Alcanivorax dieselolei KS 293 (Barbato et al., 2015) was isolated from surface waters collected from the Levantine basin of the Mediterranean Sea (Mapelli et al., 2013) after enrichment with ONR7a mineral growth medium and diesel oil 1% (v/v) as the sole carbon source. Alcanivorax borkumensis SK2 type strain was chosen as a control strain. Strains were grown axenically in 250 mL glass bottles containing 100 mL of ONR7a media supplied with either 1% n-dodecane (C12) (v/v) or pyruvate (PYR) (w/v) as the sole carbon and energy source, at room temperature, for 7 days without mixing. All Alcanivorax cultures were inoculated at an optical density (OD) at 610 nm equal to 0.05. Experiments were conducted in eight independent replicates. In an additional experiment, Alcanivorax cells of either SK2 or KS 293 were grown to the late exponential phase on either pyruvate or C12, collected by centrifugation and re-suspended in three independent replicates in a carbon-free ONR7a medium to test their capacity to grow with no added carbon source.

Cell growth was assessed through flow cytometry (FC) analyses. Flow cytometry was carried out according to De Roy et al. (2012). Utilization of the supplied carbon sources was indirectly evaluated by measuring pH variation in the growth media at the end of the incubation time, provided that either C12 or pyruvate were supplied as sole carbon source. pH values were determined with a 744 pH meter (Herisau, Metrohm, Switzerland). Cell hydrophobicity was evaluated through the Microbial Adhesion To Hydrocarbon (MATH) test, modified after Rosenberg et al. (1980). Cells were pelleted at 1699 g for 10 min and rinsed twice in PUM buffer (22.2 g/L K₂HPO₄.3H₂O; 7.26 g/L KH₂PO₄; 1.8 g/L UREA; 0.2 g/L MgSO₄.7H₂O; pH was adjusted to 7.1). Finally, cells were resuspended in PUM buffer to a final volume of 1.2 mL and OD₆₁₀ value of about 1. After measuring the rinsed-cells OD₆₁₀ (OD_i), cells were supplied with 0.1 mL n-hexadecane (C16) and incubated at 28°C for 10 min. Afterwards, cells were mixed for 2 min and left to rest for 15 min. Finally, the OD₆₁₀ of the aqueous phase was measured again (OD_f). The relative amount of cells bound to n-hexadecane was calculated as follows:

The extraction and quantification of phospholipid fatty acids (PLFAs) were performed following the method described by Denef et al. (2007). Total lipids were extracted from the cell pellet using phosphate buffer/chloroform/methanol at a 0.9:1:2 ratios and partitioned into neutral, glyco- and phospho-lipids by solid phase extraction. Phospholipids were methylated by mild alkaline methanolysis (using methanolic KOH) to form fatty acid methyl esters (FAMEs), which were analyzed by capillary gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS) (GC-C/Delta PLUS XP Thermo Scientific) via a GC/C III interface as described by Denef et al. (2007). Standard fatty acid nomenclature was used to describe PLFAs. For each sample, the abundance of individual PLFAs was calculated in absolute amounts of C and then converted to mole percentage PLFA-C.

Cells were collected at the stationary phase of growth. Three independently grown cultures were pooled together to average the cells response to the carbon source. Pooled cultures were centrifuged at 4°C for 5 min 17,949 g (Sorval RC5c PLUS, Beckman, Suarlée, Belgium) and the pellet immediately stored at −80°C. Later on, pellets were thawed and RNA quickly extracted.

Results were expressed as mean values of experiments made in 3–8 independent replicates. Bars in the graphs indicate a 95% confidence interval (95% CI) calculated using a Student t-test with a two-sided distribution. Statistical significance was assessed using a non-parametric test (Mann-Whitney test) which considered a two-sided distribution with 95% CI.

KS 293 (Barbato et al., 2015) and SK2 genomes (Golyshin et al., 2003) have been compared through the RAST platform (Overbeek et al., 2014). KS 293 showed larger genome and higher %GC and coding sequence number respect to SK2 (Table 1). Despite these differences, the two genomes shared a functional identity of 80.3%, equivalent to 1952 functional gene categories having the same function over a total of 2413. 340 gene categories were peculiar of KS 293 while only 139 of SK2, but none of them was specifically correlated to alkane metabolism (Table S1). In particular, the amino acid sequence identity of genes directly involved in the first step of alkane sensing and degradation was analog in both strains (Table S2).

