Secretion systems found in bacteria are classified cur- rently into seven major classes, among which type IV secretion (T4S) systems form the most functionally ver- satile class [2–7]. The variety of substrates and their nature (single proteins, protein complexes, DNA and nucleoprotein complexes) secreted by the T4S systems indeed single out this class of secretion systems from the others. The T4S systems are classified into three func- tional groups. The first group mediates the transfer of DNA from one bacterial cell to another in a process called conjugation that plays an important role in bacterial genome plasticity and diversity. Another group of T4S systems mediates the translocation of proteins, ranging from small protein effectors to large protein complexes. Pathogenic Gram-negative bacteria such as Helicobacter pylori, Brucella suis and Legionella pneumophila use the T4S system to inject virulence proteins into mammalian host cells [8–10] and Bordetella pertussis use the T4S systems to secrete pertussis toxin into the extracellular milieu . The third group mediates DNA release and uptake. H. pylori and Neisseria gonorrhoeae typify bacteria with this type of T4S systems . T4S systems of the first class represent an enormous public-health problem as they are involved in the rapid dissemination of antibiotic-resist- ance genes and other virulence traits among pathogens. While the fact that DNA can move from one cell to another has been established a long time ago [12,15], the mechanism of secretion was poorly understood since no structural information was available until very recently.
Following our determination of the structure of the ﬂagellar export gate complex, we expressed a variety of virulence export gate complexes using the same strategy of expression of the complete operon with a Dual-Strep tag on the C terminus of the SctT (FliR) component. Many of the systems proved fragile, with little to no SctS (FliQ) associated with the puriﬁed complexes (17), but the Shigella ﬂexneri export gate (Spa24, Spa9, and Spa29 — here referred to as SctRST) could be puriﬁed at sufﬁcient levels to allow structure determination by single-particle cryo-EM (Fig. 2A). As previously proposed (17) based on the sequence conservation in all three components (33% identity to the S. Typhimurium FliPQR across the operon), the structure conclusively demonstrates the structural conservation at the core of type III secretion systems (Fig. 2B and C).
ABSTRACT Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system inter- changeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Barto- nella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential ex- pression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs im- plies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatio- temporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium.
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Type IV secretion systems (T4SSs) are large protein complexes which traverse the cell enve- lope of many bacteria. They contain a channel through which proteins or protein–DNA complexes can be translocated. This translocation is driven by a number of cytoplasmic ATPases which might energize large conformational changes in the translocation complex. The family of T4SSs is very versatile, shown by the great variety of functions among family members. Some T4SSs are used by pathogenic Gram-negative bacteria to translocate a wide variety of virulence factors into the host cell. Other T4SSs are utilized to mediate horizontal gene transfer, an event that greatly facilitates the adaptation to environmental changes and is the basis for the spread of antibiotic resistance among bacteria. Here we review the recent advances in the characterization of the architecture and mecha- nism of substrate transfer in a few representative T4SSs with a particular focus on their diversity of structure and function.
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Finally, the TSA structure demonstrates that transloca- tion signals can be part of defined, well-characterized and larger structures supporting completely different function. This is also an unprecedented observation. Translocation signals are usually self-contained functional entity with no associated function other than mediating specific sub- strate recruitment to cognate transporters. Here we dem- onstrate that TSA is part of a vestigial helicase structure. Interestingly, TSB is also part of a helicase domain, but this time, the domain has retained helicase activity. These structural and functional features are clearly conserved among relaxases. Whether they will turn out to be found in other proteins remains to be demonstrated; however, TSA provides a template that might well prove of general use by transporters and secretion systems for interactions with their cognate substrates.
