Adaptive laboratory evolution to generate salt-resilient strain
To generate Z. mobilis strains that can grow and ferment under saline stress condition, we adopted an adaptive laboratory evolution strategy. The approach was previously employed by Wang et al., however the Z. mobilis culture with excessive NaCl concentration was not viable for long term due to a toxic effect, and the approach was not successful in generating resilient strain [13]. Therefore, we introduced a bias in the serial transfer on a following ground, to direct evolution preferable to our goal (Fig. 1).
Previously, it was shown that salt condition induces filamentation of Z. mobilis cell [11]. We observed the filamentation in our experimental setting, using our standard complex medium supplemented with 0.225 M NaCl (Fig. 2). Interestingly, almost all cells exhibited an abnormal bulge at single cell pole, with variant cell length and width (Fig. 2 top right). These cells were found as thick sedimentation in the fully-grown culture (Fig. 1), however, we did not observe any floc formation which was shown to be beneficial for stress resistance in Z. mobilis [25]. Based on cell shape and growth profile (Fig. 2, Fig. 3), we speculated that the filamentous shape with a bulged pole is a consequence of stress, rather than an adaptation to environment. We then thought the filamentation to be exploited as a biomarker to identify the stressed cells that should be avoided for serial transfer. Although the sedimentation might have involved other factors, microscopic observations led us to conclude that filamentation of cells facilitated a formation of sediment.
Following the rationales, we performed lab-directed evolution with a bias in transfer. In practical, we collected cells only from an upper layer in the fully-grown culture as an inoculum for next round (Fig. 1). Zm6 strain was evolved in the complex medium supplemented with 0.2 M NaCl for 13 transfers, then continued in the medium with 0.225 M NaCl for another 8 transfers. After about first 10 transfers, it was apparent from turbidity of cultures that the strains improved growth. Strains after 13th transfer was designated as KFS1 and after 21st transfer as KFS2. To clarify if the improved growth was due to temporal physiological adaptation without genetic mutation or stable phenotype arose from mutations, we performed a pilot experiment. The evolved strain culture without adaption, i.e., grown in the medium without additional salt, was used as an inoculum for measuring growth profile under salt condition (0.225 M NaCl). We observed the improved growth, excluding the possibility of transient adaptation and showing that mutation in the genome was the cause.
Evolved strains exhibited two characteristics in cell shape under salt condition. The strains less frequently formed bulged pole than parental strain did (Fig. 2, bottom panels), indicating that the cells were less stressed under salt condition. The cells were also found fragmented or long filament shaped which was rather contrary to what we expected (Fig. 2).
Characterization of evolved strains
Next, we characterized phenotypes of presumably evolved strains by measuring growth curve, glucose consumption and ethanol production by strains in the presence of salt. It is to be noted that used inoculum for the culture was not adapted to salt condition. (See material and method).
As shown in the figure 3, parental strain Zm6 did not consume all available glucose in the medium with salt, failing completion of fermentation due to an arrest of growth. In a sharp contrast, the evolved strains drastically improved growth and ethanol production under the same condition (Fig. 3). Remarkably, final biomass of evolved strain was about 2.5 and 2.7 times higher than that of Zm6, respectively for KFS1 and KFS2 (Fig. 3). Total ethanol production by KFS1 and KFS2 was also significantly improved, 2.74 and 2.69 times higher than by parental strain. (Fig. 3). It is to be noted that the final biomass (CDW mg/mL) and ethanol yield [EtOH(g)/Glucose(g)] by evolved strains under salt condition was close to those by parental strain under non-salt condition.
To characterize the strains further, growth profiles of all strains in the medium without supplement of salt were recorded (Fig. 3). Both evolved strains showed slight retarded growth and ethanol production under non-salt condition, and their final biomass of evolved strains was significantly lower than that of parental strain. This was somewhat expected by us, considering that the improvement of growth in salt medium was drastic and likely involved physiological alternation. However, final ethanol production by all strains was nearly same (Fig. 3), showing that final ethanol production per cell dry weight was, interestingly, higher in the evolved strain. This implies that cellular activity was spatially more ‘packed’ in the evolved strain. Along with this line, we observed that the evolved cells exhibited smaller cell size than parental strain, as shown by light microscopic images (Fig. 4).
Quantitative Metabolomics
Bacterial osmotic response involves accumulation of osmoprotectant to counter external osmolality, to maintain turgor. Proline and betaine-glycine are examples of well characterized osmoprotectant in several bacteria [26]. Such a response, accumulating specific compounds as osmolyte, may result in dynamic metabolic change and could perturb production of desirable compounds by Z. mobilis. To determine if any metabolomic adjustments played a significant role in acquired resilience in the evolved strains, we performed quantitative targeted metabolic profiling of central carbon metabolites and free amino acids in all strains.
