Martin
Herzberg
a,
Dirk
Dobritzsch
b,
Stefan
Helm
b,
Sacha
Baginsky
b and
Dietrich H.
Nies
*a
aMolecular Microbiology, Institute for Biology/Microbiology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle/Saale, Germany. E-mail: d.nies@mikrobiologie.uni-halle.de
bPlant Biochemistry, Institute for Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle/Saale, Germany
First published on 24th September 2014
Zinc is a central player in the metalloproteomes of prokaryotes and eukaryotes. We used a bottom-up quantitative proteomic approach to reveal the repository of the zinc pools in the proteobacterium Cupriavidus metallidurans. About 60% of the theoretical proteome of C. metallidurans was identified, quantified, and the defect in zinc allocation was compared between a ΔzupT mutant and its parent strain. In both strains, the number of zinc-binding proteins and their binding sites exceeded that of the zinc ions per cell, indicating that the totality of the zinc proteome provides empty binding sites for the incoming zinc ions. This zinc repository plays a central role in zinc homeostasis in C. metallidurans and probably also in other organisms.
We applied a systems biology approach, connecting and integrating cellular pathways and processes involving transition metal ions, to understand bacterial zinc homeostasis.7 The beta-proteobacterium Cupriavidus metallidurans is adapted to high concentrations of transition metals, and is able to maintain its zinc homeostasis even under adverse conditions.8,9 This is accomplished by a battery of highly redundant metal cation uptake systems with only minimal cation selectivity. These operate in combination with heavy metal efflux systems that export surplus cations10 and transport e.g. Zn(II) from the cytoplasm or periplasm to the outside.11–13 The only known import system with some specificity for zinc in C. metallidurans is ZupT of the ZIP protein family {TC#1.A.35; TC, Transporter classification14,15}, which is a protein family ubiquitous from bacteria to humans.
In C. metallidurans ZupT is needed to provide zinc under conditions of low availability.10 While the C. metallidurans parent strain keeps its zinc content at 70000 Zn(II) per cell in growth media with nM zinc concentrations, ΔzupT mutant cells are only able to accumulate 20000 Zn(II) per cell.5 Other unspecific uptake systems may provide Zn(II) to C. metallidurans cells at moderate zinc availability,10,16 filling up the cellular zinc pool to about 125000 atoms per cell in the ΔzupT mutant and its parent strain.5 Although the number of zinc atoms per cell is similar in both strains, the ΔzupT mutant nevertheless suffers from several defects resulting from disturbed zinc homeostasis,5 indicating that ZupT is also required for efficient zinc allocation at higher zinc concentrations. Cells of a quadruple mutant devoid of the most important zinc efflux systems accumulate 250000 Zn(II) per cell when 10 μM Zn(II) is added to the growth medium. When more zinc is provided, these cells were not able to grow.5 Thus, 250000 Zn(II) per cell might be the maximum zinc content of C. metallidurans, 125000 Zn(II) the optimum adjusted by efflux systems, and 20000 Zn(II) the minimum.
Since the molecular entities responsible for adjusting the zinc concentrations to the above-mentioned number should be different in the ΔzupT mutant and its parent strain, we used a bottom-up quantitative approach to compare the proteomes of both strains. We identified the zinc repository in C. metallidurans, the container of its cytoplasmic zinc pool. This zinc repository buffers the incoming zinc and other transition metal ions, thereby mollifying the differences between the mutant and the wild type. Moreover, as the tip of the iceberg, the zinc depository is also involved in regulation of synthesis of the nickel-dependent hydrogenase in the ΔzupT mutant strain.
Out of the 5804 proteins in the theoretical proteome of C. metallidurans, 3469 (60%) were found in at least one experiment (Table S1, ESI†). No lower detection limit was evident although the percent deviation increased with the decrease in the number of individual proteins per cell (Fig. S1, ESI†). The quantities of each protein in the supernatant and solubilized ultracentrifugation sediment were summed up and the resulting sum was normalized to an overall number of 1.86 million proteins per cell, which was derived from the experimentally determined average protein content. This number was lower than 2.6 million proteins per E. coli cell, although E. coli has a 1.5-fold lower cell volume and should have lower dry mass.18–21 Alternatively, the number of proteins per cell was also calculated from the dry mass of 0.6 pg of the C. metallidurans cell derived from the cellular dimensions,20 50% protein in the dry mass, an estimated mean molecular mass of 40 kDa per average protein, all together resulting in 7.5 amol protein per cell. Multiplication with the Avogadro number gave 4.5 million proteins per cell. The actual in vivo number per C. metallidurans cell could thus be higher than the 1.86 million proteins per cell used for normalization. Out of the 1.86 million proteins per cell assumed, only 20000 were encoded by the plasmids in the comparison between the CH34 wild type strain and its plasmid-free derivative AE104 (8000 pMOL28 and 12000 pMOL30, 1.07% of the proteome, data not shown).