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In Gram-negative bacteria, 6 secretion systems are known and named as Type I to Type VI. Each system has its own different components, compounds and the mechanisms. In these bacteria, materials must pass through both the inner and outer membranes or certain substances should enter into the host cell and therefore various molecules and mechanisms are required for this purpose. Gram-positive bacteria are common by Gram-negative bacteria in some secretion systems and pathways; although, most of them benefit from Sec and Tat secretion pathways to discharge materials through the single-layer membrane width 1 . Systems I, III, IV, VI are single-stage
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During the early stages of infection, extracellular infec- tious but metabolically inactive elementary bodies (EBs) adhere to the plasma membrane of the host cell and induce their own actin-dependent uptake into endocytic vacuoles. These early vacuoles coalesce and traffic to the microtubule-organising centre, forming a specialised membrane-bound compartment termed an inclusion. Within the inclusion, EBs differentiate into non-infec- tious but metabolically active reticulate bodies (RBs). RBs undergo a series of cell divisions before converting back into EBs, which are subsequently released from the cell by inclusion extrusion, or upon cell lysis . EBs and RBs are not only distinct in function, but also in morphology. While both forms of the bacterium are coc- coid, they differ significantly in size; EBs are 0.3–0.4 mm in diameter in comparison to RBs at 1 mm. Substantial changes in bacterial architecture therefore occur during EB–RB and RB–EB inter-conversion, which remain incompletely understood . The most obvious dis- tinguishing structural characteristic is the outer membrane, which is almost twice the thickness in EBs . This is attributed to a disulphide-cross-linked network of major outer membrane proteins that confer the osmotic stability and rigidity of EBs . By contrast, the disulphide bonds are reduced in RBs, allowing for greater membrane flexi- bility to facilitate cell division . Both EBs and RBs harbour type III secretion systems (T3SSs), nanomachines conserved among diverse Gram-negative bacterial patho- gens. T3SSs translocate virulence effector proteins directly into host cells, where they subvert cellular processes to promote pathogen entry, survival or replication . In this review, we will explore the relationship between the EB and RB T3SSs, their supramolecular organisation in contact with host membranes, and their contribution to sustaining the chlamydial lifecycle.
type III systems of animal pathogens and the flagellum, implying the presence of a common architectural theme present in type III systems. Based on the sequence similarities observed between FliN and FliM, it has been suggested that the two proteins might share some common structural features that participate in the formation of the C-ring, possibly by means of the occupation of quasiequivalent positions (25). Consistent with this hypothesis, we also observe a significant homology (20.5% identity and 54.5% similarity; Fig. 3B) between the HrcQ B -C and the C-
VirB3 is a small inner-membrane protein that binds to VirB4, and, intriguingly, in some T4S systems, the two are fused into a single protein . Interaction of VirB3 with VirB4 might assist membrane localization of VirB4. VirB8 subunits are bitopic proteins with a short cytoplasmic N- terminal domain, a TM region, and a large C-terminal periplasmic domain . X-ray structures have been solved for the VirB8 periplasmic fragments of Brucella suis (PDB: 2BHM; Figure 3b)  and A. tumefaciens (PDB: 2CC3) . VirB8 interacts with many other VirB proteins, including VirB4 and VirB10 , and is likely central for the assembly of the IMC. VirB6 proteins from A. tumefaciens and Helicobacter pylori are polytopic inner- membrane proteins with a periplasmic N-terminus, five TM segments, a large central periplasmic loop and a cytoplasmic C-terminus [19,20]. Gold labeling of the VirB6 N-terminus demonstrated that it is located on
In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5 144 392 bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2 803 932 bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577 351 bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157 : H7; therefore, it is not an ancestral state for the species.
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One of the earliest uses of L. lactis in the livestock industry was reported a decade ago, where GroEL heat shock protein from Brucella abortus was expressed and secreted as a vaccine candidate. However, its intra- cellular expression was shown to be unstable with a low secretion efficiency . Through technological advancements in expression and secretion systems, con- secutive attempts were proven more successful when oral administration of recombinant lactococcal strains secreting Cu–Zn superoxide dismutase (SOD) of B. abortus was found to render protective immunity against brucellosis when tested in mice . Very recently, oral administration of recombinant insulin-like growth fac- tor I (IGF-I) expressed in L. lactis also reported good biological activity, where symptoms and development of dextran sodium sulphate (DSS)-induced colitis in mice were attenuated . Use of L. lactis has also made hall- marks in the aquaculture industry where lactococcal- based vaccines against Aeromonas hydrophila using D1 and D4 aerolysin genes were developed with increased survival in tilapia fish when administered intraperito- neally and orally . Lactococcal expression of the SiMA antigen, a Streptococcus iniae membrane protein, has also incurred significant vaccinative and probiotic effects in olive flounders .
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There are several highly adapted E. coli clones that have acquired specific virulence attributes, which confers an increased ability to adapt to new niches and allows them to cause a broad spectrum of disease. Virulence factor in E. coli include the ability to resist phagocytosis, to tolerate an extremely low pH (highly acidic environment) by using multiple acid resistance mechanisms, utilization of highly efficient iron acquisition systems, expression of different adhesion proteins to prevent their removal by the peristaltic flow following passage through the stomach, the type 3 secretion systems, production of toxins (heat stable toxin, heat labile toxins and Vero/Shiga toxins) and acquisition of different pathogenecity islands that encode a variety of different virulence factors including adhesins, toxins, invasins, protein secretion systems, iron uptake systems, and others.