Both strains were able to grow with either C12 or pyruvate as sole carbon source (Figure 1A). The total cell number at the stationary phase, after 7 days of incubation, was assessed through flow cytometry. All the conditions achieved statistically similar final cell numbers (p > 0.05), with the exception of strain SK2 supplied with pyruvate (Figure 1A), which showed higher growth capacity when growing on this carbon source (p < 0.05).

The initial pH of the media (7.5) remained constant throughout the experiment in abiotic controls, while it decreased in all inoculated samples (Figure 1B): in both strains pH values significantly decreased when C12 was supplied (p < 0.05). In particular, pH was significantly lower in SK2 growing on C12 as compared to pyruvate (p < 0.05).

Final consumption capacity of either pyruvate or C12 was assessed by evaluating the grams of carbon per liter of culture (g_carbon L⁻¹) at the end of the incubation time (Table S3). The results showed no statistical difference (p > 0.05) neither between strains growing on the same carbon source, nor when comparing the same strain with either carbon source.

At the end of the incubation time, also the production of organic acids was evaluated. Neither strains produced these compounds when growing on C12 (detection limit 1 mg L⁻¹), but they produced lactate, formate and acetate after growth on pyruvate (Table S4).

Hydrophobicity tests performed on cell cultures grown on either pyruvate or C12 indicated no significant changes in A. dieselolei KS 293 cells (p > 0.05), which were always highly hydrophobic. Conversely, A. borkumensis SK2 cells were highly hydrophilic when grown on pyruvate and switched to a hydrophobic behavior on C12 (Figure 1C). These results were further investigated by assessing the capacity of the cells to carry over C12. Cells grown on C12 and subsequently re-inoculated in mineral medium with no carbon source showed growth both in the case of SK2 (2 generations) and even more in KS 293 (5 generations), suggesting that a fraction of the C12 previously supplied remained stored intracellularly or was externally attached to the cells due to their superficial hydrophobicity. No growth was observed with cells previously grown on pyruvate when re-suspended in a carbon-free medium, despite accumulation of intracellular reservoirs (Figures 2B,D). The surface tension of the culture medium showed lower values for both strains growing on C12, possibly due to the production of biosurfactant molecules. The decrease was particularly significant in SK2 where such value reached the maximum reduction possible to surface tension in the water-based broth medium equal to ≈27 mN/m, while in KS 293 it reached a value of ≈48 mN/m (Figure 1D).

The impact of different carbon sources on membrane PLFA profiles (C11–C30) was investigated. The two strains showed minor differences in PLFA profiles. In both bacteria, the total amount of unsaturated, rather than saturated PLFAs was higher with the two carbon sources; however, we observed a slight increase of saturated PLFA in both bacteria when growing with C12 if compared to pyruvate (Figure 2A).

Also, ultra-thin sections of SK2 and KS 293 cells grown on C12 and pyruvate revealed significant differences in membrane morphology (Figures 2B–E). Cell membranes in both the strains appeared rather indented (Figures 2B,C,E), while only SK2 grown with pyruvate showed neat outer profile (Figure 2D). Provided that in this condition SK2 showed highly hydrophilic cells with the lowest capacity to adhere to hydrocarbons (Figure 1C), the neat membrane morphology could be related to cell surface hydrophilicity. Furthermore, the two strains showed electron-transparent inclusion bodies attributable to carbon reservoirs only when growing on pyruvate (Figures 2B,D), showing a massive accumulation in SK2.

In each dataset a total of 2307 gene clusters were identified. Their annotation contained 1487 sequences belonging to the SSD ortholog class, which were further associated to different functional pathways in the KEGG database. Of the remaining gene clusters, 47, 57, and 1 sequences were classified as borderline-SSD, divergent non-SSD and similar non-SSD, respectively, with 715 gene clusters not classified.

The hydrophobic nature of hydrocarbons limits their water solubility and, therefore, bacterial access to oil. One of the most common strategies to counteract this problem is to increase hydrocarbon availability by producing biosurfactants, molecules composed of both lipophilic and hydrophilic moieties (Banat et al., 2010). Both A. dieselolei and A. borkumensis are described in the literature to produce biosurfactant molecules in order to increase the bioavailability of lipophilic substrates (Marchant and Banat, 2012). In A. borkumensis SK2 many different glycolipid biosurfactants have been characterized, and their most common structure is an anionic glucose lipid composed by a glucose molecule connected to tetrameric β-hydroxy-decanoic acid (Abraham et al., 1998; Yakimov et al., 1998). The only characterized biosurfactant produced by A. dieselolei strain B5, is a lipopeptide composed by a proline residue bound to three different fatty acids (C_14:0, C_16:0, and C_18:0) (Qiao and Shao, 2010). In the present work, reduced surface tension in cells growing on C12 in both strains confirmed the production of surface-active molecules, although in SK2 a more pronounced effect was assessed (Figure 1D). Decrease in surface tension during SK2 growth on C12 is coherent to the enhanced expression level of genes involved in glycerolipid and glycerophospholipid metabolism (Figure 5, Table S5). On the contrary, KS 293 upregulated the genes involved in proline metabolism (Figure 5, Table S5) together with the gene yciA (Figure S1, Table S6), responsible for the synthesis of the unsaturated fatty acid C_16:0, confirming the production of a specific proline lipid biosurfactant which synthesis was observed exclusively in the A. dieselolei species (Qiao and Shao, 2010).

A key feature of hydrocarbonoclastic bacteria is their membrane composition. Depending on its hydrophobicity, the cell membrane may become more lipophilic when hydrocarbons are present, thus enhancing their capability to adhere to hydrophobic compounds (Ron and Rosenberg, 2002; Wick et al., 2002; de Carvalho et al., 2009). In accordance with previous reports (Naether et al., 2013), SK2 surface hydrophobicity was increased when growing on C12 as compared to pyruvate, inducing a net shift in cell capacity to adhere to n-hexadecane, and a change in membrane morphology from neat to indented (Figures 2B–E). An indented cell membrane could be related to a higher capability to adhere to hydrophobic substances, which would be an advantageous trait for hydrocarbonoclastic bacteria. In the present study, this was reflected in the capacity to store C12 molecules by the cell, allowing degradation at a later stage when cells are moved in a carbon-free medium. On the contrary, KS 293 did not show morpho-physiological adaptations induced by the supplied carbon source, as growth on pyruvate and C12 yielded comparable membrane hydrophobicity, cell adherence to hydrocarbons and membrane morphology.

Linear alkanes are lipophilic substances that, due to their nature, can easily insert through cell membrane double phospholipid layer, potentially resulting in cellular death by lysis (Heipieper and Martinez, 2010). Both KS 293 and SK2 showed similar PLFA response to C12, with a slightly increasing saturated fatty acid relative abundance (Figure 2A). This may reflect an adaptation strategy to C12 as an attempt to reduce cell membrane fluidity, thus preventing the uncontrolled entrance of the linear alkane. The similar physiologic and morphologic cellular membrane features of the two strains growing with C12 are indicative of analogous adaptation strategies of the membrane toward the presence of hydrocarbons, as also confirmed by the comparable transcription of the genes involved in the biosynthesis of cellular membrane constituents (Figure S1, Table S6).

Hydrocarbons are processed and degraded inside the cells, nonetheless the mechanism by which linear alkanes cross the cellular membrane is still unclear, and in recent years the involvement of transmembrane transporters has been suggested (Sabirova et al., 2011; Wang and Shao, 2014). A. dieselolei B5 possesses transporter proteins specific for alkane of different lengths: OmpT-1, OmpT-2, and OmpT-3, respectively allowing long-, medium- and short-chain alkanes to enter the cells (Wang and Shao, 2014). While genes encoding for OmpT-2 and OmpT-3 could not be detected in KS 293 transcriptome, the expression of the ompT-1 gene in cells grown on C12 showed higher values than SK2 (Figure 4, Table S2). Wang and Shao (2014) suggested that OmpT-1 alone is not sufficient to sustain A. dieselolei B5 growth in the presence of C12 and, although Sabirova et al. (2011) proposed the outer membrane lipoprotein Blc to be involved in alkane uptake in SK2, the corresponding gene was not detected in the present transcriptomic analyses. The mechanisms responsible for short-medium length alkane transport in Alcanivorax representatives need therefore further consideration.

