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Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen \(\textit {Agrobacterium tumefaciens}\). Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in \(\textit {A. tumefaciens.}\)

The outer membrane protein OmpA is a virulence factor in many mammalian pathogens. In previous global RNA structure probing studies, we found evidence for a temperature-modulated RNA structure in the 5'-untranslated region (5'-UTR) of the \(\textit {Yersinia pseudotuberculosis ompA}\) transcript suggesting that opening of the structure at host-body temperature might relieve translational repression. Here, we support this hypothesis by quantitative reverse transcription PCR, translational reporter gene fusions, enzymatic RNA structure probing, and toeprinting assays. While \(‌\it ompA\) transcript levels decreased at 37°C compared to 25°C, translation of the transcript increased with increasing temperature. Biochemical experiments show that this is due to melting of the RNA structure, which permits ribosome binding to the 5'-UTR. A point mutation that locks the RNA structure in a closed conformation prevents translation by impairing ribosome access. Our findings add another common virulence factor to the growing list of pathogen-associated genes that are under RNA thermometer control.

The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes \(\it copA\) and \(\it cueO\) via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that \(\textit {Escherichia coli}\) CueR is prone to proteolysis by the \(AAA^{+}\) proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in \(\textit {E. coli}\) by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in \(\it hfq\) mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen \(\textit {Agrobacterium tumefaciens}\). 136 of 2544 proteins identified by iTRAQ (\(\underline {i}\)sobaric \(\underline {t}\)ags for \(\underline {r}\)elative and \(\underline {a}\)bsolute \(\underline {q}\)uantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded \(Hfq^{3xFlag}\) revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that \(\textit {A. tumefaciens}\) Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in \(\textit {A. tumefaciens}\)-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in \(\textit {A. tumefaciens}\).

The bacterial small heat shock protein IbpA protects client proteins from aggregation. Due to redundancy in the cellular chaperone network, deletion of the \(\it ibpA\) gene often leads to only a mild or no phenotypic defect. In this study, we show that a \(\textit {Pseudomonas putida ibpA}\) deletion mutant has a severe growth defect under heat stress conditions and reduced survival during recovery revealing a critical role of IbpA in heat tolerance. Transcription of the \(\it ibpA\) gene depends on the alternative heat shock sigma factor \(\sigma^{32}\). Production of IbpA protein only at heat shock temperatures suggested additional translational control. We conducted a comprehensive structural and functional analysis of the 5' untranslated regions of the \(\it ibpA\) genes from \(\textit {P. putida}\) and \(\textit {Pseudomonas aeruginosa}\). Both contain a ROSE-type RNA thermometer that is substantially shorter and simpler than previously reported ROSE elements. Comprised of two hairpin structures only, they inhibit translation at low temperature and permit translation initiation after a temperature upshift. Both elements regulate reporter gene expression in \(\textit {Escherichia coli}\) and ribosome binding \(\textit {in vitro}\) in a temperature-dependent manner. Structure probing revealed local melting of the second hairpin whereas the first hairpin remained unaffected. High sequence and structure conservation of pseudomonal \(\it ibpA\) untranslated regions and their ability to confer thermoregulation \(\textit {in vivo}\) suggest that short ROSE-like thermometers are commonly used to control IbpA synthesis in \(\it Pseudomonas\) species.