A Micrarchaeon isolate is covered by a proteinaceous S-Layer

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated “ Candidatus Micrarchaeum harzensis A_DKE” possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in “ Ca. Micrarchaeum harzensis A_DKE” and in silico characterisation of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated and the Micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described co-culture, containing Micrarchaeum A_DKE and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of “ Ca. Micrarchaeum harzensis A_DKE”. Both organisms are clearly distinguishable by cell size, shape and surface structure. Importance Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member “ Candidatus Micrarchaeum harzensis A_DKE”, an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of “ Ca. Micrarchaeum harzensis A_DKE” is an important step towards understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

ZipA Uses a Two-Pronged FtsZ-Binding Mechanism Necessary for Cell Division

The tubulin homolog FtsZ plays a central early role in organizing bacterial cell division proteins at the cytoplasmic membrane. However, FtsZ does not directly interact with the membrane itself, instead relying on proteins such as FtsA to tether it to the membrane.

Mechanosensitive channels mediate hypoionic shock-induced aminoglycoside potentiation against bacterial persisters by enhancing antibiotic uptake

Improving the efficacy of existing antibiotics is a promising strategy for combating antibiotic-resistant/tolerant bacterial pathogens that have become a severe threat to human health. We previously reported that aminoglycoside antibiotics could be dramatically potentiated against stationary-phase Escherichia coli cells under hypoionic shock conditions (i.e., treatment with ion-free solutions), but the underlying molecular mechanism remains unknown. Here we show that mechanosensitive (MS) channels, a ubiquitous protein family sensing mechanical forces of cell membrane, mediate such hypoionic shock-induced aminoglycoside potentiation. Two-minute treatment under conditions of hypoionic shock (e.g., in pure water) greatly enhances the bactericidal effects of aminoglycosides against both spontaneous and triggered E. coli persisters, numerous strains of Gram-negative pathogens in vitro , and Pseudomonas aeruginosa in mice. Such potentiation is achieved by hypoionic shock-enhanced bacterial uptake of aminoglycosides and is linked to hypoionic shock-induced destabilization of the cytoplasmic membrane in E. coli . Genetic and biochemical analyses reveal that MscS-family channels directly and redundantly mediate aminoglycoside uptake upon hypoionic shock and thus potentiation, with MscL channel showing reduced effect. Molecular docking and site-directed mutagenesis analyses reveal a putative streptomycin-binding pocket in MscS, critical for streptomycin uptake and potentiation. These results suggest that hypoionic shock treatment destabilizes the cytoplasmic membrane and thus changes the membrane tension, which immediately activates MS channels that are able to effectively transport aminoglycosides into the cytoplasm for downstream killing. Our findings reveal the biological effects of hypoionic shock on bacteria and can help to develop novel adjuvants for aminoglycoside potentiation to combat bacterial pathogens via activating MS channels.

Identification of New In Vivo TonB-FepA Rendezvous Sites

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

Since the characterization of cytochrome c552 as a multiheme nitrite reductase, research on this enzyme has gained major interest. Today, it is known as pentaheme cytochrome c nitrite reductase (NrfA). Part of the NH4+ produced from NO2− is released as NH3 leading to nitrogen loss, similar to denitrification which generates NO, N2O, and N2. NH4+ can also be used for assimilatory purposes, thus NrfA contributes to nitrogen retention. It catalyses the six-electron reduction of NO2− to NH4+, hosting four His/His ligated c-type hemes for electron transfer and one structurally differentiated active site heme. Catalysis occurs at the distal side of a Fe(III) heme c proximally coordinated by lysine of a unique CXXCK motif (Sulfurospirillum deleyianum, Wolinella succinogenes) or, presumably, by the canonical histidine in Campylobacter jejeuni. Replacement of Lys by His in NrfA of W. succinogenes led to a significant loss of enzyme activity. NrfA forms homodimers as shown by high resolution X-ray crystallography, and there exist at least two distinct electron transfer systems to the enzyme. In γ-proteobacteria (Escherichia coli) NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a pentaheme electron carrier (NrfB), in δ- and ε-proteobacteria (S. deleyianum, W. succinogenes), the NrfA dimer interacts with a tetraheme cytochrome c (NrfH). Both form a membrane-associated respiratory complex on the extracellular side of the cytoplasmic membrane to optimize electron transfer efficiency. This minireview traces important steps in understanding the nature of pentaheme cytochrome c nitrite reductases, and discusses their structural and functional features. Graphical abstract

Structural and functional state of blood lymphocytes in patients with infectious mononucleosis with different course

The article presents the results of our own studies. The aim was to determine the structural and functional status of blood lymphocytes in patients with acute and prolonged course of infectious mononucleosis (IM) in children. Materials and methods. 102 children were under clinical and laboratory-instrumental supervision, the children were divided into groups: group 1 – 65 children with IM with an acute course of the disease; group 2 – 37 children with a prolonged course of the disease. All children underwent standard clinical laboratory and instrumental laboratory examinations. The diagnosis of IM was confirmed by PCR (detection of EBV DNA in the blood) and ELISA (anti-EBV IgM and IgG). Research results. In the study of the structural state of the cytoplasmic membrane of the lymphocytes in the blood of patients with MI in the onset of the disease, it was found that the average values of penetration rate of the electron paramagnetic resonance of spin probes (PR EPR s. p.) in children of both groups were significantly higher than normal (P < 0.001). There are also differences between groups of patients. In this case, the value of PR EPR s. p. in patients with a prolonged course by 15.8 % exceeded those in patients with acute IM (P < 0.001). According to the rate of microviscosity of the intracellular content (MV IC), its values were reduced compared with the control – by 22.1 % (P < 0.001) in patients with acute course of the disease and by 25.1 % – with a prolonged course of IM). In addition, in patients with a prolonged course of the disease, the values were 9 % lower than in the group with acute infectious mononucleosis. When considering immunological parameters, it was found that the indicators of the T-immune system for patients with a prolonged course of the disease in comparison with the alternative group was characterized by a decrease in the content of CD3 <50 % (respectively in 51.3 % and 26.2 % of patients; P < 0.05); CD4 <31 % (62.1 % and 32.4 %, respectively; P < 0.05) and CD8 <15 % (37.8 % and 10.8 %, respectively; P < 0.01). With regard to the cytokine profile, the level of IL-1 <20.0 pg/ml was determined 3.5 times more often in patients with a prolonged course of the disease compared to the acute course (64.8 % and 18.5 % of patients, respectively); TNFα <20.0 pg/ml 1.9 times more often (48.6 % and 24.6 %, respectively) and a very high (>30.1 pg/ml) level of IL4 in 40.5 % and 20 %). From the B-system of immunity in patients with a prolonged course of IM in comparison with the acute course increased content of CD22 was more often determined, as well as low levels of IgA, IgM <1.1 g/l and IgG <10.0 g/l. Conclusions. According to the results of observations, the pathogenetic role of the violation of the structural organization of blood lymphocytes in the formation of IM is established. It was found that these disorders in the form of increased permeability of their cytoplasmic membrane and reduced viscoelastic properties of their intracellular environment are more pronounced with a prolonged course of the disease, which is a factor in the prolongation of the disease. It is determined that the indicators of cellular and humoral parts of the immune system affect the course of IM. During formation of an acute course of IM in children already in the acute period of a disease activation of both cellular and humoral links of immunity, which is shown in the form of increase in relative content of CD3+, CD4+, CD8+ and CD22+ and levels of immunoglobulins M, A, is noted. For the prolonged course of the disease depression of T-cell immunity in the form of a decrease in the relative content of CD3+, CD4+ and CD8+ lymphocytes and an increase in CD22+, as well as inhibition of antibody genesis are characteristic. It was found that the variant of IM depends on the type of reaction of T-helper clones, namely – in the initial period of manifestation of IM with its acute course there is activation of T1 and T2 helper response, which manifests itself in a significant increase in IL-1, TNFα and moderate IL-4. Prolonged course of the disease is formed against the background of weak activation of pro-inflammatory interleukins (IL-1, TNFα) and significant – anti-inflammatory IL-4.

New Phenotype and Mineralization of Biogenic Iron Oxide in Magnetotactic Bacteria

Many magnetotactic bacteria (MTB) biomineralize magnetite crystals that nucleate and grow inside intracellular membranous vesicles originating from invaginations of the cytoplasmic membrane. The crystals together with their surrounding membranes are referred to as magnetosomes. Magnetosome magnetite crystals nucleate and grow using iron transported inside the vesicle by specific proteins. Here, we tackle the question of the organization of magnetosomes, which are always described as constituted by linear chains of nanocrystals. In addition, it is commonly accepted that the iron oxide nanocrystals are in the magnetite-based phase. We show, in the case of a wild species of coccus-type bacterium, that there is a double organization of the magnetosomes, relatively perpendicular to each other, and that the nanocrystals are in fact maghemite. These findings were obtained, respectively, by using electron tomography of whole mounts of cells directly from the environment and high-resolution transmission electron microscopy and diffraction. Structure simulations were performed with the MacTempas software. This study opens new perspectives on the diversity of phenotypes within MTBs and allows to envisage other mechanisms of nucleation and formation of biogenic iron oxide crystals.

Bacterial competition systems share a domain required for inner membrane transport of the nuclease bacteriocin pyocin G

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent amongst these are contact-dependent inhibition (CDI), type VI secretion (T6SS) and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact dependent- and contact-independent means.

Blood bank storage of red blood cells increases RBC cytoplasmic membrane order and bending rigidity

Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells’ function and eventually the recipients’ health. We extracted the RBC’s cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes’ molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm’s bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms.