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.

Genetic Responses of Metabolically Active Limnospira indica Strain PCC 8005 Exposed to γ-Radiation during Its Lifecycle.

Two morphotypes of the cyanobacterial Limnospira indica (formerly Arthrospira sp.) strain PCC 8005, denoted as P2 (straight trichomes) and P6 (helical trichomes), were subjected to chronic gamma radiation from spent nuclear fuel (SNF) rods at a dose rate of ca. 80 Gy·h-1 for one mass doubling period (approximately 3 days) under continuous light with photoautotrophic metabolism fully active. Samples were taken for post-irradiation growth recovery and RNA-Seq transcriptional analysis at time intervals of 15, 40, and 71.5 h corresponding to cumulative doses of ca. 1450, 3200, and 5700 Gy, respectively. Both morphotypes, which were previously reported by us to display different antioxidant capacities and differ at the genomic level in 168 SNPs, 48 indels and 4 large insertions, recovered equally well from 1450 and 3200 Gy. However, while the P2 straight type recovered from 5700 Gy by regaining normal growth within 6 days, the P6 helical type took about 13 days to recover from this dose, indicating differences in their radiation tolerance and response. To investigate these differences, P2 and P6 cells exposed to the intermediate dose of gamma radiation (3200 Gy) were analyzed for differential gene expression by RNA-Seq analysis. Prior to batch normalization, a total of 1553 genes (887 and 666 of P2 and P6, respectively, with 352 genes in common) were selected based on a two-fold change in expression and a false discovery rate FDR smaller or equal to 0.05. About 85% of these 1553 genes encoded products of yet unknown function. Of the 229 remaining genes, 171 had a defined function while 58 genes were transcribed into non-coding RNA including 21 tRNAs (all downregulated). Batch normalization resulted in 660 differentially expressed genes with 98 having a function and 32 encoding RNA. From PCC 8005-P2 and PCC 8005-P6 expression patterns, it emerges that although the cellular routes used by the two substrains to cope with ionizing radiation do overlap to a large extent, both strains displayed a distinct preference of priorities.

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.

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.

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.

Genomic and Transcriptomic Changes that Mediate Increased Platinum Resistance in Cupriavidus metallidurans.

The extensive anthropogenic use of platinum, a rare element found in low natural abundance in the Earth's continental crust and one of the critical raw materials in the EU innovation partnership framework, has resulted in increased concentrations in surface environments. To minimize its spread and increase its recovery from the environment, biological recovery via different microbial systems is explored. In contrast, studies focusing on the effects of prolonged exposure to Pt are limited. In this study, we used the metal-resistant Cupriavidus metallidurans NA4 strain to explore the adaptation of environmental bacteria to platinum exposure. We used a combined Nanopore⁻Illumina sequencing approach to fully resolve all six replicons of the C. metallidurans NA4 genome, and compared them with the C. metallidurans CH34 genome, revealing an important role in metal resistance for its chromid rather than its megaplasmids. In addition, we identified the genomic and transcriptomic changes in a laboratory-evolved strain, displaying resistance to 160 µM Pt4+. The latter carried 20 mutations, including a large 69.9 kb deletion in its plasmid pNA4_D (89.6 kb in size), and 226 differentially-expressed genes compared to its parental strain. Many membrane-related processes were affected, including up-regulation of cytochrome c and a lytic transglycosylase, down-regulation of flagellar and pili-related genes, and loss of the pNA4_D conjugative machinery, pointing towards a significant role in the adaptation to platinum.