The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

On the Roles of the Nuclear Non-Coding RNA-Dependent Membrane-Less Organelles in the Cellular Stress Response.

At the beginning of the 21st century, it became obvious that radical changes had taken place in the concept of living matter and, in particular, in the concept of the organization of intracellular space. The accumulated data testify to the essential importance of phase transitions of biopolymers (first of all, intrinsically disordered proteins and RNA) in the spatiotemporal organization of the intracellular space. Of particular interest is the stress-induced reorganization of the intracellular space. Examples of organelles formed in response to stress are nuclear A-bodies and nuclear stress bodies. The formation of these organelles is based on liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and non-coding RNA. Despite their overlapping composition and similar mechanism of formation, these organelles have different functional activities and physical properties. In this review, we will focus our attention on these membrane-less organelles (MLOs) and describe their functions, structure, and mechanism of formation.

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii.

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with KD ~ 1 µM. Proteins co-localize in the soluble fraction of the cell at 30-37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpAHis6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30-37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.

Stress-Induced Membraneless Organelles in Eukaryotes and Prokaryotes: Bird's-Eye View.

Stress is an inevitable part of life. An organism is exposed to multiple stresses and overcomes their negative consequences throughout its entire existence. A correlation was established between life expectancy and resistance to stress, suggesting a relationship between aging and the ability to respond to external adverse effects as well as quickly restore the normal regulation of biological processes. To combat stress, cells developed multiple pro-survival mechanisms, one of them is the assembly of special stress-induced membraneless organelles (MLOs). MLOs are formations that do not possess a lipid membrane but rather form as a result of the "liquid-liquid" phase separation (LLPS) of biopolymers. Stress-responsive MLOs were found in eukaryotes and prokaryotes, they form as a reaction to the acute environmental conditions and are dismantled after its termination. These compartments function to prevent damage to the genetic and protein material of the cell during stress. In this review, we discuss the characteristics of stress-induced MLO-like structures in eukaryotic and prokaryotic cells.

Cell-in-Cell Phenomena in Wall-Less Bacteria: Is It Possible?

This work describes curious structures formed by the mainly phytopathogenic mycoplasma Acholeplasma laidlawii, as well as the human pathogen Ureaplasma parvum cells which resemble cell-in-cell structures of higher eukaryotes and protists. The probable significance of such structures for the mycoplasma cell is discussed. The possibility of their formation in nature and their potential role in the transformation of genetic material, for example, by maintaining (on the one hand) the stability of the genome in the line of generations during asexual reproduction or (on the other hand) the genome plasticity, are substantiated. It should be especially noted that all the arguments presented are based only on morphological data. However, closer attention to unusual structures, the existence of which was shown by electron microscopy images in this case, may prompt researchers to analyze their data more carefully and find something rare and non-trivial among seemingly trivial things. If it is proven by additional methods that cell-in-cell structures can indeed be formed by prokaryotes without a cell wall, this phenomenon may acquire general biological significance.

Maturation of Pseudo-Nucleus Compartment in P. aeruginosa, Infected with Giant phiKZ Phage.

The giant phiKZ phage infection induces the appearance of a pseudo-nucleus inside the bacterial cytoplasm. Here, we used RT-PCR, fluorescent in situ hybridization (FISH), electron tomography, and analytical electron microscopy to study the morphology of this unique nucleus-like shell and to demonstrate the distribution of phiKZ and bacterial DNA in infected Pseudomonas aeruginosa cells. The maturation of the pseudo-nucleus was traced in short intervals for 40 min after infection and revealed the continuous spatial separation of the phage and host DNA. Immediately after ejection, phage DNA was located inside the newly-identified round compartments; at a later infection stage, it was replicated inside the pseudo-nucleus; in the mature pseudo-nucleus, a saturated internal network of filaments was observed. This network consisted of DNA bundles in complex with DNA-binding proteins. On the other hand, the bacterial nucleoid underwent significant rearrangements during phage infection, yet the host DNA did not completely degrade until at least 40 min after phage application. Energy dispersive x-ray spectroscopy (EDX) analysis revealed that, during the infection, the sulfur content in the bacterial cytoplasm increased, which suggests an increase of methionine-rich DNA-binding protein synthesis, whose role is to protect the bacterial DNA from stress caused by infection.

Effects of Mycoplasmas on the Host Cell Signaling Pathways.

