Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in  Escherichia coli and many other bacteria, the gene encoding for RavA is part of a two-gene operon along with the gene encoding for the protein von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA belongs to the AAA+ family of ATPases, known for using the energy harnessed by hydrolysis of nucleotide triphosphates in remodelling molecular substrates (Regulatory ATPase variant A (RavA) is a chaperone conserved across several pathogenic clades of the Gammaproteobacteria . In Eschbstrates , 3. Likebstrates , 4, 5.E. coli RavA protomer (A): a AAA+ domain, a β-barrel–like LARA domain, and a discontinuous triple-helical bundle that links the AAA+ domain and the LARA domains (A). The LARA domain mediates the interaction of hexameric RavA with the dodecameric lysine decarboxylase enzyme, LdcI, involved in the bacterial acid stress response , which is a von-Willebrand factor A (VWA) domain. Biochemical studies showed that ViaA utilizes its VWA domain to mediate protein–protein interactions between RavA and its potential substrates  complex, an inner membrane–associated respiratory complex consisting of four proteins FrdABCD  to facilitate the interaction of RavA with Frd complex through the FrdA subunit , 12. WitE. coli , experimE. coli MG1655 was obtained from the American Type Culture Collection . The ravAviaA KO (ΔravAviaA) was previously constructed in our lab  of 0.4 units as measured using a Bio-Rad SmartSpec 3000 spectrophotometer. Cultures were then incubated at 18 °C for 16 to 20 h before being harvested and stored at −20 °C.The various RavA and ViaA constructs  were pro6-tag followed by a tobacco etch virus nuclear-inclusion-a endopeptidase (TEV) cut site, NusA-His12-tag followed by a TEV cut site, or His6-SUMO-tag as indicated in 6 or NusA-His12 were incubated with 0.1 mg/ml TEV protease, while those tagged with His6-SUMO were incubated with 0.01 mg/ml of ULP-1 protease. After protease incubation, protein samples were run through Ni-NTA affinity column a second time to separate the solubility/affinity tag fragments from the desired proteins as the tags were retained on the affinity beads. As the used TEV and ULP-1 proteases also contain H6-tags, they were also retained on the Ni-NTA beads. The resulting flow-through samples, containing the desired purified protein, were collected.Protein expression constructs contained at the N-terminus Hisprevious NTV (ViaA residues M1-H311), and NusA-CTV were further purified using fast-purification liquid chromatography as follows. Ion exchange chromatography with Mono S 5/50 HR column (Cytiva) was performed to further purify RavA. ViaA protein was separated from traces of NusA using the Hi-Trap Heparin HP column (Cytiva). The Superdex 200 10/300 column (Cytiva) was used for the previous NTV and NusA-CTV constructs. Elution fractions were collected and visualized using SDS-PAGE to verify purity. All proteins were stored at −80 °C in buffer containing 50 mM Hepes (pH 7.5), 200 mM NaCl, 1 mM MgCl2, 1 mM DTT, and 5% glycerol.RavA, ViaA, previous NTV), was dissolved in incubation buffer . The protein was diluted to a final concentration of 1 mg/ml for digestion with trypsin. Commercially available bovine trypsin (Sigma-Aldrich) was dissolved in incubation buffer and added to a final reaction concentration of 20 μg/ml. The digestion sample was incubated at 20 °C for 24 h. Postincubation, a fraction of the sample was dissolved in Laemmli buffer and visualized via SDS-PAGE. Following SDS-PAGE, a prominent band was visible, estimated to be <25 kDa when compared to the protein molecular weight markers. The band was isolated from the SDS-PAGE gel and submitted to a commercial vendor for Edman sequencing of the first five amino acids.The 311-aa long ViaA fragment  acetonitrile:water ratio and 0.1% trifluoroacetic acid. The solution was centrifuged for 1 min at 1000 rpm. The supernatant was collected. A 1 μl droplet of the collected solution was deposited onto a steel plate. One microliter of the posttrypsin-treated sample was mixed with the matrix solution. The sample was mixed with a pipette and allowed to air dry. Once the sample was dry, the steel plate was loaded onto the Bruker Microflex MALDI-ToF machine. The sample spectrum was obtained using a 20% laser power setting with a frequency setting of 200 Hz. FlexAnalysis software was used to generate the mass list and for spectrum analysis.A fraction of the remaining sample was assessed 2, 50 mM Hepes (pH 7.5), and 50 mM NaCl. Reactions were performed in triplicate, each in a volume of 150 μl in clear 96-well plate using the SpectraMax 340PC384 microplate reader. The absorbance at 340 nm was measured for a 10-min interval. The average and SD of the slopes were then calculated to obtain the ATP hydrolysis rate. The student’s t test was used to measure statistical significance and p-values < 0.05 were labeled as significant.The ATPase activity for various constructs of RavA were tested using the ATP-NADH coupled assay as previously described . The reaE. coli strain was streaked onto an LB-agar plate. After incubation of the plates at 37 °C for 16 to 20 h, three colonies were picked and each was inoculated into 5 ml of LB media. The cultures were grown overnight at 37 °C. The overnight cultures were diluted 100-fold into 3 ml of fresh LB media. The cultures were shaken at 150 rpm at 37 °C for 2 h till they reached an A600 absorbance of 0.4, measured using Bio-Rad SmartSpec 3000 spectrophotometer. Once the desired A600 was reached, the 3 ml cultures were spun at 5000g for 5 min to collect the cells. Cells were resuspended into 3 ml of fresh LB media in the presence or absence of 30 μg/ml of kanamycin. Cultures were then grown for an additional 9 h. To perform colony counts, 1 ml of culture was spun down, washed with, and resuspended in PBS buffer. Five-fold serial dilutions were performed for each culture using pH 7.5 PBS. The diluted samples were then plated onto LB-agar plates and allowed to incubate at 37 °C for 16 h prior to counting of colony-forming units. For each strain/condition, three colonies were selected at random and grown in LB-kanamycin media. These colonies were verified to be susceptible to the antibiotic when inoculated in LB-kanamycin media. The numerical data for each strain represents three colony-level replicates derived from one bacterial stock. The data are presented as means and SDs. The student’s t test was used to measure statistical significance and p-values < 0.05 were labeled as significant.Frozen stock of the relevant 15N/13C-labeled protein using the automated FMCGUI method (https://cyana.org/wiki/Index.php/Main_Page). The best 20 of 100 CYANA-calculated structures were refined with CNSsolve  was determined by NMR spectroscopy. NTV samples were prepared at concentrations ranging from 200 to 500 μM and buffered in 50 mM Hepes, pH 7.5, 100 to 200 mM NaCl, and 10 mM β-mercaptoethanol. Spectra were acquired at 313 K on Bruker spectrometers operating at 600 or 800 MHz, equipped with triple-resonance inverse cryoprobes. Initial partial resonance assignments were determined for I method  based onI method . ResonanI method , interfaI method . Peak piI method . TorsionI method . H-bond I method   . VAST caRavA and ViaA are functionally linked proteins , 13. We A). Unlike RavA, the structure for the ViaA protein has remained elusive. Based primarily on sequence alignment . However, structure prediction using AlphaFold  : a highly alpha helical N-terminal domain, a flexible middle domain (MD), and a C-terminal VWA domain.RavA forms a hexameric complex with high ATPase activity , 10, 12.Fig. 1/P0ADN0) , for exaA). The molecular weight of the generated fragment was confirmed by MALDI-ToF analysis . N-terminal EDMAN sequencing confirmed that the trypsin-stable domain spans residues 1 to 191 of ViaA. Therefore, in this work, we refer to residues 1 to 191 as the NTV, while residues 192 to 311 are referred to as the MD, and residues 312 to 483 as CTV (B). Note that, in our previous published work . Hence, we are redefining NTV in the current study.To further establish the domain arrangement of ViaA, the N-terminal 311-residue fragment of the protein was isolated and treated with trypsin. The trypsin digestion of the 311-residue fragment (∼35 kDa) led to the identification of a ∼23 kDa domain as shown on SDS-PAGE gel A. The mo3 as CTV B. Note tin vitro ATPase activity of RavA was tested in the presence of the different domains of ViaA. Figure 2C shows the results for the ATPase activity of full-length RavA protein in the presence of the three different ViaA domains. It should be noted that CTV was purified with an N-terminal NusA fusion protein as it could not be maintained in solution without the added solubility of the fused NusA. The NusA protein was purified and used as a control. There is a 3-fold increase in RavA ATPase activity in the presence of the full-length ViaA protein (C). When the isolated domains of ViaA were added, only MD led to an increase in RavA ATPase, similar to the effect of the full-length protein itself. NTV, NusA-CTV, and NusA had no effect on RavA ATPase (C). Therefore, MD appears to be the domain in ViaA responsible for the enhancement of RavA ATPase activity.Subsequently, the  protein C. When tA ATPase C. TherefA). D and E, show the ATPase activity for these constructs in the presence of the three ViaA domains. Unexpectedly, RavAΔLARA is more than 2-fold more active than the full-length protein (C vs. D). This could be indicative of the potential physical constraints or autoinhibitory activity imposed by the LARA domain in the full-length RavA. Despite the overall increased activity, the effect of the ViaA domains on the ATPase activity of RavAΔLARA presents trends similar to the full-length protein. RavAΔLARA ATPase activity is enhanced by ViaA and by the MD of ViaA (D). In contrast, the ATPase activity of the AAA+ domain, which lacks the triple-helical bundle and the LARA domains, is not influenced by ViaA or its domains (E). Note that the AAA+ domain of RavA has about half the ATPase activity of the full-length protein (C vs. E)To further localize the region of interaction between the two proteins, two additional constructs of RavA were tested: RavAΔLARA (RavA lacking the LARA domain) and RavA-AAA+ (residues 1–306 of RavA) A. Figure protein , C vs. D of ViaA D. In con domains E. Note t protein , C vs. ECollectively, the data of E. coli’s as well as other Gram-negative bacteria’s sensitivity toward antibiotics , a strain deleted of the RavA-ViaA (ΔravAviaA), a strain deleted of FrdABCD and RavA-ViaA (Δfrd ΔravAviaA). All the deletion strains lack resistance markers . The deletion of frd shows similar levels of persistence as WT (A). Importantly, though the deletion of ravAviaA increases persistence, the deletion of frd in addition to ravAviaA results in a strain having WT levels of persistence. Therefore, RavA-ViaA appear to play a negative role on the persistence of WT E. coli under these growth conditions. Furthermore, this influence of RavA-ViaA is observed only in the presence of the Frd enzyme, consistent with previous observations showing that Frd activity is reduced by the presence of RavA-ViaA .WT WT cells A. The dece as WT A. ImportB). Plasmids expressing RavA-ViaA or Frd under their respective native promoters were introduced into mutant strains missing either one or both protein complexes. Mock expression plasmid p11 was also introduced into the strains as negative control. It should be noted that the use of p11 plasmid led to a decrease in the basal level of persistence for the WT strain. This is evident from the comparison between the persistence of WT strain in Figure 3A and WT + p11 in Figure 