Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies.

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity.

Harnessing the central dogma for stringent multi-level control of gene expression.

Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided, but without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple solution to this challenge.

The iron-sulfur scaffold protein HCF101 unveils the complexity of organellar evolution in SAR, Haptista and Cryptista.

Background: Nbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea.

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH.

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity.

Mechanochemical control of epidermal stem cell divisions by B-plexins.

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear.

Efficient antisense inhibition reveals microRNA-155 to restrain a late-myeloid inflammatory programme in primary human phagocytes.

A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells.

A Stringent-Response-Defective Bradyrhizobium diazoefficiens Strain Does Not Activate the Type 3 Secretion System, Elicits an Early Plant Defense Response, and Circumvents NHNO-Induced Inhibition of Nodulation.

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria.

Transcriptional analysis identifies potential biomarkers and molecular regulators in acute malaria infection.

Aims: Malaria is a serious health threat in tropical countries. The causative parasite of Malaria tropica, the severe form, is the protozoan Plasmodium falciparum. In humans, it infects red blood cells, compromising blood flow and tissue perfusion.

The two-component system ActJK is involved in acid stress tolerance and symbiosis in Sinorhizobium meliloti.

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain.

Carbon Source-Dependent Reprogramming of Anaerobic Metabolism in Staphylococcus aureus.

To be a successful pathogen, has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, uses glycolysis to generate ATP via substrate-level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by reoxidation of NADH equivalents. However, it is less clear how proceeds under anoxic conditions and glucose limitation, likely representing the bona fide situation in the host.

Single-Molecule Dynamics at a Bacterial Replication Fork after Nutritional Downshift or Chemically Induced Block in Replication.

Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium , we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift.

Stable inheritance of Sinorhizobium meliloti cell growth polarity requires an FtsN-like protein and an amidase.

In Rhizobiales bacteria, such as Sinorhizobium meliloti, cell elongation takes place only at new cell poles, generated by cell division. Here, we show that the role of the FtsN-like protein RgsS in S. meliloti extends beyond cell division.

Aethionema arabicum genome annotation using PacBio full-length transcripts provides a valuable resource for seed dormancy and Brassicaceae evolution research.

Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed (development, regulation, germination, dormancy) and fruit (development, dehiscence mechanisms) characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae.

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis.

The hydrothermal vent tubeworm hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication.

Expansion and re-classification of the extracytoplasmic function (ECF) σ factor family.

Extracytoplasmic function σ factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As a result, we identified an average of ∼10 ECFs per bacterial genome and 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism.

Disease-Causing Mutations and Rearrangements in Long Non-coding RNA Gene Loci.

The classic understanding of molecular disease-mechanisms is largely based on protein-centric models. During the past decade however, genetic studies have identified numerous disease-loci in the human genome that do not encode proteins. Such non-coding DNA variants increasingly gain attention in diagnostics and personalized medicine.

Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon .

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology.

Advances and opportunities in image analysis of bacterial cells and communities.

The cellular morphology and sub-cellular spatial structure critically influence the function of microbial cells. Similarly, the spatial arrangement of genotypes and phenotypes in microbial communities has important consequences for cooperation, competition, and community functions. Fluorescence microscopy techniques are widely used to measure spatial structure inside living cells and communities, which often results in large numbers of images that are difficult or impossible to analyze manually.

Awakening a latent carbon fixation cycle in Escherichia coli.

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes.

Do FeS clusters rule bacterial iron regulation?

For decades, the bacterial ferric uptake regulator (Fur) has been thought to respond to ferrous iron to transcriptionally regulate genes required for balancing iron uptake, storage, and utilization. Because iron binding to Fur has never been confirmed , the physiological iron-sensing mechanism remains an open question. Fontenot now show that Fur purified from binds an all-Cys-coordinated [2Fe-2S] cluster.

mutations conferring rifampicin-resistance affect growth, stress response and motility in .

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, . Different mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects.

Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation.

Background: MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles.

Results: Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm.

The alarmone (p)ppGpp confers tolerance to oxidative stress during the stationary phase by maintenance of redox and iron homeostasis in Staphylococcus aureus.

Slow growing stationary phase bacteria are often tolerant to multiple stressors and antimicrobials. Here, we show that the pathogen Staphylococcus aureus develops a non-specific tolerance towards oxidative stress during the stationary phase, which is mediated by the nucleotide second messenger (p)ppGpp. The (p)ppGpp mutant was highly susceptible to HOCl stress during the stationary phase.