Mycoplasmas are the smallest free-living organisms. Reduced sizes of their genomes put constraints on the ability of these bacteria to live autonomously and make them highly dependent on the nutrients produced by host cells. Importantly, at the organism level, mycoplasmal infections may cause pathological changes to the host, including cancer and severe immunological reactions. At the molecular level, mycoplasmas often activate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) inflammatory response and concomitantly inhibit the p53-mediated response, which normally triggers the cell cycle and apoptosis. Thus, mycoplasmal infections may be considered as cancer-associated factors. At the same time, mycoplasmas through their membrane lipoproteins (LAMPs) along with lipoprotein derivatives (lipopeptide MALP-2, macrophage-activating lipopeptide-2) are able to modulate anti-inflammatory responses via nuclear translocation and activation of the Nrf2 (nuclear factor-E2-related anti-inflammatory transcription factor 2). Thus, interactions between mycoplasmas and host cells are multifaceted and depend on the cellular context. In this review, we summarize the current information on the role of mycoplasmas in affecting the host's intracellular signaling mediated by the interactions between transcriptional factors p53, Nrf2, and NF-κB. A better understanding of the mechanisms underlying pathologic processes associated with reprogramming eukaryotic cells that arise during the mycoplasma-host cell interaction should facilitate the development of new therapeutic approaches to treat oncogenic and inflammatory processes.

N- and C-terminal regions of the small heat shock protein IbpA from Acholeplasma laidlawii competitively govern its oligomerization pattern and chaperone-like activity.

Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones preventing the irreversible denaturation of proteins. While in Escherichia coli two sHSPs IbpA and IbpB work in strong cooperation, the sole Mollicute with free-living ability Acholeplasma laidlawii carries a single gene encoding the sHSP protein AlIbpA. In vitro, independently of the temperature, AlIbpA forms a heterogeneous mixture of approximately 24-mer globules, fibrils and huge protein aggregates. The removal of either 12 or 25 N-terminal amino acids led to the formation of fibrils and enhanced the protein ability to prevent the temperature-induced aggregation of insulin, assuming the fibrillar form as an active protein. In turn, the deletion of the C-terminus or substitution of C-terminal LEL motif by SEP decreased the temperature stability of AlIbpA and eliminated its chaperone function completely, although the protein remained predominantly in a globular state. This suggests that the C-terminal LEL motif is necessary for the chaperon-like activity of AlIbpA and fibril formation. Double N- and C-terminal truncations abolished both the chaperone-like activity and huge oligomer formation. Since the globular form of sHSPs is considered as their inactive form, our data suggest that the N-terminus of AlIbpA is responsible for the huge globule (low-active form) formation and behaves as an intramolecular inhibitor of the fibrils (active form) formation and substrates binding. Taken together these data demonstrate non-trivial properties of AlIbpA, in which the competitive action of N- and C-termini governs the equilibrium between either fibrillar or globular structures representing a possible molecular mechanism of the AlIbpA activity regulation.

Outer membrane vesicles secreted by pathogenic and nonpathogenic Bacteroides fragilis represent different metabolic activities.

Numerous studies are devoted to the intestinal microbiota and intercellular communication maintaining homeostasis. In this regard, vesicles secreted by bacteria represent one of the most popular topics for research. For example, the outer membrane vesicles (OMVs) of Bacteroides fragilis play an important nutritional role with respect to other microorganisms and promote anti-inflammatory effects on immune cells. However, toxigenic B. fragilis (ETBF) contributes to bowel disease, even causing colon cancer. If nontoxigenic B. fragilis (NTBF) vesicles exert a beneficial effect on the intestine, it is likely that ETBF vesicles can be utilized for potential pathogenic implementation. To confirm this possibility, we performed comparative proteomic HPLC-MS/MS analysis of vesicles isolated from ETBF and NTBF. Furthermore, we performed, for the first time, HPLC-MS/MS and GS-MS comparative metabolomic analysis for the vesicles isolated from both strains with subsequent reconstruction of the vesicle metabolic pathways. We utilized fluxomic experiments to validate the reconstructed biochemical reaction activities and finally observed considerable difference in the vesicle proteome and metabolome profiles. Compared with NTBF OMVs, metabolic activity of ETBF OMVs provides their similarity to micro reactors that are likely to be used for long-term persistence and implementing pathogenic potential in the host.

Corrigendum: Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

[This corrects the article on p. 2 in vol. 7, PMID: 28144586.].

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction.

Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins.

Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.

The identification and characterization of IbpA, a novel α-crystallin-type heat shock protein from mycoplasma.

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.