To work upon the hypothesis, four hydrogenases operon deletion mutants were exposed to a dose of antibiotic (ampicillin) at their mid-exponential phase. Ampicillin was chosen as it is widely used for persister isolation (Wang et al. 2017; Lewis 2019). The cells before and after antibiotic treatment were evaluated for a certain persistence associated with metabolic phenomenon.
Hydrogenase operon deletion enhanced cell viability:
To evaluate the hydrogenase mediated cell viability during the antibiotic-induced persistence, the colony-forming unit (cfu) of the four hydrogenase mutants was estimated by counting the colonies before and after antibiotic treatment, as follow:
Along with the operon deleted mutants their respective large subunit gene deleted mutants were also included for the comparative analysis and to predict the impact of complete operon deletion over a single gene. As a result, the cellular viability of the mutants was found increased from the antibiotic-treated cultures of all the hydrogenase large subunit gene and operon mutants compared to the PS (Fig. 1A). However, no significant change was observed in the number of viable cells before antibiotic treatment from all the mutants, the cell viability was found comparatively more in the case of large subunit gene deletion mutants, indicating operon deletions are more deleterious than single-gene deletions, as shown by our previous study (Shekhar et al. 2021). From both types of deletions, hyb and hyc were noted vital for bacterial survival since the viabilities were 20- and 18-fold increased respectively from the PS.
Along with their viabilities under micro-aerobic (oxygen deprived, explained later) conditions, hyb and hyc operon deletions were also found significant under strict anaerobic conditions, due to their comparable more cellular viabilities (Fig. 1B). hya, and hyf deletions also showed slightly increased viability as compared to the PS, although it was lower than hyb, hyc deletions. Importantly, cellular viabilities were noted reduced under anaerobic conditions than micro-aerobic ones (Fig. 1A and 1B). Additionally, results showed that the influence of the four hydrogenases is not similar indicating their different physiological functioning under similar conditions.
Impact of antibiotic on the growths of the mutants and the role of the dissolved oxygen in hydrogenase-mediated persistence:
Since persister level increases during growth (Keren et al. 2004; Lewis 2019), it is important to evaluate the growth of the strains in the presence and absence of the antibiotic. The growth of each hydrogenase operon mutant was monitored during the persister assay. A control group of the same strains was simultaneously monitored where no antibiotic was supplemented. As result, in the absence of antibiotic, relatively reduced growth of the mutants was observed compared to the PS, specifically, hyb and hyf mutants (Fig. 2A). Although not significant, a reduction in the growth of each mutant was noted when grown in the presence of antibiotic (data not shown). The growth differences between antibiotic-treated and untreated states of the mutants were minimum in the case of hyb mutant compared to other strains (data not shown), which may indicate a relatively more ability of this mutant to grow in presence of antibiotic. As E. coli grows, the level of persisters dramatically increases (Keren et al. 2004; Lewis 2019), therefore, hyb mutant is supposed to give a greater number of persisters as supported by its high cellular viability (Fig. 1B). However, hyf mutant showed similar differences as other strains, indicating despite its lower growth, it might not be significant in persistence, since ampicillin treatment leads to the persisters formation by killing cells that are unable to obtain the persister state or less fit to fight the effects of the antibiotic to enter a state of persistence (Hong et al. 2012).
Furthermore, the concentration of the DO in the cultures was monitored at different time points during the persister assay where all the strains showed rapid consumption of oxygen (Fig. 2B). In a result, the oxygen level found dropped near to zero indicating an oxygen deprived state of the culture (referred to as micro-aerobic state, Shan at al. 2012) at their mid-exponential phase i.e., the point at which antibiotic was added to the culture of each mutant. Oxygen consumption rate observed dissimilar among them, where hyf mutant found slower and hyb mutant found relatively faster indicating higher oxygen exhaustion rate. As reported, the survival of persisters requires a small (20%) drop in DO saturation, and at high DO concentration levels, the persister population is killed over time (Grant et al. 2012). The cultural conditions, in this study, before and after antibiotic treatment, representing nearly zero oxygen concentrations, may indicate the presence of that subpopulation that is susceptible to act as a persister population.
Hydrogenase expressions elevated after antibiotic treatment:
The test conditions of cell populations before and after antibiotic treatment were micro-aerobic (oxygen deprived), as confirmed by DO assay (Fig. 2B). Although hydrogenases functions were mostly reported from anaerobic conditions (Maeda et al. 2018; Vardar-Schara et al. 2008; Trchounian et al. 2012), some reports are indicating their activity under aerobic or micro-aerobic conditions also (Olson et al. 2002; Cordero et al. 2019). Therefore, this was interesting to evaluate the expression of hydrogenases in the absence and the presence of the antibiotic. In this regard, the expression of the gene encoding the large subunit of each hydrogenase was evaluated from each hydrogenase operon mutant. As a result, as expected, the expression of hydrogenases was found very low in the cultures of initial cells (indicated with ‘- ‘) (Fig. 3). Previously, the expression of Hyd-1 and Hyd-2 was found to be maximum under anaerobic conditions (Richard et al. 1999; Maeda et al. 2018); Hyd-3, repressed by oxygen (Pecher, 1983); and Hyd-4, normally do not transcribe or shows very low transcription (Trchounian et al. 2012). These reports support the lower or almost no expression obtained, in this study.
Interestingly, hydrogenase expression was found elevated in the antibiotic-treated (indicated with ‘+ ‘) mutant cells (specifically hya, hyb, and hyc) (Fig. 3). Antibiotic treated hya mutant cells showed relatively higher levels of hydrogenase expressions than other strains, however, hyf mutant did not show any change in the expression of hydrogenases before and after antibiotic treatment. These elevated hydrogenase expressions under the influence of antibiotic may indicate a significant association of hydrogenases with persistence.