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The sigma-E response (Figure 18) protects Gram-negative bacteria from envelope stresses that result in misfolding of OMPs and accumulation of periplasmic LPS intermediates (Lima et al., 2013). This response consists of a number of sigma-E-stimulated genes, including the rpoE-rseABC cluster and the sigma-E sigmulon (the complete set of genes regulated, directly or indirectly, by a given sigma factor) composed of a number of genes and operons. While sigma-E was found to be required for secretion of effectors through type 2 and 3 secretion systems in V. cholerae and Y. enterocolitica (Carlsson et al., 2007, Zielke et al., 2014), activation of the sigma-E response by the secretion system alone (sans secreted effector proteins) or by the secretins alone (without the accompanying type 2 or 3 secretion system machinery) has not been demonstrated. Findings presented here using the RNA-seq approach are consistent with the microarray data (Lloyd et al., 2004, Jovanovic et al., 2006) in that the sigma-E response was not stimulated by the wildtype pIV secretin. Furthermore, a question addressed here was whether a leaky secretin, which is expected to impose an increased envelope stress, like that imposed by type 3 secretin YscC, would also result in activation of the sigma-E response.
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Major progress has been made in unravelling the underlying molecular mechanisms for spontaneous symmetry breaking. In contrast to traditional models of cell polarization, which follow hierarchical principles, spontaneous cell polarization relies on efficient feed- back loops that lead to amplification of stochastic fluctuations. In migrating neutrophils, a feedback loop is established through the polarized accumulation of Phosphatidylinositol- (3,4)-bisphosphate (PIP2) and Phosphatidylinositol-(3,4,5)-triphosphate (PIP3). PIP3 polar- ization is then stabilized through activation of the small GTPases Rac1, Cdc42 and their downstream targets, which trigger actin polymerization and the Phosphatidyl-3-Kinase (PI3 Kinase). In order to generate an asymmetric distribution, PIP3 needs to be inacti- vated in other parts of the cell by its global inhibitor PTEN (Meinhardt 2000; Altschuler et al. 2008; Weiner et al. 2002). This example of global inhibition and local activation can explain the spontaneous and also robust polarization in neutrophils but can also be ap- plied on other polarizing systems such as S. cerevisiae. Recent studies in yeast suggest that actin-mediated transport plays a major role in establishing and stabilizing positive feedback loops (Wedlich-Soldner et al. 2003). Furthermore, it has been shown that this mechanism is sufficient to break symmetry in G1 arrested cells expressing a constitutively active Cdc42 mutant (Wedlich-Soldner et al. 2003).
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In most Nepenthes species, the pitcher is composed of a lid, a peristome (ribbed upper rim of the pitcher), a waxy zone and a digestive zone with a pool of digestive fluid (Adams and Smith, 1977; Owen and Lennon, 1999). These zones differ greatly in their geometry, structure, surface architecture and functions. The lid and the peristome of pitchers are supposed to be involved in animal attracting and trapping (Jebb and Cheek, 1997; Owen and Lennon, 1999; Gaume et al., 2002), and the waxy zone has been widely studied for its implications in trapping and preventing the escape of prey (Lloyd, 1942; Juniper and Burras, 1962; Juniper et al., 1989; Gaume et al., 2002, 2004). By contrast, the digestive zone is believed to contribute solely to prey utilisation. For this reason, the digestive zone has been studied mostly for its chemical function in digestion, absorption and transport of the insect- derived nitrogen compounds (Juniper et al., 1989; Schulze et al., 1999; Owen et al., 1999; An et al., 2001). However, this zone may also influence the trapping efficiency of the pitcher, since animals are usually captured and drawn into the digestive fluid (Juniper et al., 1989; Adams and Smith, 1977; Owen and Lennon, 1999). Recent experiments with flies (Drosophila melanogaster) and ants (Iridomyrmex humilis) on epidermal surfaces of the Nepenthes alata pitchers showed that the glandular secretion probably acts mechanically, like a glue, and impedes insect locomotion (Gaume et al., 2002).
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stickiness. Needle posts with a length of approximately 2·mm and an outer diameter of 0.2mm were the broken tips of jeweler’s brooches (purchased from local cooperative jewelers), which were also used to trim the ends of Malpighian tubules. The brooches are made of hardened steel that resists bending when the tips are filed to sharpness. Two such sharpened jewelers brooches are routinely used to fray open the end of Malpighian tubules for in vitro microperfusion and for fluid secretion assays by the method of Ramsay. The use of blunt instruments tends to crush the tubule and seal the lumen.