3B. This decrease was seen across all the strains containing the p11 plasmid except for the ΔravAviaA strain that has enhanced persistence. While not further investigated, this could be due to the presence of the ampicillin resistance marker on the plasmid. The frd deletion mutant has near WT persistence and is not influenced by the introduction of Frd (B). The strain lacking RavA-ViaA is orders of magnitude more persistent than the WT strain. This increase in persistence is completely abrogated by the introduction of RavA-ViaA (B). This complementation is consistent with the observation that RavA-ViaA play a negative role in persistence. Finally, introduction of Frd but not RavA-ViaA into the triple mutant KO Δfrd ΔravAviaA was sufficient to increase persistence in the absence of RavA-ViaA (B). These results clearly indicate that RavA-ViaA are acting on Frd to modulate persistence of the cells.To verify the importance of the interaction between RavA-ViaA and Frd in this observed persistence phenotype, complementation assays were performed B. Plasmin of Frd B. The stavA-ViaA B. This cavA-ViaA B. These C). Initially, the ΔravAviaA strain was complemented with one of the following plasmids: p11 (mock expression plasmid), pRavAViaA (expresses RavA and ViaA), pRavA , pRavAmViaA (expresses RavA ATPase–deficient mutant K52Q and WT ViaA). In all plasmids, the proteins are expressed under the endogenous promoter of the ravAviaA operon. While the expression of RavA and ViaA in ΔravAviaA strain resulted in the reduction in persistence compared to that of ΔravAviaA + p11 strain, the expression of RavA alone or RavA ATPase–deficient mutant with WT ViaA in ΔravAviaA strain resulted in similar persistence level as that of ΔravAviaA + p11 strain (C). These results demonstrate that the presence of ViaA and ATPase active RavA are required for this persistence phenotype.Next, an assessment of the importance of the RavA ATPase activity on the effect of RavA-ViaA in this fumarate-dependent persistence phenotype was performed C. Initia1 strain C. These ravAviaA strain was complemented with RavA and ViaA or RavA and ViaA deleted of each one of its three domains  and PrDOS , while residues L169-L191 are disordered (A and B). Fig. S2B), which define their relative position and packing within the tertiary structure. Each of the helices also have corresponding minima in the random coil index plot . NTV exhibits a novel fold that has not been observed before (see below) that we name the VAN fold for ViaA alpha helical N-terminal domain fold.In order to verify the predicted structure of ViaA, we initially attempted to obtain the X-ray structure of the full-length protein as well as of NTV+MD fragment of the protein. However, our efforts were not successful. Subsequently, we concentrated on obtaining the structure of NTV using NMR spectroscopy see , which wFigure 4 M1-Q168 B, while sordered , A and BC). Interestingly, there is a negatively charged belt generated by glutamic acid residues 20, 21, 69, 149, 150, 153, 158, and 161 that runs around most of the middle of NTV. This might suggest a potential affinity to a positively charged surface of another protein or biomolecule, yet to be determined. On the other hand, there are far fewer positively charged sidechains available on the surface of NTV to form interactions with other molecules. Residues Lys38 and Arg41 (C), which are solvent exposed in both the NMR and AlphaFold structures, provide the highest concentration of positive charge on the surface of NTV. Another interaction site could be formed by a significantly hydrophobic groove formed by residues Pro86, Ile89, Val90, Leu92, Pro93, and Leu96 (C) on the solvent-facing end of NTV.The surface of NTV exhibits both positive and negative surface potential C. Interend Arg41 C, which nd Leu96 C on the D). In core 2, there is a cysteine residue (Cys76) that forms sulfur–aromatic (S–π) interaction with Trp132 that has S to ring centroid distance of 5.5 Å inclined at an angle of 75° from the aromatic ring plane. This geometry of the interaction is generally consistent with previous observations on S–π interactions in the core region of protein structures. In such protein cores, the S–π interactions display a broad distance distribution of 4 to 11 Å and angles ranging from 0 to 90°  (A). The Sp18 is a sperm protein which, due to amphipathic properties, displays fusogenic abilities  . This protein is known to form complexes with protein partners and assist in phosphotransfer relays. Interestingly, Hpt has an unusually high fraction of Met and Cys residues with several of the Cys residues involved in S–π interactions  A. The Spbilities . For fra B7FGU6)  . The NMR and AlphaFold structures show close backbone alignment throughout much of the length of the domain with the exception of residues lying at the very C terminal in NTV. The RMSD of Cα atoms is 3.96 Å for residues 1 to 167, with the strongest agreement between the two models in fragment 2, as exemplified by the lower RMSD value of 2.79 Å. The most salient differences between the two structures lie in fragment 1, which has an RMSD compared to the NMR structure of 4.70 Å (C). The models display a noticeable divergence in the direction of the bend at the midway point of helix α8, which runs through fragments 1 and 2 (see B). The difference in the orientation of helix α8 coincides with a disparity in the hydrophobic packing arrangement and relative orientations of the smaller helices α3 and α4. When the fragments 1 and 2 of the two models were aligned independently, the RMSD values were reduced to 3.31 Å and 1.23 Å, respectively, indicting greater similarity between the two fragments than the global alignment would suggest. The other significant difference between the two models appears in the region pertaining to residues 170 to 190, with AlphaFold of the full-length protein predicting this region to be alpha-helical, while the NMR-derived structure identified it as a random coil (A and B). This could be due to the possibility that this region of NTV is stabilized by interaction with CTV (B). Hence, the structural model of NTV predicted by AlphaFold is generally consistent with the experimentally derived structure, with the most significant differences corresponding to the lowest confidence levels within the NTV domain of the AlphaFold model. It will be interesting to learn whether these differences highlight an artifact in the computational modeling, a structural influence imparted onto the purified domain separated from the rest of the ViaA protein, or a degree of flexibility inherent in the protein structure.In Figure 5f 4.70 Å C. The mond 2 see B. The didom coil , A and Bwith CTV B. Hence,C–E clearly demonstrate that the binding between MD of ViaA and the triple-helical bundle domain of RavA results in the enhancement of the ATPase activity of RavA  that runs through both fragments. It is unlikely that these two fragments would fold independently into their native structures without the support of these interconnected interactions. As such, we believe that the full-length NTV constitutes a distinct folding unit. Since no structurally similar matches to the folded region of NTV could be found in the PDB we propose that VAN is indeed a novel fold.E. coli against antibiotics. Results show that the presence or absence of RavA-ViaA can have a significant influence on the level of antibiotic persistence . The persister counts not only increased for the ΔravAviaA strain but returned to WT levels once Frd was also deleted in the Δfrd ΔravAviaA knockout (A). The deletion of Frd produced a similar number of persisters as WT. Since we previously observed that RavA-ViaA inhibit the activity of the Frd enzyme  or a plasmid-expressing WT RavA and ViaA deleted of any one of its three domain (D) did not complement the persister phenotype of the ΔravAviaA strain. Also, a plasmid-expressing WT RavA and WT ViaA did not complement the persister phenotype of the Δfrd ΔravAviaA strain (B). These results clearly demonstrate that the persister phenotype depends on a direct interaction between RavA-ViaA and the Frd complex in LB-fumarate media. El Khoury et al. (It should be noted that our observations contradict the recent results of El Khoury et al. , who proet al. , we cleaet al. C or a ple domain D did notA strain B. These y et al.  used difet al. (Adding to the complexity of the RavA-ViaA function is the observation of the tight binding between RavA and LdcI, mediated by the LARA domain of RavA, to form a cage-like structure consisting of two LdcI decamers and five RavA hexamers  and S3. et al.  found maAll data are presented in the article. Materials are available from the corresponding author upon reasonable request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


An in situ gear test rig has been developed at the Institute of Polymer Technology (LKT) to characterize the deformation of plastic gears during operation. It analyses timing differences between following index pulses of rotary encoders on the input and output shaft. This measurement principle enables the continuous measurement of the elastic tooth deformation and permanent deformations and wear at operating speed by switching between a high and low torque. Gear tests using a steel-polybutylene terephthalate (PBT) gear set were performed at different rotational speeds and tooth temperatures to analyze the tooth deformation during operation. The results were compared to the calculated deformation according to gear design guideline VDI 2736. Moreover, the results of the gear tests were correlated with the results of a dynamomechanical analysis (DMA). Both, the DMA and the in situ gear tests show that the effect of temperature on deformation is much higher than the effect of frequency or rotational speed. However, the experimentally measured tooth deformation is significantly higher (up to 50%) than the calculated at lower speed. Thus, the check calculation according to VDI 2736 underestimates the actual tooth deformation at lower speeds. Therefore, the guideline should be adjusted in the future. Thermoplastic gears show many advantages compared to metal gears due to the specific properties of polymers. These are, for instance, noise reduction , dry runHowever, until now, the current design guideline VDI 2736  has not VDI 2736 Part 2 contains design and check calculation formulas for the determination of the load-carrying capacity of plastic gears. Apart from the tooth deformation calculation, there are also suggestions for the calculation of the tooth root load-carrying capacity, the frictional wear load capacity and the tooth temperature. Moreover, VDI 2736 Part 2 provides a check calculation formula for peak loads.According to VDI 2736 Part 2 , the teeThe tooth deformation λ depends on the nominal tangential force etime is :(2)λzul=VDI 2736 Part 1 [However, there is no data available for the elastic modulus measured at different frequencies. Moreover, VDI 2736 Part 1 [Further calculation methods for the tooth root stress due to the bending of the plastic teeth are described in the literature. These calculation procedures are adjustments to the Lewis Equation (3). According to the Lewis Equation (3), the strength of a gear tooth can be determined as shown below :(3)Ws=s·Walton and Shi  comparedIn conclusion, it can be stated that analytic calculations of the tooth deformation and the resulting root stresses can only simplify describing the actual tooth deformation of plastic gears during operation. Actual experimental tooth deformation data for validation is lacking.A simulative approach using finite element analysis  shows thExperiments for the tooth deflection and the rotational delay of the steel gear performed by