Mechanism of the Spontaneous and Directional Membrane Insertion of a 2-Transmembrane Ion Channel.

Protein insertion into membranes is a process occurring in every cell and every cellular compartment. Yet, many thermodynamic aspects of this fundamental biophysical process are not well understood. We investigated physicochemical parameters that influence protein insertion using the model protein KcsA, a 2-transmembrane ion channel.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence.

Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110.

The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5' Leader Featuring the sRNA UpsM.

Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division.

Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110.

Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution.

Rhodobacter sphaeroides CryB is a bacterial cryptochrome with (6-4) photolyase activity.

Photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers or (6-4) photoproducts in a light-dependent cleavage reaction. The closely related classical cryptochrome blue light photoreceptors do not repair DNA lesions; instead they are involved in regulatory processes. CryB of Rhodobacter sphaeroides was until now described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition.

Protein Translocation: SecA-SecY Conformational Crosstalk Opens Channel.

A new study of the bacterial Sec translocase complex reports that ADP/ATP binding to SecA triggers multiple conformational changes in the SecYEG channel that may allow the passive directional movement of the polypeptide chain.

Membrane Targeting and Insertion of the C-Tail Protein SciP.

C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-terminal part of the SciP protein sequence are present. Two short hydrophobic sequences were identified that targeted a green fluorescent protein-SciP fusion protein to the membrane involving the signal recognition particle.

Antimicrobial susceptibility and body site distribution of community isolates of coagulase-negative staphylococci.

The primary aim of this study was to determine antimicrobial resistance in coagulase-negative staphylococci (CoNS) from healthy adults in the community. Healthy adults (n = 114) were swabbed on six body sites; both armpits, both knee pits and both sides of the groin. Species determination was performed using Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) and susceptibility testing for 11 relevant antimicrobials was performed by the disc diffusion method and minimal inhibitory concentration gradient test.

T7 ejectosome assembly: A story unfolds.

T7 phage DNA is transported from the capsid into the host cytoplasm across the cell wall by an ejectosome comprised of the viral proteins gp14, gp15 and gp16. Prior to infection, these proteins form the so-called internal core in the mature virion. Gp16 was shown to associate with pure phospholipid bilayers while gp15 bound to DNA.

Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation.

Background: Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters.

The T7 ejection nanomachine components gp15-gp16 form a spiral ring complex that binds DNA and a lipid membrane.

Bacteriophage T7 initiates infection by ejecting several internal capsid proteins into the host cell; these proteins then assemble into a nanomachine that translocates the viral genome from the phage head into the cytoplasm. The ejected proteins are thought to partially unfold as they pass through the lumen of the portal and the short stubby T7 tail during their entry into the cell. In vivo, the internal proteins gp15 and gp16 assemble into a tubular structure that spans the periplasm and cytoplasmic membrane.

Membrane Insertases Are Present in All Three Domains of Life.

In this issue of Structure, Borowska et al. (2015) report the crystal structure and provide experimental evidence on an archaeal membrane insertase, the DUF106 protein from Methanocaldococcus jannaschii, demonstrating that YidC/Oxa1/Alb3-like insertases exist in the archaeal plasma membrane.

The sRNA SorY confers resistance during photooxidative stress by affecting a metabolite transporter in Rhodobacter sphaeroides.

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ((1)O2) in the facultative photosynthetic bacterium Rhodobacter sphaeroides. We have identified SorY as an sRNA, which is induced under several stress conditions and confers increased resistance against (1)O2. SorY by direct interaction affects the takP mRNA, encoding a TRAP-T transporter.

Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli.

Background: Recombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required.

The role of the strictly conserved positively charged residue differs among the Gram-positive, Gram-negative, and chloroplast YidC homologs.

Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3.

Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome.

The archaeal exosome is a phosphorolytic 3'-5' exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG.

Riboregulation in plant-associated α-proteobacteria.

The symbiotic α-rhizobia Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium etli and the related plant pathogen Agrobacterium tumefaciens are important model organisms for studying plant-microbe interactions. These metabolically versatile soil bacteria are characterized by complex lifestyles and large genomes. Here we summarize the recent knowledge on their small non-coding RNAs (sRNAs) including conservation, function, and interaction of the sRNAs with the RNA chaperone Hfq.

Structure and function of the archaeal exosome.

The RNA-degrading exosome in archaea is structurally very similar to the nine-subunit core of the essential eukaryotic exosome and to bacterial polynucleotide phosphorylase (PNPase). In contrast to the eukaryotic exosome, PNPase and the archaeal exosome exhibit metal ion-dependent, phosphorolytic activities and synthesize heteropolymeric RNA tails in addition to the exoribonucleolytic RNA degradation in 3' → 5' direction. The archaeal nine-subunit exosome consists of four orthologs of eukaryotic exosomal subunits: the RNase PH-domain-containing subunits Rrp41 and Rrp42 form a hexameric ring with three active sites, whereas the S1-domain-containing subunits Rrp4 and Csl4 form an RNA-binding trimeric cap on the top of the ring.

RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels.

The membrane insertase YidC.

The membrane insertases YidC-Oxa1-Alb3 provide a simple cellular system that catalyzes the transmembrane topology of newly synthesized membrane proteins. The insertases are composed of a single protein with 5 to 6 transmembrane (TM) helices that contact hydrophobic segments of the substrate proteins. Since YidC also cooperates with the Sec translocase it is widely involved in the assembly of many different membrane proteins including proteins that obtain complex membrane topologies.

Beyond catalysis: vitamin B12 as a cofactor in gene regulation.

Vitamin B12 is well known as an enzyme cofactor in the catalysis of many important biological reactions, and the role of B12 in regulation of bacterial gene expression as a ligand of riboswitches is well established. Only recently evidence has emerged that B12 can also affect bacterial gene expression by acting as a cofactor of regulatory proteins. In 2011 a role of B12 as a cofactor of the transcriptional repressor of carotenogenesis, CarH, in Myxococcus xanthus was reported.

The C-terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli.

The marine Gram-negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C-terminal regions similar to the YidC homologues in mitochondria and Gram-positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C-terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane.

DegS and RseP homologous proteases are involved in singlet oxygen dependent activation of RpoE in Rhodobacter sphaeroides.

Singlet oxygen ((1)O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to (1)O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR.