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

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 D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery. IMPORTANCE: Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

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. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBC via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened, 'stalled' at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.

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. Our data indicate that FlhAB-FliPQR gate opening, which is triggered by substrate export signals, is energised by FlhA in a proton motive force-dependent manner. We present evidence that the export substrate and the FliJ stalk of the flagellar ATPase provide mechanistically distinct, non-redundant gate-activating signals that are critical for efficient export.

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. FliJ binds unladen chaperones and, by using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf-driven export by the fT3SS.

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. It also explains the interventions that can be used to prevent and manage catheter blockages and describes the role of the nurse in supporting patients with a long-term catheter in situ in community settings.

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. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. RESULTS: We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture. CONCLUSIONS: We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.

Long-term habitat changes in a protected area: Implications for herpetofauna habitat management and restoration.

Point Pelee National Park, located at the southern-most tip of Canada's mainland, historically supported a large number of herpetofauna species; however, despite nearly a century of protection, six snake and five amphibian species have disappeared, and remaining species-at-risk populations are thought to be in decline. We hypothesized that long-term changes in availability and distribution of critical habitat types may have contributed to the disappearance of herpetofauna. To track habitat changes we used aerial image data spanning 85 years (1931-2015) and manually digitized and classified image data using a standardized framework. Change-detection analyses were used to evaluate the relative importance of proportionate loss and fragmentation of 17 habitat types. Marsh habitat diversity and aquatic connectivity has declined since 1931. The marsh matrix transitioned from a graminoid and forb shallow marsh interspersed with water to a cattail dominated marsh, altering critical breeding, foraging, and overwintering habitat. Reduced diversity of marsh habitats appears to be linked to the expansion of invasive Phragmites australis, which invaded prior to 2000. Loss of open habitats such as savanna and meadow has reduced availability of high quality thermoregulation habitat for reptiles. Restoration of the northwestern region and tip of Point Pelee National Park to a mixed landscape of shallow wetlands (cattail, graminoid, forb, open water) and eradication of dense Phragmites stands should improve habitat diversity. Our results suggest that long-term landscape changes resulting from habitat succession and invasive species can negatively affect habitat suitability for herpetofauna and protection of land alone does not necessarily equate to protection of sensitive herpetofauna.

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. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.

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. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.

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. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

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. Recent work published by Evans et al. [Nature (2013) 504: 287-290], has gone some way towards solving this puzzle, presenting a simple and elegant transit mechanism in which growth is powered by the subunits them selves as they link head-to-tail in a chain that is pulled through the length of the growing structure to the tip. This new mechanism answers an old question and may have resonance in other assembly processes.

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.

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. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip.

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. Whereas most target sites (C(m)CWGG) are fully methylated in stationary phase cells, many sites with an extended CC(m)CWGG motif are only partially methylated in exponentially growing cells. We speculate that these partially methylated sites may be selected, as these are slightly correlated with the risk of spontaneous, non-synonymous conversion of methylated cytosines to thymines. Microarray analysis in a cytosine methylation-deficient mutant of E. coli shows increased expression of the stress response sigma factor RpoS and many of its targets in stationary phase. Thus, DNA cytosine methylation is a regulator of stationary phase gene expression in E. coli.

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. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ∼30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.

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. The findings from this work demonstrate that when both cognitive and interpersonal variables are entered into one model to predict bipolar high risk, direct effects are observed for the interpersonal factors, which then have a fully mediational effect on the cognitive factors. This work proposes that interpersonal factors develop and maintain cognitive risk factors and that future formulations and treatment of bipolar disorder need to focus on addressing such interpersonal issues.