Name | Complex−1 | WT, cell−1 | Mut., cell−1 | Da | Description |
---|---|---|---|---|---|
a The number of proteins per AE104 cell (WT) or ΔzupT cell (Mut.) is shown plus the number of monomers per F1F0 complex (unknown for the c subunit, 10 is assumed, also unknown for AtpI) and the predicted molecular mass is in Da. “NeF”, never found in the proteome analysis. The numbers in italic font indicate down-regulation and in bold font up-regulation. | |||||
Rmet_3493, AtpC | 1 | 2930 ± 246 | 2976 ± 271 | 14958 | Q1LHL1 ATP synthase epsilon chain |
Rmet_3494, AtpD | 3 | 7664 ± 709 | 6955 ± 667 | 50120 | Q1LHL0 ATP synthase subunit beta |
Rmet_3495, AtpG | 1 | 2406 ± 139 | 2347 ± 487 | 32320 | Q1LHK9 ATP synthase gamma chain |
Rmet_3496, AtpA | 3 | 5312 ± 439 | 4656 ± 229 | 55410 | Q1LHK8 ATP synthase subunit alpha |
Rmet_3497, AtpH | 1 | 1892 ± 978 | 1624 ± 453 | 20753 | Q1LHK7 ATP synthase delta subunit |
Rmet_3498, AtpF | 2 | 1934 ± 177 | 1021 ± 354 | 17384 | Q1LHK6 ATP synthase b chain |
Rmet_3499, AtpE | 10? | 601 ± 87 | 104 | 9676 | Q1LHK5 ATP synthase c subunit |
Rmet_3500, AtpB | 1 | 778 ± 37 | 274 ± 171 | 30968 | Q1LHK4 ATP synthase a chain |
Rmet_3501, AtpI | ? | NeF | NeF | 18707 | Q1LHK3 ATP synthase I chain |
Name | WT, cell−1 | Mut., cell−1 | Description |
---|---|---|---|
a The number of proteins per AE104 cell (WT) or ΔzupT cell (Mut.) is shown; “NeF”, never found in the proteome analysis; “NF”, not found in this strain. The numbers in italic font indicate down-regulation and in bold font up-regulation of strain AE104 compared to CH34 and the AE104ΔzupT mutant compared to AE104. | |||
Rmet_3291, RpoA | 7174 ± 1044 | 7959 ± 1314 | Q1LI63 DNA-directed RNA polymerase subunit alpha |
Rmet_3334, RpoB | 4908 ± 340 | 5411 ± 962 | Q1LI20 DNA-directed RNA polymerase subunit beta |
Rmet_3333, RpoC | 4709 ± 128 | 4801 ± 1105 | Q1LI21 DNA-directed RNA polymerase subunit beta' |
Rmet_0857, RpoZ | 508 ± 287 | 569 ± 365 | B3R3N5, DNA-directed RNA polymerase subunit omega |
Rmet_2606, RpoD | 1927 ± 96 | 1521 ± 394 | Q1LK43 RNA polymerase sigma factor |
Rmet_4661, RpoD2 | 233 ± 70 | 1066 ± 814 | Q1LEA3 RNA polymerase sigma factor |
Rmet_0303, RpoN | 330 ± 29 | 332 ± 23 | Q1LRN7 Sigma-54 (RpoN) |
Rmet_0272, RpoH | 119 | NF | Q1LRR8 RNA polymerase sigma factor |
Rmet_2115, RpoS | 252 ± 160 | 200 ± 80 | Q1LLI2 RNA polymerase sigma factor |
Rmet_2425, RpoE | 273 ± 43 | 415 ± 198 | Q1LKM4 RNA polymerase sigma factor. ECF:RpoE cluster |
Rmet_3702, FliA | NeF | NeF | Q1LH02 RNA polymerase sigma factor |
Rmet_1120, RpoI | NeF | NeF | Q1LPC0 Sigma-24, ECF:FecI1 cluster |
Rmet_4499, RpoJ | 53 | 151 ± 56 | Q1LER2 Sigma-24, ECF:FecI1 cluster |
Rmet_4001, RpoK | NeF | NeF | Q1LG57 Sigma-24, ECF:FecI1 cluster |
Rmet_0597, RpoO | NF | NF | Q1LQU3 Sigma-24, ECF:FecI2 cluster |
Rmet_3280, RpoL | 279 ± 14 | NF | Q1LI74 Sigma-24, ECF:FecI2 cluster |
Rmet_5400, RpoM | 10 | 150 | Q1LC67 Sigma-24, ECF:FecI2 cluster |
Rmet_1648, RpoP | NeF | NeF | Q1LMU5 Sigma-24. ECF:RpoE cluster |
Rmet_4686, RpoQ | NF | NF | Q1LE78 Sigma-24, “ECF” cluster |
Rmet_0910, RpoR | 149 ± 3 | 150 ± 49 | Q1LPY0 Sigma-24, “ECF” cluster |
Rmet_3844, “SigJ” | NeF | NeF | Q1LGL4 RNA polymerase, Sigma-24 subunit, ECF subfamily |
The number of F1F0 complexes in E. coli is 3200 per cell22 in the composition α3β3γδε of the F1 part and ab2c10 of the F0 part. In C. metallidurans, 3000 F1 complexes were identified with a ratio of 2.0 ± 0.5:3.0 ± 0.8:0.9 ± 0.2:0.7 ± 0.3:1 normalized to subunit ε (Table 1) as a mean value of all experiments performed. Soluble proteins could be accurately counted and the ratio of the subunits was correctly obtained. Of the F0 complex, ab2 was found in the correct ratio but in 5-fold lower numbers compared to the F1 subunits, ab2cn = 0.2 ± 0.1:0.4 ± 0.2:0.1 ± 0.1 (Table 1). We conclude that membrane proteins were difficult to count, especially when they were small and multimeric such as the c ring.
Judged from the number of RpoB and RpoC subunits, C. metallidurans should contain 5000 RNA polymerase core proteins per cell (RNAP, Table 2). RpoA, present in two copies in the RNAP, was found in a ratio of 1.53:1. The values for E. coli were from 1500 in slow-growing cells up to 11400 molecules in cells with a doubling time of 2.5 h−1,23,24 with the growth rate determining the number of molecules and their deployment.25 A number of 5000 RNAP molecules per cell in the 1.5-fold larger C. metallidurans cell growing slower (μ = 0.23 h−1, Fig. S2, ESI†) than E. coli (doubling time 0.5 h−1) again was a fair approximation of reality. Although the ΔzupT mutant strain contains a high number of misfolded RpoC subunits in inclusion bodies,5 these additional copies were not found here and probably removed during the low touring centrifugation of the cell-free extract or not solubilized from the ultracentrifugation sediment (Table 2). The number of RpoZ, responsible for the quality control of the RNAP assembly,26 was only 540 copies. This protein thus did not remain associated with the mature RNAP complexes after assembly. The number of RpoD subunits for the main housekeeping sigma factor was 1720 in both strains, twice as high as in E. coli.27,28 Of the remaining 16 sigma factors, 5 were not found in this experimental series, among those 5 the flagellum sigma factor FliA and RpoI were responsible for siderophore biosynthesis in C. metallidurans.29,30
A number of 51 ribosomal proteins were reproducibly identified in cells of strain AE104 and the ΔzupT mutant with numbers between 140 per cell and 9560 per cell with not many differences between the strains (Fig. 1). The mean value of all ribosomal proteins was 5701 ± 774 per cell, however, some of the smaller ribosomal proteins gave low copy numbers (Fig. 1). When only the 25 largest ribosomal proteins (>14 kDa) were counted, the result was 7182 ± 905 ribosomes per cell. E. coli is slightly smaller but grows faster than C. metallidurans and would contain 6800 ribosomes per cell at a growth rate of 0.6 h−1.19
This all indicated that the individual protein numbers obtained were fair approximations of the in vivo numbers and their stoichiometry.
The ΔzupT mutant strain and its parent differed only in the lag phase of growth (Fig. S2, ESI†) and consequently no difference between the proteomes of both cells was evident at the top KO level (Fig. 2 and Table S2, ESI†), indicating the presence of some zinc-buffering system in mutant and parent cells that mollified the global effect of the zinc starvation. Despite this, the ΔzupT mutant cells synthesized 40651 proteins involved in hydrogenase formation and Calvin cycle enzymes that were not found in the parent strain AE104 (Table S3, ESI†).
