flores-Langarica, Jennifer L. Marshall, Saeeda Bobat, Elodie Mohr, Jessica Hitchcock, Ewan A. Ross, Ruth E. Coughlan, Mahmood Khan, Nico Van Rooijen, Ian R. Henderson, Ian C. M. MacLennan and Adam F. Cunningham. Unfortunately, an error was introduced into Fig. 4C of the above article. The correct figure is shown below. Correction

Adherent-invasive Escherichia coli strains are increasingly being associated with intestinal pathologies. Here we present the genome sequence of E. coli HM605, a strain isolated from colonic biopsy specimens of a patient with Crohn's disease.

De novo DNA methylation and the maintenance of DNA methylation in asymmetrical sequence contexts is catalyzed by homologous proteins in plants (DRM2) and animals (DNMT3a/b). In plants, targeting of DRM2 depends on small interfering RNAs (siRNAs), although the molecular details are still unclear. Here, we show that two SRA-domain proteins (SUVH9 and SUVH2) are also essential for DRM2-mediated de novo and maintenance DNA methylation in Arabidopsis thaliana. At some loci, SUVH9 and SUVH2 act redundantly, while at other loci only SUVH2 is required, and this locus specificity correlates with the differing DNA-binding affinity of the SRA domains within SUVH9 and SUVH2. Specifically, SUVH9 preferentially binds methylated asymmetric sites, while SUVH2 preferentially binds methylated CG sites. The suvh9 and suvh2 mutations do not eliminate siRNAs, suggesting a role for SUVH9 and SUVH2 late in the RNA-directed DNA methylation pathway. With these new results, it is clear that SRA-domain proteins are involved in each of the three pathways leading to DNA methylation in Arabidopsis., Author Summary Our genetic heritage plays an important role in determining who we are and the characteristics we possess. However, in the past decade it has become increasingly clear that in addition to the genes we inherit, a second level of information is critical for expression of these genes. This information takes the form of modifications to either the DNA (DNA methylation) or the proteins that package the DNA (histones). These modifications can determine whether a gene is expressed or silenced. In this paper, we identify two new genes that are part of a DNA methylation–targeting pathway in the model plant A. thaliana. Disruption of these two closely related genes prevents DNA methylation by one of the cellular DNA methyltransferases. However, these genes are not simply redundant. They are both capable of binding methylated DNA, but differ in their preference for specific sequences in the genome. This ability to bind to methylated DNA suggests that these proteins help target or retain the modification apparatus at particular regions of the genome. These results are important in that they identify two new players in this vital cellular process and bring us closer to understanding how epigenetic modifications can be targeted to specific genes.

Bacterial pathogenesis research often focuses on virulence determinants that are secreted to the bacterial cell surface or released into the external environment. Despite the prodigious number of recognized secreted proteins, thus far only five basic protein-secretion mechanisms have been identified in Gram-negative bacteria. However, recently some confusion has arisen in the literature concerning the nomenclature of these secretion systems. This confusion stems from the near-simultaneous publication of papers and reviews designating two different secretion mechanisms as the type IV secretion system1xBiochemistry of type IV secretion. Burns, D.L. Curr. Opin. Microbiol. 1999; 2: 25–29Crossref | PubMed | Scopus (85)See all References, 2xCommon themes in microbial pathogenicity revisited. Finlay, B.B. and Falkow, S. Microbiol. Mol. Biol. Rev. 1997; 61: 136–169PubMedSee all References, 3xThe great escape: structure and function of the autotransporter proteins. Henderson, I.R. et al. Trends Microbiol. 1998; 6: 370–378Abstract | Full Text | Full Text PDF | PubMed | Scopus (389)See all References, 4xType III protein secretion systems in bacterial pathogens of animals and plants. Hueck, C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379–433PubMedSee all References, 5xSecretion of proteins and assembly of bacterial surface organelles: shared pathways of extracellular protein targeting. Lory, S. Curr. Opin. Microbiol. 1998; 1: 27–35Crossref | PubMedSee all References. Thus, both the autotransporter secretion system6xSecretion of virulence factors by Escherichia coli. China, B. and Goffaux, F. Vet. Res. 1999; 30: 181–202PubMedSee all References, 7xThe major phase-variable outer membrane protein of Escherichia coli structurally resembles the immunoglobulin A1 protease class of exported protein and is regulated by a novel mechanism involving Dam and OxyR. Henderson, I.R. and Owen, P. J. Bacteriol. 1999; 181: 2132–2141PubMedSee all References, 8xA novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa. Wilhelm, S. et al. J. Bacteriol. 1999; 181: 6977–6986PubMedSee all References and the apparatus involved in the secretion of the Bordetella pertussis toxin (Ptl) and T-DNA in Agrobacterium tumefaciens9xGenetic and sequence analysis of the pTiC58 trb locus, encoding a mating-pair formation system related to members of the type IV secretion family. Li, P.L. et al. J. Bacteriol. 1998; 180: 6164–6172PubMedSee all References, 10xAssembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells. Zupan, J.R. et al. Curr. Opin. Microbiol. 1998; 1: 649–655Crossref | PubMedSee all References have been designated as type IV secretion mechanisms. Although differences exist between Ptl and T-DNA secretion (and similar secretion mechanisms of other Gram-negative bacteria), there are sufficient similarities between the different components of the secretion apparatus to group them as a single mechanism. To clarify the situation and prevent further confusion in the literature and the reference databases, we propose that the type IV nomenclature be reserved for the Ptl and homologous systems found in A. tumefaciens and other Gram-negative bacteria. Currently, we favor the use of the term ‘autotransporter’ to represent the self-mediated secretion of protein molecules across both inner and outer membranes11xCommon structural features of IgA1 protease-like outer membrane protein autotransporters. Jose, J. et al. Mol. Microbiol. 1995; 18: 378–380Crossref | PubMedSee all References11 as this designation is more descriptive of the secretion process. However, we suggest that when it is absolutely essential to apply a numerical value to the autotransporter secretion system that the mechanism should be designated the type V secretion system.It is additionally worth noting that nomenclature allows scientists to infer a relationship among various members of a category and ideally it should comprise a descriptive framework. Therefore, we propose that future secretion systems be designated by descriptive epithets that readily communicate the defining features of a category and that facilitate communication and recall.

