Cadmium resistance and its possible mechanism(s) have been reported in bacteria from a wide range of taxonomies and habitats (Khan et al. 2016; Qin et al. 2019; Wang et al. 2019). The majority of organisms that fit this profile can withstand concentrations of cadmium between 1 and 7 mM (Izrael-Živković et al. 2018), possibly through adsorption or efflux mechanisms. Twenty-six cadmium-resistant bacteria were isolated from Malda, West Bengal, India. Out of these, 10 exhibited significant resistance to cadmium (15 mM CdCl2. H2O) when grown in a traditional bacteriological medium (LA). However, the bioavailability of heavy metals in solid media can pose a challenge, leading to a potential overestimation of Cd2+resistance when measured on bacteriological media gelled with agar (Agarwal et al. 2020). This has induced the investigators of this study to raise doubts about the methodology and inferences drawn from the experiments that have been reported earlier in terms of determining actual MIC of cadmium salts. Hence, the foremost challenge was to determine the actual maximum tolerated concentration of cadmium salts for "cadmium-resistant" isolates. The initial strategy adopted in this study was to shift from a solid medium to a liquid medium for determining the MIC of Cd2+. Eventually, we encountered a remarkable problem while conducting experiments in conventional rich growth media like Luria-broth (LB), Nutrient broth (NB), Plate count broth (PCB), and Tryptone Soya broth (TSB). In each of this media, adding cadmium salt turned the medium opaque due to formation of insoluble fine suspension with time, leading to deceptive interpretations. Therefore, in order to address the issue of media opaqueness, we searched for alternative media where precipitation and cross-reaction of media components in presence of cadmium salt could be avoided (Fig. S3). Several bacteriological media were tried to find a most suitable medium that would support the maximum bio-availability of cadmium and enable bacteria to experience the highest uptake of dissolved cadmium. While using standard mineral salts medium (MSM) for MIC determination, free phosphate in MSM reacted with cadmium and formed a precipitate in the medium. It was also revealedthat the presence of organic compounds in the media leads to the chelation of heavy metals. Additionally, the degree of chelation or precipitation is directly proportional to the amount of organic compound in the medium (Angle and Chaney 1989). After careful adjustments, we modified the organic ingredient (yeast extract) content in the formulated or modified medium to the ideal concentration of 0.1 g/L. This ensured that the cadmium chloride monohydrate remained dissolved completely and was thus readily available for bacterial growth and MIC determination, even at concentrations as high as 100 mM. When the newly modified medium was used, out of the 10 isolates initially screened to have been cadmium resistant, five showed resistance to cadmium up to 9 mM.
The selected cadmium-resistant strains have also shown resistance to Ni2+, Cr6+, Zn2+, Co2+, As (V), and As (III). Isolate CD3 could tolerate a comparatively higher concentration for arsenic (in terms of MIC) than cadmium. This is because arsenic is a common metalloid in the environment where bacteria have evolved much stronger mechanism to evade their toxic effects. The global average arsenic content in soil is 5 mg/ Kg, in open sea water 1–2 µg/l (0.001–0.002 ppm), and in unpolluted surface and ground water is below 10 µg/l (< 0.01 ppm) (Raju 2022). In contrast, Cd2+ concentration in unpolluted water is usually below 1 µg/l (< 0.001 ppm) (Friberg et al. 1986). Again, cadmium is extremely toxic and has not been associated with any known biological functions in living organisms, except for diatoms in rare cases (Templeton 2023). Understanding the binding of metal ions to the biological system relies on factors such as the electronegativity and ionic radius of the metal ions(Naja et al. 2010). The size of an ion plays a significant role in its adsorption strength, while high electronegativity helps the ion bind more securely to surface functional groups (Sulaymon et al. 2011). Based on the findings of the multi-metal/metalloid resistance study, it can be inferred that isolate CD3 demonstrates a high level of tolerance to every tested metal/metalloid.
