Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination.

The soil-dwelling α-proteobacterium Sinorhizobium meliloti serves as model for studies of symbiotic nitrogen fixation, a highly important process in sustainable agriculture. Here, we report advancements of the genetic toolbox accelerating genome editing in S. meliloti.

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network.

The cell envelope stress response (CESR) encompasses all regulatory events that enable a cell to protect the integrity of its envelope, an essential structure of any bacterial cell. The underlying signaling network is particularly well understood in the Gram-positive model organism Bacillus subtilis. It consists of a number of two-component systems (2CS) and extracytoplasmic function σ factors that together regulate the production of both specific resistance determinants and general mechanisms to protect the envelope against antimicrobial peptides targeting the biogenesis of the cell wall.

Design and Assembly of DNA Sequence Libraries for Chromosomal Insertion in Bacteria Based on a Set of Modified MoClo Vectors.

Efficient assembly of large DNA constructs is a key technology in synthetic biology. One of the most popular assembly systems is the MoClo standard in which restriction and ligation of multiple fragments occurs in a one-pot reaction. The system is based on a smart vector design and type IIs restriction enzymes, which cut outside their recognition site.

Engineering Hybrid Chemotaxis Receptors in Bacteria.

Most bacteria use transmembrane sensors to detect a wide range of environmental stimuli. A large class of such sensors are the chemotaxis receptors used by motile bacteria to follow environmental chemical gradients. In Escherichia coli, chemotaxis receptors are known to mediate highly sensitive responses to ligands, making them potentially useful for biosensory applications.

Microalgae as Solar-Powered Protein Factories.

Microalgae have an enormous ecological relevance as they contribute significantly to global carbon fixation. But also for biotechnology microalgae became increasingly interesting during the last decades as many algae provide valuable natural products. Especially the high lipid content of some species currently attracts much attention in the biodiesel industry.

The magic dance of the alarmones (p)ppGpp.

The alarmones (p)ppGpp are important second messengers that orchestrate pleiotropic adaptations of bacteria and plant chloroplasts in response to starvation and stress. Here, we review our structural and mechanistic knowledge on (p)ppGpp metabolism including their synthesis, degradation and interconversion by a highly diverse set of enzymes. Increasing structural information shows how (p)ppGpp interacts with an incredibly diverse set of different targets that are essential for replication, transcription, translation, ribosome assembly and metabolism.

DistAMo: A Web-Based Tool to Characterize DNA-Motif Distribution on Bacterial Chromosomes.

Short DNA motifs are involved in a multitude of functions such as for example chromosome segregation, DNA replication or mismatch repair. Distribution of such motifs is often not random and the specific chromosomal pattern relates to the respective motif function. Computational approaches which quantitatively assess such chromosomal motif patterns are necessary.

A Non-Stem-Loop CRISPR RNA Is Processed by Dual Binding Cas6.

A subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site.

Anatomy of the bacitracin resistance network in Bacillus subtilis.

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well-characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle-inhibiting AMP bacitracin.

Transcription-coupled DNA supercoiling dictates the chromosomal arrangement of bacterial genes.

Over the recent decade, the central importance of DNA supercoiling in chromosome organization and global gene regulation of bacteria became more and more visible. With a regulon comprising more than 2000 genes in Escherichia coli, DNA supercoiling is among the most influential regulators of gene expression found in bacteria so far. However, the mechanism creating thousands of diverse temporal gene expression patterns coordinated by DNA supercoiling remains unclear.

Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD.

Importance of Multiple Methylation Sites in Escherichia coli Chemotaxis.

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors' activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors' sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.

Deterministic Assembly of Complex Bacterial Communities in Guts of Germ-Free Cockroaches.

The gut microbiota of termites plays important roles in the symbiotic digestion of lignocellulose. However, the factors shaping the microbial community structure remain poorly understood. Because termites cannot be raised under axenic conditions, we established the closely related cockroach Shelfordella lateralis as a germ-free model to study microbial community assembly and host-microbe interactions.

Ustilago maydis produces itaconic acid via the unusual intermediate trans-aconitate.

Itaconic acid is an important biomass-derived chemical building block but has also recently been identified as a metabolite produced in mammals, which has antimicrobial activity. The biosynthetic pathway of itaconic acid has been elucidated in the ascomycetous fungus Aspergillus terreus and in human macrophages. In both organisms itaconic acid is generated by decarboxylation of the tricarboxylic acid (TCA) cycle intermediate cis-aconitate.

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model.

The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches.

Metagenomic analysis of the microbiota in the highly compartmented hindguts of six wood- or soil-feeding higher termites.

Background: Termites are important contributors to carbon and nitrogen cycling in tropical ecosystems. Higher termites digest lignocellulose in various stages of humification with the help of an entirely prokaryotic microbiota housed in their compartmented intestinal tract. Previous studies revealed fundamental differences in community structure between compartments, but the functional roles of individual lineages in symbiotic digestion are mostly unknown.

Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti.

Unlabelled: Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3',5'-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S.

Structures and functions of mitochondrial ABC transporters.

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis.

Bacillus subtilis Bactofilins Are Essential for Flagellar Hook- and Filament Assembly and Dynamically Localize into Structures of Less than 100 nm Diameter underneath the Cell Membrane.

Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies.

Bacterial Networks in Cells and Communities.

Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks (BacNet15) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (-omics) approaches and mathematical modeling can help understand design principles relevant in microbiology.

Circularization restores signal recognition particle RNA functionality in Thermoproteus.

Signal recognition particles (SRPs) are universal ribonucleoprotein complexes found in all three domains of life that direct the cellular traffic and secretion of proteins. These complexes consist of SRP proteins and a single, highly structured SRP RNA. Canonical SRP RNA genes have not been identified for some Thermoproteus species even though they contain SRP19 and SRP54 proteins.

Undiscovered regions on the molecular landscape of flagellar assembly.

The bacterial flagellum is a motility structure and one of the most complicated motors in the biosphere. A flagellum consists of several dozens of building blocks in different stoichiometries and extends from the cytoplasm to the extracellular space. Flagellar biogenesis follows a strict spatio-temporal regime that is guided by a plethora of flagellar assembly factors and chaperones.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP.