Investigation of cobalt resistance in Rhodobacter sphaeroides at molecular level

Abstract

Rhodobacter sphaeroides, a Gram-negative α-proteobacterium, is able to perform photosynthesis and possesses a diverse array of metabolic capabilities. These capabilities include lithotrophy, respiration in both oxygen-rich and oxygen-poor environments, nitrogen-fixation, as well as the production of tetrapyrroles, chlorophylls, vitamin B12 and heme. R. sphaeroides possesses the ability to modify its metabolism, its metabolism can adapt to changes in environmental conditions and nutrient availability. This metabolic flexibility, combined with its fully sequenced genome and its ability to thrive under normal growth conditions, makes it a desirable choice for various biotechnological applications. It serves as a valuable model organism for the development and investigation of different protein expression systems. Particularly, it excels in the study of membrane protein complexes that convert light energy into electrical energy. This bacterium offers various advantages, including a fully sequenced genome, having an adaptive and well-known metabolism, and an enhanced membrane surface area when cultivated in oxygen-depleted environments. In a previous study, using evolutionary engineering, a R. sphaeroides mutant population resistant to cobalt chloride stress was obtained. This was achieved by subjecting the initial population to batch selection under gradually increased cobalt chloride stress conditions, without applying random mutagenesis prior to the selection process. Remarkably, the final mutant population exhibited resistance to cobalt levels as high as 15 mM, which has not been previously observed in R. sphaeroides. Seven mutant individuals selected from the final population were investigated, and mutant individuals were physiologically characterized. After characterization, the most resistant individual mutant (G7) with superior resistance properties was selected for more detailed analysis. Additional analysis of the G7 individual mutant from the final population revealed its ability to exhibit cross-resistance against various compounds like nickel (ІІ) chloride (2.2 mM, 2.4 mM), ethanol (8% v/v), sodium chloride (0.5 M), and aluminum chloride (5 mM), magnesium chloride (750 mM, 1M), iron (ІІ) chloride (5 mM), boric acid (30 mM, 50 mM), caffeine (20 mM) and ammonium iron (II) sulfate (5 mM). However, the underlying genomic causes of this physiological resistance capability remained unknown. The main aim of this study was to detect and analyze specific variations in the genetic makeup of the cobalt-resistant R. sphaeroides strain, known as single nucleotide polymorphisms (SNPs), which have the potential to significantly influence their resistance to cobalt chloride stress. By comprehensively exploring the interplay between these SNPs and their potential role in cobalt chloride stress resistance, this study aimed to shed light on the underlying mechanisms and pathways involved, ultimately contributing to a deeper understanding of bacterial adaptation and the development of effective strategies to combat cobalt chloride stress resistance. In this study, Flame Atomic Absorption Spectrometry (FAAS) method was used as a first step to gain insight into the cobalt resistance mechanism of G7 to determine if the mutant individual G7 retains cobalt ions inside the cell or not. As a result of the FAAS analyses, it was found that G7, which can survive in the presence of cobalt stress conditions, takes cobalt ions into/onto the cell. Moreover, to gain a detailed understanding of the underlying mechanisms behind cobalt tolerance, comparative Whole Genome Re-sequencing analysis was performed with G7 to identify and determine single nucleotide polymorphisms (SNPs) in this strain. Specifically, the G7 mutant individual and the Reference Strain (RS) were sequenced which allowed the identification of specific SNPs that potentially play a crucial role in the ability of G7 to resist cobalt chloride. By delving into the genetic variations and their potential implications, this approach aims to unravel the intricate mechanisms that contribute to cobalt resistance, thus paving the way for targeted interventions and strategies to combat this stress. According to whole genome re sequencing results, 11 missense mutations were found in various genes of the G7 mutant individual which were not present in the RS. Known mutated genes include mviN, hutC, rpoD, nifB and nhaD. Further genomic and proteomic studies would be necessary to understand the role of these genes and mutations in the cobalt chloride stress resistance of R. sphaeroides. To summarize, the comprehensive evaluation of cross-resistance tests, growth physiology observations, and genome sequencing data yielded significant insights into the genetic basis of cobalt stress resistance observed in the G7 strain. Through the identification of variations in different genes, this investigation has provided valuable information regarding the underlying mechanisms that contribute to the G7 strain's ability to withstand cobalt and other heavy metal stresses. These findings contribute to the understanding of the genetic background of heavy metal resistance and offer potential avenues for further research and targeted interventions in this field that involve R. sphaeroides.