Exploration of microbial resistance to heavy metals, in particular cadmium, zinc, and cobalt, is paramount to deciphering the role of inoculum density on microbial tolerance levels. We hypothesized that net availability of metal salt molecules in a dissolved state, through active or passive diffusion, per bacterial cell would determine the tolerance limit vis-a-vis the resistance phenotype of a given bacterium towards a particular heavy metal. In other words, when cell density in a metal-containing medium is high and the net available number of metal salt molecules per cell is less, the MIC value will be high and vice-versa. We tested this hypothesis in the present study by exposing cadmium-resistant isolate CD3 at varying cell densities to a varying concentration of CdCl2. H2O or CoCl2. 6H2O or ZnSO4. 7H2O. We have presented the CD3’s results in comparison to the control strain, E. coli K12 MTCC 1302 to validate our hypothesis.
It was revealed that there is a significant effect of inoculum density on the MIC values of Cd, Zn, and Co. The resistance levels of the CD3 or the ability of CD3 to grow in presence of increasing metal salt concentrations, expressed in MIC values, increased with increasing initial cell numbers (= inoculum density) in a given test medium. Consequently, the intracellular metal ion concentration per cell reduces at high initial cell density, which is accompanied by reduced metal ion toxicity in an individual cell. As a result, the overall resistance of the population increases as they divide. At a lower density of initial cell input in the growth medium and a high import of cadmium ions into the cell, the influence of efflux pumps and other mechanisms aimed at metal sequestration fails to bring down the intracellular cadmium concentration to a level that can prevent Cd2+ ions from out-competing other divalent cations to bind to their respective enzymes. In low cell densities, the influx of the number of Cd2+, or Zn2+ and Co2+ ions per cell increases in liquid medium containing salts of the heavy metal following the principle of diffusion and randomness of interaction between cells and dissolved or bio-available metal ions. Hence, the intracellular metal ion concentration may overwhelm the cell’s detoxification capacity, in contrast to the condition when intracellular concentration per cell goes down under an identical concentration of heavy metal in the medium when the initial input cells are log-fold higher. In fact, we have demonstrated that MIC values of heavy metal(s) are indirectly proportional to the inoculum density, establishing enhanced collective defence in higher numbers of cells. Furthermore, this phenomenon is not limited to the CD3 strain but has been unequivocally demonstrated by E. coli K12 MTCC 1302, indicating that the biological principles are universal. The results, therefore, have novel implications for the traditional microbiological methods for determining MIC of heavy metals shown by any experimental bacterium. Future research directions, therefore, must involve system biology tools in understanding the molecular mechanisms that drive density-dependent resistance. Additionally, it would be valuable to investigate how these findings can be applied to bioremediation strategies and the enhancement of microbial strains for heavy metal detoxification.
In order to explore more about the physiological basis of cadmium resistance, it was found that pre-exposing CD3 cells to low concentrations of cadmium ions can induce resistance to higher concentrations of cadmium. Induced cells (cells pre-grown in the presence of low concentrations of cadmium salt) enter the logarithmic phase of growth earlier than the un-induced cells (cells grown in absence of cadmium salt). The CD3 cells pre-grown separately at increasing concentrations of CdCl2. H2O from 0.03125 to 0.5 mM has shown a gradual reduction of the lag phase when grown in the presence of 1.5 mM CdCl2. H2O. The cellular response in making the defence system ready for cadmium assaults is modulated differentially when grown in lower concentrations of Cd2+, indicating that lower cadmium concentrations can activate efflux mechanisms more effectively in lesser preparatory time (Chen et al. 2022), especially when shifted to a fresh medium with abruptly high cadmium (1.5 mM).This study has also described the effect of zinc or cobalt concentrations in the pre-culture (i.e., zinc or cobalt-induced cells) on cadmium resistance in CD3. It was shown that the cells' resistance to cadmium was influenced by pre-growing them in the presence of ZnSO4.7H2O (0.25 mM). Nevertheless, cells previously cultured in 0.25 mM zinc or 0.5 mM cadmium have demonstrated a somewhat comparable impact on bacterial growth in relation to the stimulation of cadmium resistance. Therefore, cells induced by 0.25 mM zinc have a greater impact on cadmium resistance in terms of reduction in lag-phase duration when compared to cells induced by an equivalent concentration of cadmium. The Cd2+ transporting P-type ATPase, also known as the CadA transporter, facilitates the movement of Zn2+ ions from the cytoplasm to the periplasm. The periplasmic adapter domain of CzcS forms a strong bond with Zn2+ ions, exhibiting a high affinity. This interaction leads to the activation of the adaptor domain, which in turn activates CzcR. Subsequently, CzcR stimulates the transcription of the czcCBA operon, facilitating the expulsion of Cd2+ ions from the cytoplasm and periplasm of CD3 cells (Liu et al. 2021). However, the impact of cobalt-induced cells mirrored that of cadmium-induced cells (Fig. S1).The findings of the AAS analyses indicate that efflux pumps are pivotal in the cadmium resistance of CD3. The significant extracellular accumulation of Cd2+(average 85.33 ppm) observed after exposure to 1 mM CdCl2.H2O suggests that CD3 efficiently transports cadmium out of the cell into the surrounding environment. This process is crucial for maintaining a lower intracellular cadmium concentration (average 13 ppm) despite external exposure, highlighting the effectiveness of efflux mechanisms in resisting cadmium toxicity. Despite this fact, it is also important to note that this intracellular accumulation suggests that CD3 is capable of taking up cadmium from its environment. The CD3 genome contains several zinc transporters, metallothionein, and some thiol-rich proteins that help in cadmium sequestration (Table S8).
