Bacterial Cytoplasm Research Articles

Dietary Isothiocyanates, Sulforaphane and 2-Phenethyl Isothiocyanate, Effectively Impair Vibrio cholerae Virulence.

Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.

Challenges in Drug Discovery for Intracellular Bacteria.

Novel drugs are needed to treat a variety of persistent diseases caused by intracellular bacterial pathogens. Virulence pathways enable many functions required for the survival of these pathogens, including invasion, nutrient acquisition, and immune evasion. Inhibition of virulence pathways is an established route for drug discovery; however, many challenges remain. Here, we propose the biggest problems that must be solved to advance the field meaningfully. While it is established that we do not yet understand the nature of chemicals capable of permeating into the bacterial cell, this problem is compounded when targeting intracellular bacteria because we are limited to only those chemicals that can permeate through both human and bacterial outer envelopes. Unfortunately, many chemicals that permeate through the outer layers of mammalian cells fail to penetrate the bacterial cytoplasm. Another challenge is the lack of publicly available information on virulence factors. It is virtually impossible to know which virulence factors are clinically relevant and have broad cross-species and cross-strain distribution. In other words, we have yet to identify the best drug targets. Yes, standard genomics databases have much of the information necessary for short-term studies, but the connections with patient outcomes are yet to be established. Without comprehensive data on matters such as these, it is difficult to devise broad-spectrum, effective anti-virulence agents. Furthermore, anti-virulence drug discovery is hindered by the current state of technologies available for experimental investigation. Antimicrobial drug discovery was greatly advanced by the establishment and standardization of broth microdilution assays to measure the effectiveness of antimicrobials. However, the currently available models used for anti-virulence drug discovery are too broad, as they must address varied phenotypes, and too expensive to be generally adopted by many research groups. Therefore, we believe drug discovery against intracellular bacterial pathogens can be advanced significantly by overcoming the above hurdles.

Transcription-dependent confined diffusion of enzymes within subcellular spaces of the bacterial cytoplasm

BackgroundKnowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo.ResultsWe have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles.ConclusionsOur results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Lactococcus lactis Expressing Type I Interferon From Atlantic Salmon Enhances the Innate Antiviral Immune Response In Vivo and In Vitro.

In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens.

Bacterial gene expression is under the control of a large set of molecules acting at multiple levels. In addition to the transcription factors (TFs) already known to be involved in global regulation of gene expression, small regulatory RNAs (sRNAs) are emerging as major players in gene regulatory networks, where they allow environmental adaptation and fitness. Developments in high-throughput screening have enabled their detection in the entire bacterial kingdom. These sRNAs influence a plethora of biological processes, including but not limited to outer membrane synthesis, metabolism, TF regulation, transcription termination, virulence, and antibiotic resistance and persistence. Almost always noncoding, they regulate target genes at the post-transcriptional level, usually through base-pair interactions with mRNAs, alone or with the help of dedicated chaperones. There is growing evidence that sRNA-mediated mechanisms of actions are far more diverse than initially thought, and that they go beyond the so-called cis- and trans-encoded classifications. These molecules can be derived and processed from 5' untranslated regions (UTRs), coding or non-coding sequences, and even from 3' UTRs. They usually act within the bacterial cytoplasm, but recent studies showed sRNAs in extracellular vesicles, where they influence host cell interactions. In this review, we highlight the various functions of sRNAs in bacterial pathogens, and focus on the increasing examples of widely diverse regulatory mechanisms that might compel us to reconsider what constitute the sRNA.

Conjugates of Ciprofloxacin and Amphiphilic Block Copoly(2-alkyl-2-oxazolines)s Overcome Efflux Pumps and Are Active against CIP-Resistant Bacteria.

Conjugation of antibiotics with polymers is an emerging strategy to improve the performance of these important drugs. Here, the antibiotic ciprofloxacin (CIP) was conjugated with amphiphilic poly(2-oxazoline) (POx) block copolymers to investigate whether the activity of the antibiotic was enhanced due to additionally induced membrane activity. The resulting polymer-antibiotic conjugates (PACs) are an order of magnitude more active against the bacterial strain Staphylococcus aureus than CIP and show high activities against numerous pathogenic bacterial strains. Their high activity depends on an optimal hydrophobic/hydrophilic balance (HHB) of the POx tail. Mechanistic studies revealed that the derivatization of CIP required for the polymer conjugation lowers the affinity of the antibiotic to its target topoisomerase IV. However, the amphiphilic PACs are most likely concentrated within the bacterial cytoplasm, which overcompensates the loss of affinity and results in high antibacterial activity. In addition, the development of resistance in S. aureus and Escherichia coli is slowed down. More importantly, the amphiphilic PACs are active against CIP-resistant S. aureus and E. coli. The PACs with the highest activity are not cytotoxic toward human stem cells and do not lyse blood cells in saturated solution.

Host - Bacterial Pathogen Communication: The Wily Role of the Multidrug Efflux Pumps of the MFS Family.