Once entering the cell, a three-step activation process initiates hydrocarbon degradation, where alkanes are first converted into alcohols, then to aldehydes and finally to fatty acids (Rojo, 2009). The first reaction is carried out by alkane hydroxylases enzymes (mainly AlkB and Cyp450) specific for alkanes of different length (Syutsubo et al., 2001; Hara et al., 2004; Rojo, 2009; Wang and Shao, 2014). This enzymatic apparatus has been thoroughly investigated in the last years, and its action mechanism uncovered in A. dieselolei, A. borkumensis and many other hydrocarbon degraders (Van Beilen et al., 2003; Rojo, 2009; Liu et al., 2011). KS 293 and SK2 genomes harbor both alkB and cyp450 genes, but they were differentially transcribed and regulated (Figure 4, Table S2).

The subsequent conversion of alcohol to aldehyde relies on a dehydrogenase (adhC) whose transcription was upregulated in SK2 with respect to KS 293 (Figure 4, Table S2). The aldehyde is then converted to fatty acid, which can be degraded to acetyl-CoA through β-oxidation, constitute cellular membranes and exopolymeric substances, or be converted into storage materials (Rojo, 2009; Naether et al., 2013). Expression level of β-oxidation related genes in SK2 was equal or reduced as compared to KS 293. KS 293 also showed higher transcription levels for most of the fatty acid biosynthesis related genes and for the genes involved in Krebs cycle and oxidative phosphorylation (Figure 4, Table S2). Therefore, the main destiny of fatty acids directly deriving from C12 utilization in SK2 appears to be the formation of membrane structures or specific metabolites such as biosurfactant molecules, while in KS 293 it appears to be the production of ATP. Overall, genes involved in C12 mineralization and CO₂ production were transcribed at higher levels in KS 293 than in SK2, in contrast to (1) the much lower pH value of SK2 cell supernatant at the end of the incubation time with C12 (Figure 1B) and (2) the statistical similarity between the carbon consumption capacity (g_carbon L⁻¹; Table S3). We analyzed the amount of fermentation products accumulated by the cultures at the end of the incubation to test whether other factors could have accounted for the pH change. While cultures grown on C12 did not produce any organic acid (detection limit 1 mg L⁻¹), they released traces of lactate, formate and acetate when grown on pyruvate (Table S4). The pKa of these organic acids is very low as compared to ONR7a medium initial pH (3.86, 3.77, and 4.76 vs. 7.5, respectively). However, cultures grown on pyruvate were statistically more alkaline than those grown on C12 (p < 0.05), indicating that the strong decrease in pH with either carbon source was due to other factors. In light of the transcriptomic results, the observed pH changes in SK2 growth media may be dependent on extracellular molecules, with biosurfactants a possible candidate.