Terashima, Tsukamoto and Shi  show thaHowever, the test setup used by Terashima, Tsukamoto and Shi  does notAs summarized, mostly analytical and simulative approaches exist in the literature. However, there are considerably fewer experimental studies, especially for tooth deformation during operation.In general, two main categories of in situ gear test rigs can be distinguished: test rigs for condition monitoring and test rigs for wear and deformation measurement. Condition monitoring aims for the efficient planning of maintenance and the mitigation of downtime due to failure of machine elements . These tSince condition monitoring test rigs typically do not directly quantify the underlying process of gear failure, such as deformation, there are in situ gear test rigs particularly designed for deformation and wear measurement.Tooth deformation and wear are measured by using tactile sensors , quantifConsidering the limitations of current in situ gear test rigs for measuring the tooth deformation during operation, a new testing principle has been developed at the LKT. The principle is described in The main components of the LKT in situ gear test rig  are a thThe motor on the input side drives the shaft on which the steel pinion is mounted. The loading torque is applied to the plastic gear by the hysteresis brake on the output side. Torque fluctuations due to vibrations during the test run and the related frequency spectrum on the input and output sides are measured for the pinion and gear separately by the torque transducers. For continuously tracking the tooth root temperature of the plastic gear, a thermocouple, type K, is placed in a drilled hole with a diameter of 0.6 mm in the tooth root of the plastic gear. Telemetry, type TEL1-PLM-IND, by Kraus Messtechnik GmbH, Otterfing, Germany, is used to transfer the temperature information to a data logging PC. The in situ wear and deformation measurement is based on the rotary encoders on the input and output shafts.t0 < t1), as shown in The measurement principle developed at the LKT is similar to a single-flank rolling test. However, instead of directly evaluating the angular displacement between pinion and gear, the index pulses of the rotary encoders on the input and output shafts are evaluated. This enables measurement at high operating speeds with high resolution. The idea behind the concept is that progressing wear and deformation of the plastic gear leads to a delay in the index pulse of the plastic gear on the output side. The steel pinion remains unchanged; thus, the time period between the input and output index pulses increases  was subtracted from the calculated tooth tip deformations at the different load levels. The temperature was set at 23 ± 2 °C and 80 ± 4 °C using compressed or heated air. For the calculations, the elastic modulus of the steel pinion E1 was assumed to be 210.000 N/mm2. The elastic moduli E2 of the PBT gear at 23 °C and 80 °C were graphically evaluated using 2 = 2700 N/mm2 at 23 °C and E2 = 650 N/mm2 at 80 °C were assumed for the elastic moduli.The results were compared to the calculation of the tooth tip deformation λ according to Equation (1). The used torque levels of the gear tests were converted to the corresponding nominal tangential forces of 25.6 N, 38.5 N, 51.3 N, 64.1 N, 76.9 N, 89.7 N and 102.6 N using the pitch circle diameter dThe DMA was performed under a very small torsional load. The measured deformation was 0.02%. A temperature sweep between 25 °C and 80 °C (in steps of 5 °C) and a frequency sweep between 0 Hz and 20 Hz (in steps of 1 Hz) were conducted to evaluate the thermal-mechanical behavior of PBT Ultradur B4520 in the relevant ranges.The calibration tests with the steel gear show increasing deformation with increasing load. Since the deformation is quite similar for each rotational speed, the average deformation was used for the correction of the results of the plastic gear tests. The measurement offset is a result of the deformation of the test rig components . g of PBT, and a rotational gear speed of 1000 min−1 the tooth tip deformation increases from 14.1 ± 2.6 µm at 0.50 Nm loading torque to 18.5 ± 5.3 µm at 0.75 Nm. At 1.0 Nm loading torque, the tooth deformation is 26.6 ± 12.6 µm. It increases further to 37.7 ± 5.8 µm at 1.25 Nm. After a slightly lower average deformation of 33.8 ± 5.8 µm at 1.5 Nm the deformation increases from 43.9 ± 1.4 µm at 1.75 Nm loading torque to 34.9 ± 7.7 µm at 2.00 Nm. Considering the standard deviations, the tooth deformation increases roughly linearly with increasing loading torque. At a tooth temperature of 80 °C above Tg, the tooth tip deformation at 0.50 Nm loading torque is 35.0 ± 4.1 µm. It increases up to 196.4 ± 32.4 µm at 1.50 Nm loading torque. At even higher torques of 1.75 Nm and 2.00 Nm the gears fail before the end of the test run due to tooth root breakage. However, microscopy shows no signs of initial cracks in the tooth root area at 32 × magnification after the tests at 80 °C and 1.50 Nm loading torque. The comparison with the calculation of the tooth tip deformation according to VDI 2736 (Equation (1)) shows very good consistency with the experimental data at 80 °C. At 23 °C, the calculated deformation is also quite similar to the experimentally measured deformation. The load-dependent increase rate is roughly identical. The calculated deformation is slightly lower, but mostly within the range of the standard deviations of the experiments.The results of the in situ gear tests under variations in rotational speed and temperature are summarized in −1 the average tooth tip deformation is 13.8 ± 7.5 µm, which is approximately the same as at 1000 min−1 rotational speed. However, it increases roughly linearly up to 62.9 ± 6.7 µm, which is approximately 50% higher than the calculated results and significantly higher than the deformation at 1000 min−1 gear speed. Moreover, the deformation is already within the range of the maximum allowed deformation λzul = 70 µm according to VDI 2736 part 2 (Equation (2)) at a loading torque of 2.00 Nm.At a rotational gear speed of 510 minThe outcome of the gear tests corresponds well to the results of the DMA . The infg (between 40 °C and 60 °C). The DMA results also show that the effect of temperature on the elastic material behavior is significantly higher than the effect of frequency, as shown in With increasing temperature, the torsion storage modulus G’, which corresponds to the elastic modulus, decreases severely, according to the DMA results. G’ increases with higher frequency. The effect of the load frequency on G’ is especially prominent at low frequencies between 0 Hz and 3 Hz and around the glass transition temperature T−1) G’ is at approximately 890 N/mm2 and increases only slightly to approximately 894 N/mm2 at 16.7 Hz (1000 min−1). The temperature increase from 23 °C to 80 °C causes a severe reduction in G’ from 894 N/mm2 to approximately 224 N/mm2.At 8.5 Hz  than at higher speeds (34.9 ± 7.7 µm at 2.00 Nm loading torque). In addition, the actual tooth deformation is significantly higher (roughly 50%) than the calculated one at lower speeds and high loads. In this case, the actual tooth deformation is already within the range of the maximum allowed deformation at high loads.Current and future research at the LKT focuses on the effect of the injection molding process on the crystallinity of the plastic gear’s teeth and the interaction with temperature- and frequency-dependent tooth deformation behavior. This research aims for an improved calculation formula for tooth tip deformation.


A protein fold is defined as a structural arrangement of a secondary structure in a three-dimensional space. It would be interesting to know whether a particular fold can be assigned to certain features of the corresponding folding/unfolding transitions. To understand the underlying principles of the manifold folding transitions in more detail, single-molecule FRET is the method of choice. Taking the two-domain protein phosphoglycerate kinase (PGK) as an example, we investigated denaturant-induced unfolded states of PGK using the above method. For this purpose, different intramolecular distances within the two domains were measured. In addition to the known two-state transition, a transition with a compact folding intermediate was also identified in each of the two domains. Based on the structural homology of the domains (characterized by a Rossmann fold) and the striking similarity in the features of the measured distance changes during unfolding, clear evidence emerged that the underlying domain topology plays an important role in determining the observed structural changes. When analyzing the folding of proteins, fluorescent dyes are typically attached at different positions in the protein so that single-molecule Förster resonance energy transfer (smFRET) can be used to measure intramolecular distances during folding/unfolding transitions ,2,3. EspTaking the two-domain protein phosphoglycerate kinase from yeast (yPGK) as an example, we aim to investigate the properties of distance changes within each domain under denaturing conditions. The PGK enzyme (415 residues) is composed of two structurally homologous domains, the N- and the C-terminal domains .The topology, i.e., the sequential order of the secondary structure elements and their spatial arrangement, is nearly identical for both domains and displays a Rossmann-like α/β/α sandwich fold ,12. As iHere, we prepared additional cysteine double mutants to measure corresponding denaturant-induced intra-domain distance changes within the N-terminal domain. Using such a more extended data set  from smFRET measurements, the goal was to identify regularities in the distance changes and understand them with respect to the underlying topology.Saccaharomyces cerevisea were produced as described in [E. coli using BL21(DE3) competent cells . DE3 lysogen expresses T7 RNA polymerase (RNAP) from the bacterial genome under the control of the lac repressor. Cells were grown at 37 °C in Terrific Broth medium (TB)  with shaking from OD600nm = 0.2 up to OD600nm = 1. Afterwards, protein expression was induced by addition of 1 mM Isopropyl s-D-1-thiogalactopyranoside (IPTG), and incubation was continued for 3 h at the same temperature. Finally, protein expression was stopped by harvesting cells at 6000× g for 15 min at 4 °C. The cell pellet was resuspended in lysis buffer  supplemented with cOmplete™ Protease Inhibitor Cocktail (1 tablet/50 mL) . Cells were opened using a cell disruptor by passing them 2 times at a pressure of 1.7 mbar to increase protein recovery. Cell lysate was centrifuged at 30,000× g for 45 min to remove cell debris. A salting-out step using Ammonium sulphate precipitation followed. First, the clear lysate was mixed with 40% (w/v) (NH4)2SO4  for 1 h at 4 °C. Non-precipitated proteins were recovered after centrifuging the solution at 5000× g for 45 min. The process was repeated with 60% (w/v) of (NH4)2SO4. Soluble proteins were further purified with Nickel-Nitriloacetic acid (Ni-NTA) affinity chromatography. Batch purification was performed using 5 mL of resin from a 50% (w/v) suspension of ProtinoR Ni-NTA agarose . Beads were equilibrated in lysis buffer before incubating them for 1 h with the protein solution. Two washes each with 5 column volumes (CVs) using incremental amounts of imidazole in lysis buffer, namely 10 mM and 20 mM, followed. Lastly, the protein of interest (POI) was eluted after passing 5 CVs of lysis buffer supplemented with 250 mM imidazole. The elution was dialyzed overnight in 400× volume excess of lysis buffer plus 1:10 (w/w) of TEV-Hisx6 protease (produced in house). TEV protease and uncut proteins were removed through reverse Ni-NTA affinity