Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These alphaproteobacteria endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg incompatibility. They can also move horizontally across species boundaries, resulting in a widespread and global distribution in diverse invertebrate hosts. Here, we review the basic biology of Wolbachia, with emphasis on recent advances in our understanding of these fascinating endosymbionts.

Wolbachia: master manipulators of invertebrate biology.

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 A. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

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Structures of the bacterial ribosome at 3.5 Å resolution

Despite their name, synonymous mutations have significant consequences for cellular processes in all taxa. As a result, an understanding of codon bias is central to fields as diverse as molecular evolution and biotechnology. Although recent advances in sequencing and synthetic biology have helped to resolve longstanding questions about codon bias, they have also uncovered striking patterns that suggest new hypotheses about protein synthesis. Ongoing work to quantify the dynamics of initiation and elongation is as important for understanding natural synonymous variation as it is for designing transgenes in applied contexts.

/pdf/synonymous-but-not-the-same-the-causes-and-consequences-of-4xxuk0p9fz.pdf

Synonymous but not the same: the causes and consequences of codon bias.

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

https://science.sciencemag.org/content/sci/313/5795/1935.full.pdf

Structure of the 70S Ribosome Complexed with mRNA and tRNA

Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

/pdf/sticking-together-building-a-biofilm-the-bacillus-subtilis-59fj6f3u5v.pdf

Sticking together: building a biofilm the Bacillus subtilis way

Bacteria growing under different conditions experience a broad range of demand on the rate of protein synthesis, which profoundly affects cellular resource allocation. During fast growth, protein synthesis has long been known to be modulated by adjusting the ribosome content, with the vast majority of ribosomes engaged at a near-maximal rate of elongation. Here, we systematically characterize protein synthesis by Escherichia coli, focusing on slow-growth conditions. We establish that the translational elongation rate decreases as growth slows, exhibiting a Michaelis-Menten dependence on the abundance of the cellular translational apparatus. However, an appreciable elongation rate is maintained even towards zero growth, including the stationary phase. This maintenance, critical for timely protein synthesis in harsh environments, is accompanied by a drastic reduction in the fraction of active ribosomes. Interestingly, well-known antibiotics such as chloramphenicol also cause a substantial reduction in the pool of active ribosomes, instead of slowing down translational elongation as commonly thought.

/pdf/reduction-of-translating-ribosomes-enables-escherichia-coli-3tbk94qgu9.pdf

Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth.

https://digitalcommons.chapman.edu/cgi/viewcontent.cgi?article=1359&context=sees_articles

How the Sequence of a Gene Can Tune Its Translation

The widely used antibiotic spectinomycin inhibits bacterial protein synthesis by blocking translocation of messenger RNA and transfer RNAs on the ribosome. Here, we show that in crystals of the Escherichia coli 70S ribosome spectinomycin binding traps a distinct swiveling state of the head domain of the small ribosomal subunit. Spectinomycin also alters the rate and completeness of reverse translocation in vitro. These structural and biochemical data indicate that in solution spectinomycin sterically blocks swiveling of the head domain of the small ribosomal subunit and thereby disrupts the translocation cycle.

/pdf/a-steric-block-in-translation-caused-by-the-antibiotic-5b5qi2ef00.pdf

A steric block in translation caused by the antibiotic spectinomycin.

During protein synthesis, transfer RNAs (tRNAs) are translocated from the aminoacyl to peptidyl to exit sites of the ribosome, coupled to the movement of messenger RNA (mRNA), in a reaction catalyzed by elongation factor G (EF-G) and guanosine triphosphate (GTP). Here, we show that the peptidyl transferase inhibitor sparsomycin triggers accurate translocation in vitro in the absence of EF-G and GTP. Our results provide evidence that translocation is a function inherent to the ribosome and that the energy to drive this process is stored in the tRNA-mRNA-ribosome complex after peptide-bond formation. These findings directly implicate the peptidyl transferase center of the 50S subunit in the mechanism of translocation, a process involving large-scale movement of tRNA and mRNA in the 30S subunit, some 70 angstroms away.

https://science.sciencemag.org/content/sci/300/5622/1159.full.pdf

Catalysis of ribosomal translocation by sparsomycin.

The recognition of promoters by RNA polymerase (RNAP) requires an associated ς specificity factor (9, 13). In Bacillus subtilis, the principle ς subunit, ςA, can be substituted by any of a number of alternate ς factors (11). Alternate ς factors control the expression of stress responses (ςB), flagellar motility, chemotaxis, and cell wall-related functions (ςD), and the gene expression cascade leading to formation of a dormant endospore (ςH, ςE, ςF, ςG, and ςK). Alternate ς subunits may interact with a small fraction of the available core enzyme to activate transcription of a subset of genes or, as appears to happen during sporulation, become the predominant specificity factor present at a given time. The mechanisms acting to control ς factor activity are remarkably diverse and include the production and regulation of specific anti-ς factors, proteolytic processing of active ς factors from inactive precursors, and regulated turnover.

The sequencing of the B. subtilis genome has led to the identification of seven putative ς factors of the extracytoplasmic function (ECF) subfamily (18). ECF ς factors are found in a wide range of bacteria and control the uptake or secretion of specific molecules or ions and responses to a variety of extracellular stress signals (20). For example, ECF ς factors regulate ferric citrate uptake and heat shock responses in Escherichia coli (2, 7), alginate and exotoxin secretion in Pseudomonas aeruginosa (14, 21), antibiotic production in Streptomyces antibioticus (17), hairpin protein secretion in Erwinia amylovora (25), and carotenoid biogenesis in Myxococcus xanthus (8). The roles of the seven B. subtilis ECF ς factors, and the extent to which their functions may overlap, are largely unknown.

We have previously reported studies of one of the B. subtilis ECF ς factors, ςX. Mutants lacking this protein have an increased sensitivity to elevated temperatures (15). We have determined the consensus sequence for recognition by holoenzyme containing ςX (EςX) and, using this information, were able to identify several target genes recognized by EςX in vivo and in vitro (16). In the course of this work, we noticed a similar promoter element preceding the gene encoding another putative ECF ς factor designated ςW.

In this report, we demonstrate that the promoter element (PW) preceding the sigW-ybbM operon is transcribed by EςW both in vivo and in vitro. In addition, EςW recognizes a subset of promoters that are partially dependent on ςX for expression, consistent with previous in vivo data suggesting that these regulons overlap (16).

Kurt Fredrick

Papers

Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth.

How the Sequence of a Gene Can Tune Its Translation

A steric block in translation caused by the antibiotic spectinomycin.

Catalysis of ribosomal translocation by sparsomycin.

Promoter recognition by Bacillus subtilis sigmaW: autoregulation and partial overlap with the sigmaX regulon.