Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function

Expression and Assembly of a Functional Type IV Secretion System Elicit Extracytoplasmic and Cytoplasmic Stress Responses in Escherichia coli

Conditions perturbing protein homeostasis are known to induce cellular stress responses in prokaryotes and eukaryotes. Here we show for the first time that expression and assembly of a functional type IV secretion (T4S) machinery elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. After induction of T4S genes by a nutritional upshift and assembly of functional DNA transporters encoded by plasmid R1-16, host cells activated the CpxAR envelope stress signaling system, as revealed by induction or repression of downstream targets of the CpxR response regulator. Furthermore, we observed elevated transcript levels of cytoplasmic stress genes, such as groESL, with a concomitant increase of σ(32) protein levels in cells expressing T4S genes. A traA null mutant of plasmid R1-16, which lacks the functional gene encoding the major pilus protein pilin, showed distinctly reduced stress responses. These results corroborated our conclusion that the activation of bacterial stress networks was dependent on the presence of functional T4S machinery. Additionally, we detected increased transcription from the rpoHp(1) promoter in the presence of an active T4S system. Stimulation of rpoHp(1) was dependent on the presence of CpxR, suggesting a hitherto undocumented link between CpxAR and σ(32)-regulated stress networks

GroEL Plays a Central Role in Stress-Induced Negative Regulation of Bacterial Conjugation by Promoting Proteolytic Degradation of the Activator Protein TraJ▿

Transcription of DNA transfer genes is a prerequisite for conjugative DNA transfer of F-like plasmids. Transfer gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on transfer gene expression and DNA transfer in Escherichia coli. Raising the growth temperature from 22°C to 43°C transiently reduced transfer gene expression to undetectable levels and reduced conjugative transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive groEL44 allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in groEL(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type E. coli cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in groEL(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis

Growth Phase- and Cell Division-Dependent Activation and Inactivation of the σ32 Regulon in Escherichia coli▿ †

Alternative sigma factors allow bacteria to reprogram global transcription rapidly and to adapt to changes in the environment. Here we report on growth- and cell division-dependent σ32 regulon activity in Escherichia coli in batch culture. By analyzing σ32 expression in growing cells, an increase in σ32 protein levels is observed during the first round of cell division after exit from stationary phase. Increased σ32 protein levels result from transcriptional activation of the rpoH gene. After the first round of bulk cell division, rpoH transcript levels and σ32 protein levels decrease again. The late-logarithmic phase and the transition to stationary phase are accompanied by a second increase in σ32 levels and enhanced stability of σ32 protein but not by enhanced transcription of rpoH. Throughout growth, σ32 target genes show expression patterns consistent with oscillating σ32 protein levels. However, during the transition to early-stationary phase, despite high σ32 protein levels, the transcription of σ32 target genes is downregulated, suggesting functional inactivation of σ32. It is deduced from these data that there may be a link between σ32 regulon activity and cell division events. Further support for this hypothesis is provided by the observation that in cells in which FtsZ is depleted, σ32 regulon activation is suppressed

Thirty-Eight C-Terminal Amino Acids of the Coupling Protein TraD of the F-Like Conjugative Resistance Plasmid R1 Are Required and Sufficient To Confer Binding to the Substrate Selector Protein TraM

Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (ΔN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 × 10(7) liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP

Microbial induced acid corrosion from a field perspective - Advances in process understanding and construction material development

Microbial induced acid corrosion is accounted for ~40 % of the degradation of subsurface wastewater infrastructure globally. While fundamental process understanding has increased significantly within the last decades, to date no sustainable building material exists, which meets the long-term requirements in such aggressive and corrosive environments. This work describes a novel model based on field studies, conducted in various Austrian sewer networks, intertwining biological, mineralogical and hydro-chemical factors. Additionally, an extensive field testing campaign over the duration of 18 months, conducted on different geopolymer concretes (GPC), particularly designed for the latter environments will be presented. Innovative GPCs were tested regarding their microstructural behavior, microbial accessibility and hydro-chemical alterations over time and compared to commercially produced cement based products, including OPC and CAC concretes. Additionally, the system specific environmental parameters, such as relevant gas concentrations (H2S, CO2, CH4), relative humidity and temperature were constantly monitored