Complete genome sequence of Serratia ureilytica KML.E1, a copper-tolerant strain isolated from a disused tungsten mine.

Serratia ureilytica KML.E1 was recovered from a disused tungsten mine in Hong Kong and can tolerate copper(II) concentrations up to 90 mM. Its complete genome, a single chromosome of 5,094,661 bp (59.68% G+ C), was established through hybrid assembly.

Insights into copper sensing and tolerance in Pneumocystis species.

Introduction: Pneumocystis species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of Pneumocystis murina, a model organism that infects mice, in the context of its copper sensing and tolerance. Methods and results: The query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in P. murina, significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of P. murina exposed to various CuSO4 concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO4 concentration probed (100 µM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies - homology-based annotation, SEC-ICP-MS/IMAC/LC-MS/MS, and RNA-seq - yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods. Discussion: The insights contribute to the broader knowledge of Pneumocystis copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of Pneumocystis pathogenicity and copper stress survival should consider the entire spectrum of identified genes.

Comparison of Copper-Tolerance Genes between Different Groups of Acidovorax citrulli.

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.

Early transcriptional changes of heavy metal resistance and multiple efflux genes in Xanthomonas campestris pv. campestris under copper and heavy metal ion stress.

BACKGROUND: Copper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to copper stress in Xcc and resistance to metal induced damage is not well understood. However, homologs of heavy metal efflux genes from the related Stenotrophomonas genus are found in Xanthomonas which suggests that metal related efflux may also be present. METHODS AND RESULTS: Gene expression in Xcc strain BrA1 exposed to 0.8 mM CuSO4.5H2O for 15 minutes was captured using RNA-seq analysis. Changes in expression was noted for genes related to general stress responses and oxidoreductases, biofilm formation, protein folding chaperones, heat-shock proteins, membrane lipid profile, multiple drug and efflux (MDR) transporters, and DNA repair were documented. At this timepoint only the cohL (copper homeostasis/tolerance) gene was upregulated as well as a chromosomal czcCBA efflux operon. An additional screen up to 4 hrs using qPCR was conducted using a wider range of heavy metals. Target genes included a cop-containing heavy metal resistance island and putative metal efflux genes. Several efflux pumps, including a copper resistance associated homolog from S. maltophilia, were upregulated under toxic copper stress. However, these pumps were also upregulated in response to other toxic heavy metals. Additionally, the temporal expression of the coh and cop operons was also observed, demonstrating co-expression of tolerance responses and later activation of part of the cop operon. CONCLUSIONS: Overall, initial transcriptional responses focused on combating oxidative stress, mitigating protein damage and potentially increasing resistance to heavy metals and other biocides. A putative copper responsive efflux gene and others which might play a role in broader heavy metal resistance were also identified. Furthermore, the expression patterns of the cop operon in conjunction with other copper responsive genes allowed for a better understanding of the fate of copper ions in Xanthomonas. This work provides useful evidence for further evaluating MDR and other efflux pumps in metal-specific homeostasis and tolerance phenotypes in the Xanthomonas genus. Furthermore, non-canonical copper tolerance and resistance efflux pumps were potentially identified. These findings have implications for interpreting MIC differences among strains with homologous copLAB resistance genes, understanding survival under copper stress, and resistance in disease management.

Activation of CzcS/CzcR during zinc excess regulates copper tolerance and pyochelin biosynthesis of Pseudomonas aeruginosa.

Zinc is an important transition metal that is essential for numerous physiological processes while excessive zinc is cytotoxic. Pseudomonas aeruginosa is a ubiquitous opportunistic human pathogen equipped with an exquisite zinc homeostatic system, and the two-component system CzcS/CzcR plays a key role in zinc detoxification. Although an increasing number of studies have shown the versatility of CzcS/CzcR, its physiological functions are still not fully understood. In this study, transcriptome analysis was performed, which revealed that CzcS/CzcR is silenced in the absence of the zinc signal but modulates global gene expression when the pathogen encounters zinc excess. CzcR was demonstrated to positively regulate the copper tolerance gene ptrA and negatively regulate the pyochelin biosynthesis regulatory gene pchR through direct binding to their promoters. Remarkably, the upregulation of ptrA and downregulation of pchR were shown to rescue the impaired capacity of copper tolerance and prevent pyochelin overproduction, respectively, caused by zinc excess. This study not only advances our understanding of the regulatory spectrum of CzcS/CzcR but also provides new insights into stress adaptation mediated by two-component systems in bacteria to balance the cellular processes that are disturbed by their signals. IMPORTANCE: CzcS/CzcR is a two-component system that has been found to modulate zinc homeostasis, quorum sensing, and antibiotic resistance in Pseudomonas aeruginosa. To fully understand the physiological functions of CzcS/CzcR, we performed a comparative transcriptome analysis in this study and discovered that CzcS/CzcR controls global gene expression when it is activated during zinc excess. In particular, we demonstrated that CzcS/CzcR is critical for maintaining copper tolerance and iron homeostasis, which are disrupted during zinc excess, by inducing the expression of the copper tolerance gene ptrA and repressing the pyochelin biosynthesis genes through pchR. This study revealed the global regulatory functions of CzcS/CzcR and described a new and intricate adaptive mechanism in response to zinc excess in P. aeruginosa. The findings of this study have important implications for novel anti-infective interventions by incorporating metal-based drugs.