chromatography, as described above, where the protein of interest was recovered in the flow through. Finally, protein preparation was polished in lysis buffer with size-exclusion chromatography using preparative HiLoad 16/600 Superdex 200 pg . The presence of protein in the fractions of interest was confirmed by SDS-PAGE and Western blotting. The cysteine double mutants of the gene of phosphoglycerate kinase from ribed in . In addiThe labeling reaction was performed simultaneously for both positions by mixing equal parts of 10× final molar excess of maleimide dyes  with 10 µM yPGK in lysis buffer. Changes in the labeling scheme were used for some PGK variants by mixing different parts of donor and acceptor into the protein solution in order to maximize labeling yield. The reaction was incubated for 2 h in the dark at room temperature and continued overnight at 4 °C. Excess of free dye was removed by passing the labeling mixture through long Zeba™  dye and biotin-removal 2 mL spin column . Sample was kept at 4 °C until used in the confocal microscope.Fluorescently labeled samples were measured on a confocal fluorescence microscope Microtime 200 . First, the confocal volumes of each laser excitation (485 nm and 640 nm) were determined using standard fluorophores (Atto 655-NHS ester or Alexa488-NHS ester) with known diffusion coefficients. For this purpose, 30 µL of the sample solution was pipetted on a small cover slide of 22 × 22 mm with a thickness of 0.17 mm  on top of a water-immersion high-numerical-aperture objective . For single-molecule FRET measurements, 100 µL of 13 pM of fluorescently labeled sample were pipetted on a cover slide sealed with parafilm to reduce sample evaporation. Measurements were performed in PIE mode with a 20 MHz repetition rate . Laser pE>. The latter were used to determine the inter-dye distance withR0 represents the Förster radius which was determined for each measuring condition individually. The obtained values for R0 ranged between 51.8 and 53.2 Å; for details, see [Data from the measurements were analyzed with the PAM toolkit . Furtherils, see . Three-dils, see , and topils, see . In order to extend our understanding of denaturant-induced unfolding transitions in yPGK, we designed new cysteine double mutants for measuring intra-domain distance changes. Since we already analyzed three different distances within the C-terminal domain in a previous work see . In totaFirst, we observed the already often described two-state transition (TS), where a compact native state was more or less well separated from a less compact unfolded state. Here, the native state was gradually depopulated at the expense of the population of the unfolded state. The second type of transition was characterized by a compact intermediate state (CI). Remarkably, the compacted intermediate had an inter-dye distance that was smaller than that in the native protein structure. Under high denaturant concentrations (>1 M), the compact intermediate state disappeared more or less completely, and only a fully unfolded state remained. Finally, we can report on another unfolding transition which showed only one population with a largely unchanged inter-dye distance during the whole transition, here called “no transition” (NT). Since the C1-variant also unfolded very well during the transition in this case . Forn gives the number of residues and l is a persistence length [DA values for the unfolded state . Here, a transient compacting of the structure during the unfolding transition was assumed. Furthermore, additional refolding studies indicated that the compact intermediate state occurred not only during unfolding but also during refolding. A similar reversible unfolding/refolding behavior was already observed earlier for yPGK between the native and the fully unfolded state [The most remarkable aspect of the data obtained is given by the fact that the intermediate state was characterized by an inter-dye distance which was even smaller than that of the native state Red state . It shoued state . In thised state . Also, ied state ,24. The Finally, it is worth noting that for the three intra-domain distances within each domain, we obtained exactly the three types of unfolding transitions mentioned above. A more detailed discussion of this aspect follows in the subsequent subsection. In order to establish a possible link between the folding schemes found and the structural elements enclosed between the two dye attachment positions, we chose a special point of view of the yPGK structures involved. Here, the enclosed structural elements were highlighted and positioned within the corresponding domain for each variant . A closeEssentially, the different inter-dye distances measure structural properties in different parts of the protein and along different spatial directions. We can assume that the macromolecular structure of a protein with thousands of atoms shows a rather complex unfolding behavior on the atomic level. As a consequence, we observed part of this complexity (in terms of three different types of unfolding transitions) in the measured data because we sampled conformational changes with a sensitivity approaching the atomic level, here with smFRET with a spatial resolution in the nanometer to sub-nanometer regimes. The striking similarity in unfolding properties observed for the distances between the dyes within the N-terminal and C-terminal domains can best be understood by taking a closer look at the domain topology see .1/2, the intermediate states started to disappear at the expense of the unfolded state. As in Taking these facts into account, a comparison of the folding transition types  with theAll enclosed structure elements represent, at least a part, i.e., one to four strands out of the six parallel β-strands, of the Rossmann fold, in most cases sandwiched by the accompanied α-helices.Significant inter-dye distance changes between the different states  were only observed if one of the dye attachment positions was close to a further secondary structure element ). This condition was fulfilled for the N3-and C-3 variants (TS) and for the N1-and C2-variants (CI). Importantly, it was not fulfilled for the N2- and C1-variants (NT).Looking at the domain topologies, differences between the two-state and compact intermediate transitions seem to be related to which position within the Rossmann fold one of the two dye attachment sites was located in. A more upstream position of the dye attachment site appeared to reveal the compact intermediate, whereas a more downstream attachment site did not (position 1 versus position 34 for the N-variants and position 202 versus position 256 for the C-variants).These results indicate that in the compact intermediate transition, one β-strand (for the C-domain 281–286) or two β-strands (for the N-domains 128–132 and 135–139) was at a non-native  distance from the elements of the Rossmann fold. Thus, a wrong secondary structure packing seemed to occur transiently, which needed to be “unfolded” again before the correct structure could be formed (off-pathway intermediate), see for example . In the two-domain protein studied here, measuring six different intra-domain distances during the unfolding transition allowed the folding behavior of PGK to be studied in detail. Three distances each were measured in the N- and C-terminal domains. For all distances within a domain, different properties of the unfolding transitions were observed, which were able to be classified into three types of unfolding transitions. It was found that all three types were observed for each of the two domains. This result can certainly be explained by the large structural homology of the two domains. Our data suggest that a compact intermediate transiently appears in both domains during the folding/unfolding transition, but it is only visible in one of the variants  for each domain. Unfortunately, only the six variants presented here could be produced for our smFRET analyses . FurtheAnother important aspect that has also not been addressed in this study is the extent to which the presence of neighboring domains (in the case of multi-domain proteins) has an impact on the folding properties of a selected domain. An answer to this question can be obtained from studies with isolate N- and C-terminal domains (truncated versions of yPGK). Comparison with the results from studies with full-length variants may then improve the understanding of how inter-domain interactions contribute to the folding of individual domains.


While cooking yield increased in beef patties containing almond flour, shrinkage decreased. In addition, the cooking process caused decreases in L*, a*, and b* values. Using almond flour in beef patties decreased the SFAs and increased the amounts of oleic and linoleic acids. Hardness, cohesiveness, resilience, and chewiness were significantly affected by the use of almond flour (p < .01), and higher hardness and chewiness, and lower cohesiveness and resilience were determined in the samples containing almond flour compared to the control. On the other hand, the use of almond flour instead of animal fat in beef patties did not have a significant effect on the determined sensory properties (p > .05).Almond is rich in vitamins, minerals, and dietary fiber and contains high fat and protein. For this reason, almond flour can be used as an additive in the production of various foods to increase nutritional value, improve texture and flavor, and produce healthier products. The purpose of this study is to determine the availability of almond flour as an animal fat replacer in the production of beef patties. For this purpose, beef patties were produced in five different formulations containing animal fat and/or almond flour, and pH, moisture content, color, and TBARS values were detected in both raw and cooked samples. In addition, cooking yield and shrinkage were calculated and fatty acid composition, texture profile, and sensory analyses were performed on cooked samples. Replacing animal fat with almond flour increased pH in raw and cooked beef patties but decreased moisture content,  Consumers' interest in nutrition and health has led the meat and meat product industry to produce functional products, as in other branches of the food industry. In this study, almond flour was used as a fat replacer in beef patties to obtain a healthier meat product, and the physicochemical, textural, and sensory properties of beef patties were investigated. It was concluded that almond flour could be used in beef patties as an animal fat replacer. Fresh meat can be marketed as pieces or minced meat, or it can be processed into various products and offered for consumption. Beef patty, one of these products, is a meat product obtained by mixing minced meat, animal fat, and flavorings with some other additives and shaping this mixture into small portions. In this context, beef patty can be prepared and consumed in different compositions and forms throughout the world  and beef fat  obtained from middle‐aged beef carcasses that have completed the rigor‐mortis stage were purchased from a local butcher shop. Almond flour  obtained by grinding after peeling was purchased from a commercial company (Wefood). After beef meat and beef fat were minced separately through a 3‐mm plate with a grinder  and kept overnight in the refrigerator (4°C), five different groups of patties were produced. In the control group, 80% beef meat, 20% beef fat, and 1.5% NaCl were used. In other groups, beef fat was completely or partially replaced with almond flour  color measurements using a colorimeter  having a two‐standard observer, 8‐mm aperture and diffuse illumination. L* defines the color lightness (ranging from 0 for black to 