Interrogation of chemical changes on, and through, the bacterial envelope of Escherichia coli FabF mutant using time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) using a primary ion beam of (CO2)6k+ was used to analyse chemical changes in the bacterial envelope of a fabF knock‐out Escherichia coli strain. fabF is the gene coding for FabF, the enzyme involved in the elongation of FA(16:1) to FA(18:1) and has been associated with plasmid transfer that can lead to acquired multiantibiotic resistance. Comparison of the membrane composition between fabF mutant E. coli and wild type E. coli during the logarithmic and stationary growth phases at two culture temperatures (37°C and 30°C) revealed substantial depletion of FA(18:1) in the fabF mutant during logarithmic growth that resulted in a correlated reduction in FA(cp19:0) during stationary phase. While no clear temperature dependence on the effect of the fabF mutation was found, a reduction in cyclopropanation was observed at lower culture temperature in the wild type strain. Additionally, depth profile analysis revealed a ‘thickening’ of the lipid A layer on the surface of the bacteria during stationary phase and also the appearance of cyclic enterobacterial common antigen (ECACYC) below the surface of the bacteria upon the shift from logarithmic to stationary growth phase.


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Conjugation, the process of direct cell-to-cell DNA transfer between bacteria, is a particularly hazardous route through which bacteria can obtain multiantibiotic resistance. The conjugation process in ToF-SIMS also has a record of being used in bacterial analyses to classify bacteria responsible for urinary tract infection, classify Bacillus strains, and has been proven to be especially sensitive to secreted molecules with roles as antibiotics or in signalling. [9][10][11][12][13][14] ToF-SIMS has also been used to image antibiotics at subcellular scale in single bacterial cells. 15 The advent of gas cluster ion beams (GCIBs) has increased the sensitivity of ToF-SIMS to analyse larger mass molecules of up to several thousand kDa. 16,17 while maintaining good surface sensitivity and depth resolution. 18 The benefits of GCIBs have been demonstrated many times not only on tissue samples such as breast cancer tissue, mouse heart and skin cancer tissue but also on bacterial samples where lipid changes during the stringent response in E. coli with and without the global signalling molecule ppGpp were studied. [19][20][21][22] In the ToF-SIMS study by Dimovska Nilsson et al., the mutated E. coli strains were analysed using a 40 keV GCIB with clusters of (CO 2 ) 6k + both on the surface to investigate any lipid changes observed in the outer membrane of the cells and depth profiled to probe parts of the bacterial envelope. 7 The depth profiles allowed detection of the higher mass species lipid A, the anchoring point of the larger molecule lipopolysaccharide (LPS), at m/z 1796, and cyclic enterobacterial common antigen (ECA CYC ), a molecule found in the periplasm, at m/z 2428. It was also found that the fabF knock out strain was depleted in the cyclopropanated fatty acid FA(cp19:0), a surprising find because FabF is expected to be involved in the elongation of FA(16:1) to FA(18:1), where only a small effect was found, not cyclopropanation.

Further, this effect was observed in cells grown at 37 C although
FabF is reported to be largely responsible for elongation of FA (16:1) at lower temperatures.
In this study, we further investigate the influence of FabF on membrane composition. Bacterial arrays of wild type (WT) and fabF E. coli strains were analysed using ToF-SIMS. Cells were cultured at two different temperatures, 37 C and 30 C and analysed at both logarithmic (log) and stationary growth phase to investigate the temperature dependence of the findings and see if the effects were found at both points of the growth curve.
2 | EXPERIMENTAL 2.1 | Culturing of E. coli strains A strain of E. coli with a knock-out mutation in the fabF gene (ΔfabF::kan/F 0 ), that will be referred to as fabF mutant, and a WT control strain with a neutral mutation, that will be referred to as WT (Table 1), were cultured aerobically in LB medium supplemented with kanamycin (50 μg/ml) and tetracycline (10 μg/ml) to select for the plasmid. The cells were cultured overnight in a rotary shaker at either 37 C or 30 C for the stationary phase sample. The overnight culture was diluted and grown in the same medium in a rotary shaker at 37 C or 30 C to log phase (OD600 = 0.5) for the log phase samples. In total, eight samples were taken: the fabF mutant and WT bacteria grown at two temperatures and at two growth phases.

| Preparation of E. coli samples for ToF-SIMS analysis
Before ToF-SIMS analysis, the cells were washed using a washing procedure reported in previous work. 7  Two technical replicates from each culture were deposited on the silicon wafer. Visual inspection of the droplets indicated that the sample drops were several bacteria thick with small variation between the different samples. The droplets were left to air dry and before inserting the samples into the ToF-SIMS instrument; they were placed in a desiccator for approximately 30 min.