SummaryThe outer membrane β-barrel trans-membrane proteins in gram-negative bacteria are folded into the membrane with the aid of polypeptide transport-associated (POTRA) domains. These domains occur, and probably function, as a tandem array situated on the periplasmic side of the outer membrane. Two crystal structures and one NMR study have attempted to define the structure and articulation of the POTRA domains of the Escherichia coli, prototypic Omp85 protein BamA. We have used pulsed electron paramagnetic resonance (EPR) to determine the distance and distance distribution between (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSSL), placed across the domain interface of the first two POTRA domains of BamA. Our results show tightly defined interdomain distance distributions that indicate a well-defined domain orientation. Examination of the known structures revealed that none of them fitted the EPR data. A combination of EPR and NMR data was used to generate converged structures with defined domain-domain orientation.

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3′ end-processing factor, Pfs2p. FY regulates RNA 3′ end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3′ end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3′ end processing.

During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection.

The hexaploid bread wheat genome comprises over 16 gigabases of sequence across 21 chromosomes. Meiotic crossovers are highly polarized along the chromosomes, with elevation in the gene-dense distal regions and suppression in the Gypsy retrotransposon-dense centromere-proximal regions. We profiled the genomic landscapes of the meiotic recombinase DMC1 and the chromosome axis protein ASY1 in wheat and investigated their relationships with crossovers, chromatin state, and genetic diversity. DMC1 and ASY1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed strong co-enrichment in the distal, crossover-active regions of the wheat chromosomes. Distal ChIP-seq enrichment is consistent with spatiotemporally biased cytological immunolocalization of DMC1 and ASY1 close to the telomeres during meiotic prophase I. DMC1 and ASY1 ChIP-seq peaks show significant overlap with genes and transposable elements in the Mariner and Mutator superfamilies. However, DMC1 and ASY1 ChIP-seq peaks were detected along the length of each chromosome, including in low-crossover regions. At the fine scale, crossover elevation at DMC1 and ASY1 peaks and genes correlates with enrichment of the Polycomb histone modification H3K27me3. This indicates a role for facultative heterochromatin, coincident with high DMC1 and ASY1, in promoting crossovers in wheat and is reflected in distalized H3K27me3 enrichment observed via ChIP-seq and immunocytology. Genes with elevated crossover rates and high DMC1 and ASY1 ChIP-seq signals are overrepresented for defense-response and immunity annotations, have higher sequence polymorphism, and exhibit signatures of selection. Our findings are consistent with meiotic recombination promoting genetic diversity, shaping host–pathogen co-evolution, and accelerating adaptation by increasing the efficiency of selection.

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. Exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks (DSBs), catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterisation of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation and fertility and hence opens a route to modifying recombination in this agronomically important crop.

Reciprocal DNA crossovers between chromosomes form new allelic combinations and contribute to the formation of novel genetic diversity. Crossovers are formed during meiosis of germ cells and these recombination events have influenced plant genome evolution and are used during breeding to create improved plant varieties. Meiotic crossovers are not uniformly formed across the genome but instead occur in regions with low nucleosome density. The recombination landscape differs between the model plant organism Arabidopsis thaliana and crops such as rice and maize. Genotyping-by-sequencing is a technique that can detect crossover location and provide information on the recombination landscape genome-wide. This technique can be used to compare crossover position between ecotypes, species, and mutant lines to gain information on factors controlling meiotic recombination. In this protocol, we describe the steps to purify DNA from plant tissue, prepare 96 DNA libraries in parallel and perform quality control before next-generation sequencing.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3′-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.