100 for white), a* indicates the degree of color between red and green , and b* indicates color degree between yellow and blue . Color measurement was performed from both surfaces of beef patties. Thiobarbituric acid‐reactive substance (TBARS) analysis was performed according to Lemon  was used for texture profile analysis (TPA). Cylindrical samples (2 cm diameter and 1 cm thickness) extracted from beef patties cooled to 25–30°C were analyzed at room temperature with two consecutive compression cycles using a 50.8‐mm‐diameter cylindrical probe . One millimeter per second pretest speed, 2 mm/s test, and posttest speed, 3‐s recovery time, and 50% target strain were used in the analysis. Texture profiles of the samples  were calculated from the force‐time curves.2.4Sensory evaluation was conducted according to the procedures of AMSA  and IFT 2.5p < .05) (IBM SPSS Statistics 20). All data were given as mean values ± standard error in the tables and figures.Treatments and cooking process were taken as factors in the study, and experiments were conducted according to the completely randomized design. Two measurements were performed in the physicochemical analyses. However, six measurements were performed in the TPA analysis. The analysis of variance was applied to the obtained data (one‐way ANOVA), and differences between means were compared by Duncan's multiple comparison test at the 95% confidence level (33.1p < .01). The pH value of both raw and cooked beef patties increased with the use of almond flour instead of animal fat, and the highest mean values were determined in the T4 and T5 groups, in which the animal fat was replaced with almond flour at the ratios of 75% and 100%, respectively (p < .05). Similarly, Rajkumar et al. . However, the moisture contents in the cooked samples, ranging from 54.88% to 57.28%, were affected by the animal fat replacement significantly (p < .01). Moisture content in cooked samples decreased by substituting animal fat with almond flour (p < .05), but no significant change was observed after the 50% substitution level (p > .05). The decrease in moisture content of beef patties formulated with almond flour was probably the result of an increase in solid content due to almond flour having lower moisture content than animal fat. Some other authors also reported that replacing animal fat with oat bran . As the amount of almond flour used in production increased, the cooking yield increased, and the shrinkage decreased (Table p < .05). The lower cooking yield in control beef patties was probably due to the excessive fat separation and water release during cooking. The increase in cooking yield with fat substitution might be explained by the low fat in the samples and dietary fiber in almond flour. It was also reported that dietary fibers increased cooking yield and reduced shrinkage in meat products providing a high ability to keep moisture and fat in the meat matrix  while causing a significant change in the b* parameter (p < .05) in both raw and cooked beef patties. The b* value of the beef patties using almond flour was higher in the raw samples and lower in the cooked ones than the control , and significant differences were detected between the mean values (p < .05). Lower TBARS values were obtained in beef patties containing almond flour compared to the control for raw and cooked samples. This effect could be due to the natural antioxidants contained in almond flour. Indeed, it was reported that almonds contain a variety of antioxidant phytochemicals, including phenolic compounds and α‐tocopherol , it was not significant in the other groups (p > .05). This is probably because, as mentioned above, almond flour contains antioxidant substances. It was also reported in some other studies that the TBARS value of beef patties increased because of the cooking process . Although there was no significant change in the total monounsaturated fatty acids (MUFA), the oleic acid (C18:1) amount in the samples increased significantly (p < .01) with the use of almond flour, and the highest oleic acid amount was determined in the T5 group. This could be mainly due to the predominant oleic acid content of the almond flour. This result indicates that the nutritional and health values of beef patties are enriched as a result of using almond flour instead of animal fat. There were significant differences in the polyunsaturated fatty acids (PUFAs) among the groups (p < .01). The PUFAs were increased with the increasing levels of almond flour, and the highest amount of PUFAs was determined in the T5 group. This was probably due to the linoleic acid content of the almond flour. Already, a similar increase was detected in the amount of linoleic acid (C18:2) in the samples. However, the α‐linolenic acid (C18:3) amount decreased significantly with the increasing levels of almond flour. As a result, replacing animal fat with almond flour decreased the SFAs and increased the amounts of oleic and linoleic acids in beef patties. This is important for obtaining healthier products in terms of fatty acid profile. Rajkumar et al. , while adhesiveness and springiness were not (p > .05). Using almond flour increased the hardness and chewiness compared to the control; the highest values for these parameters were obtained in the group containing 100% almond flour instead of animal fat (T5). Textural properties of meat products strongly depend on the protein, fat, and moisture contents as well as the presence of nonmeat ingredients. In this study, it is thought that decreasing fat, increasing protein, and lower moisture contents due to fat replacement with almond flour caused higher hardness and chewiness in beef patties. In addition, the dietary fiber content of almond flour might be contributed to high hardness and chewiness. Dietary fibers are reported to increase the consistency of meat products by modifying the rheological properties . This remarkable result showed that using almond flour in manufacturing beef patties as a fat replacer did not cause a significant change in consumer demand. A similar result was also reported by Rajkumar et al. ; formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Ahmet Akköse: Conceptualization ; investigation ; methodology ; supervision ; writing – original draft ; writing – review and editing .This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.The authors declare that they do not have any conflict of interest.


The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains  and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes.Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of  Geobacter bacteria are predominant in several natural habitats due to their high respiratory versatility, including the use of either intracellular or extracellular terminal electron acceptors to generate the necessary metabolic energy from the degradation of organic compounds [Geobacter sulfurreducens, which can couple the oxidation of organic compounds to the reduction of insoluble metal oxides, some of them pollutants and radioactive [Geobacter cells in the fields of bioenergy and bioremediation [G. sulfurreducens results from the intricate electron transfer network involving c-type cytochromes, most of them containing multiple heme groups. Indeed, these cytochromes are found at the inner membrane, periplasm and at the outer membrane . Recenteme OmcE , the hexeme OmcE  and the eme OmcE .Geobacter cells encode for monomeric periplasmic c-type cytochromes with unusual high numbers of hemes, previously designated as nanowires of hemes [G. sulfurreducens genome—GSU2210 (with 27 hemes), GSU0592 and GSU1996 (both with 12 hemes) [G. metallireducens and G. uraniireducens [Geobacter cells, since continuous production of energy is warranted even when extracellular electron acceptors are exhausted, thus providing enough survival time for the bacteria to find new and appropriate environments [Contrasting with the polymeric assembly of mature cytochromes, the presence of which cannot be anticipated by genomic analysis, of hemes . Three w2 hemes) . Homologreducens ,17. The reducens ,14. Instreducens ,19. Thesronments .c-type cytochromes [The structure of the monomeric dodecaheme cytochrome GSU1996 (42 kDa) has been determined by X-ray crystallography . The hemochromes ,21. HoweLengthy cytochromes containing many heme groups are unquestionably important either for long-range electron transfer, as is the case of the bacterial electric conductive filaments OmcE, OmcS and OmcZ, or to function as capacitors as in the case of cytochrome GSU1996. These properties can be conceivably explored for practical biotechnological applications. Yet, this would require detailed knowledge of the functional proprieties of these nanowires.The functional characterization of multiheme cytochromes, including the determination of the heme’s reduction potential values, their modulation by the oxidation state of their neighbors (redox-interactions), as well as its modulation by the pH (redox-Bohr interactions) cannot be assessed by simple potentiometric or voltametric redox titrations. In fact, such approaches fail in discriminating the individual hemes and do not provide the necessary mechanistic information to properly describe the functional behavior of the redox centers in multiheme cytochromes (for a review see ). This c1H-nuclear Overhauser effect spectroscopy (NOESY) and 2D 1H-total correlation spectroscopy (TOCSY) or 2D 1H correlation spectroscopy (COSY) for samples in the reduced state [1H,13C heteronuclear multiple quantum coherence (HMQC) for samples in the oxidized state [c. The same approach was later implemented to assign these signals in a tetraheme cytochrome c3 from Desulfovbrio vulgaris [13C-enriched porphyrin samples have also been described in the literature to assist in the assignment of some heme substituents in monoheme cytochromes or to obtain diverse structural information [To distinguish the individual oxidation profiles of the hemes, it is first necessary to assign the heme proton signals. The most cost-effective manner to assign these signals encompasses the use of natural abundance samples. In these samples, it is usually sufficient to combine the information obtained from 2D ed state  or by coed state . In thised state . Althouged state ,30. Thered state ,31,32,33vulgaris ,28. Sincvulgaris ,34). Alt1H-NMR spectra for the dodecaheme cytochrome GSU1996 (~42 kDa) and triheme cytochrome PpcA (~10 kDa) are represented in The high number of heme groups and the concomitant high molecular weight of nanowire cytochromes considerably decrease the NMR spectral quality due to signal broadness. Consequently, the necessary assignment of the heme proton signals is compromised, as it is the subsequent strategy necessary for the characterization of the proteins’ functional mechanism. To illustrate the effect of the increase in the molecular weight on the spectra quality, the 1D Given the potential of nanowire cytochromes to be explored as capacitors, key components for bioenergy production and even to act as scaffolds for the next generation of biogenic electronic nanomaterials, efforts need to be undertaken to functionally characterize these biological systems. The modular deconstruction of nanowires of hemes and electric conductive bacterial filaments can conceivably be explored to assist in their characterization. As mentioned above, the number of hemes and the molecular weight of the full-length proteins will often impair their detailed functional characterization. Indeed, such characterization has been, to date, limited to cytochromes containing four heme groups. In the case of bacterial filaments, efforts should be directed toward the expression and biochemical characterization of their