In contrast, (i) the glycerophospholipid and glycerolipid metabolism; (ii) chemotaxis, synthesis of the flagellum, components for twitching motility and of pili; (iii) two-component regulatory systems, and (iv) transport proteins were at lower abundance in the ΔzupT mutant (Table 3). The periplasmic CuZn superoxide dismutase SodC (Rmet_2757) was also found in lower abundance in the ΔzupT mutant cell (164 ± 43 proteins in AE104, 98 ± 12 in ΔzupT), explaining the lower tolerance of the ΔzupT mutant to reactive oxygen species.5
Name | WT, cell−1 | Mut., cell−1 | Description |
---|---|---|---|
a The number of proteins per AE104 cell (WT) or ΔzupT cell (Mut.) is shown. The numbers in italic font indicate down-regulation and in bold font up-regulation. “NF”, not found in one strain. “NeF”, never found in all strains. | |||
Secondary uptake systems | |||
Rmet_2621, ZupT | NeF | NeF | Q1LK28 zinc/iron permease |
Rmet_1973, PitA | NeF | NeF | Q1LLX4 phosphate transporter |
Rmet_3052, CorA1 | 117 ± 8 | 50 ± 8 | Q1LIV2 magnesium and cobalt transport protein CorA |
Rmet_0036, CorA2 | 38 ± 19 | NF | Q1LSF4 Mg2+ transporter protein, CorA-like protein |
Rmet_3287, CorA3 | 37 ± 5 | NF | Q1LI67 Mg2+ transporter protein, CorA-like protein |
Rmet_0549, ZntB | NeF | NeF | Q1LQZ1 Mg2+ transporter protein, CorA-like protein |
Primary uptake systems | |||
Rmet_2211, MgtA | 51 | 44 | Q1LL86 ATPase, P-type |
Rmet_5396, MgtB | NF | 104 | Q1LC71 ATPase, P-type |
Efflux systems/CDF | |||
Rmet_0198, DmeF | NeF | NeF | Q1LRZ2 cation diffusion facilitator family transporter |
Rmet_3406, FieF | 42 ± 5 | NF | Q1LHU8 cation diffusion facilitator family transporter |
Efflux systems/P-type | |||
Rmet_4594, ZntA | 396 ± 288 | NF | Q1LEH0 zinc translocating PIB2-type ATPase |
Rmet_2303, CadA | 82 | NF | A7HYL0 cadmium translocating PIB2-type ATPase |
Rmet_3524, CupA | 75 ± 12 | NF | Q1LHI0 copper translocating PIB1-type ATPase |
Rmet_2379, CtpA1 | NF | NF | Q8GQ88 copper translocating PIB1-type ATPase |
Rmet_2046, RdxI | NF | 516 | Q1LLQ1 copper translocating PIB1-type ATPase |
Efflux systems/RND | |||
Rmet_5319, ZniA | 76 ± 42 | NF | Q1LCE8 heavy metal efflux pump HME3a group |
Rmet_4123, HmyA | 182 ± 112 | 55 | Q1LFT6 heavy metal efflux pump HME3c group |
Efflux systems/ABC | |||
Rmet_0391, AtmA | 38 ± 3 | NF | Q1LRE9 ABC transporter-related protein |
With 15000 copies per cell, the elongation factor Tu was the most abundant protein, followed by the GroEL chaperonin (11000), ribosomal proteins, alkyl hydroperoxide reductase and citrate synthase (Data base, ESI†). The second and fourth most abundant proteins, however, were surprisingly two periplasmic binding proteins for citrate and related compounds (Rmet_0521, 13000 per cell) and branched-chain amino acids (Rmet_2480, 11000), respectively. These numbers were not different between mutant and parent strains.