The relatively small but detectable fraction of Cd2+ (average 9 ppm) bound to the cell surface underscores the role of efflux pumps in preventing excessive accumulation within the cell. This binding likely represents an equilibrium between cadmium uptake and efflux, with efflux pumps continuously exporting cadmium ions to maintain cellular homeostasis. The mechanisms by which cell surface sorption occurs are not influenced by cell metabolism. Instead, they rely on the physicochemical interactions between heavy metal ions and the functional groups present on the cell walls of microorganisms. Biomass possesses the characteristic of working as a chemical substance and a biological ion exchanger. The effect was revealed to be caused by the specific cell wall structure of some bacteria (Hassan et al. 2010). Furthermore, the cell wall of the microbe mostly comprises polysaccharides, lipids, and proteins, which offer several opportunities for metal binding. These compounds have many functional groups, such as carboxylate, hydroxide, amine, imidazole, sulfate, and sulfhydryl, with different charge distributions and geometries. Functional groups have the ability to selectively attach to specific metal ions. In this section, the process of binding is ascribed to many mechanisms such as ion exchange, adsorption, complexing, microprecipitation, and crystallization, which take place on the cell wall (Veglio’ and Beolchini 1997; Davis et al. 2003; Malik 2004; Sheng et al. 2004). The elements of the cell wall are pivotal in the sequestration of metals due to the intricate nature of the biomaterials used.
Dead cells often exhibit a lower isoelectric point compared to living cells(Çolak et al. 2011; Huang et al. 2013). This difference in isoelectric point may be a key factor contributing to the increased biosorption of Cd2+ in heat-killed CD3 cells. Electrostatic contact is a crucial factor in the biosorption process(Huang et al. 2013). Additionally, it was shown that deceased bacterial cells had a higher level of negative charge on their cell surface and demonstrated a larger capacity for biosorption compared to living cells. This indicates that deceased cells possess a higher affinity for Cd2+ binding compared to live cells.
