The pathway of sulfide oxidation to octasulfur globules in the cytoplasm of aerobic bacteria

Sulfur-oxidizing bacteria can oxidize hydrogen sulfide (H 2 S) to produce sulfur globules. Although the process is common, the pathway is unclear. In recombinant Escherichia coli and wild-type Corynebacterium vitaeruminis DSM20294 with SQR but no enzymes to oxidize zero valence sulfur, SQR oxidized H 2 S into short-chain inorganic polysulfide (H 2 S n , n≥2) and organic polysulfide (RS n H, n≥2), which reacted with each other to form long-chain GS n H (n≥2) and H 2 S n before producing octasulfur (S 8 ), the main component of elemental sulfur. GS n H also reacted with GSH to form GSnG (n≥2) and H 2 S; H 2 S was again oxidized by SQR. After GSH was depleted, SQR simply oxidized H 2 S to H 2 S n , which spontaneously generated S 8 . S 8 aggregated into sulfur globules in the cytoplasm. The results highlight the process of sulfide oxidation to S 8 globules in the bacterial cytoplasm and demonstrate the potential of using heterotrophic bacteria with SQR to convert toxic H 2 S into relatively benign S 8 globules. IMPORTANCE Our results support a process of H 2 S oxidation to produce octasulfur globules via SQR catalysis and spontaneous reactions in the bacterial cytoplasm. Since the process is an important event in geochemical cycling, a better understanding facilitates further studies and provides theoretical support for using heterotrophic bacteria with SQR to oxidize toxic H 2 S into sulfur globules for recovery.

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

Background Knowledge 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. Results We 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. Conclusions Our 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.

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.

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.

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. Importance This 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.

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2

ABSTRACT Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism. IMPORTANCE The transfer of information between DNA, RNA, and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware, i.e., the nucleoid, the RNA polymerase, and the ribosomes, are habitually taken for granted. In this work, we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida strain KT2440 through the nonhomogeneous milieu of the bacterial cytoplasm. The results expose that—similarly to computers—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code and accompanies the environmental lifestyle of this bacterium.

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.