Half of the predicted zinc-binding proteins in the strain AE104 belonged to the KO group Genetic Information Processing and here mainly zinc-binding ribosomal proteins (30000) and the RNA polymerase subunits (17000) are included. A smaller number (37.5%) were metabolic proteins, mainly involved in amino acid metabolism (19000) especially of methionine (11000), energy metabolism (7500) including carbonic anhydrases, and carbohydrate metabolism (5800). 12% of the proteins were not assigned to a KO group in the database, including the translational elongation factor Tsf (4300) and the putative metal-binding proteins CobW2 and CobW3, zinc-containing alcohol dehydrogenases and proteases (Table S4, ESI†). Since (i) the number of zinc-binding proteins was between 110000 and 120000, (ii) some of these proteins contain more than one zinc-binding site per monomer, and (iii) the assumed 1.86 million proteins per cell used for normalization was the lower limit of protein per cell, the number of zinc-binding sites in C. metallidurans AE104 and its ΔzupT mutant is higher than the number of 70000 zinc atoms per cell.5
In contrast, cells of strain AE104 contained 1302 ± 169 putative Co(II)-binding proteins per cell, and the mutant strain 1035 ± 413 (Table S4, ESI†). The number of cobalt atoms per cell was 3760 in cells of AE104 cultivated without added cobalt and 6290 in ΔzupT cells under the same conditions. C. metallidurans AE104 cells contained more cobalt atoms per cell than the known cobalt binding sites. Nevertheless, the strain could accumulate about 124000 Co(II) when 50 μM cobalt chloride was added,5 100-fold more than that available for cobalt-binding proteins. The strain AE104 contained 1000 nickel binding sites per cell but the ΔzupT mutant contained 11500 due to the increased number of hydrogenases (Data base, ESI†). The number of nickel atoms in strain AE104 was 5300 per cell.5,10 For the ΔzupT mutant the two determinations with different washing procedures resulted in 2500 ± 330 nickel atoms5 and 12500 ± 5000 nickel atoms,10 which are in agreement with the number of nickel-binding sites found. There were more zinc binding sites in C. metallidurans cells than the measured zinc ions but more cobalt and nickel ions are present than predicted by the specific binding sites for these metals when the cells were cultivated in medium without added metals.
Thus, C. metallidurans cells may contain a zinc repository formed by the totality of all zinc binding sites of its cytoplasmic proteins. According to this hypothesis, the repository is able to accommodate at least 110000 zinc ions but probably many more. The E. coli ribosome bound 8 equivalents of zinc per ribosome that was required for synthesis of zinc-containing proteins,43 and between 2000 and 70000 ribosomes, depending on the growth conditions.44 These ribosomes could bind 16000 to 560000 zinc ions. In rapidly growing E. coli cells, the number of zinc binding sites solely provided by the ribosomes would exceed the number of 114000 zinc atoms measured.10 In C. metallidurans, 8 zinc atoms per ribosome would lead to 56000 zinc ions instead of 30000 bound to the 7000 ribosomes in each cell. This alone would raise the number of binding sites to 140000. In contrast, the strain AE104 contains 70000 zinc atoms at the middle of its exponential phase of growth when only nM concentrations of zinc were added, which fills up the repository only by half. The ΔzupT mutant contains only 20000 zinc atoms per cell under these conditions, less than 1/7 of the repository is filled. Since ribosomes need the available zinc for efficient synthesis of zinc-requiring proteins,43 this explains the inefficient folding of the RpoC subunit of the RNA polymerase5 and the gratuitous synthesis of the nickel-containing hydrogenases by HypB and the zinc enzyme HypA.
When 100 μM Zn(II) were added, both strains contain 125000 zinc atoms per cell, and the repository starts to fill up. In a titration experiment, the number of zinc ions per cell increased with the external zinc chloride concentration from 50 to 100 μM but the cellular zinc content was saturated at 125000 zinc atoms per cell in strain AE104 (Fig. 3) up to 150 μM. Only mutants with deletions in zinc efflux systems contain more zinc atoms per cell, 250000,5 indicating a saturated or overflowing repository, and these cells are not able to tolerate more zinc. This indicates that the repository also serves as a counterbalance of the transport systems. Finally, 50 μM Co(II) also leads to 124000 cobalt atoms per cell,5 and similar numbers have also been observed for copper ions and even gold complexes (Wiesemann and Nies, unpublished), indicating that the zinc repository might also initially accommodate other transition metal cations until they can be suitably allocated or exported again. Moreover, misregulation of hydrogenase synthesis also indicates that allocation pipelines for other divalent transition metal cations such as Ni(II) start at the zinc repository, and that comparison of those ions with Zn(II) could be an essential part of the identification process.
