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To test if AmiDwMel has β-lactamase activity, the protein was expressed in E. coli JM83 cultures supplemented with CENTATM and incubated for 16 h. Analysis of absorbance measurements showed an increase of λ405 from 0.48 (± 0.04 SEM) to 1.36 (± 0.15 SEM) in cultures containing the positive control E. coli ML-35 pYC in six independent experiments, indicating CENTATM hydrolysis (Figure 57). In contrast, cultures expressing AmiDwMel rarely increased from λ405 =0.4 (±0.03 SEM) to 0.48 (± 0.09 SEM) in eight independent assays.
Cultures expressing the empty vector control slightly increased from λ405 =0.4 (± 0.03 SEM) to 0.58 (± 0.07 SEM) in six independent assays. In conclusion, β-lactamase activity of AmiDwMel was not observed under the conditions tested.
Figure 57: β-lactamase activity assay of AmiDwMelin vivo. E. coli JM83 with AmiDwMel in pASK-IBA2C were induced with 200 ng/ml AHT. E. coli ML35-pYC constitutively expressing a periplasmic β-lactamase were used as a positive control, pASK-IBA2C (empty vector) served as a negative control. AmiDwMel data represent means from eight independent assays, the positive and negative control were tested six times. Error bars represent ± SEM.
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PBP3wMel consists of a predicted N-terminal transmembrane domain ranging from amino acids 1–34, but no signal peptide (Supplementary Figure 14).
PBP3 wMel ---MQALLKNKLRSLCFIVPLFIFYIIIIFR-IFSLTFDQLTT---SE 41 PBP3 E. coli MKAAAKTQKPKRQEEHANFISWRFALLCGCILLALAFLLGRVAWLQVISPDMLVKEGDMR 60
::. :: ** : * : ::: . : :: * *.. . PBP3 wMel NFRKDNIVHKQPDILDRNGVVIATNVPTTSLYIDATKVKNPESIAAQLCS-TL---HDLE 97 PBP3 E. coli SLRVQQVSTSRGMITDRSGRPLAVSVPVKAIWADPKEVHDAGGISVGDRWKALANALNIP 120
.:* ::: .: * **.* :*..**..::: * .:*:: .*:. :* ::
PBP3 wMel YKNL--YRVLTSEKKFAWIKRHLTPKELLAIKNAGVPGVNFDDDIKRIYPHSNLFSHVLG 155 PBP3 E. coli LDQLSARINANPKGRFIYLARQVNPDMADYIKKLKLPGIHLREESRRYYPSGEVTAHLIG 180
.:* . : :* :: *::.*. **: :**::: :: :* ** .:: :*::*
PBP3 wMel YTDIDGNGIAGVEAYISKN---NEQEKPIILSLDTRV 189 PBP3 E. coli FTNVDSQGIEGVEKSFDKWLTGQPGERIVRKDRYGRVIEDISSTDSQAAHNLALSIDERL 240
:*::*.:** *** :.* .: : : **:* *:
PBP3 wMel QSIVHEELTKAVRRYQALGGVGIVLNVRNSEVISMVSLPDFNPNLQNKAEDVQKFNRASL 249 PBP3 E. coli QALVYRELNNAVAFNKAESGSAVLVDVNTGEVLAMANSPSYNPNNLSGTPKEAMRNRTIT 300
*::*:.**.:** :* .* .::::*...**::*.. *.:*** . : . **:
PBP3 wMel GVYEMGSVLKYFTIAAALDANATKTSDLYD---VSTPITIGKYKIQDFHKSKIPKITVQD 306 PBP3 E. coli DVFEPGSTVKPMVVMTALQRGVVRENSVLNTIPYTIPYRINGHEIKDV--ARYSELTLTG 358
.*:* **.:* :.: :**: ...: ..: : : * *. ::*:*. :: ::*: .
PBP3 wMel IFVKSSNIGAAKIAVKLGIEKQVEYFKAMKLFSPLKIEIPEKSTPI--IPDKWSETTLIT 364 PBP3 E. coli VLQKSSNVGVSKLALAMPSSALVDTYSRFGLGKATNLGLVGERSGLYPQKQRWSDIERVT 418
:: ****:*.:*:*: : . *: :. : * . :: : : : : ::**: :*
PBP3 wMel ASYGYGIAVTPIHLAQTAAALINNGIFHNATLMLNK-RSIGEQIISRRTSREMRK-LLRA 422 PBP3 E. coli FSFGYGLMVTPLQLARVYATIGSYGIYRPLSITKVDPPVPGERVFPESIVRTVVHMMESV 478
*:***: ***::**:. *:: . **:: :: . **::: . * : : : .