Bacterial pathogens are able to survive within diverse habitats. The dynamic adaptation to the surroundings depends on their ability to sense environmental variations and to respond in an appropriate manner. This involves, among others, the activation of various cell-to-cell communication strategies. The capability of the bacterial cells to rapidly and co-ordinately set up an interplay with the host cells and/or with other bacteria facilitates their survival in the new niche. Efflux pumps are ubiquitous transmembrane transporters, able to extrude a large set of different molecules. They are strongly implicated in antibiotic resistance since they are able to efficiently expel most of the clinically relevant antibiotics from the bacterial cytoplasm. Besides antibiotic resistance, multidrug efflux pumps take part in several important processes of bacterial cell physiology, including cell to cell communication, and contribute to increase the virulence potential of several bacterial pathogens. Here, we focus on the structural and functional role of multidrug efflux pumps belonging to the Major Facilitator Superfamily (MFS), the largest family of transporters, highlighting their involvement in the colonization of host cells, in virulence and in biofilm formation. We will offer an overview on how MFS multidrug transporters contribute to bacterial survival, adaptation and pathogenicity through the export of diverse molecules. This will be done by presenting the functions of several relevant MFS multidrug efflux pumps in human life-threatening bacterial pathogens as Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumoniae, Shigella/E. coli, Acinetobacter baumannii.

CyDisCo production of functional recombinant SARS-CoV-2 spike receptor binding domain.

The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.

Editorial: Bacterial Effectors as Drivers of Human Disease: Models, Methods, Mechanisms

Bacteria colonise virtually all ecological niches on earth. To interact with their surroundings, they evolved sophisticated multi-protein secretion systems that transport proteins, called effectors, from the bacterial cytoplasm into the extracellular space or directly into target bacteria or eukaryotic cells. Secretion systems therefore perform critical roles in inter-bacterial competition and the pathogenesis of many clinically important opportunistic and professional human pathogens. Generically numbered type I, type II, etc. secretion systems (T1SSs, T2SSs, …), the complex multi-protein architecture of each type is remarkably conserved in different bacteria (Costa et al., 2015). In contrast, the effector repertoires are diverse in numbers and composition reflecting adaptation to specific lifestyles. Environmental, broad host range pathogens like Legionella pneumophila encode more than 300 effectors (Qiu and Luo, 2017). These effectors, whilst not selected under evolutionary pressure by the human immune system, still allow L. pneumophila to cause opportunistic infections. In contrast, pathogens with a more restricted mammalian host range, such as Salmonella enterica, have a considerably smaller (~20-40) arsenal of effectors (Jennings et al., 2017). This Frontiers Research Topic comprises a series of reviews and original research articles highlighting common and pathogen-specific findings and approaches to dissect the function of effectors in bacterial pathogenesis.

Biogenesis of a Bacteriophage Long Non-Contractile Tail

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes’ synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.

Functional dissection of structural regions of the Thermus thermophilus competence protein PilW: Implication in secretin complex stability, natural transformation and pilus functions

Uptake of DNA from the environment into the bacterial cytoplasm is mediated by a macromolecular transport machinery that is similar in structure and function to type IV pili (T4P) and, indeed, DNA translocator and T4P share common components. One is the secretin PilQ which is assembled into homopolymeric complexes forming highly dynamic outer membrane (OM) channels mediating pilus extrusion and DNA uptake. How PilQ interacts with the motor is still enigmatic. Here, we have used biochemical and genetic techniques to study the interaction of PilQ with PilW, a unique protein which is essential for natural transformation and T4P extrusion of T. thermophilus. PilQ and PilW form high molecular mass complexes in the OM of T. thermophilus. When pilW was deleted, PilQ complexes were no longer observed but only PilQ monomers, accompanied by a loss of DNA uptake as well as a loss of T4P and twitching motility. Piliation of a ΔpilT2/ΔpilW double mutant suggests that PilW is important for stable assembly of PilQ complexes. To analyze the role of different regions of PilW, partial deletions (pilW∆2–40, pilW∆50–150, pilW∆163–216 and pilW∆216–292) were generated and the effect on DNA uptake, PilQ complex formation and T4P functions such as twitching motility, biofilm formation and cell-cell interaction was studied. These studies revealed that a central disordered region in PilW is required for pilus dynamics. We propose that PilW is part of a protein network that connects the transport ATPase to drive different functions of the DNA translocator and T4P.

Starvation induces shrinkage of the bacterial cytoplasm

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

Photoelectric Bacteria Enhance the In Situ Production of Tetrodotoxin for Antitumor Therapy.