Lowering the pH of the growth medium from 7 to 6 resulted in a shorter lag phase for isolate CD3. Previously, a decrease in the uptake of cadmium, cobalt, copper, manganese, and nickel by encapsulated Klebsiella pneumoniae in acidic pH was reported (Rudd et al. 1983). It remains uncertain whether a decrease in pH solely decreases cadmium accumulation and/ or enhances cadmium efflux from within the cell. Specific metal efflux pumps are powered by the proton motive force, while others rely on adenosine triphosphate (ATP) for their function (Nies 1999). Decreasing the pH raises the concentration gradient of protons across the bacterial cell wall, enhancing the proton motive force and enabling faster ATP synthesis. In addition, earlier it was observed that exposure of E. coli K12 to cadmium in an acidic pH environment for 5 minutes resulted in the activation of many stress response genes (Worden et al.2008). Another explanation was proposed, stating that the higher concentration of hydrogen ions at low pH levels intensifies the rivalry between hydrogen and metal ions for attachment sites on the cell surface. This ultimately results in decreased toxicity of cadmium(Franklin et al. 2000) .It remains uncertain how the increase in pH enhances metal toxicity, but it could be related to metal speciation into a more harmful form(Babich and Stotzky 1985) and/or an elevation in metal adsorption and uptake, specifically by microorganisms. As an illustration, the theory suggests that the transformation of cadmium into a single-charged, hydroxylated form is responsible for the enhanced toxicity of cadmium to fungi, bacteria, and actinomycetes at alkaline pH. Other cadmium species, apart from Cd2+, could potentially enhance cadmium toxicity at pH ≥ 7. As the pH increases, concentrations of cadmium species like CdOH+ also increase. Studies have shown that certain ions, such as CdOH+, can be more hazardous compared to the more commonly found Cd2+ ions (Babich and Stotzky 1985; Collins and Stotzky 1992; Ivanov et al. 1997). It is believed that the alteration in charge leads to the destabilization of the bacterial cell membrane, resulting from CdOH+ toxicity.
Microbial biofilm is widely recognized as a crucial factor in bacterial heavy metal resistance(Patel et al. 2016; Yang et al. 2018). Microorganisms generate extracellular polysaccharides (EPSs), a fundamental element of the biofilm that provides protection against heavy metal stress (Nocelli et al. 2016). Enhancing EPS production can increase heavy metal resistance in certain strains. Studies have shown that the environment's heavy metal content can impact the development of biofilms in specific types of bacteria, as evidenced by earlier research (Oknin et al. 2015; Hao et al. 2016; Nocelli et al. 2016; Alviz-Gazitua et al. 2019). However, the effect of heavy metals on biofilm formation can differ based on the unique interaction between a particular heavy metal and bacterial species. For example, divalent cations such as Mg2+ and Ca2+ can significantly impact the development of biofilms. They have the ability to directly alter electrostatic interactions and indirectly influence attachment processes based on physiology. They play crucial roles as cellular cations and are necessary for enzyme function, as mentioned by various researchers (Fletcher 1988; Malik and Kakii 2003; Song and Leff 2006). Microorganisms in biofilms can shield themselves from the harmful effects of heavy metals(Nocelli et al. 2016). Additionally, biofilms can even absorb certain heavy metals (Azizi et al. 2016). Despite these findings, the impact of heavy metal ions on biofilm formation still needs to be fully understood. According to a study, it was found that cadmium, a frequently found soil-contaminant, has the ability to directly hinder the growth of bacteria, leading to a reduction in biofilm formation (Rau et al. 2009). Our study revealed that at lower concentrations of CdCl2. H2O (up to 0.75 mM), there was an observed increase in biofilm formation. However, as the concentration of CdCl2. H2O exceeded 0.75 mM, a decrease in biofilm formation was observed (Fig. 3b). Similar kind of results were observed when studying the impact of Cd2+ on the biofilm formation of the Bacillus subtilis strain 1JN2(Yang et al. 2018). When bacterial cells are damaged or injured, their functions can be disrupted or inhibited, causing the contents inside the cells to leak or be released. This disruption can impede the bacteria's capacity to generate additional polymeric substances (Bouhdid et al. 2010). The presence of heavy metals can have a negative impact on bacterial biofilms. This is because they can disrupt the water channels that are essential for the transportation of nutrients within the biofilm (Syed et al. 2021). At higher concentrations of Cd2+, a reduction in the expression of genes associated with biofilm formation was observed (Yang et al. 2022).
Studying the whole genome sequence (WGS) is an incredibly valuable approach to assessing genetic potential of a bacterium to combat metal assault and corroborate with the phenotypic and physiological data. WGS analysis pinpoints specific genes that may play different role in developing resistance to toxic metals, metalloids, and antibiotics and helps understand adapting to different environments (Adetunji et al. 2022). Comparative WGS analyses enable valuable insights into the evolutionary dynamics of bacteria, enhancing our understanding of microbial interactions in diverse ecosystems. It also enables the identification of new resistance mechanisms against antibiotics and, as emphasized in recent studies, heavy metals, which present significant challenges in environmental and clinical settings (Garza-Ramos et al. 2023).