PBP3 wMel AVTDGTGRKAKIKAYSIGGKTGSAEKVVDGKYSKDANIASFIGVLTMLDPRYIVLIAIDE 482 PBP3 E. coli ALPGGGGVKAAIKGYRIAIKTGTAKKVGPDGRYINKYIAYTAGVAPASQPRFALVVVIND 538
*: .* * ** **.* *. ***:*:** . : ** ** :**: :::.*::
PBP3 wMel PQGMHHTGGIIAAPIVKNIINRIAPILNVTPEM--- 515 PBP3 E. coli PQAGKYYGGAVSAPVFGAIMGGVLRTMNIEPDALTTGDKNEFVINQGEGTGGRS 592
**. :: ** ::**:. *:. : :*: *:
Figure 58: Amino acid alignment of PBP3wMel and E. coli PBP3. PBP3wMel (WP_010963147.1) shares 26 % identity to E. coli PBP3 (ARB43848.1). Conserved SXXK, SX(D/N) and K(S/T)G motifs found in PBP3wMel are written in bold letters, motifs which align to E. coli PBP3 are additionally framed in black. The predicted transmembrane domain is highlighted gray. * fully conserved residue; : conservation between groups of strongly similar properties; . conservation between groups of weakly similar properties.
3.5.2 Secondary structure analysis of PBP3wMel
To get a deeper insight into the putative active site serine of the SXXK motif, secondary structure analysis was performed. Here, the three conserved motifs were localized based on sequence alignments to E. coli. The SXXK motifs S107EKK and S339PLK are predicted to be located outside the active site (Figure 59). S445AEK is located in a β-sheet next to the K442TG motif, while S256VLK is found in an α-helix. Thus, one of these SXXK motifs might build the catalytic center together with S311SN (α-helix and loop) and K442TG (β-sheet). The gene pbp3 from wMel was cloned into pASK-IBA2C, transformed into different E. coli strains and tested in in vivo and in vitro activity assays.
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Figure 59: Secondary structure of PBP3wMel as predicted by Phyre2. S256VLK or S445AEK (blue) might, together with S311SN (green) and K442TG (red), build the catalytic center of the enzyme, while S107EKK, S339PLK are predicted to be located outside the active site (blue). The molecular structure was illustrated by Jmol.
3.5.3 Characterization of PBP3wMel and active site analysis in vivo
Since PBP3wMel is the homolog of E. coli PBP3, it was examined whether overexpression of PBP3wMelis sufficient to restore cell division in E. coli MCI23 at the non-permissive temperature of 42 °C. To test dependency on functional SXXK motifs, site-directed mutagenesis was performed. Substitution of S107, S256, and S339 to alanine and in vivo activity assays of these mutants were part of a master thesis (Ritzmann, 2016). Mutagenesis PCR of serine from the fourth SXXK motif S445 was part of this thesis resulting in a PBP3wMel S107A-S256A-S339A-S445A quadruple mutant (Figure 60). Successful amino acid substitution was confirmed by nucleotide sequencing of the PBP3wMel gene (Supplementary Figure 15).
Figure 60: Primary structure analysis of PBP3wMel in pASK-IBA2C after site-directed mutagenesis. Serine residues from SXXK motifs were substituted by alanine (red bold letters). The amino acid sequence is shown in single-letter code, the predicted transmembrane domain is highlighted in gray.
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PBP3wMel expression at 42 °C led to a mixed set of phenotypes of E. coli MCI23 (Figure 61A). Quantitative analysis of six independent assays showed that 66.7 % (± 27.5 SD) of observed cells expressing PBP3wMel were single and dividing cells, while 33.3 % (± 27.5 SD) were elongated. Neither single nor simultaneous mutation of the serines S107, S256, and S339 of SXXK motifs had an impact on PBP3wMel activity at 42 °C (Ritzmann, 2016). However, site-directed mutagenesis of all four SXXK motifs including S445 showed a decrease of PBP3wMel in vivo activity resulting in 21.2 % (± 14.3 SD) single and dividing cells and concomitant increase of elongated cells to 78.8 % (± 14.3 SD) (Figure 61B,D). Statistical analysis of cell size showed that PBP3wMel expressing E. coli were significantly shorter than the quadruple SXXK mutant and the empty vector control (Figure 61E).
Figure 61: Complementation assay to test for the ability of PBP3wMel and PBP3wMel active site mutants to rescue cell division in a temperature sensitive E. coli MCI23 mutant. E. coli MCI23 were grown in LB medium containing chloramphenicol until OD600=0.4. Cultures were induced with 100 ng/ml tetracycline and incubated for 120 min 42 °C. A) E. coli MCI23 expressing recombinant PBP3wMel are mainly short and dividing, while B) cells expressing PBP3wMel S107A-S256A-S339A-S445A are predominantly filamentous. C) E. coli MCI23 expressing the empty vector pASK-IBA2C have filamentous cells. Scale bars = 20 µm. D) Cell size of at least 960 cells from 30 pictures taken from six independent assays was measured by Image J. Boxes extend from the 25th to the 75th percentile of cell size distribution. The line in the middle of the box is the median. Whiskers represent 1st and 99th percentiles. Dots represents outliers. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s comparison post-hoc test, ***=P ≤ 0.001. E) Columns represent the mean ± SD occurrence of different phenotypes at 42 °C from five randomly chosen pictures from each of the six experiments.