monomers. On the other hand, for monomeric nanowires of hemes, the expression of individual domains, for which characterization is relatively straightforward, followed by the characterization of different combinations of these domains, is a feasible strategy.In the present work, we used the monomeric dodecaheme cytochrome GSU1996 as a model to illustrate a strategy that can be explored in the future to characterize nanowire cytochromes at the microscopic level. We used a deconstruction-based biochemical approach in which the triheme domains (A–D) and hexaheme bi-domains (AB and CD) of the dodecahemic protein were independently produced. Enough protein was obtained for the individual domains C/D and bi-domain CD, which were then used to illustrate the methodology developed in this work. The successful assignment of the heme proton signals in the triheme domains C and D were then used as a guide to assign the signals from the hexaheme bi-domain CD. The presented strategy paves the way for a future characterization of the electron transfer mechanisms in periplasmic nanowires and electric conductive bacterial filaments.The cytochrome GSU1996 and respective domains were expressed and purified as previously described in the literature see .1H-NMR spectra of domains C, D and bi-domain CD are characteristic of low-spin c-type cytochromes in both the reduced and oxidized states , S = 0) and paramagnetic (Fe(III), S = 1/2) in the reduced and oxidized forms, respectively.The crucial step underlying the characterization of the functional mechanisms of multiheme cytochromes requires the assignment of the heme protonsto the specific hemes in the structure, namely the methyl groups. The 1D d states . The NMRcf. top and bottom panels in 209 and Met287 in domains C and D, respectively). The pattern for the NMR signals of axial methionine typically has a three-proton intensity peak at approximately −3 ppm and up to four one-proton intensity peaks in the same spectral region [Analysis of the more shifted signals in either the reduced or oxidized spectra of the three proteins show that the NMR signals in the hexaheme bi-domain CD closely follow the spectral distribution of each individual domain  the diamagnetic state of the hemes—ensuring that less signal broadening will be observed when the two domains are studied as a whole; and (ii) the very well-defined spectral regions covered by the heme signals according to their type—the only exception is the propionate groups, since they are more structurally variable.The NMR heme proton signals of domain C in the reduced state were previously assigned and are not discussed here (for a review see ). The da1H and 81H); 5–2.5 ppm for heme methyls  and 3–(−1.0) ppm for thiother methyls (32CH3 and 82CH3) (1H/32CH3 and 81H/82CH3) groups are scalar coupled. Thus, the first step of the assignment encompasses the identification of these pairs in the 2D 1H-TOCSY NMR spectra (1H-NOESY NMR spectra acquired with short mixing-times (50–100 ms). Meso protons 20H are only connected to two heme methyls (21CH3 and 181CH3) and meso protons 15H show no connections to heme methyls or thioether substituents  A [27,37, spectra B. Then, 1H-NOESY experiments performed with a mixing time in the range of 150–400 ms allowed for the observation of the long-range intraheme connectivities and further confirmed the assignment of the signals. It is worth mentioning that the mixing-time ranges should be considered as a guide and it is recommended that, in absence of previous knowledge of the systems, NOE buildup curves to assist the selection of the mixing time should be investigated. However, triheme cytochromes have been extensively studied by our group, including the abovementioned domain C  and theAfter assigning the heme signals, we then moved to their specific assignment in the protein structure. To achieve this, the observed chemical shifts were compared to those calculated from the crystal structure of bi-domain CD .I, 20HIII, 21CH3III, 121CH3I, 82CH3I and 82CH3IV . The RMSD of the 36 shifts was 0.10 ppm, with deviations of 0.02 (heme I), 0.04 (heme III) and 0.04 (heme IV). As in the case of domain C , the obsH3IV cf. .1H-NOESY spectra, which confirms that both crystal and solution structures are similar.The assignment of domain D heme signals was further tested by examination of the interheme NOE connectivities and their comparison with the distances obtained from the crystal structure of bi-domain CD. All NOE connectivities between protons up to 3 Å were observed in the 2D 1H-NOESY spectra of the bi-domain  [E. coli strains harbor the plasmid pEC86 containing the c-type cytochrome maturation gene cluster ccmABCDEFGH [The full-length cytochrome GSU1996, each triheme domain (A–D) and the hexaheme bi-domains AB and CD were expressed and purified as previously described ,47,48,49 JCB7123 , while AABCDEFGH . The strIn each case, the fractions with the protein of interest were pooled, concentrated and loaded onto a HiLoad 16/600 Superdex 75 column , equilibrated with 20 mM sodium phosphate buffer pH 8.0 with 100 mM NaCl. The presence of the desired proteins was confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue. Both chromatography steps were performed in an ÄKTA Prime Plus FPLC System .The triheme domains C and D, as well as the correspondent hexaheme bi-domain CD, showed the highest levels of expression. On the other hand, domain B was poorly expressed. For this reason, the triheme domains C/D and the hexaheme bi-domain CD were used to illustrate and validate the deconstruction-based biochemical strategy.2H2O (CIL), through ultrafiltration procedures with Amicon Ultra Centrifugal Filter Units (Millipore). Protein concentrations were determined by UV-visible spectroscopy with the specific absorption coefficient of the α-band at 552 nm determined for the reduced triheme cytochrome PpcA [2 and reduced in the presence of catalytic amounts of Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) [The buffer used in the last step of the purification was exchanged for 80 mM sodium phosphate buffer pH 8.0  in 99.9% ome PpcA ,52. Protborough) .1H-TOCSY and 2D 1H-NOESY NMR spectra were recorded with standard pulse techniques and with mixing times of 50 and 80/200 ms, respectively. The spectra were acquired with 4096 (t2) × 512 (t1) data points, with 256 scans per increment, covering a sweep width of 20 kHz. The 1H chemical shifts are reference to sodium dodecyl sulfate (DSS) at 0 ppm, by using the residual water signal as an internal secondary reference [TM software  and analyzed with Sparky .The NMR spectra were acquired on a Bruker Avance 600 MHz spectrometer at 288 K. To assist the assignment of the heme proton signals of domains C, D and CD in the reduced state, a series of 2D eference . All NMRThe ring-current shifts were calculated from the crystal structure of bi-domain CD  followinc-type cytochromes forming electric conductive filaments are involved in extracellular electron transfer pathways in which they can transfer electrons to long range distances to outside of the cells or act as cellular capacitors. While structural models for these full-length proteins have been successfully obtained by X-ray crystallography, or more recently by Cryo-EM, the determination of their heme redox properties and the concomitant deciphering of their mechanistic and functional properties remains elusive.Nanowires of hemes and the polymeric assembly of c-type heme groups. To date, detailed thermodynamic characterization, and hence, mechanistic information, have only been obtained for multiheme cytochromes containing up to four heme groups [The main reason for the lack of precise functional mechanistic information is explained by the high molecular weight and number of e groups . The same groups .The exemplified deconstruction-based strategy provides an effective tool by which to study the redox properties of the individual hemes in nanowires or electrically conductive filaments. In the case of nanowires of hemes with all redox centers connected by a unique polypeptide chain, as it is in the case of GSU1996, the expression of individual domains is an appropriate approach. In the case of electrically conductive bacterial filaments made of the polymeric assembly of cytochromes, as in the case of OmcE, OmcS and OmcZ filaments, the expression and characterization of their individual monomers can be similarly attained.


The modular design broke the pathway to synthesize γ-polyglutamic acid (γ-PGA) using CO2 and N2, attributing to the artificially constructed direct interspecific substance and electron transfer. So, the intracellular ATP and NADPH were enhanced by 69 and 30%, respectively, and the produced γ-PGA was enhanced by 104%. The strategy was further extended to produce a commercial antibiotic of bacitracin A. These achievements improve the selectivity and yield of functional polypeptides with one click by CO2 and N2, and also provide an innovative strategy for creating photosynthetic systems on demand.Developing artificial symbionts beyond natural synthesis limitations would bring revolutionary contributions to agriculture, medicine, environment, etc. Here, we initiated a solar-driven multi-organism symbiont, which was assembled by the CO 2 and N2.A solar-powered multi-organism symbiont system based on energy transfer is constructed to synthesize polypeptides from CO PCC6803 (Syn), N2 fixation module of Rhodopseudomonas palustris, γ-PGA synthesis module of Bacillus licheniformis, and the electron transfer module of conductive polymer [PFP: cationic poly(fluorene-co-phenylene) derivative] , and improve photoelectric efficiency by hybridization with biological systems , and for the nitrogen fixation module, R. palustris fixes N2 through nitrilase (N2ase) into dissolving NH4+. For the electron transfer module, conductive polymer PFP shortens the distance among species by aggregating them to form a conduction network. By expanding the light capture into the ultraviolet (UV) range by PFP, the photosynthetic efficiency and photogenerated electron’s ability of Syn and R. palustris are both enhanced. The photogenerated electrons are transmitted to B. licheniformis for synthesis module through PFP to improve the yields of intracellular adenosine triphosphate (ATP) and NADPH [reduced form of nicotinamide adenine dinucleotide phosphate (NADP+)]. B. licheniformis uses carbohydrates and NH4+ to synthesize γ-PGA through the metabolic pathway in a controllable and programmable manner. This method is easily extended to the synthesis of antibiotic bacitracin A using CO2 and N2 as carbon and nitrogen sources through DISET among organism species with different input instructions.The controlled synthesis of γ-PGA using COSyn, R. palustris, and B. licheniformis were −51, −37, and −44 mV, respectively, while their zeta potentials were all positively shifted to −23, −28, and −32 mV, respectively, after incubation with PFP (fig. S4A). The ITC results showed that the binding constants (Ka) of Syn/PFP and R. palustris/PFP were 2.59 × 105 and 2.15 × 105 M−1 , respectively. Hydrophobic fusion existed between positive entropy and positive enthalpy change , which demonstrated the hydrophobic action in the second half of the process. The Ka values of B. licheniformis/PFP in two stages were calculated as 1.56 × 108 and 6.61 × 106 M−1, respectively. The early stage of the interaction was the electrostatic interaction between positively charged PFP and negatively charged outer membrane of B. licheniformis. Then, the hydrophobic side chain of PFP was combined with the outer membrane of B. licheniformis through hydrophobic interaction. These results showed that PFP could stably bind upon the membranes of all three kinds/organisms by electrostatic and hydrophobic interactions.Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were performed to directly observe the interactions between the three organisms and PFP, respectively. After coincubation with PFP, the blue signals performed on the three organisms are from the fluorescent emission of PFP, and their surfaces were also much rougher than the PFP-free groups in SEM images (figs. S2 and S3). Zeta potential and isothermal microthermal titration (ITC) were measured to explore their binding modes. The zeta potentials of Syn and R. palustris, while PFP emission (400 to 550 nm) overlapped with absorption spectra of Syn and R. palustris , photochemical quenching coefficient (qP), and electron transfer rate were increased by 15.9, 19.0, and 15.9%, respectively, after binding with PFP. In addition, the increased ratios of Fv/Fm , Pl total (comprehensive performance index), ABS/RC (absorbed energy per reaction center), TRo/RC (trapped energy per reaction center), ETo/RC (energy used for electron transfer per reaction center), REo/RC (energy transported to PSI per reaction center), ABS/CSm (absorbed energy per unit area), TRo/CSm (trapped energy per unit area), ETo/CSm (energy used for electron per unit area), REo/CSm (energy transported per unit area), and Δ(Ro) (the efficiency of electron transfer to PSI). showed that the energy absorption, capture, and electron transfer chain efficiency of Syn/PFP were all enhanced than the free Syn  of PFP were complementary to those of the photosynthetic alustris . In the d 640 nm . It demooduction . The flufree Syn . Referrifree Syn , all theB. licheniformis could use the external electrons to produce γ-PGA, the improved generation and transfer of photoelectrons were subsequently studied by chronoamperometry. As shown in Syn/PFP electrode (0.92 mA/m2) and R. palustris/PFP electrode (0.53 mA/m2) exhibited more substantial photocurrent enhancement than the syn electrode (0.19 mA/m2) and R. palustris electrode (0.20 mA/m2) under white light illumination. As per the cyclic voltammetry of B. licheniformis and B. licheniformis/PFP shown in B. licheniformis was not disturbed by PFP, and Cyt c could also be worked as the medium for electron transfer from Syn and R. palustris to B. licheniformis. Moreover, Rct of organism electrodes of Syn, B. licheniformis, and R. palustris binding with PFP were respectively decreased from 125, 390, and 550 ohms to 110, 125, and 205 ohms  images shown in figs. S6 to S8, the observed blue fluorescent signal, coarser bacterial surfaces, and certain height of bulge on bacterial cells indicated that PFP was evenly distributed in the organism clusters (figs. S6 to S8). The closed connection between organisms would favor substance communication, and the effect of direct interspecific electron transfer was investigated by Kelvin probe force microscopy. As shown in fig. S9, the potential difference between the bacterial cluster with PFP and the substrate was 5 mV, which was much smaller than the control group without PFP (22 mV). It means that PFP with satisfying conductivity could contribute to the better direct interspecific electron transfer by forming a network of assembled organisms.Syn, B. licheniformis, and R. palustris were investigated by observing the growth situation. As shown in Syn was contained, while B. licheniformis and R. palustris could not survive on their own. Moreover, PFP have no adverse impact on the symbiont. According to the carbohydrate concentration in Syn could fix and convert CO2 due to the photosynthetic process. The calculation results showed that the carbohydrate of Syn alone was 242.9 mg/liter, and it was further increased by 14% to 275.7 mg/liter after adding PFP. For both B. licheniformis and R. palustris to reduce the carbohydrate generated by Syn, the carbohydrate concentration was decreased to 65 and 55.4 mg/liter in three-organism symbiont with or without PFP. It means that the consumed carbohydrate in three-organism systems was increased from 186.6 to 210.6 mg/liter after adding PFP. The other two photosynthetic carbon fixation units of Chlorella pyrenoidosa and Synechococcus were also respectively cocultured with B. licheniformis instead of Syn. However, they could not coexist with B. licheniformis because of the quorum sensing and interactions. The selected Syn was expected to form the symbiotic relationship for γ-PGA synthesis. In addition to the carbohydrate, the abundant ammoniums from R. palustris were another direct element for confirming the γ-PGA generation. Similarly, the NH4+ yield produced by photosynthetic R. palustris was increased from 7.3 to 11.4 mg/liter with the addition of PFP . Therefore, the acetic acid concentration in the Syn/B. licheniformis system decreased from 5.43 to 1.83 mM after the introduction of R. palustris. To sum up, Syn, R. palustris, and B. licheniformis, with mutually beneficial relationships, formed an artificial symbiotic system by PFP, where PFP could promote the carbon and nitrogen source generation, communication, and utilization for γ-PGA synthesis efficiently.Subsequently, the most important symbiotic relationship and certain influence of n of PFP . In addiSyn/B. licheniformis were respectively increased from 11.8 μA/cm2 and 1.82 μW/cm2 to 20.8 μA/cm2 and 4.78 μW/cm2 once PFP was added. The MFC of R. palustris and B. licheniformis with PFP performed a similar increased tendency, which was increased from 11.2 μA/cm2 and 0.52 μW/cm2 to 14.7 μA/cm2 and 1.95 μW/cm2. Meanwhile, all the power of MFCs was substantially decreased when the organisms were dead, and the electron transfer between Syn and R. palustris was hardly observed . The MFC of R. palustris and B. licheniformis increased from 2.30 to 4.65 μW/cm2 with the acetic acid due to the good ability of R. palustris/PFP to oxidize acetic acid (fig. S12). The increase of biocurrents depended on the enhancement of photogenerated electrons and the improvement of electron transport ability. PFP enhanced Syn and R. palustris photosynthesis to produce more photoelectrons. Meanwhile, conductive polymer could also form the conductive network, and it is beneficial for the efficient electron transfer between microorganisms and electrodes .The increased electron transfer between bacterial species by PFP was another reasonable factor for the more γ-PGA generation. So two-chamber microbial fuel cells (MFCs) were fabricated to determine the influence of PFP on direct interspecific electron transfer efficiency (fig. S11). The maximum current density and power of B. licheniformis was only about 10.7 mg/liter, while those in the symbiotic system of Syn/B. licheniformis and Syn/R. palustris/B. licheniformis were increased to 55.9 and 70.7 mg/liter. After assembly with PFP, the γ-PGA yield in Syn/B. licheniformis/PFP pair was further increased to 97.8 mg/liter and finally arrived to 144.2 mg/liter (104% increase) in Syn/R. palustris/B. licheniformis/PFP, although the B. licheniformis/PFP group had a similar concentration of γ-PGA as the B. licheniformis alone.The successful generation of γ-PGA in the constructed symbiont was verified by the standard glutamate monomer spacing of 129 in the mass spectrometry, and the advantaged symbiotic effect was further detailed by quantitative analysis in 2 conversion to NH4+ increased from 0.20 to 0.31% in our system, and that of the CO2 conversion to carbohydrate increased from 1.97 to 2.23%. The introduction of PFP increased the conversion rate of γ-PGA from 36 to 64%. The photosynthetic efficiency of artificial solar-powered symbiont system with the introduction of PFP increased from 0.71 to 1.43%, which further indicated that PFP could promote the photosynthetic efficiency and product selectivity of symbiont by expanding the light trapping range and accelerating electron transfer. To further expose the mechanism in the symbiont of multi-organisms and PFP, the expression levels of key genes related to CO2 fixation, N2 conversation, and γ-PGA synthesis were analyzed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The expression levels of rbcL and prk, which are the subunits of Rubisco and essential enzyme in the Calvin cycle, are up-regulated by 374 and 148%, respectively, in Syn after adding PFP  . R. palustris BNCC3366448 was purchased from Beina Chuanglian Biological Technology Co. Ltd. Toray carbon paper (TGP-H-090), Ag/AgCl (saturated in KCl solution), and platinum wire electrodes were purchased from Shanghai Chuxi Industrial Co. Ltd. Amplite fluorimetric NADP+/NADPH ratio assay kit 15264 and enhanced ATP assay kit were purchased from AAT Bioquest and Beyotime Biotechnology, respectively.All chemicals and solvents used in the experiments were bought from Acros, Sigma-Aldrich Chemical Company, or Beijing Chemical Works. PFP was prepared on the basis of the literature  and Autolab PGATAT302N . UV-visible absorption measurements were taken on a JASCO V-550 spectrophotometer. Fluorescence spectra were conducted with a Hitachi F-4500 spectrofluorometer equipped with a xenon lamp excitation source. SEM images were measured on a JSM 6700F SEM . AFM images were viewed with Bruker MultiMode 8. The optical source was produced by Xenon fiber optic lamp . Chlorophyll fluorescence parameters were measured by Handy PEA chlorophyll fluorimeter and FMS-2 pulse-modulated fluorimeter . The illumination intensity was regulated by a radiometer . The quantitative measurement of glutamate was performed with an HPLC III 400 MHz HD spectrometer (Waters 2535Q system). The acetic acid was measured by gas chromatography (GC)  equipped with a flame ionization detector (FID). The zeta potential was measured with Malvern Zetasizer Nano ZS90 (ZEN3600). CLSM images were viewed with a CLSM . Ultrapure water (resistivity >18.2 megohm·cm at 25°C) obtained from a Milli-Q water purification system  was used for all the experiments. All experiments and measurements were carried out at room temperature unless indicated otherwise.Synechocystis sp. PCC6803 (Syn). The cultured medium of R. palustris was K2HPO4 (1.0 g/liter), MgSO4 (0.5 g/liter), and yeast extract (10 g/liter). The pH of medium was adjusted to 7.4 with NaOH. Under the continuous irradiation of 1500 lux white light at 25°C, the light was cycled for 12 hours and dark for 12 hours. R. palustris required closed tubes filled with medium for culture. The conical flasks were shaken three times a day. The cultured medium of B. licheniformis CICC 10099 was K2HPO4 (0.5 g/liter), MgSO4 (0.24 g/liter), citric acid (2.0 g/liter), ferriamine citrate (0.5 g/liter), glycerin (20 g/liter), and agar (13 g/liter), and the pH was adjusted to 7.4 with NaOH. The medium and bottles were autoclaved for 20 min at 121°C and cooled to room temperature. The bacterial powder was dissolved in sterile water in suspension form and transplanted into a solid culture tube, which was placed diagonally at 30°C for culture. After the inclined surface grew into colonies, B. licheniformis were scraped into a liquid culture medium without agar and cultured at 30°C, 180 rpm for 24 hours. B. licheniformis were washed with 1× phosphate-buffered saline (PBS) phosphoric acid buffer solution (pH 7.4) and centrifuged at 7500 rpm for 3 min. Then, optical density at 600 nm (OD600) was adjusted to 1.0, and the mutagenesis was carried out by UV irradiation for 30 min. The bacterial solution (100 μl) was diluted 50,000 times and taken into the prepared bacterial plate. The bacteria stick was evenly coated and grown at 30°C for 48 hours. The sticky, smooth, and opaque colonies were selected to culture in liquid medium for 24 hours and preserved at −80°C with 30% sterilized glycerin as cryoprotectant. The cultured medium of B. licheniformis CICC 23642 was peptone (5.0 g), beef extract (3.0 