Engineered bacteria are promising bioagents to synthesize antitumor drugs at tumor sites with the advantages of avoiding drug leakage and degradation during delivery. Here, we report an optically controlled material-assisted microbial system by biosynthesizing gold nanoparticles (AuNPs) on the surface of Shewanella algae K3259 (S. algae) to obtain Bac@Au. Leveraging the dual directional electron transport mechanism of S. algae, the hybrid biosystem enhances in situ synthesis of antineoplastic tetrodotoxin (TTX) for a promising antitumor effect. Because of tumor hypoxia-targeting feature of facultative anaerobic S. algae, Bac@Au selectively target and colonize at tumor. Upon light irradiation, photoelectrons produced by AuNPs deposited on bacterial surface are transferred into bacterial cytoplasm and participate in accelerated cell metabolism to increase the production of TTX for antitumor therapy. The optically controlled material-assisted microbial system enhances the efficiency of bacterial drug synthesis in situ and provides an antitumor strategy that could broaden conventional therapy boundaries.

Structural Insights of Shigella Translocator IpaB and Its Chaperone IpgC in Solution.

Bacterial Type III Secretion Systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or deliver Type III Secretion effectors directly into eukaryotic host cell cytoplasm. Shigella, the causing agent of bacillary dysentery or shigellosis, bears a set of T3SS proteins termed translocators that form a pore in the host cell membrane. IpaB, the major translocator of the system, is a key factor in promoting Shigella pathogenicity. Prior to secretion, IpaB is maintained inside the bacterial cytoplasm in a secretion competent folding state thanks to its cognate chaperone IpgC. IpgC couples T3SS activation to transcription of effector genes through its binding to MxiE, probably after the delivery of IpaB to the secretion export gate. Small Angle X-ray Scattering experiments and modeling reveal that IpgC is found in different oligomeric states in solution, as it forms a stable heterodimer with full-length IpaB in contrast to an aggregation-prone homodimer in the absence of the translocator. These results support a stoichiometry of interaction 1:1 in the IpgC/IpaB complex and the multi-functional nature of IpgC under different T3SS states.

HrpB4 from Xanthomonas campestris pv. vesicatoria acts similarly to SctK proteins and promotes the docking of the predicted sorting platform to the type III secretion system.

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates bacterial effector proteins into plant cells. At least nine membrane-associated and cytoplasmic components of the secretion apparatus are homologous to corresponding Sct (secretion and cellular translocation) proteins from animal pathogens, suggesting a similar structural organisation of T3S systems in different bacterial species. T3S in X. campestris pv. vesicatoria also depends on non-conserved proteins with yet unknown function including the essential pathogenicity factor HrpB4. Here, we show that HrpB4 localises to the cytoplasm and the bacterial membranes and interacts with the cytoplasmic domain of the inner membrane (IM) ring component HrcD and the cytoplasmic HrcQ protein. The analysis of HrpB4 deletion derivatives revealed that deletion of the N- or C-terminal protein region affects the interaction of HrpB4 with HrcQ and HrcD as well as its contribution to pathogenicity. HrcQ is a component of the predicted sorting platform, which was identified in animal pathogens as a dynamic heterooligomeric protein complex and associates with the IM ring via SctK proteins. HrcQ complex formation was previously shown by fluorescent microscopy analysis and depends on the presence of the T3S system. In the present study, we provide experimental evidence that the absence of HrpB4 severely affects the docking of HrcQ complexes to the T3S system but does not significantly interfere with HrcQ complex formation in the bacterial cytoplasm. Taken together, our data suggest that HrpB4 links the predicted cytoplasmic sorting platform to the IM rings of the T3S system.

VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm.

VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.ImportanceThis study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions.

The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.

Dehalogenases for pollutant degradation in brief: A mini review

Dehalogenases are microbial enzyme catalysed the cleavage of carbon-halogen bond of halogenated organic compounds. It has potential use in the area of biotechnology such as bioremediation and chemical industry. Halogenated organic compounds can be found in a considerable amount in the environment due to utilization in agriculture and industry, such as pesticides and herbicides. The presence of halogenated compound in the environment have been implicated on the health and natural ecosystem. Microbial dehalogenation is a significant method to tackle this problem. This review intends to briefly describe the microbial dehalogenases in relation to the environment where they are isolated. The basic information about dehalogenases in relation to dehalogenation mechanisms, classification, sources and the transportation of these pollutants into bacterial cytoplasm will be described. We also summarised readily available synthetic halogenated organic compound in the environment.

Multi-Scale Simulations and Neutron Scattering Experiments Reveal Dynamical Properties of the Bacterial Cytoplasm Near Cell-Death Temperature

Life on Earth exhibits an amazing adaptive capacity to a vast range of temperatures. While the molecular mechanisms underlying such adaptability are not yet fully understood, it has been proposed that the cell-death temperature coincides with a denaturation catastrophe of the proteome. Here we use multi-scale simulations and neutron scattering experiments to describe the dynamical properties of proteins inside a bacterial cytoplasm when approaching thermal denaturation. Through a combination of extensive coarse-grain and all-atomistic simulations, we characterize distinct contributions to the picosecond- and nanosecond dynamics of proteins immersed in a crowded and heterogeneous environment mimicking the composition of E.