Additionally, growth of E. coli was monitored to exclude that the different observed phenotypes resulted from potential cell arrests induced by protein induction. In three independent measurements, all cultures were growing exponentially after induction (Figure 62).
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Figure 62: Growth kinetics of E. coli MCI23 during periplasmic expression of PBP3wMel. Protein expression was induced at an OD600 of 0.5 with 100 ng/ml tetracycline. OD600 was measured every hour. Each point represents mean ± SD (n=3).
3.5.4 Aztreonam treatment of PBP3wMelin vivo
Aztreonam is a β-lactam with high affinity for PBP3 leading to arrested cell division and a filamentous phenotype (Weiss et al., 1997). To examine its effect on PBP3wMel activity, the antibiotic was added to exponentially growing E. coli MCI23 cultures at 30 °C. E. coli MCI23 overexpressing PBP3wMel were still able to partially maintain cell division in the presence of aztreonam indicating that PBP3wMel activity cannot be blocked by aztreonam (Figure 63A). In contrast, addition of aztreonam to E. coli MCI23 overexpressing the empty vector at 30 °C resulted in filamentous cells indicating inhibition of the native E. coli PBP3 activity and divisome function (Figure 63B). Analysis of six independent assays revealed that untreated cells expressing PBP3wMel or the empty vector had similar cell sizes at 30 °C (Figure 63C). In contrast, cells with the induced empty vector were significantly larger than cells expressing PBP3wMel in the presence of aztreonam.
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Figure 63: Cell division of E. coli MCI23 expressing PBP3wMel is not inhibited by aztreonam, a specific inhibitor of PBP3. E. coli MCI23 harboring either PBP3wMel or the empty vector were grown in LB medium at 30 °C containing chloramphenicol until an OD600 of 0.4. Cultures were induced with 100 ng/ml tetracycline and after 30 min, 8 µg/ml aztreonam were added. A) PBP3wMel overexpressing cells partially divide in the presence of aztreonam, while B) cells expressing the empty vector pASK-IBA2C are elongated. Cultures without induction or without aztreonam served as controls. C) Cell size of at least 787 cells from 30 pictures taken from six independent assays was measured by Image J. Boxes extend from the 25th to the 75th percentile of cell size distribution. The line in the middle of the box is the median. Whiskers represent 1st and 99th percentiles, dots represent outliers.
Statistical analysis was performed using Kruskal-Wallis test and Dunn’s comparison post-hoc test, ns =not significant, ***=P ≤ 0.001.
3.5.5 In vivo β-lactamase activity assay of PBP3wMel
As PBP3wMel was resistant to aztreonam in vivo (see chapter 3.5.4) and no penicillin-binding was observed in vitro (Ritzmann, 2016), a potential β-lactamase activity of this enzyme was examined. PBP3wMel was expressed in E. coli C43 cultures supplemented with CENTATM and incubated for 16 h. In six independent experiments, absorbance λ405 increased of from 0.48 (± 0.04 SEM) to 1.36 (± 0.15 SEM) in cultures containing the positive control E. coli ML-35 pYC in six independent experiments, indicating CENTATM hydrolysis (Figure 64). In contrast, cultures expressing PBP3wMel remained constant around λ405 =0.49 (± 0.05 SEM). Cultures expressing the empty vector control slightly increased from λ405 =0.4 (± 0.03 SEM) to 0.58 (± 0.07 SEM). In conclusion, no β-lactamase activity of PBP3wMel was detected under the conditions tested.
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Figure 64: β-lactamase activity assay of PBP3wMelin vivo. E. coli C43 carrying PBP3wMel in pASK-IBA2C were induced with 200 ng/ml AHT. E. coli ML35-pYC constitutively expressing a periplasmic β-lactamase were used as a positive control, pASK-IBA2C (empty vector) served as a negative control. Data represent means from six independent assays. Error bars represent ± SEM.
3.5.6 In silico modeling of PBP3wMel
In silico analysis predicted a binding of the β-lactam antibiotic cefoxitin at two serines (S256 and S445) of the four SXXK motifs (Figure 65). In vivo activity assays implied that SXXK motifs can substitute each other and recombinant PBP3wMel was not impaired by the β-lactam aztreonam. Thus, PBP3wMel might be functional in Wolbachia even in the presence of a β-lactam.
Figure 65: 3D structure of PBP3wMel bound to cefoxitin as predicted by 3DLigandSite. The residues S256, K259, F294, K296, S311, N313, Y369, K442, T443, G444, S445, M486, H488 putatively involved in binding to cefoxitin (green) are marked in blue. Arrows point to the active site serines S256 and S445 of the SXXK motifs predicted to be involved in binding.
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