g), NaCl (5.0 g), and agar (13 g/liter), and pH was adjusted to 7.0 with NaOH. The medium and bottles were autoclaved for 20 min at 121°C and cooled to room temperature. The bacterial powder was dissolved in sterile water in suspension form and transplanted into a solid culture tube, which was placed diagonally at 30°C for culture. After the inclined surface grew into colonies, B. licheniformis were scraped into a liquid culture medium without agar and cultured at 30°C and 180 rpm for 24 hours. The defined photosynthesis medium (DPM) was NaNO3 (5 g/liter), K2HPO4 (0.04 g/liter), MgSO4 (0.15 g/liter), citric acid (1.0 g/liter), ferriamine citrate (0.25 g/liter), EDTANa2 (1 mg/liter), and Na2CO3 (0.02 g/liter). DPM was sterilized by passage through a 0.2-μm surfactant-free cellulose acetate (SFCA) filter. The concentration of Syn and R. palustris was determined by OD730, and the concentration of B. licheniformis was determined by OD600.BG11 was used as the sterile medium of Syn/PFP or R. palustris/PFP electrode is as follows: 100 μl of PFP, OD730 = 2.0 Syn (100 μl) or R. palustris, and 40 μl of 0.5% Nafion were pipetted into the active section of carbon paper sequentially. The electrode was dried in air between settling of each liquid settled. Similarly, Syn or R. palustris electrode was prepared by adding 100 μl of deionized water, 100 μl OD730 = 2.0 of Syn or R. palustris, and 40 μl of 0.5% Nafion to the substrate. Syn, PFP, Syn/PFP, R. palustris, and R. palustris/PFP coated with carbon papers (1 cm2) were used as working electrodes. Pt and Ag/AgCl electrode were used as the counter and reference electrode, respectively. The electrolyte was PBS (pH 7.4), and the illumination intensity was 60 mW/cm2. The photocurrents were measured at the bias voltage of 0.2 V (versus Ag/AgCl) under periodic light (10 s) and dark (10 s). Cyclic voltammetries were measured in 0.01 M PBS by removing the oxygen (pH 7.4) solution at a scan rate of 0.1 V s−1. Linear sweep voltammetry was conducted with a CHI1040C electrochemical workstation at a scan rate of 50 mV s−1.Electrochemical measurements including chronoamperometry, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS) were measured with a standard three-electrode system. Photocurrents and cyclic voltammetry were conducted with a CHI1040C electrochemical workstation. The process of working electrode for Syn/PFP or R. palustris/PFP electrode is as follows: 100 μl of PFP (50 μM), OD730 = 2.0 Syn (100 μl) or R. palustris, and 40 μl of 0.5% Nafion were pipetted into the active section of carbon paper sequentially. The Syn, R. palustris, and PFP electrode were the control group. One hundred microliters of PFP (50 μM), OD600 = 2.0 B. licheniformis (100 μl), and 40 μl of 0.5% Nafion were pipetted into the active section of carbon paper sequentially. The B. licheniformis electrode was the control group. The electrolyte was 1 mM Fe(CN)64−/3− solution. EIS measurements were used with a standard three-electrode system. Pt and Ag/AgCl were used as the counter and reference electrode, respectively.EIS was measured with an Autolab PGATAT302N electrochemical workstation. The process of working electrode for B. licheniformis, Syn, and R. palustris samples were incubated with PFP solution (10 μM) at 10 min at 30°C. The free polymer was removed by centrifuging at 7200 rpm for 3 min. The obtained bacterial were washed with ultrapure water and then resuspended in ultrapure water for zeta potential measurements. The untreated B. licheniformis, Syn, and R. palustris were measured as control.The B. licheniformis, Syn, or R. palustris (200 μl) was added to the sample tank and stirred continuously with a blender, adding 50 μM PFP at regular intervals. During the test, the temperature was maintained at 25°C.The binding parameters were obtained by fitting the ITC curves.PBS, B. licheniformis (OD600 = 0.2), Syn (OD730 = 0.5), R. palustris (OD730 = 0.1), and B. licheniformis/Syn/R. palustris were incubated with PFP solution (10 μM) at 10 min at 30°C. The unbound PFP was removed by centrifuging at 7500 rpm for 3 min. The samples were dropped on clean silicon slices and allowed to evaporate at 25°C. After the specimens were dried, 2.5% glutaraldehyde was added for fixation overnight. When drying out in the air, samples were fixed with glutaraldehyde (2.5%) in ultrapure water for 8 hours. Ethanol was added in a graded series  followed by natural drying in the air. Last, the specimens were sprayed with platinum before characterization by SEM.B. licheniformis (OD600 = 0.2), Syn (OD730 = 0.5), R. palustris (OD730 = 0.1), and B. licheniformis/Syn/R. palustris were incubated with PFP solution (10 μM) at 10 min at 30°C. The unbound PFP was removed by centrifuging at 7500 rpm for 3 min. The mixtures were washed with deionized water twice and then mounted on a glass slide with a coverslip on top and examined with confocal lasers to excite PFP and Syn, which were 405 and 488 nm, respectively.Syn and Syn/PFP were measured by Handy PEA chlorophyll fluorescence meter and FMS-2 pulse modulation fluorescence meter. Syn and Syn/PFP (1.5 ml) were added to the sample bottle, and the photosynthetic parameters were measured. Then, after the dark adaption for 30 min, the PSII reaction center was fully opened, and FMS-2 and Handy PEA fluorometer were measured.The chlorophyll fluorescence parameters of B. licheniformis (OD600 = 0.2), Syn (OD730 = 0.5), and R. palustris (OD730 = 0.1) samples were mixed evenly and incubated with PFP solution (10 μM) at 10 min at 30°C. The unbound PFP was removed by centrifuging at 7500 rpm for 3 min. Then, 10 μl of suspensions was spread on a mica sheet. The samples were imaged with AFM, and the untreated mixed bacteria were imaged in the same conditions. In addition, to test the surface potential of the samples, it is necessary to coat the mica with conductive platinum before sample preparation.The B. licheniformis (OD600 = 0.1), Syn (OD730 = 0.1), and R. palustris (OD730 = 0.3) were diluted for the growth curve in graded PFP concentration series . Syn and R. palustris were cultured in an illumination incubator, with continuous irradiance of white light (1500 lux) on a cycle of 12-hour light and 12-hour dark. B. licheniformis was cultured at 30°C. The concentration of samples was determined with an Evolution 201 spectrophotometer.After the fermentation, the fermented liquid was centrifuged at 8000 rpm for 5 min to collect the supernatant, and anhydrous ethanol with three times the volume was added. The γ-PGA precipitate was obtained by lyophilization overnight and by centrifugation at 11,000 rpm for 10 min. The precipitation was dissolved into ultrapure water and centrifuged at 11,000 rpm for 10 min to obtain the supernatant as γ-PGA solution. The turbidity of γ-PGA reaction with cetyltrimethylammonium bromide (25 g/liter)–2% NaOH solution could be reflected by the absorbance of the reaction system, and then the content of γ-PGA can be calculated by the linear relationship between turbidity and γ-PGA concentration.B. licheniformis CICC 10099 or 23642 (OD600 = 0.2), Syn (OD730 = 0.5), R. palustris (OD730 = 0.1), Syn/B. licheniformis, B. licheniformis/R. palustris, Syn/R. palustris, and Syn/R. palustris/B. licheniformis were incubated with PFP solution (10 μM) for 10 min at 30°C. Untreated samples of various bacteria were used as controls. The coculture systems were cultured in an illumination incubator with DPM, with a continuous irradiance of white light (1500 lux) on a cycle of 12-hour light and 12-hour dark for 10 days at 25°C.3COOH solution was measured by GC and 1H-NMR in D2O. CH3COOH standard curve  was detected through GC. The amount of CH3COOH was determined using a GC equipped with a FID and a DB-WAX column. N2 was used as mobile phase. The initial temperature of the capillary column was 150°C, and the retention time was 2 min. In addition, the injection temperature and FID temperature were 200°C.Samples were taken from the six-hole plate and filtered through a 0.22-μm cellulose membrane filter. The CHSyn/PFP or R. palustris/PFP electrode is as follows: 100 μl of PFP (50 μM), OD730 = 2.0 Syn (100 μl) or R. palustris, and 40 μl of 0.5% Nafion were pipetted into the active section of carbon paper sequentially. The electrode was dried in air between each liquid settled. Similarly, the Syn or R. palustris electrode was prepared by adding 100 μl of deionized water, OD730 = 2.0 Syn (100 μl) or R. palustris, and 40 μl of 0.5% Nafion to the substrate. Syn, Syn/PFP, R. palustris, and R. palustris/PFP coated on carbon papers (1 cm2) were used as working electrodes. The carbon paper was incubated in B. licheniformis and B. licheniformis/PFP for 24 hours at 30°C to form the biofilm. The control group of dead bacteria was obtained by ultrasonic crushing, and other operations were the same as the above method. Pt and Ag/AgCl electrode were used as the counter and reference electrode, respectively. The electrolyte was PBS (pH 7.4), and the illumination intensity was 60 mW/cm2. The polarization curves of the cells by two kinds of bacteria were measured. The power curves were obtained by calculating the polarization curve.The process of working electrode for 2SO4). The mixture was boiled in a boiling bath for 10 min, and the absorbance at 625 nm was detected. The carbohydrate content was calculated via the standard curve (a series concentration of glucose solutions). NH4+ was measured by a Solarbio ammonium nitrogen kit.The cocultured samples were centrifuged at 8000 rpm for 10 min to obtain the supernatant. The supernatant (0.5 ml) was added into the 2.5-ml anthrone solution (1 mg/ml/80% HSyn (OD730 = 1.0) or R. palustris (OD730 = 1.0) was incubated with PFP (10 μM) at room temperature for 10 min. As control, 5 ml of cell resuspensions was added to equal volume of ultrapure water. B. licheniformis (OD600 = 0.2), Syn (OD730 = 0.5), and R. palustris (OD730 = 0.1) were mixed evenly and incubated with PFP solution (10 μM) for 10 min at 30°C. Untreated samples of various bacteria were used as controls. The prepared samples were measured under the light intensity of 10 mW cm−2 for 60 min. The samples (5 ml) were collected by centrifuging at 7500 rpm for 3 min. A total of 0.5 ml of cell lysis buffer was added, and ultrasound was performed for 30 min. The NADPH/NADP+ ratio and ATP were measured by an Amplite fluorimetric NADPH/NADP+ ratio assay kit and an enhanced ATP bioluminescent assay kit (Beyotime Biotechnology), respectively.The physically separated system was used by dialysis membrane. The physically separated setup was a semipermeable membrane between bacterial species. Five milliliters of The quantum yield was determined by comparison of the initial rate of products with the measured photon flux.m is the number of electrons for per mole product, C is the carbohydrate or NH4+ concentration, V is the total suspension volume, ϕph cm−2 s−1 is the photo flux, A is the area of illumination, t is the reaction time, and NA is Avogadro’s number.The photosynthetic efficiencies (PE) equation is as followsThe following calculation example is based on the dataV = 5 ml, ϕph = 1.144 × 1015 s−1 cm−2, A = 10 cm2, NA = 6.022 × 1023, t = 120 hours × 3600 s = 432,000 s, and m = 24 (carbohydrate) or 8 (NH4+).Syn, three nitrogen-fixing key genes of R. palustris, and three γ-PGA synthesis key genes of B. licheniformis) were designed (table S3). The housekeeping gene β-actin and S RNA16 served as internal control.SYBR Green I real time PCR was used to detect the mRNA transcription of target genes in the mixed samples of bacteria and algae. The specific primers of eight genes . The system was operated in isocratic mode using 35% water/60% methanol/5% acetonitrile as mobile phase at a flow rate of 1 ml min