A cytoplasmic chemoreceptor and reactive oxygen species mediate bacterial chemotaxis to copper.

Chemotaxis is a widespread strategy used by unicellular and multicellular living organisms to maintain their fitness in stressful environments. We previously showed that bacteria can trigger a negative chemotactic response to a copper (Cu)-rich environment. Cu ion toxicity on bacterial cell physiology has been mainly linked to mismetallation events and reactive oxygen species (ROS) production, although the precise role of Cu-generated ROS remains largely debated. Here, using inductively coupled plasma optical emission spectrometry on cell fractionates, we found that the cytoplasmic Cu ion content mirrors variations of the extracellular Cu ion concentration. ROS-sensitive fluorescent probe and biosensor allowed us to show that the increase of cytoplasmic Cu ion content triggers a dose-dependent oxidative stress, which can be abrogated by superoxide dismutase and catalase overexpression. The inhibition of ROS production in the cytoplasm not only improves bacterial growth but also impedes Cu chemotaxis, indicating that ROS derived from cytoplasmic Cu ions mediate the control of bacterial chemotaxis to Cu. We also identified the Cu chemoreceptor McpR, which binds Cu ions with low affinity, suggesting a labile interaction. In addition, we demonstrate that the cysteine 75 and histidine 99 within the McpR sensor domain are key residues in Cu chemotaxis and Cu coordination. Finally, we discovered that in vitro both Cu(I) and Cu(II) ions modulate McpR conformation in a distinct manner. Overall, our study provides mechanistic insights on a redox-based control of Cu chemotaxis, indicating that the cellular redox status can play a key role in bacterial chemotaxis.

Characteristics of the copper-induced viable-but-non-culturable state in bacteria.

The antimicrobial applications of copper (Cu) are exploited in several industries, such as agriculture and healthcare settings. While Cu is capable of efficiently killing microorganisms, sub-lethal doses can induce a viable-but-non-culturable (VBNC) state in bacteria of many distinct clades. VBNC cells cannot be detected by standard culture-based detection methods, and can become a threat to plants and animals as they often retain virulent traits upon resuscitation. Here we discuss the putative mechanisms of the Cu-induced VBNC state. Common observations in Cu-induced VBNC cells include a cellular response to reactive oxygen species, the exhaustion of energy reserves, and a reconfiguration of the proteome. While showing partial overlap with other VBNC state-inducing stressors, these changes seem to be part of an adaptive response to Cu toxicity. Furthermore, we argue that Cu resistance mechanisms such as P-type ATPases and multicopper oxidases may ward off entry into the VBNC state to some extent. The spread of these mechanisms across multi-species populations could increase population-level resistance to Cu antimicrobials. As Cu resistance mechanisms are often co-selected with antibiotic resistance mechanisms, this threat is exacerbated.

Refolding and biophysical characterization of the Caulobacter crescentus copper resistance protein, PcoB: An outer membrane protein containing an intrinsically disordered domain.

Copper cations play fundamental roles in biological systems, such as protein folding and stabilization, or enzymatic reactions. Although copper is essential to the cell, it can become cytotoxic if present in too high concentration. Organisms have therefore developed specific regulation mechanisms towards copper. This is the case of the Pco system present in the bacterium Caulobacter crescentus, which is composed of two proteins: a soluble periplasmic protein PcoA and an outer membrane protein PcoB. PcoA oxidizes Cu+ to Cu2+, whereas PcoB is thought to be an efflux pump for Cu2+. While the PcoA protein has already been studied, very little is known about the structure and function of PcoB. In the present work, PcoB has been overexpressed in high yield in E. coli strains and successfully refolded by the SDS-cosolvent method. Binding to divalent cations has also been studied using several spectroscopic techniques. In addition, a three-dimensional structure model of PcoB, experimentally supported by circular dichroism, has been constructed, showing a ß-barrel conformation with a N-terminal disordered chain. This peculiar intrinsic disorder property has also been confirmed by various bioinformatic tools.

Environmental Conditions Modulate the Transcriptomic Response of Both Caulobacter crescentus Morphotypes to Cu Stress.

Bacteria encounter elevated copper (Cu) concentrations in multiple environments, varying from mining wastes to antimicrobial applications of copper. As the role of the environment in the bacterial response to Cu ion exposure remains elusive, we used a tagRNA-seq approach to elucidate the disparate responses of two morphotypes of Caulobacter crescentus NA1000 to moderate Cu stress in a complex rich (PYE) medium and a defined poor (M2G) medium. The transcriptome was more responsive in M2G, where we observed an extensive oxidative stress response and reconfiguration of the proteome, as well as the induction of metal resistance clusters. In PYE, little evidence was found for an oxidative stress response, but several transport systems were differentially expressed, and an increased need for histidine was apparent. These results show that the Cu stress response is strongly dependent on the cellular environment. In addition, induction of the extracytoplasmic function sigma factor SigF and its regulon was shared by the Cu stress responses in both media, and its central role was confirmed by the phenotypic screening of a sigF::Tn5 mutant. In both media, stalked cells were more responsive to Cu stress than swarmer cells, and a stronger basal expression of several cell protection systems was noted, indicating that the swarmer cell is inherently more Cu resistant. Our approach also allowed for detecting several new transcription start sites, putatively indicating small regulatory RNAs, and additional levels of Cu-responsive regulation.

Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP.

BACKGROUND: When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active. RESULTS: We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates. CONCLUSION: The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

The Transcriptomic Landscape of Cupriavidus metallidurans CH34 Acutely Exposed to Copper.

Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5' end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.

Copper Resistance Mediates Long-Term Survival of Cupriavidus metallidurans in Wet Contact With Metallic Copper.

Metallic copper to combat bacterial proliferation in drinking water systems is being investigated as an attractive alternative to existing strategies. A potential obstacle to this approach is the induction of metal resistance mechanisms in contaminating bacteria, that could severely impact inactivation efficacy. Thus far, the role of these resistance mechanisms has not been studied in conditions relevant to drinking water systems. Therefore, we evaluated the inactivation kinetics of Cupriavidus metallidurans CH34 in contact with metallic copper in drinking water. Viability and membrane permeability were examined for 9 days through viable counts and flow cytometry. After an initial drop in viable count, a significant recovery was observed starting after 48 h. This behavior could be explained by either a recovery from an injured/viable-but-non-culturable state or regrowth of surviving cells metabolizing lysed cells. Either hypothesis would necessitate an induction of copper resistance mechanisms, since no recovery was seen in a CH34 mutant strain lacking metal resistance mechanisms, while being more pronounced when copper resistance mechanisms were pre-induced. Interestingly, no biofilms were formed on the copper surface, while extensive biofilm formation was observed on the stainless steel control plates. When CH34 cells in water were supplied with CuSO4, a similar initial decrease in viable counts was observed, but cells recovered fully after 7 days. In conclusion, we have shown that long-term bacterial survival in the presence of a copper surface is possible upon the induction of metal resistance mechanisms. This observation may have important consequences in the context of the increasing use of copper as an antimicrobial surface, especially in light of potential co-selection for metal and antimicrobial resistance.

The asymmetric spatial distribution of bacterial signal transduction proteins coordinates cell cycle events.

The polar localization of signaling proteins that are essential for Caulobacter cell cycle control is temporally regulated. Here we provide evidence that phosphorylation of the essential response regulator, DivK, is required for both its function and its cell cycle-regulated localization. The asymmetric location of the DivJ and PleC histidine kinases and their antagonistic activities on the cellular concentration of phosphorylated DivK provide positional and temporal information for the ordered sequence of DivK localization during the cell cycle. DivJ activity on DivK affects its correct localization, which, in turn, is required for PleC function. Since DivJ and PleC regulate different cell cycle events, the interconnected function of these two histidine kinases through localization of a common response regulator provides a mechanism for coordinating cell cycle progression. Study of a DivK homolog in the morphologically symmetric bacterium Sinorhizobium meliloti suggests that this type of cell cycle mechanism is widespread in prokaryotes.

An interdisciplinary study around the reliquary of the late cardinal Jacques de Vitry.

The reliquary of Jacques de Vitry, a prominent clergyman and theologian in the early 13th century, has experienced several transfers over the last centuries, which seriously question the attribution of the remains to the late Cardinal. Uncertainty about the year of his birth poses an additional question regarding his age at death in 1240. The reliquary, located in the Saint Marie d'Oigines church, Belgium, was reopened in 2015 for an interdisciplinary study around his relics as well as the Treasure of Oignies, a remarkable cultural heritage notably built from Jacques de Vitry's donation. Anthropological, isotopic and genetic analyses were performed independently on the remains found in the reliquary. Results of the analyses provided evidence that the likelihood that these remains are those of Jacques de Vitry is very high: the remains belong to the same human male individual and the historical tradition about his age is confirmed. In addition, a separate relic (left tibia) was analysed and found to match with the remains of the reliquary (right tibia). The unique Jacques de Vitry's mitre, made of parchment, was sampled non-destructively and the extracted parchment collagen was analysed by a proteomic method in order to determine the animal species. The results showed that, surprisingly, not all parts of the mitre were made from the same species. All together, these findings are expected to fertilize knowledge carried by historical tradition around the relics of Jacques de Vitry and his related cultural heritage.

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy.

Photodynamic therapy (PDT) is a minimally invasive treatment for cancer and several noncancerous proliferating cell diseases. PDT relies on the uptake of a photosensitizing compound by the pathologic tissue followed by a selective irradiation with visible light, which leads to oxidative stress-mediated cell death. However, some studies showed that PDT induces the release of proangiogenic factors, such as vascular endothelial growth factor, and/or cyclooxygenase-2 (COX-2), thereby promoting cancer cell regrowth following PDT. In this work, we focused on the molecular mechanisms regulating COX-2 expression after low-dose PDT in two cancer cell lines, namely HeLa and T24. We report that PDT induces COX-2 expression in these cells and this expression is mainly due to nuclear factor kappa B (NF-kappaB)-dependent transcription of cox-2 gene without any post-transcriptional regulation. However, the transduction mechanism leading to NF-kappaB activation and subsequent cox-2 gene transcription differs in both cell types. In T24, NF-kappaB activation occurs through a protein kinase C (PKC)alpha- and phosphoinositide-3-kinase (PI3K)-dependent I kappa B kinase (IKK) complex activation, whereas in HeLa cells, NF-kappaB activation is mediated by PKC- and PI3K-independent IKK complex activation.

NF-kappaB in photodynamic therapy: discrepancies of a master regulator.

Tumor eradication by photodynamic therapy (PDT) results from the onset of distinct killing processes. In addition to the well-known necrotic and apoptotic mechanisms, PDT initiates an inflammatory response that will indirectly contribute to tumor clearance. The NF-kappaB transcription factor is a major regulator of inflammation modulating the expression of cytokines, chemokines, and adhesion molecules in various cell types in response to a large number of stimuli. Besides, NF-kappaB regulates the expression of antiapoptotic genes, cyclooxygenases (COXs) and metalloproteinases (MMPs) as well, thereby favoring tumor cell proliferation and dissemination. In the present review, we aim to summarize the current knowledge on NF-kappaB status following photosensitization of cancer cells and endothelial cells. In order to unravel the NF-kappaB impact in PDT tumorigenicity and recurrences, we will stress the discrepancies of this major transcription factor relative to the signaling cascades underlying its activation and the cellular effects triggered by its translocation into the nucleus and its binding to its target genes.

Caulobacter crescentus intrinsic dimorphism provides a prompt bimodal response to copper stress.

Stress response to fluctuating environments often implies a time-consuming reprogramming of gene expression. In bacteria, the so-called bet hedging strategy, which promotes phenotypic stochasticity within a cell population, is the only fast stress response described so far(1). Here, we show that Caulobacter crescentus asymmetrical cell division allows an immediate bimodal response to a toxic metals-rich environment by allocating specific defence strategies to morphologically and functionally distinct siblings. In this context, a motile swarmer cell favours negative chemotaxis to flee from a copper source, whereas a sessile stalked sibling engages a ready-to-use PcoAB copper homeostasis system, providing evidence of a prompt stress response through intrinsic bacterial dimorphism.

Brucella abortus Cell Cycle and Infection Are Coordinated.

Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection.

Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers.

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting ("high dose PDT"). When one of these components is limiting ("low dose PDT"), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents, i.e. pyropheophorbide and hypericin.

Involvement of oxidative stress in NF-kappaB activation in endothelial cells treated by photodynamic therapy.

In human endothelial cells ECV 304 and HMEC-1 photosensitized by pyropheophorbide-a methylester (PPME) in sublethal conditions transcription factor Nuclear Factor kappa B (NF-kappaB) activation takes place for several hours. Activated NF-kappaB was functional because it stimulated the transcriptional activation of either a transfected reporter gene or the endogenous gene encoding interleukin (IL)-8. Concomitant with NF-kappaB activation, inhibitor of NF-kappaB alpha (IkappaB alpha) was degraded during photosensitization and IkappaB beta, p100, p105 and IkappaB epsilon were slightly modified. Reactive oxygen species (ROS) were shown to be crucial intermediates in the activation because antioxidants strongly decreased NF-kappaB activation. Using both a fluorescent probe and isotope substitution, it was shown that ROS, and especially singlet oxygen (1O2), were important in the activation process. Because NF-kappaB activation in the presence of ROS was suspected to proceed through a pathway independent of the IkappaB kinases (IKK), we demonstrated that the IKK were indeed not activated by photosensitization but required an intact tyrosine residue at position 42 on IkappaB alpha, suggesting the involvement of a tyrosine kinase in the activation process. This was further reinforced by the demonstration that herbimycin A, a tyrosine kinase inhibitor, prevented NF-kappaB activation by photosensitization but not by TNF alpha, a cytokine known to activate NF-kappaB through an IKK-dependent mechanism.

Phosphate starvation triggers production and secretion of an extracellular lipoprotein in Caulobacter crescentus.

Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS) is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conclusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment.

Induction of heme-oxygenase 1 requires the p38MAPK and PI3K pathways and suppresses apoptotic cell death following hypericin-mediated photodynamic therapy.

Photodynamic therapy (PDT) is an established anticancer modality utilizing the photogeneration of reactive oxygen species (ROS) to kill the cancer cells and hypericin is a promising photosensitizer for the treatment of bladder tumors. In this paper we characterize the signaling pathways and the mechanisms leading to the up-regulation of the antioxidant enzyme heme oxygenase (HO-1) in PDT treated cancer cells. We show that PDT engages the p38(MAPK) and PI3K signaling cascades for HO-1 induction. p38(MAPK) inhibitors or small interfering RNA (siRNA) for p38(MAPK) suppress HO-1 induction after PDT and complete repression is attained when p38 and PI3K antagonists are combined. Blocking these signaling pathways increases additively the propensity of the cells to undergo PDT-induced apoptosis, mirroring the effect of HO-1 silencing. Conversely, increasing HO-1 protein level by hemin prior to irradiation is cytoprotective. HO-1 stimulation by PDT is dependent on transcription and de novo protein synthesis and it is preceded by the nuclear accumulation of the Nrf2 transcription factor, which is reduced by inhibitors of p38(MAPK) and PI3K. Altogether these results indicate that stimulation of HO-1 expression by hypericin-PDT is a cytoprotective mechanism governed by the p38(MAPK) and PI3K pathways, likely through the control of the nuclear availability of the Nrf2 pool.

Cytokinesis monitoring during development; rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter.

For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development.