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Although the stomach is mainly known for its ability to secrete hydrochloric acid, there is increasing evidence that the gastric mucosa also secretes bicarbonate. A simple method for simultaneous measurement of gastric HCO-3 secretion and H+ secretion was developed from a two-component model of gastric secretion. The method, which is based upon gastric juice volume, H+ concentration, and osmolality, was validated both in vitro and in vivo. In 14 healthy human beings, basal gastric HCO-3 secretion averaged 2.6 mmol/h (range, 0.7-8.7 mmol/h). Basal HCO-3 secretion was approximately 50% of basal H+ secretion and there was a significant correlation between basal HCO-3 and H+ secretion in individual subjects (r = 0.79). HCO-3 was secreted in basal nonparietal secretion at a concentration of
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The secretion of calcitonin by slices of porcine thyroid glands has been investigated. Calcitonin in the incubation medium was determined by radioimmunoassay. Secretion of calcitonin was diminished when calcium or magnesium was omitted and was increased stepwise as the concentration of calcium or magnesium in the incubation medium was increased. Calcitonin secretion was augmented substantially when either the quantity of thyroid tissue or volume of incubation medium was increased. Secretion of calcitonin was stimulated by glucagon, theophylline, and dibutyryl cyclic 3¢,5¢-adenosine monophosphate. It is concluded that calcitonin secretion is regulated by the concentration of calcium and magnesium, that secretion may be inhibited by calcitonin or a precursor and that secretion can be stimulated by increasing the concentration of cyclic 3¢,5¢-adenosine monophosphate in the parafollicular cells of the thyroid gland.
and K12(LV2) similar with BL21(LV2) but with parental E. coli K-12 W3310 genetic background. The IgG sin- gle chain antibody fragment genes (anti-β-galactosidase scFv13R4, anti-histone, and anti-tetanus toxin single- chain Fv) were synthesised by GeneART (Thermo Fisher) and sub-cloned into pENTRY and pDEST vectors, con- taining the kanamycin resistance marker. For strain benchmarking, the pETORS-eGFP expression vector  was used. For the expression in BL21(DE3) stain, the pET28 vector (no-riboswitch control), with the sig- nal peptide and the scFv13R4 were used. For secretion constructs, synthetic DNA containing the riboswitch and signal peptide sequences (DsbA WT, PelB WT, Piii WT and yBGL2 WT) synthesized by GeneART (Thermo Fisher), and cloned upstream of the scFv gene by restric- tion digest and ligation via NdeI/SpeI sites. Inducers: IPTG (Isopropyl β-D-1-thiogalactopyranoside) (Sigma), PPDA (Pyrimido[4,5-d]pyrimidine‐2,4‐diamine) (Peak- dale Molecular). A CLARIOstar Microplate Reader (BMG) was used to measure the eGFP fluorescence and cell density (OD 600 ) for intact cells.
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signal peptide from + 3 to + 2 resulted in the doubling of the amounts of tendamistat in the supernatant of the het- erologous expression host S. lividans, whereas increasing the positive net charge to + 4 or + 6 had an adverse effect . Similarly, a saturation mutagenesis of the positions 2–7 of the n-region of the B. subtilis α-amylase AmyE signal peptide revealed that three out of four isolated mutant signal peptides that significantly increased the amounts of the heterologous target protein cutinase from F. solani pisi in the B. subtilis culture supernatant like- wise led to a reduction of the net charge of the n-region from + 3 to + 2 . The analysis of the export kinetics of the respective precursor proteins by pulse-chase experi- ments interestingly revealed that some of the mutated signal peptides actually slowed down the translocation of the cutinase across the cytoplasmic membrane. It was speculated that a highly efficient targeting and transloca- tion of the cutinase might result in an overloading of the extracytosolic folding catalyst PrsA , thereby leading to the accumulation of misfolded cutinase on the trans- side of the membrane and to the subsequent induc- tion of cell wall stress-induced proteases (i.e. HtrA and HtrB) which then reduce the overall secreted amount of the cutinase. Such a scenario in fact could explain why, in some cases, decreasing the export rate can lead to a higher amount of a secreted heterologous protein in the culture supernatant of the respective expression host . Altering the net charge of the n-region has been also shown to improve the performance of a Tat-specific signal peptide. Replacement of the lysine at position 38 in the very long n-region of the xylanase signal peptide by a negatively charged glutamate residue resulted in an 219% increase of xylanase secretion by S. lividans. Strik- ingly, also a replacement of a negatively charged aspartate residue at position 41 by an asparagine residue more than doubled the amounts of the xylanase in the S. lividans culture supernatant. It was concluded that the number of positively or negatively charged amino acid residues in the n-region is less important than their distribution in the n-domain of the Tat-specific xylanase signal peptide .
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