Fig. 3 Titration of the cellular zinc content with the external zinc concentration. The cellular zinc content of strain AE104 (closed circles) and its Δe4 deletion strain (open circles) that has the four most important zinc efflux systems deleted (ΔzntA ΔcadA ΔdmeF ΔfieF) were cultivated in Tris-buffered mineral salt medium containing various concentrations of added zinc chloride, and the cellular zinc content was determined by ICP-MS as published.5 |
The hypothesis of the zinc repository predicts a high number of unsaturated metal-binding proteins in C. metallidurans. These binding sites might be required for rapid binding of transition metal cations in a tetrahedral complex, using only the 4sp orbitals and no 3d orbitals of the respective metal for binding to keep this process high-rate albeit of low specificity. These metals could be just sequestered or even act as metal cofactors. Indeed, enzymes with a low metallation quotient in vivo have been described.45 Alternatively, some enzymes may be promiscuous with regard to their metal cofactor4 or use in vivo Fe(II), which is present as 750000 Fe atoms per cell in strain AE104 (ref. 5) but renders the respective enzyme strongly sensitive to H2O2.46 It is, however, unclear at this stage if iron also needs the zinc repository for initial binding and sorting. C. metallidurans uses nearly no manganese, does not contain a Mn-dependent superoxide dismutase and no MntH uptake system to limit the import of cadmium as one pre-requisite of its cadmium resistance.10
Regulatory zinc binding sites responsible for flux control of transport proteins2 or for transcriptional control such as ZntR1 but also primary substrate binding sites of efflux systems can be pictured as part of the zinc repository, which also explains the zinc content of bacterial cells despite the femtomolar sensitivity of zinc regulatory proteins.1 Incoming zinc is immediately accommodated by empty binding sites of the repository, traverses down the affinity gradient from loosely to more strongly bound as shown for copper,47 thereby filling up the levels of tightly bound zinc first, e.g. in the interior of the proteins such as RpoC {PDB: 4IGC48}. With an increase in the filling levels, regulatory sites and the substrate binding sites of efflux systems are being filled, leading to a decreased import and an increased export of zinc, which keeps the number of zinc atoms in the cytoplasm at the same level as observed (Fig. 3). Additionally, low weight thiols such as glutathione in E. coli or bacillithiol in Bacillus subtilis may also play a role as a zinc pool, almost certainly at least under conditions of metal excess.49–51 Transport systems and the zinc repository are thus the key players of zinc homeostasis in C. metallidurans, and may be also in other organisms.
Nano-LC-HD-MSE data were acquired on a SYNAPT G2-S (Waters, Eschborn, Germany) (same protein amount per sample, three biological repeats for AE104 and ΔzupT, one for CH34, at least three technical repeats per biological repeat). Glycogen phosphorylase B (P00489) was used as an internal standard at a concentration of 10 fmol (Waters, Eschborn, Germany) as well as common contaminants (http://ftp://ftp.thegpm.org/fasta/cRAP/crap.fasta). 1 μL of each in-solution digest (containing ∼400 ng of protein) were separated on an ACQUITY nanoUPLC System (Waters, Eschborn, Germany). Peptides were trapped for 5 min at 5 μL min−1 at 1% B (0.1% formic acid in ACN) and 99% A (0.1% trifluoroacetic acid in water) on a 200 mm × 180 μm fused silica trap column packed with 5 μm Symmetry C18 (Waters, Eschborn, Germany), and separated at 300 nL min−1 in a linear gradient of 7–35% B (0.1% formic acid in ACN, A: 0.1% formic acid in water) within 140 min on a 250 mm × 75 μm fused silica separation column packed with 1.8 μm HSS T3 C18 (Waters, Eschborn, Germany).
The resulting cellular numbers and deviations for each protein per biological repeat and bacterial strain were used to calculate the mean protein numbers per bacterial cell and strain. The deviation was the mean value of the deviations of the biological repeats. Finally, the protein numbers of the strains were compared. A protein number was judged as up- or down-regulated, if the quantities were at least 2-fold lower or higher in the mutant-derived extract, respectively. These values were labeled in italic font (down-regulated) or bold font (up-regulated). Additionally, a “D” value was calculated as a measure of the significance of the difference. The “D” value gives the difference in the protein numbers, mutant minus parent, divided by the sum of the deviation of these mean values. If D > 1, the deviation bars of the mean values do not overlap, leading to a significant result (>95%) if n > 3 as published.54 If a protein was up- and down-regulated, respectively, and the difference between the protein numbers was significant (D ≥ 1), the value was judged as significantly different (bold font). If not, the value was counted as not significant (italic font). All values are provided in the supplementary data base (ESI†).
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4mt00171k |
This journal is © The Royal Society of Chemistry 2014 |