Fatty acids resulting from hydrocarbon metabolism can also be stored as carbon reservoirs in intracellular bodies. To build intracellular reservoirs of carbon is a common strategy among bacteria when the environment has an unbalanced C:N ratio, i.e., when the reduced N supply is perceived as limiting for protein synthesis and cell replication (Zinn et al., 2001), a condition demonstrated in KS 293 with a high transcription level of ntrB that acts as sensor for low nitrogen availability (Figure S2, Table S8). The oligotrophic marine environments not chronically polluted by hydrocarbons offer limited opportunities to hydrocarbonoclastic bacteria with a narrow metabolic range apart from hydrocarbons (Yakimov et al., 1998; Liu and Shao, 2005), and the capability to store carbon reservoirs intracellularly could significantly increase their chances to survive. Accumulation of hexadecane-derived wax esters (WEs) has been indeed observed in Marinobacter hydrocarbonoclasticus and A. borkumensis (Kalscheuer et al., 2007; Grossi et al., 2010). In our experimental setting, a rather abundant carbon load was supplied to the strains in order to simulate culture conditions resulting from a spill (Sabirova et al., 2006). The phaC2 gene involved in the synthesis of poly-β-hydroxybutyrate (PHB) was upregulated in KS 293, when growing on C12 respect to pyruvate, and not affected by carbon source in SK2. However, in both strains no PHB accumulation was detected, as previously revealed for A. borkumensis (Sabirova et al., 2006; Kalscheuer et al., 2007; Manilla-Pérez et al., 2010). The gene atfA1 was suggested to encode for an enzyme involved in the synthesis of triacyl glycerols (TAGs) and WEs in A. borkumensis (Kalscheuer et al., 2007). Transcription levels of these genes were indeed much higher in SK2 growing on pyruvate rather than C12 (Figure 6), in agreement with an overabundant accumulation of electron-transparent intracellular grains in these cells (Figure 2D). Previous studies on A. borkumensis indicated the existence of alternative, uncharacterized metabolic routes for TAGs and WEs production, as intracellular grains of neutral lipids were observed notwithstanding a complete atfA1 knock out (Kalscheuer et al., 2007). These findings may account for the small but detectable electron-transparent grains accumulated in KS 293 grown on pyruvate (Figure 2B), in which transcription levels of both phaC2 and atfA1 were particularly low (Figure 6). The expression level of this gene was, nevertheless, comparable in both KS 293 and SK2 when growing on C12, and the lack of inclusion bodies when growing on this substrate suggests that this alkane may not trigger intracellular carbon accumulation. Moreover, the positive transcriptional response of PHB synthesis related genes was not followed by their actual accumulation in the cell, nor it was for TAGs or WEs, whose target gene expression level was unaffected, indicating that excess carbon might not be a sufficient condition to trigger the synthesis of intracellular storage of carbon.

Chemiotaxis has been recognized as an advantage for hydrocarbonoclastic bacteria such as A. dieselolei B5 to detect the entrance of petroleum-hydrocarbons in the cell environment (Wang and Shao, 2014). Genes related to this function (ompS, Wang and Shao, 2014) are present in the genome of both strains, and when growing on C12 they were transcribed at significantly higher levels in KS 293 (Figure S2, Table S8). This suggests that other functions not directly linked to alkane metabolism could diversify the response of different hydrocarbonoclastic bacteria to hydrocarbon substrates.

Taken together, these data bring evidence of many common and some exclusive responses to oil in hydrocarbonoclastic isolates harboring the same genomic background. When using an alkane as carbon and energy source, the two strains showed comparable growth capacity and surface hydrophobicity as a strategy to increase the interaction with the lipophilic substrate. Furthermore, they shared the same cellular membrane composition and physiological properties toward C12 utilization, with similar pathways of alkane activation, and both overexpressed genes related to biosurfactant production as a strategy to increase alkane availability. These evidences confirm that the two strains share the same functions in terms of metabolic pathways sustaining the capacity to grow on alkanes. Nonetheless, their transcription patterns showed functional redundancy in key metabolic pathways, as differential responses in the genes expressed for alkane activation and regulation and for the production of surfactant molecules (glycolipids in SK2, lipopeptides in KS 293). The most remarkable difference between the strains resided in their adaptation mode to the different substrates. SK2 had significantly higher efficiency to grow on substrates alternative to alkane (pyruvate) and changed many key physiological aspects when growing on C12: the outer membrane changed from neat to indented and became highly hydrophobic to better interact with the lipophilic substrate; moreover, a significant decrease in surface tension of the cell supernatant was assessed, putatively due to the production of surfactant molecules. On the contrary, KS 293 did not show any significant adaptation mechanism in its capacity to grow on the different substrates, with analogous physiological responses to pyruvate and C12. This relevant difference between the strains was reflected in a differential expression of genes related to carbon utilization pathways: despite comparable growth efficiency on C12, SK2 mostly overexpressed genes related to fatty acids biosynthesis and elongation, while KS 293 overexpressed genes related to β-oxidation, Krebs cycle and oxidative phosphorylation. The capacity of the strains to utilize carbon sources alternative to hydrocarbons would sustain them to permanently colonize pristine marine environments, while their differential transcriptomic responses to hydrocarbons could make them a potential functionally redundant and plastic reservoir of oil-degrading players, able to activate in the case of oil-spill occurrence, sustaining natural attenuation and biostimulation based clean-up processes.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HFS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.