Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria.

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

The non-pathogenic Escherichia coli strain W secretes SslE via the virulence-associated type II secretion system beta.

BACKGROUND: Many pathogenic E. coli strains secrete virulence factors using type II secretory systems, homologs of which are widespread in Gram-negative bacteria. Recently, the enteropathogenic Escherichia coli strain E2348/69 was shown to secrete and surface-anchor SslE, a biofilm-promoting virulence factor, via a type II secretion system. Genes encoding SslE and its associated secretion system are conserved in some non-pathogenic E. coli, including the commonly-used W (Waksman) strain. RESULTS: We report here that E. coli W uses its type II secretion system to export a cognate SslE protein. SslE secretion is temperature- and nutrient-dependent, being robust at 37°C in rich medium but strongly repressed by lower temperatures or nutrient limitation. Fusing either of two glycosyl hydrolases to the C-terminus of SslE prevented it from being secreted or surface-exposed. We screened mutations that inactivated the type II secretion system for stress-related phenotypes and found that inactivation of the secretion system conferred a modest increase in tolerance to high concentrations of urea. Additionally, we note that the genes encoding this secretion system are present at a hypervariable locus and have been independently lost or gained in different lineages of E. coli. CONCLUSIONS: The non-pathogenic E. coli W strain shares the extracellular virulence factor SslE, and its associated secretory system, with pathogenic E. coli strains. The pattern of regulation of SslE secretion we observed suggests that SslE plays a role in colonization of mammalian hosts by non-pathogenic as well as pathogenic E. coli. Our work provides a non-pathogenic model system for the study of SslE secretion, and informs future research into the function of SslE during host colonization.

Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions.

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay "Congo Red Analysis of Cellulose Concentration," or "CRACC." CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.

Structural insights into the Type II secretion nanomachine.

The Type II secretion nanomachine transports folded proteins across the outer membrane of Gram-negative bacteria. Recent X-ray crystallography, electron microscopy, and molecular modeling studies provide structural insights into three functionally and spatially connected units of this nanomachine: the cytoplasmic and inner membrane energy-harvesting complex, the periplasmic helical pseudopilus, and the outer membrane secretin. Key advances include cryo-EM reconstruction of the secretin and demonstration that it interacts with both secreted substrates and a crucial transmembrane clamp protein, plus a biochemical and structural explanation of the role of low-abundance pseudopilins in initiating pseudopilus growth. Combining structures and protein interactions, we synthesize a 3D view of the complete complex consistent with a stepwise pathway in which secretin oligomerization defines sites of nanomachine biogenesis.

Competition favours reduced cost of plasmids to host bacteria.

Conjugative plasmids of Gram-negative bacteria have both vertical and horizontal modes of transmission: they are segregated to daughter cells during division, and transferred between hosts by plasmid-encoded conjugative machinery. Despite maintaining horizontal mobility, many plasmids carry fertility inhibition (fin) systems that repress their own conjugative transfer. To assess the ecological basis of self-transfer repression, we compared the invasion of bacterial populations by fin(+) and fin(-) variants of the plasmid R1 using a computational model and co-culture competitions. We observed that the fin(+) variant had a modest cost to the host (measured by reduction in growth rate), while the fin(-) variant incurred a larger cost. In simulations and empirical competitions the fin(-) plasmid invaded cultures quickly, but was subsequently displaced by the fin(+) plasmid. This indicated a competitive advantage to reducing horizontal transmission and allowing increased host replication. Computational simulations predicted that the advantage associated with reduced cost to the host would be maintained over a wide range of environmental conditions and plasmid costs. We infer that vertical transmission in concert with competitive exclusion favour decreased horizontal mobility of plasmids. Similar dynamics may exert evolutionary pressure on parasites, such as temperate bacteriophages and vertically transmitted animal viruses, to limit their rates of horizontal transfer.

Roles of active site residues and the HUH motif of the F plasmid TraI relaxase.

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.

In vivo oligomerization of the F conjugative coupling protein TraD.

Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.

General mutagenesis of F plasmid TraI reveals its role in conjugative regulation.

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.