Through a comprehensive examination of the CD3 genome, it was determined that it belongs to a Pseudomonas species known for its remarkable resistance and ability to produce biofilms. Additionally, the analysis revealed the presence of a unique enzymatic system that enables the strain to efficiently remove cadmium ions, further enhancing its resistance to this particular metal. Minor variations were observed in the genes associated with cadmium efflux when compared to the type strain DSM50071. The whole-genome tree generated by the Type Strain Genome Server (TYGS) offers a state-of-the-art method for comprehending the relationships and evolution of microorganisms. With the help of whole-genome sequencing data, TYGS can generate phylogenetic trees that depict the genetic relationships between different microbial strains, including type strains. This methodology provides a precise and thorough approach to categorising and recognising microorganisms, surpassing conventional methods that depend on restricted genetic markers or phenotypic characteristics. The utilization of the whole-genome tree approach allows researchers to track the evolutionary lineage of microorganisms, discover new species, and gain insights into the genomic aspects of microbial diversity. Using whole-genome (WG)- based phylogenetic studies, strain CD3 was found to be the closest relative to P. aeruginosa DSM50071T among the type strains of Pseudomonas spp., as determined by the TYGS method (Fig. 4).
The SNP tree is an essential tool for studying intraspecies genetic variations. It provides a detailed view of the subtle genetic differences within a species, allowing for greater comprehension of these variations. By examining SNPs, which are genetic variations at individual nucleotide positions in the genome, scientists can create phylogenetic trees that unveil the genetic connections and evolutionary past of diverse populations within a given species. This approach offers valuable insights into the genetic diversity, population structure, and evolutionary dynamics of species, showcasing the role of genetic variations in shaping phenotypic diversity, adapting to environmental changes, and combating diseases. In microbial genomics, SNP trees play an integral part in monitoring the transmission of microbial pathogens, gaining insights into the spread of antibiotic resistance, and pinpointing the genetic factors behind virulence. SNP trees are pivotal in revealing the genetic foundation of variations within a species, which has far-reaching implications for disciplines such as evolutionary biology, epidemiology, and conservation (Zhang and Liu 2023; Bu et al. 2023). Through SNP-based phylogenetic analysis, strain CD3 was found to be genetically closely related to P. aeruginosa strains LESB58 and LES431. On the other hand, P. aeruginosa PA01 was found to be genetically more distant, residing on a distinct branch in the evolutionary tree. Although there is significant sequence homology with PA01, the analysis reveals that strain CD3 has a distinct genetic composition compared to PA01. This revelation emphasizes the distinct genetic identity of CD3 within the P. aeruginosa lineage, showcasing its unique evolutionary path (Fig. 5).
The czcCBA sequence-based phylogenetic analysis and gene comparison between strain CD3 and other cadmium-resistant bacteria enabled us to contextualize these findings regarding evolution of bacterial metal-resistance mechanisms. The phylogenomic investigation using the concatenated nucleotide and translated amino acid sequences of the czcC, czcB, czcA genes has revealed valuable insights into the evolutionary relationships and genetic preservation of these resistance mechanisms within the Pseudomonadaceae family (Fig. 6a and 6b). The remarkable similarity between strain CD3 and P. aeruginosa PA01 in the CzcCBA pump sequence indicates a significant evolutionary conservation of the cadmium resistance mechanism in certain strains of P. aeruginosa. This finding highlights the vital role of the CzcCBA efflux pump in conferring bacterial resistance to cadmium, an essential adaptive benefit in habitats polluted with heavy metals.
The convergence of all members of the Pseudomonadaceae family, including strain CD3, into a single cluster, highlights their collective evolutionary background and potential occupation of similar ecological niches. The unique placement of Cupriavidus necator strain N1, the pioneer strain in possessing the CzcCBA pump, emphasizes the evolutionary divergence and possible horizontal gene transfer incidences that could have permitted the dissemination of cadmium resistance genes among several bacterial families (Fig. 6a and 6b).