| ToF-SIMS analysis
The ToF-SIMS analyses were performed using a J105-3D Chemical imager (Ionoptika Ltd, UK). The J105 is a nonconventional ToF-SIMS instrument that has previously been described in detail. 23,24 In short, the J105 uses a semicontinuous primary ion beam that allows for use of large clusters as primary ion projectile while also generating shorter acquisition time. The J105 used in this work is fitted with a 40 keV GCIB, and in this study, an ion projectile consisting of (CO 2 ) 6k + was Ar ð Þ n + or, as in this work, CO 2 ð Þ n + generates less fragmentation resulting in higher signal from molecular ions and higher mass species compared with using more traditional ion beams such as C 60 + . 25,26 The J105 utilises a buncher to bunch the secondary ions as they are injected into a reflectron ToF-analyser, thereby creating a time focus that makes this instrument less sensitive to topographical differences resulting in consistent mass resolution and improved mass accuracy.
Surface and depth analyses of the cells were performed in both positive and negative ion mode; however, here the focus will be on the data collected in negative ion mode where the fatty acid signals asso-  In our previous work where only stationary phase bacteria were analysed, it was also found that while the amount of FA(18:1) was just moderately affected by the deletion of the FabF gene, it appeared that the larger effect was found in the cyclopropanated fatty acid FA(cp19:0) signal. A significant depletion of FA(cp19:0) in the fabF mutant was found also in this work. In Figure 1, it can be seen how the signal intensity of this fatty acid is very low for both WT and the fabF mutant in the cells analysed in log phase. We can also see how both WT and the fabF mutant show a higher signal intensity of FA(cp19:0) in the cells cultured at 37 C, although the difference between the temperatures is clearer for WT due to the low signal found in the fabF mutant. Cyclopropanation of unsaturated fatty acids mainly takes place as the culture enters stationary phase. 32 Cyclopropanated fatty acids are produced by methylation, using S-adenosylmethionine, of cis-unsaturated fatty acids, that is, FA(16:1) or FA(18:1). 33 This could explain why a large effect is seen not only on the direct product of FabF, that is, FA(18:1) but also the product of a reaction where FA(18:1) is reactant, that is, The data indicate that, at the two temperatures used in this study, FabF plays a major role in the generation of FA(18:1) and is not only  Figure S5) and 2. in log phase cells, the signal from lipid A is highest at the very surface and then decreases as the membrane is eroded ( Figure S5). This is more clearly depicted in Figure 2a where the averaged signal for the log and stationary phase depth profiles has been compared. The data behind Figure 2 can be found in Figure S4. Comparing the absolute numbers ( Figure S5c Depth profiles of ECA CYC were generated showing how ECA CYC appears to be absent in log phase ( Figure S6). While the signal is increased with depth until a signal maximum is reached at approximately layer 50 (i.e., a PIDD of 4.61 × 10 12 ions/cm 2 ) ( Figure S6) in stationary phase. The increase is lower for WT cultured at 37 C, however, this strain has a higher overall signal ( Figure S6). No obvious signal maxima can be found in log phase ( Figure S6a,c). This finding is more clearly illustrated in Figure 2b where the average signals for the log and stationary phase data are compared. The data behind Figure 2 can be found in Figure S4. These results indicate that ECA CYC is not produced or transported to the periplasm in log phase but only in F I G U R E 3 (A) Schematic of elongation and cyclopropanation seen in wild type (WT) and the fabF mutant in logarithmic (log) and stationary (stat) phase. (B) Illustration of part of the bacterial envelope based on the findings of this work in log and stat phase stationary phase. Previous results have shown that stationary phase cells have a less permeable membrane and this is regulated by the RpoS sigma factor. 38 We hypothesise that the cyclic form of ECA is produced primarily in stationary phase and contributes to this strengthened impermeability. The RpoS dependence of this effect will be tested in future experiments.
A possible explanation for the temperature dependence of the cyclopropanation illustrated in Figure 1 is that the 30 C cultures grew slower compared with the ones cultured at 37 C and therefore An illustrative summary of the results from the study is shown in

| CONCLUSIONS
ToF-SIMS has developed to become a powerful technique for analysis of biological samples such as the bacterial envelope of E. coli. GCIBs, here coupled to a J105 instrument, have demonstrated once again that it is possible not only to detect high mass species and intact molecular ions on the very surface of a sample but also to depth profile them using GCIB ToF-SIMS.
A fabF knock-out mutant strain was analysed and compared with a WT strain. The cultures were grown at 37 C and 30 C and analysed in log and stationary phase to investigate the temperature and growth phase dependence, respectively, on some of the FabF specific findings of our previous publication. In the previous work by our group, it was found how the amount of FA(18:1) was not significantly reduced in the fabF mutant, even though it was expected to due to FabF's involvement in the production of FA(18:1) through elongation of FA(16:1). Instead, the larger effect was found on the cyclopropanated fatty acid, FA(cp19:0).
In this work, an effect on the amount of fatty acids were found in both FA(18:1), contrary to what was found in the previous work, and in FA(cp19:0), in line with our previous findings.
In depth profiles of the fabF mutant and WT, it was observed how lipid A, in log phase, showed a signal maximum at the first analysed layer compared with stationary phase where the signal maximum was found at approximately layer 15 (i.e., PIDD of 1.38 × 10 12 ions/cm 2 ). It was also found how ECA CYC was not present in log phase but appeared first in stationary phase. While this study showed clear trends that could be linked to biological processes, once improvements in the metrology concerning spot-to-spot signal variation and accurate measurement erosion rate are made, the approach may become applicable to detecting more subtle changes in bacterial membranes with improved relative quantitation.