Actividad de cobre sobre cocáceas grampositivas multi-resistentes a los antibióticos aisladas en hospitales chilenos / Copper activity against multiresistant Gram-positive cocci isolated in Chilean hospitals

Background: Nosocomial infections caused by multiresistant Gram-positive cocci are a serious problem for public health systems worldwide. The use of copper surfaces in hospital environments has proven to be an effective alternative for the control of various microorganisms, including multiresistant nosocomial pathogens. Aim: To determine the association between antibiotic multiresistance and higher levels of copper tolerance in Gram-positive cocci isolated from Chilean hospitals, which might confer a selective advantage in environments with copper. Methods: The ionic copper tolerance levels were evaluated using the Mueller Hinton agar dilution method, in S. aureus and Enterococcus spp. strains with different levels of susceptibility to clinically relevant antibiotics. Results: A statistically significant association between higher levels of tolerance to copper ion and multi-resistance to antibiotics in Enterococcus spp. was observed.

Introducción: Las infecciones nosocomiales, producidas por bacterias cocáceas grampositivas multi-resistentes constituyen un serio problema para los sistemas de salud pública mundial. El uso de superficies de cobre en ambientes hospitalarios ha demostrado ser una alternativa efectiva para el control de diversos microorganismos, incluyendo patógenos nosocomiales multi-resistentes. Objetivo: Determinar la asociación entre la multi-resis-tencia a antimicrobianos y mayores niveles de tolerancia a cobre en bacterias cocáceas grampositivas aisladas de hospitales chilenos, que podrían conferir una ventaja selectiva en ambientes cobrizados. Material y Métodos: Se evaluó los niveles de tolerancia a cobre iónico en grupos de cepas de Staphylococcus aureus y Enterococcus spp. con distintos grados de susceptibilidad a antimicrobianos de relevancia clínica, mediante el método de dilución en agar Mueller Hinton. Resultados: Se observó una asociación estadísticamente significativa entre mayores niveles de tolerancia a cobre iónico y la multi-resistencia a antimicrobianos en Enterococcus spp.

Actividad de cobre sobre bacilos gramnegativos multi-resistentes aislados en hospitales chilenos / Copper activity against multiresistant Gram negative bacilli isolated from Chilean hospitals

Introduction: Multiresistant nosocomial pathogens, especially Gram-negative bacilli (GNB), are a serious problem for public health systems worldwide. Due to their antimicrobial properties, copper alloys have been suggested as an alternative for the control of bacterial burden in surfaces in hospital environment. However, antibiotic multiresistance and copper resistance could be associated in GNB, and there is evidence that both kind of resistance genes (antibiotic and copper) can be located on the same genetic structures. For this reason antibiotic-multiresistant strains could survive in the presence of copper, selecting for bacterial phenotypes resistant to both antibacterial agents. Aim: To evaluate antibacterial activity of copper against nosocomial extended-spectrum β-lactamases (ESBL) (+) and ESBL (-) GNB, and carbapenems resistant or susceptible strains. Material and Method: This study included 390 strains of GNB isolated from Chilean hospitals: Acinetobacter baumannii and Pseudomonas aeruginosa resistant (CAR R) and susceptible (CAR S) to carbapenem antibiotics, and Klebsiella pneumoniae and Escherichia coli producers and non-producers of ESBL. Susceptibility levels to cupric sulphate were determined by agar dilution method and statistical analysis were used to determine the significance of the differences in the copper tolerance levels between the strains groups. Results: Statistically superior copper tolerance levels were found in the CAR R and ESBL producing strains of A. baumannii and K. pneumoniae, in relation with the CAR S and ESBL not-producing strains. Conclusion: A relation between a diminished susceptibility to ionic copper and to recent generation antimicrobial agents was observed in K. pneumoniae y A. baumannii strains.

Introducción: Los patógenos intrahospitalarios multi-resistentes constituyen un grave problema mundial de salud pública, especialmente los bacilos gramnegativos (BGN). El uso de cobre como antimicrobiano de superficie en hospitales se postula como una alternativa para el control de microorganismos en estos ambientes. Sin embargo, la multi-resistencia a antimicrobianos en BGN hospitalarios puede asociarse con la tolerancia a cobre, ya que existe evidencia que genes que codifican tolerancia a este metal pueden encontrarse en elementos genéticos que confieren resistencia a antimicrobianos. Por esta razón, cepas multi-resistentes a antimicrobianos podrían sobrevivir en presencia de cobre, seleccionando bacterias resistentes a ambos agentes antibacterianos. Objetivo: Investigar la actividad de cobre sobre BGN hospitalarios productores y no productores de β-lactamasas de espectro extendido (BLEE), y resistentes o susceptibles a antimicrobianos carbapenémicos. Material y Métodos: Se estudió 390 cepas de BGN aisladas en hospitales chilenos: Acinetobacter baumannii y Pseudomonas aeruginosa resistentes (CAR R) y susceptibles (CAR S) a carbapenémicos y Klebsiella pneumoniae y Escherichia coli productoras y no productoras de BLEE. Se investigó los niveles de susceptibilidad a sulfato cúprico, mediante dilución seriada en agar y se evaluó la significancia estadística de la diferencia de estos niveles entre los distintos grupos de cepas. Resultados: Se encontraron niveles de tolerancia a cobre superiores en cepas de A. baumannii y K. pneumoniae, CAR R y productoras de BLEE respectivamente, con respecto a sus pares CAR S y no productoras de BLEE. Conclusión: Observamos una relación entre la disminución de la susceptibilidad a cobre iónico y a antimicrobianos de última generación en K. pneumoniae y A. baumannii.