The complete matching of the intragenic nucleotide region between czcR and czcC in strain CD3 and strain PA01 indicates a strongly conserved regulatory mechanism for the activation of the CzcCBA efflux pump in P. aeruginosa. The conservation of this region indicates its crucial function in controlling resistance to cadmium, possibly through shared transcriptional regulatory mechanisms in various strains. Regulatory elements play an essential part in enabling bacteria to adjust and endure in environments polluted with heavy metals. In addition, a deep understanding of the genetic and regulatory mechanisms that drive heavy metal resistance can provide valuable insights for devising effective strategies to address the proliferation of resistance genes (Demircan and Memon 2022; Fardami et al. 2023; Garg et al. 2024). This is especially crucial in environments where antibiotic and heavy metal resistance work together to create multidrug-resistant pathogens.
Investigating the genetic diversity and mutation landscape of cadmium resistance genes in P. aeruginosa strain CD3, alongside strains PA01, MR41, and San_ai, highlights the intricate processes in which bacteria adapt and evolve in the face of environmental challenges like exposure to heavy metals. Through careful analysis of nucleotide sequences for important cadmium resistance-related genes, the mutation spectrum within these loci has been revealed. This sheds light on commonalities as well as uniqueness in genetic responses to cadmium-mediated stress among various strains of P. aeruginosa. The abundance of various mutations, encompassing both transverse and transition types, within the cadmium resistance genes, along with a distinct pattern of dominant and silent mutations, suggests an array of genetic variation (Fig. S2). This restraineddiversity is most likely a result of the evolutionary stresses caused by exposure to cadmium, which requires genetic versatility for survival. The prevailing mutations, which lead to alterations in amino acids, may play a crucial role in modifying the function or effectiveness of the related mechanisms of resistance, potentially increasing the bacterium's ability to detoxify or sequester cadmium ions (Yu et al. 2022; Zhao et al. 2023). The rate at which a population approaches its fitness optimum can be significantly impacted by the presence of even a few substantial effect mutations. However, the finding that the dN/dS ratio is less than 1 for all the genes examined implies a situation of purifying selection rather than positive selection. This contradicts the initial assumption of adaptive evolution caused by cadmium exposure (Wolf et al. 2009; Roy et al. 2020; Zwonitzer et al. 2023). Based on the synonymous mutations, it appears that most of the mutations in the cadmium resistance genesdid not affect the amino acid sequences of the proteins they encode. Nevertheless, there are some non-synonymous mutations that may potentially enhance protein function, which is yet to be explored. It seems that the core functions of the proteins coded by these genes remained unchanged in the face of sustained selective pressure. This conservation may be attributed to their crucial role in maintaining cellular homeostasis when exposed to toxic cadmium concentrations. It could indicate a delicate equilibrium between the need to adjust to the environment and the importance of maintaining essential cellular processes, ensuring the bacterium's survival while maintaining its overall fitness (Sendolo et al. 2022). In simple words, studying the evolution of the CD3 genome in relation to cadmium resistance reveals the intricate relationship between genetic diversity, types of mutations, and evolutionary forces. It highlights the intricate strategies utilised by P. aeruginosa to overcome the obstacles presented by toxic metal exposure, underscoring the significance of preserving genetic integrity to maintain vital biological processes in unfavourable circumstances.
In this study, we used the STRING database (v12.0) to uncover an intricate web of protein-protein interactions within P. aeruginosa PAO1. This provided us with valuable knowledge regarding bacterial resistance mechanisms and the formation of biofilms. Through the integration of various sources of evidence, such as genomic context, experimental data, and database mining, we have successfully identified four distinct clusters of proteins with different levels of interaction confidence (Fig. 8). These clusters showcase the multifaceted features of proteins associated with bacterial cadmium efflux, signal transduction, and biofilm formation and maturation.
The initial cluster, primarily linked to the formation and development of biofilms, highlights the keyimportance of BfmR and its associated proteins (BfmS, PA4103-PA4107) in maintaining the structural integrity and resilience of biofilms. Biofilms play a vital role in the survival and virulence of bacteria, as they help bacteria resist antibiotics and evade the host's immune responses (Al-Tayawi et al. 2023). Our research supports previous studies that highlight the importance of BfmR in regulating biofilm development in P. aeruginosa(Harmsen et al. 2010). Identification of this cluster not only confirms the important role of BfmR but also indicates potential protein interactions that could be focused on disrupting biofilm formation.