Altered ATP levels after antibiotic treatment:
It was commonly observed that the formation of persister cells is inversely correlated with metabolic activity and energy production (Lewis 2019). ATP was found as a general cause of tolerance since most bactericidal antibiotics kill by corrupting active, energy-dependent targets (Shan et al. 2017). Moreover, variation in the level of ATP served as a mechanism of persister formation in E. coli (Lewis 2019; Shan et al. 2017). Therefore, quantification of ATP was realized important, and initial, treated, and untreated cells were evaluated. As a result, the treated cells were found to have a reduced amount of ATP as compared to their untreated state (Fig. 4). The percentage decreased ATP (from untreated to treated) was found maximum in hyb and hyc mutants (31% and 27% respectively), whereas it was comparatively less in the case of hya and hyf (21% and 18%), also in the PS (18%). The relatively lower level of ATP in hyb mutant may indicate its more significance in persistence.
Altered levels of total NAD in the antibiotic-treated cells:
As reported, low metabolic activity is the key to the survival of persister cells, since in exponentially growing cultures, persisters were found significantly more abundant in the least redox-active subpopulation (Wood et al. 2013; Mehmet et al. 2015). To elucidate the role of hydrogenase operon deletions and their influence on the total NAD content of the cell, total NAD (NAD+ and NADH) and NADH were quantified from the cultures of the strains. As a result, the NAD/NADH ratios were estimated reduced in all antibiotic-treated mutant cells (Fig. 5). Although, hyb mutant with a relatively more and hyc mutant a less NAD/NADH ratio indicating their lowest and highest fold change decrease than other mutants, respectively, these results may indicate a weakened metabolism of all the antibiotic-treated strains. In support of that, the KEGG pathway enrichment analysis also showed a similar result when metabolically important pathways were considered (Fig. 6, Table S4A-4D). All the treated mutants showed reduced amino acid biosynthesis and degradation, carbohydrate biosynthesis and degradation, cell wall biogenesis, energy metabolism, glycolysis, lipid metabolism, metabolic intermediate biosynthesis, oxidative phosphorylation, and TCA cycle compared to their respective untreated ones. hyc operon mutant showed relatively more reduced metabolism than hyb operon mutant in its treated state than untreated. The reduced metabolism of the strains may indicate its facilitation to the persistence and thereby significant cellular viabilities (Fig. 1A).
Altered ROS levels in the antibiotic-treated strains:
Recent studies have suggested that ROS can provide a protective effect against the antibiotics by inducing persistence as well as contributing lethality to the antibiotics to kill them (Dwyer et al. 2014; Brynildsen et al. 2013; Kohanski et al. 2010). The ability of ROS to both protect and kill bacterial cells motivated us to quantify it in all the strains. As result, the ROS quantity which was detected relatively the same in all the strains before antibiotic treatment found significantly altered after antibiotic treatment, except hyb mutant (Fig. 7). In comparison with the untreated cells, antibiotic treatment demonstrated ROS surge in the strains.
As bacterial survival against antibiotic is mediated by the ability to detoxify ROS (Grant et al. 2012; Lewis 2019), the significantly decreased ROS in treated hyb mutant cells may be indicating its ability to detoxify or scavenge the ROS induced by the action of the antibiotic. This further justifies the high cellular viability of hyb mutant after antibiotic treatment. Contrary to hyb, the increased ROS levels in hya, hyc, and hyf operon mutant may reflect their association to the ROS-mediated protective effects against antibiotics (Dwyer et al. 2014; Brynildsen et al. 2013).
Gene ontology (GO) enrichment analyses of hydrogenase-mediated persistence:
To understand further the influential role of hydrogenases in persistence, the change in global gene expression in the antibiotic-treated mutant cells was investigated through transcriptomics analysis (Table S5A-5D). The influenced biological process (BP), cellular component (CC), and molecular function (MF) were identified using the GO enrichment analysis approach (Young et al. 2010). The top 20 GO terms ranked based on most upregulated in treated samples are presented through plots (Fig. S1-S4). In results, the translation, large ribosomal subunit, cellular response to DNA damage stimulus, carbohydrate metabolic process, protein folding and transport, cell division and cycle, response to the antibiotic, tricarboxylic acid cycle, and phosphoenol pyruvate-dependent sugar phosphotransferase system were found most commonly altered BPs in all four treated operon mutant cells (Fig. S1A, S2A, S3A, S4A). In the category of CC, cytosol, and cytoplasm, membrane and plasma membrane, integral component of membrane and plasma membrane, large and small ribosomal subunits, outer cell membrane, periplasmic space, pore complex, proton-transporting ATP synthase complex, ATP-binding cassette transporter complex, plasma membrane respiratory chain complex I or NADH dehydrogenase complex were obtained as significantly altered CCs in all treated mutant cells (Fig. S1B S2B, S3B, S4B). Whereas the common MFs observed in all treated mutant cells includes, ATP and NAD binding, DNA and RNA binding, structural constituent of ribosome, metal ion binding, magnesium, and zinc ion binding, ATPase activity, tRNA and rRNA binding, 4 iron-4 sulfur cluster binding, electron transfer activity, proton-transporting ATP synthase activity, and oxidoreductase activity (Fig. S1C, S2C, S3C, S4C). This analysis showed that the influence of hydrogenases on the persistence in E. coli is mediated by multiple factors and phenomena.