A proteomic study of Corynebacterium glutamicumAAA+ protease FtsH
© Lüdke et al; licensee BioMed Central Ltd. 2007
Received: 19 August 2006
Accepted: 25 January 2007
Published: 25 January 2007
The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis.
In contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization.
The data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria.
Corynebacterium glutamicum, is a Gram-positive soil bacterium, which is used for the industrial production of different amino acids, mainly L-glutamate and L-lysine, and of nucleotides [1, 2]. As a member of the Corynebacterinae, C. glutamicum is closely related to other mycolic acid-containing bacteria, e. g. to the amino acids producer Corynebacterium efficiens and to important pathogens such as Corynebacterium diphtheriae, Mycobacterium tuberculosis and Mycobacterium leprae . Due to the enormous industrial importance of C. glutamicum, this bacterium is very well investigated. Its genome was sequenced and published independently by different industry-supported groups recently [4, 5] and different global analyses techniques are available including transcriptome , metabolome , flux  and proteome analyses .
We are interested in nitrogen metabolism and nitrogen control in C. glutamicum (for review, see [10–12]) and recently identified proteolysis as a new regulatory mechanism in nitrogen regulation . Different proteases, namely ClpXP and ClpCP  as well as FtsH are involved in the degradation of nitrogen signal transduction protein GlnK . The identified enzymes are members of the AAA+ protease family. These proteases and protein disassembly machines are found in all kingdoms of life and often exhibit crucial regulatory functions (for recent reviews, see [15, 16]).
In C. glutamicum, an effect of FtsH on the degradation of nitrogen signal control protein GlnK was reported . The deletion of the ftsH gene is very well tolerated by C. glutamicum cells and obvious detrimental effects of an ftsH deletion could not be observed. Since we were interested in the function of this protease, we initiated a proteomic study and investigated the influence of an ftsH deletion on membrane and cytoplasmic protein profiles.
Influence of FtsH on growth of C. glutamicumstrains
Differences in the membrane proteome of wild type and ftsHdeletion strain
FtsH is a membrane-bound AAA+ protease and therefore we started our investigations with an analysis of membrane proteins. While the separation of C. glutamicum membrane proteins by 2-D PAGE is restricted to those with two or less transmembrane helices [21, 22], recently, a technique was established to separate highly hydrophobic proteins of the membrane fraction by anion exchange chromatography and 1-D SDS-PAGE . This technique was applied for the comparison of membrane proteins from wild type and corresponding ftsH deletion strain.
Protein pattern of the membrane fraction of the wild type ATCC13032 and ftsH deletion mutant.
succinate dehydrogenase CD (sdhCD)
succinate dehydrogenase A (sdhA)
succinate dehydrogenase B (sdhB)
glutamate binding protein (gluB)
ATP-dependent protease (clpC)
homocysteine methyltransferase (metE)
cell division protein
Additionally, a role of FtsH in the response to nitrogen starvation and to improving nitrogen conditions after a starvation period was tested. Compared to the wild type, NADH dehydrogenase (ndh), a putative integral membrane protein (cg0952) and the ATPase component of an ABC-type sugar transport system (msiK) were down-regulated by a factor of 0.5 (data not shown).
Comparison of cytoplasmic protein profiles
Cytoplasmic protein pattern of wild type strain ATCC13032 and ftsH deletion mutant. The listed proteins differ in their abundance of a factor of at least two.
ΔftsH/wild type ratio
biotin carboxylase/biotin carboxyl carrier protein (accBC)
glyceraldehyde-3-phosphate dehydrogenase (gap)
malate synthase (aceB)
isocitrate lyase (aceA)
conserved hypothetical protein
homocysteine methyltransferase (metE)
maltooligosyl trehalose synthase (treY)
1,4-alpha-glucan branching enzyme (glgB)
putative nicotinate-nucleotide pyrophosphorylase
nicotinic acid phosphoribosyltransferase
inositol-monophosphate dehydrogenase (guaB2)
AMP nucleosidase (amn)
transcriptional regulator, MerR family (ramB)
putative DNA helicase
sulfite reductase (hemoprotein) (cysI)
glutamine synthetase (glnA)
DNA-directed RNA polymerase beta chain (rpoB)
aspartate ammonia-lyase (aspartase) (aspA)
ATPases of the AAA+ class
polyphosphate glucokinase (ppgK)
probable formyltetrahydrofolate deformylase protein (purU)
N-acetymuramyl-L-alanine amidase (cwlM)
fumarate hydratase (fum)
aspartyl aminopeptidase (pepC)
putative L-lactate dehydrogenase (lldA)
dihydrolipoamide succinyltransferase (sucB)
glyceraldehyde-3-phosphate dehydrogenase (gap)
phosphoenolpyruvate carboxylase (ppc)
succinyl-diaminopimelate desuccinylase (dapE)
inositol-monophosphate dehydrogenase (guaB1)