Our initial goal was to obtain intracellular concentration for a comparative analysis. The challenge here was that cellular volume was highly heterogeneous in all strains under salt condition (Fig. 2), hindering intracellular concentration measurements that require defined cell volume. Therefore, we first normalized metabolite abundance in each strain by cell dried weight (Fig. 5). The normalized metabolites abundance was compared between strains and conditions, as shown by the heatmap of log2 fold change in the Fig. 5. From fixed weight of cell extract, Zm6 cells without stress (Zm6 NS) generally comprised larger pool of the metabolites than Zm6 cells under salt condition (Zm6 S) (Fig. 5, left panel). Interestingly, several free amino acids including proline are the only metabolites found upregulated under salt condition per fixed dry weight, although mildly. Similarly, extracts from evolved strains under salt condition (KFS1 S, KFS2 S) showed smaller pool size of the metabolites than from Zm6 NS (Fig. 5, middle and right panel).
Next, we stained membrane of Z. mobilis cells with a staining dye Fm4-64 to observe if there is a compartmentalization within cell. We observed no septum formation nor membrane organelle in the bulged Zm6 cells (Fig. 6). Apparently, salted Zm6 single cell volume was much larger than that of rod shape Zm6 under non-salt condition (Fig. 2, Fig. 6).
According to previous studies, Protein, DNA and RNA are the main component constituting about 70–80% of weight in bacteria [27]. It is less likely that slow growing filament (Zm6 S) possessed more dense macro molecules per fixed cell volume than actively growing small cell (Zm6 NS). We therefore speculated that Zm6 S is expected to have larger cell volume per fixed amount of CDW than that of Zm6 NS. This further leads to that the log2 ratio of Zm6 S/ Zm6 NS in Fig. 5, normalized by CDW, should decrease when intracellular concentration is deployed. Thus, our metabolomics data suggest that Zm6 cells did not drastically accumulate central metabolites or free amino acids during the stress response. Most of ED pathway metabolites and nucleosides in Zm6 S was drastically downregulated, coinciding with low growth and slow glucose uptake. Moreover, canonical osmoprotectant, for example proline, was not found significantly accumulated either. Although, it was not completely excluded that other metabolites might have been upregulated during the stress response.
KFS1 and KFS2 under saline condition exhibited heterogeneous cell shapes, ranging from fragmented cells to long extended cells (Fig. 2). Membrane staining showed that cells could produce septum at several locations, unlike Zm6. Nevertheless, KFS1 cells did not complete division and instead formed long filament, and KFS1 S cell compartment size was found overall larger than that of Zm6 S (Fig. 6). Similar to the case of comparison of Zm6 extract between saline conditions, bigger volume and slower growth of KFS1 S and KFS2 S to Zm6 NS imply that actual intracellular concentration ratio of KFS1 or KFS2 S to Zm6 NS is smaller than the log2 ratio depicted in Fig. 5. This suggests that evolved strains did not accumulate osmolytes to counter the stress either.
To further understand the resilient mechanism in evolved strains, we measured intracellular ratio of reduced/oxidized form of NAD cofactor (NADH/NAD) ratio by an enzymatic assay. Malate dehydrogenase and oxoglutarate dehydrogenase complex are not encoded in Z. mobilis genome [3, 28], which influences regeneration of NAD. Previous works suggested that maintaining low NADH/NAD ratio appear to be important in response against salt and acetic acid stress, to sustain glycolysis in the ED pathway that requires oxidized NAD [15, 29]. To see if the redox regulation of co-factor conferred the resilience, we examined the NADH/NAD ratio in the strains. The analysis showed that stress increased the ratio in Zm6 (Table 1), as it was previously shown [15]. Interestingly, upregulation of NADH/NAD ratio under saline condition was also observed in evolved strain as well, within similar range of shift to Zm6. These data indicate that modulation of NADH/NAD was not a part of resilience mechanism in the evolved strains. Although the modulation of ratio is vital in general Z. mobilis stress response, as shown by previous studies, the resilience to salt stress in the evolved appeared to be mediated by a separate mechanism.
Identification Of Gene Loci
Next, we sought to identify what mutations in genome brought the phenotype in evolved strains. The whole genomes of parental and evolved strains were sequenced and aligned against the reference genome [28]. The analysis showed that our lab stock Zm6 strain possesses several mutations (Table S2). These mutations likely arose during previous laboratory practices. We found several mutations only arose in evolved strains (Table. 2). A disruptive insertion in ZZ6_1449 coding carboxyl protease (CTP) was among them. CTP is found in all kingdom of life and mainly cleaves serine or lysine nearby at C-terminus of substrate. In bacteria, it has been shown that mutation in CTP caused alternation in cell envelop and higher sensitivity to antibiotics [30] and osmotic down-shift in Escherichia coli [31]. In Pseudomonas aeruginosa, disruption of CTP results in impaired growth in medium with low salt [32]. The phenotype in E. coli and P. aeruginosa is to some extent consistent with our results from growth profile. Other three point-mutations only found in KFS1 and KFS2 are possibly irrelevant with stress resilience, based on the annotated function. We found a point-mutation only found in KFS2, and an annotation of mutated gene is not available from the database. There are mild differences between KFS1 S and KFS2 S in morphology and growth, and it cannot be excluded that the mutation in the uncharacterized gene conferred extra resilience in KFS2. Together with available literature, it strongly suggests that disruption of ZZ6_1449 was mainly responsible for the improvement of growth. To confirm that the phenotype was due to the disruption, we attempted to perform complementation test in evolved strains. However, we did not obtain the construct due to technical challenges in cloning, and the test was not performed.