The second cluster emphasises the importance of two-component signal transduction systems, which play a decisive role in bacterial response to environmental stimuli, especially metal ions. Proteins like CopR, CopS, PA2523 (CzcR), and PA2524 (CzcS) are key players in this process. These proteins play a crucial role in regulating gene expression in response to metal ion concentrations, which is vital for the survival of bacteria in challenging environments. Our findings align with previous research that has shown the role of CzcR and CzcS in the regulation of the czc operon, facilitating the removal of heavy metals and enhancing resistance to metal toxicity (Liu et al. 2021).
The third cluster, which includes the CzcCBA pump components (CzcC, CzcB, CzcA), plays a crucial role in bacterial resistance to cadmium toxicity by directly facilitating cadmium efflux. Thenetwork analysis presented (Fig. 8) sheds light on the interaction between the CzcCBA pump and other protein clusters, indicating a coordinated response to cadmium stress and providing additional contexts in this extensively characterized resistance mechanism.
Lastly, the fourth cluster, which includes CadR and CadA, plays a crucial role in driving out cadmium to the periplasmic space, providing an additional defense mechanism against cadmium toxicity. This finding adds to our understanding of the processes behind cadmium resistance in P. aeruginosa and supports the possibility of a cooperative relationship between the CzcCBA pump and CadA-CadR mediated efflux systems.
The results of this study confirm previously reported interactions and reveals potential new connections between important proteins involved in biofilm formation, signal transduction, and heavy metal efflux. These interactions provide a more profound insight into the intricate regulatory networks that govern bacterial survival strategies. Future research should focus on conducting experiments to validate the predicted interactions and further investigate their impact on bacterial physiology and pathogenicity. In addition, focusing on particular interactions within these clusters could offer innovative methods for managing bacterial resistance and biofilm-mediated infections.
The ClueGO and CluePedia plugins were used in Cytoscape to analyze the complex network of gene interactions and functional enrichments linked to the regulatory proteins BfmR, BfmS, CzcR, and CzcS in P. aeruginosa. The analysis reveals an intricate network of regulations, highlighting the crucial influence of BfmR on the functions of CzcR and CzcS, which are essential for biofilm formation and receptor signalling pathways (Modrzejewska et al. 2021; Kim et al. 2022). It is evident that BfmR plays a crucial role in the positive regulation of single-species biofilm formation, closely interacting with CzcR.Understanding biofilm formation is essential in studying the virulence of P. aeruginosa, as it allows the bacterium to establish prolonged infections and evade antimicrobial therapies (Kristensen et al. 2022). The connection between BfmR and CzcR highlights an overlooked aspect of regulatory control, which could provide fresh perspectives on how biofilm regulatory networks incorporate environmental signals. In addition, the connection between BfmR and receptor signalling activities, which are additionally observed in BfmS, implies that BfmR may have a wider role in regulating signal transduction mechanisms that control biofilm formation and bacterial response to environmental stress. The close connection between BfmR and BfmS in receptor signalling and molecular signal transduction activities emphasises a coordinated regulatory mechanism that could finely adjust the bacterium's adaptive responses. The coordination is further demonstrated by the shared roles of BfmS and CzcS in phosphorelay sensor kinase activity, a crucial process in bacterial signal transduction pathways. Based on the results, it appears that BfmR has an impact on the activity of CzcS, possibly through its regulatory interactions with BfmS. This could potentially affect the bacterium's ability to detect and respond to heavy metal stress, considering the known involvement of CzcS in the czcoperon. Therefore, the observation of associations confirms a hierarchical regulatory connection; BfmR serves to control CzcR and CzcS activities. This hierarchy indicates a potentially vital role for BfmR in coordinating environmental cues and biofilm formation as well as heavy-metal resistance. BfmR not only positively regulates biofilm formation but also uses CzcR and CzcS receptor signalling pathways as a regulator, which means that an intricate regulatory network is at play, positioning BfmR as a central hub in coordinating the bacterium’s response to various environmental stimuli.
Summarizing all the interpretations of results obtained from this study we put forward a hypothesized model of the molecular mechanism of cadmium resistance in strain CD3 (Fig. 10).