pyruvate dehydrogenase E1 component (aceE)
acyl-CoA synthetase (fadD4)
succinate dehydrogenase A (sdhA)
predicted carbohydrate kinase
superfamily II DNA/RNA helicase, SNF2 family
dihydroxy-acid dehydratase (ilvD)
GTP cyclohydrolase (folE)
Data which hint to an involvement of FtsH in GlnK signal transduction protein degradation  prompted us to investigate the influence of this AAA+ protease on membrane and cytoplasmic protein profiles in C. glutamicum. Using a combination of anion exchange chromatography and SDS-PAGE for membrane protein analysis and 2-D PAGE for cytoplasmic proteins, we were able to show that FtsH regulates only a few proteins under the growth conditions tested. However, since the applied method only covers about 10% of the C. glutamicum membrane proteome , some FtsH targets may have been missed due to technical limitations. For example, the FtsH target GlnK is degraded depending on FtsH but proteolysis is also influenced by ClpCP and ClpXP . In contrast to the situation in E. coli (for recent review, see ), we found that deletion of the ftsH gene is tolerated by C. glutamicum cells very well, although this gene is conserved in all other corynebacterial genome sequences published so far, i. e. in the C. diphtheriae , C. efficiens  and Corynebacterium jeikeium  genome, and although no obvious paralog of the ftsH gene is encoded in the C. glutamicum genome. Obvious detrimental effects of an ftsH deletion were not observed. In this respect the C. glutamicum results resemble the situation in B. subtilis and C. crescentus. Also for these organisms, a less severe effect of ftsH mutation compared to an E. coli mutant was shown. In B. subtilis, FtsH is involved in sporulation, stress adaptation and protein secretion [18, 19], and the effect of its deletion on the cytosolic proteome has been studied . FtsH deletion resulted in increased levels of an arginase, a protein similar to a quinone oxidoreductase, and penicillin binding protein, but for the latter direct proteolytic action could be excluded and for the other two proteins it was not verified. FtsH of M. tuberculosis, which is phylogenetically closely related to C. glutamicum, was heterologously expressed in E. coli, and proteolytic activity against the known E. coli substrates heat shock transcription factor σ32 protein, protein translocation subunit SecY, and bacteriophage λCII repressor protein was observed . For M. tuberculosis no experimental verification exists if SecY is indeed a target of FtsH, and our data for C. glutamicumdoes not support this hypothesis, but it does not completely rule this out, too. For C. crescentus an involvement of FtsH in development, stress response and heat shock control was shown . The ftsH gene is expressed transiently after temperature upshift and in stationary phase in this organism, while during normal growth conditions FtsH is dispensable. In C. crescentus a mutation of ftsH influences chaperones, DnaK is derepressed under normal temperature compared to the wild type, while an influence on GroEL abundance was not observed. In contrast, the ftsH deletion in C. glutamicum had no influence on DnaK and even less GroEL was observed compared to the wild type. Further differences besides chaperone activation are sporulation and cell cycle proteins, processes which are absent in C. glutamicum. The majority of proteins identified to be differentially synthesized in dependence of FtsH C. glutamicum seem to be involved in carbon and energy metabolism.
The data obtained in this study, indicate that C. glutamicum AAA+ metalloprotease FtsH is not involved in the cellular response to heat or osmotic stress as shown in other bacteria. An astonishingly small amount of membrane and cytoplasmic proteins is affected by an ftsH deletion. From these data an involvement of FtsH in regulation of energy and carbon metabolism as well as in amino acid biosynthesis is indicated.
Strains and growth conditions
C. glutamicum type strain ATCC 13032  and ftsH deletion mutant  were routinely grown on a rotary shaker at 30°C. A fresh C. glutamicum culture in BHI medium was used to inoculate minimal medium (per litre 42 g MOPS, 20 g (NH4)2SO4, 5 g urea, 0.5 g K2HPO4 × 3 H2O, 0.5 g KH2PO4, 0.25 g MgSO4 × 7 H2O, 0.01 g CaCl2, 50 g glucose, 0.2 mg biotin, 10 mg FeSO4, 10 mg MnSO4, 1 mg ZnSO4, 0.2 mg CuSO4, 0.02 mg NiCl2 × 6 H2O, 0.09 mg H3BO3, 0.06 mg CoCl2 × 6 H2O, 0.009 mg NaMoO4 × 2 H2O; pH adjusted to pH 7.0 using NaOH; ) for overnight growth. This culture, with an overnight OD600 of approximately 25 to 30, was used to inoculate fresh minimal medium to an OD600 of approximately 1, and cells were grown for 4 to 6 hours until the exponential growth phase was reached (OD600 approximately 4–5). To induce nitrogen starvation, cells were harvested by centrifugation and the pellet was suspended in and transferred to pre-warmed minimal medium without nitrogen source. The nitrogen-deprived cells were incubated at 30°C under aeration.
