Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Unlabelled: Type III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery.

Recognition of discrete export signals in early flagellar subunits during bacterial type III secretion.

Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel.

Regulation of bacterial Type III Secretion System export gate opening by substrates and the FliJ stalk of the flagellar ATPase.

Type III Secretion Systems (T3SS) transport proteins from the bacterial cytosol for assembly into cell surface nanomachines or direct delivery into target eukaryotic cells. At the core of the flagellar T3SS, the FlhAB-FliPQR export gate regulates protein entry into the export channel whilst maintaining the integrity of the cell membrane. Here, we identify critical residues in the export gate FliR plug that stabilise the closed conformation, preserving the membrane permeability barrier, and we show that the gate opens and closes in response to export substrate availability.

Chaperone-mediated coupling of subunit availability to activation of flagellar Type III secretion.

Bacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilize the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones.

Prevention and management of urinary catheter blockages in community settings.

Self-management of long-term urinary catheters can be challenging for patients, and recurrent catheter blockages may cause concern among patients, carers and healthcare professionals. Catheter blockages are a significant challenge for nurses practising in community settings, because frequent and unplanned catheter changes can be costly to healthcare services in terms of time and resources. This article details evidence-based recommendations for the assessment and diagnosis of catheter blockages, as well as the identification of risk factors.

Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein.

Background: Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e.

Interactions of Flagellar Structural Subunits with the Membrane Export Machinery.

During assembly of the bacterial flagellum, structural subunits synthesized inside the cell must be exported across the cytoplasmic membrane before they can crystallize into the nascent flagellar structure. This export process is facilitated by a specialized Flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. Here, we describe three methods-isothermal titration calorimetry, photo-crosslinking using unnatural amino acids, and a subunit capture assay-used to investigate the interactions of flagellar structural subunits with the membrane export machinery component FlhB.

Both genome and cytosol dynamics change in E. coli challenged with sublethal rifampicin.

While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as μNS-GFP), measuring short time-scale displacements across several hours of drug exposure.

The frequency and duration of Salmonella-macrophage adhesion events determines infection efficiency.

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism.

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci.

Building a flagellum outside the bacterial cell.

Flagella, the helical propellers that extend from the bacterial surface, are a paradigm for how complex molecular machines can be built outside the living cell. Their assembly requires ordered export of thousands of structural subunits across the cell membrane and this is achieved by a type III export machinery located at the flagellum base, after which subunits transit through a narrow channel at the core of the flagellum to reach the assembly site at the tip of the nascent structure, up to 20μm from the cell surface. Here we review recent findings that provide new insights into flagellar export and assembly, and a new and unanticipated mechanism for constant rate flagellum growth.

Building a flagellum in biological outer space.

Flagella, the rotary propellers on the surface of bacteria, present a paradigm for how cells build and operate complex molecular 'nanomachines'. Flagella grow at a constant rate to extend several times the length of the cell, and this is achieved by thousands of secreted structural subunits transiting through a central channel in the lengthening flagellum to incorporate into the nascent structure at the distant extending tip. A great mystery has been how flagella can assemble far outside the cell where there is no conventional energy supply to fuel their growth.

A chain mechanism for flagellum growth.

Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure.

Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription.

DNA cytosine methylation regulates gene expression in mammals. In bacteria, its role in gene expression and genome architecture is less understood. Here we perform high-throughput sequencing of bisulfite-treated genomic DNA from Escherichia coli K12 to describe, for the first time, the extent of cytosine methylation of bacterial DNA at single-base resolution.

Genomic analysis of DNA binding and gene regulation by homologous nucleoid-associated proteins IHF and HU in Escherichia coli K12.

IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA.

Behavioural risk of bipolar disorder in an analogue population: the role of cognitive, developmental and interpersonal factors.

Research to date has identified the contribution of a number of cognitive, developmental and interpersonal risk factors in the development of bipolar affective disorder. However, further work is needed to understand the mechanisms and interactions between these risk factors in relation to bipolar mood instability. The aim of this study is to explore the possibility of identifying high risk of bipolar disorder through cognitive and interpersonal factors and to further expand our knowledge regarding the relationship between such factors.

Common variants at VRK2 and TCF4 conferring risk of schizophrenia.

Common sequence variants have recently joined rare structural polymorphisms as genetic factors with strong evidence for association with schizophrenia. Here we extend our previous genome-wide association study and meta-analysis (totalling 7 946 cases and 19 036 controls) by examining an expanded set of variants using an enlarged follow-up sample (up to 10 260 cases and 23 500 controls). In addition to previously reported alleles in the major histocompatibility complex region, near neurogranin (NRGN) and in an intron of transcription factor 4 (TCF4), we find two novel variants showing genome-wide significant association: rs2312147[C], upstream of vaccinia-related kinase 2 (VRK2) [odds ratio (OR) = 1.

Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli.

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these--H-NS and Fis--bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip.

Development of an apparatus to produce fractures from short-duration high-impulse loading with an application in the lower leg.

Axial loading of the lower leg during impact events can cause significant fractures of the tibia. The magnitude of lower leg axial loading that occurs during short-duration high-impulse events, such as antivehicular landmine blasts, can lead to life-altering injuries. These events achieve higher forces over shorter durations than car crashes, the current standard used for protective measures.

Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity.

Cyclic-di-GMP is a bacterial second messenger that controls the switch between motile and sessile states. It is synthesized by proteins containing the enzymatic GGDEF domain and degraded by the EAL domain. Many bacterial genomes encode several copies of proteins containing these domains, raising questions on how the activities of parallel c-di-GMP signalling systems are segregated to avoid potentially deleterious cross-talk.

Common variants conferring risk of schizophrenia.

Schizophrenia is a complex disorder, caused by both genetic and environmental factors and their interactions. Research on pathogenesis has traditionally focused on neurotransmitter systems in the brain, particularly those involving dopamine. Schizophrenia has been considered a separate disease for over a century, but in the absence of clear biological markers, diagnosis has historically been based on signs and symptoms.

Recruitment of the earliest component of the bacterial flagellum to the old cell division pole by a membrane-associated signal recognition particle family GTP-binding protein.

The specialised signal recognition particle family guanosine 5c-triphosphate (GTP)-binding protein FlhF is required for the correct localisation of flagella in several bacterial species. Here, we characterise the regions of Vibrio cholerae FlhF that are required for its function and targeting to the old cell pole, and we present evidence for a mechanism by which FlhF establishes flagellum polar localisation. Substitution of residues in FlhF nucleotide-binding motifs reduced GTP binding and the efficiency of flagellum biogenesis, and caused flagellum mislocalisation.