Polymerase chain reaction
To verify the deletion of the ftsH gene, PCR experiments were carried out. Primers were designed to anneal approx. 214 bps up and down stream of ftsH gene (2562 bps) (ftsH+200up fw: 5'-GTG GGC TAC GGA CTT GAT TTC G-3'; ftsH+200down rv: 5'-GAA CCA ACT CTT CAT GGC CCT C-3'). Chromosomal DNA prepared by phenol-chloroform extraction was used as template. PCR was performed using Taq polymerase and the following program: 95°C 5 min; 30 cycles (95°C 30 s; 64°C 30 s; 72°C 3 min) followed by 72°C 10 min and cooling down to 4°C. PCR products were analyzed by agarose gel electrophoresis .
SDS-PAGE and Western blotting
To demonstrate deletion of ftsH on protein level, cells were disrupted band fractionated as described below or 2-D PAGE. Cytoplasmic proteins and membrane fraction of the wild type ATCC13032 and the deletion strain were separated by Tricine-buffered 9.5% SDS gels as described . After SDS-PAGE proteins were transferred onto a polyvinylidene difluoride membrane (PVDF, Immobilon-P, pore size 0.45 μm, Millipore, Bedford, MA, USA) by semi-dry electroblotting. Immunodetection of FtsH was performed with antibodies against peptide fragments of E. coli FtsH, produced in rabbit. Antibody binding was visualised by using appropriate anti-antibodies coupled to alkaline phosphatase (Sigma-Aldrich, Traufkirchen, Germany) and the BCIP/NBT alkaline phosphatase substrate (Sigma-Aldrich, Traufkirchen, Germany).
For analysis of membrane proteins, a combination of anion exchange chromatography and SDS-PAGE was applied as described previously . For this method, cells were disrupted by French Press treatment; the membrane fraction was separated from cell debris and cytoplasm by differential (ultra)centrifugation and washed with 2.5 M NaBr to remove membrane-associated proteins. Membrane proteins were subsequently solubilised in buffer containing 2% ASB-14 and separated by anion exchange chromatography. After TCA precipitation and SDS-PAGE, gels were scanned and analyzed using the LabScan software package (Amersham Biosciences, Freiburg, Germany). The scanner was calibrated with a greyscale marker (Kodak) and the same settings applied for all gels. Scanning was carried out at 300 dpi and 8 bit greyscale. Gel bands were quantified relative to each other by densitometry using the software scion image (version beta 4.0.2; Scion Corporation). Proteins from three independent experiments (biological replicates) were regarded as regulated if a p-value < 0.1 was calculated for a Student's t-test (paired, two-tailed).
2-D PAGE, staining and protein analysis
For 2-D PAGE analyses C. glutamicum cells were disrupted using glass beads and a Q-BIOgene FastPrep FP120 instrument (Q-BIOgene, Heidelberg, Germany) by lyzing the cells four times for 30 sec and 6.5 m sec-1 in the presence of the proteinase inhibitor Complete as recommended by the supplier (Roche, Basel, Switzerland). Proteins were separated by ultracentrifugation in cytoplasmic and membrane-associated protein fractions [37, 21]. For classical 2-D PAGE, only the cytoplasmic proteins were analyzed further. Protein concentrations were determined according to Dulley and Grieve . For isoelectric focusing (IEF) 24 cm pre-cast IPG strips pI 4–7 and an IPGphor IEF unit (Amersham Biosiences, Freiburg, Germany) were used as described . 100 μg and 200 μg of protein were loaded by rehydration for 24 h in a sample buffer containing 6 M urea, 2 M thiourea, 4% CHAPS, 0.5% Pharmalyte (3–10) and 0.4% DTT. The isoelectric focusing was performed for 48 000 Vh. The run for the second dimension was carried out using 12.5% polyacrylamide gels and an Ettan Dalt II system (Amersham Biosiences, Freiburg, Germany). After electrophoresis 2-D gels were stained with Coomassie brilliant Blue . The Coomassie-stained gels were aligned using the Delta2D software, version 3.3 (Decodon, Greifswald, Germany). All samples were separated at least twice by 2-D PAGE to minimize irregularities (technical replicates). To validate the results, each comparison of interest was performed using samples from at least three independent experiments (biological replicates). The Delta2D software (version 3.3) was also used for spot quantification. Proteins were regarded as regulated if (i) the corresponding ratios referring to the relative volumes of the spots changed more than two-fold and if (ii) this regulation pattern was found in all biological and technical replicates. All other proteins were classified as "not regulated". Pearson coefficients for wild type gels were higher than 0.9962, for ftsH gels 0.9929, and for the comparisons of wild type and ftsH mutant 0.9510.
Protein spots or bands with significantly altered abundance in the ftsH mutant compared to the wild type were analyzed by trypric in-gel digest and MALDI-ToF-MS as described earlier .
FtsH-specific antibodies were kindly provided by Teru Ogura (Kumamoto University, Japan). The authors wish to thank C. Lück (Technical University Munich), U. Meyer and S. Morbach (University of Cologne) for providing unpublished data. The financial support of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 635, TP17) and the Bundesministerium für Bildung und Forschung (Neue Methoden zur Proteomanalyse: Anwendung und Verknüpfung mit Metabolomanalysen am Beispiel von Corynebacterium glutamicum) is gratefully acknowledged.
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