臺灣博碩士論文加值系統

Lon是一種ATP依賴型的蛋白酶,在分類上被歸類在AAA+ superfamily，此蛋白酶不僅在原核生物和真核生物中都呈現高度的保留性。Lon的蛋白分解活性目前已被確認在針對蛋白質的品量控制和代謝調控上皆扮演著非常重要的角色，因此，當此蛋白酶要執行其功能時，如何正確無誤地辦認其結合性蛋白或需要被分解的受質，在生物系統的交互作用上是非常重要的一個環節，這種精準的蛋白分解過程是屬於不可逆的轉錄後調控，由此可知，選擇其適當的分子目標蛋白尤其重要，因為錯誤的多胜肽鏈分解會嚴重導致細胞傷害甚至致死。
在這篇研究報告中，我們建立了一個以蛋白質體學為基礎的實驗策略來捕捉受質，藉此鑑定出大腸桿菌中Lon蛋白酶的潛在性受質，其主要生物性材料使用BW野生株大腸桿菌 (BW25113, BW)、lon基因剃除的大腸桿菌株(JW0429, JW)、以及能表現His6融合性LonS679D點突變蛋白的建構性質體 (其特性為保有ATPase活性卻失去蛋白酶活性)。因此我們將純化好的His6融合性LonS679D點突變蛋白以達到飽和的方式鍵結在鎳離子螯合層析管柱上，藉此捕捉來自BW或JW大腸桿菌細胞均質液內的Lon受質或結合性蛋白質，之後再把鍵結在LonS679D上的蛋白洗出，最後以溶液內胜肽水解法分解成短胜肽鏈，隨後經由串聯式液態層析質譜儀 (LC-MS/MS)進行分析。藉此散彈槍蛋白質體分析法以及比較來自BW或JW大腸桿菌的這些被分析鑑定到的蛋白質，此方法能夠讓我們去區分屬於Lon的分解性受質或單純只是結合性蛋白，結果我們總共找到了38個可能會被Lon分解的受質以及6個結合性蛋白質。然而，為了更進一步證實此分析結果的可信度，基於現有的研究文獻為基準，我們挑選了10個最有可能的潛在性受質，包含了XerC/D、UspG、Fis、BssR、ArnA、BasR、FabR、MukE和CbpA，藉由同時進行胞內和胞外蛋白水解分析法，結果我們確認出XerC/D、Fis、BssR、和ArnA可能直接或間接透過Lon依賴型的方式來執行蛋白質水解，但是關於這些受質被水解的生理意義在生物體內仍須更待進一步的研究。
總結我們所用的這項捕捉受質實驗，目前已經能成功驗證並且找到現階段研究皆屬未知的全新Lon分解受質，另外，在未來我們可以使用這種失去蛋白酶活性的Lon來當餌，以便在不同生長條件下幫助我們找出更多關於Lon蛋白酶的生物意義。

ATP-dependent Lon protease, which belongs to the AAA+ superfamily, is highly conserved in prokaryotes and eukaryotic organelles. Lon is important for not only protein quality control but also regulation of metabolic processes via proteolysis. The recognition of the correct partners or substrates is critical for almost all biological transactions. For irreversible posttranslational regulation like protein degradation, choosing the appropriate molecular targets is especially important because cleavage of the wrong polypeptides could be damaging or even fatal.
In this study, we utilized a proteomic-based substrate trapping strategy to identify potential substrates of E. coli Lon (EcLon). Using BW25113 (BW; a wild type strain), JW0429 (JW; a lon knockout strain), and one construct expressed His6-fused EcLonS679D (loss of protease activity but remain ATPase activity) as materials, we thereby saturated Ni2+-NTA beads with EcLonS679D to pull down the trapped substrates or associated proteins from lysates of BW and JW. The eluants were then applied to in-solution digestion for subsequent analysis via LC-MS/MS. Using shotgun proteomic approach and comparison of each pool of identified proteins from BW and JW to distinguish Lon’s substrates from associated proteins, we totally identified 38 proteins which might be subjected to Lon-mediated proteolysis, and 6 proteins might associate with Lon protease. To validate the credibility of the results, 10 potential candidates were selected, including XerC/D, UspG, Fis, BssR, ArnA, BasR, FabR, MukE, and CbpA, based on the criteria of previous studies. By both in vitro and in vivo proteolytic assays, we were able to confirm that XerC/D, Fis, BssR, and ArnA are subjected to Lon-dependent degradation through a direct or indirect manner. However, the physiological purposes for these degradation processes still remain further investigation.
Our trapping method has proven successfully validate previously unknown substrates of Lon. In the future, using protease-deficient Lon as bait under different growth conditions might help reveal even more of this versatile protease.

1 Introduction 1
1-1 AAA+ superfamily and Lon Protease 1
1-2 Structure and functionality of Lon protease 4
Figure 1. Domain organization of Lon (LonA) 4
1-3 General principles for AAA+ proteases to discriminate their substrates 6
1-4 Recognition and degradation of cytoplasmic proteins by Lon 7
Table 1. Known substrates of E. coli Lon protease 12
1-5 Candidate Substrates of EcLon 14
1-6 Aim of this Study 14
2 Materials and Methods 15
2-1 Bacterial strains and plasmids 15
2-2 Identification of Potential Substrates of EcLon 15
2-3 In-solution digestion and desalting 17
2-4 Liquid chromatography - mass spectrometric analysis 17
2-5 Cloning of EcLon, and its potential substrates 19
Table 2. Oligonucleotides used in this study 21
2-6 Protein Expression and Purification 24
2-7 In-gel digestion 24
2-8 Identification of the cleavage sites 25
2-9 LC-ESI-MS/MS Analyses 26
2-10 In vitro degradation assays 27
2-11 In vivo proteolytic assays 28
2-12 Preparation of protein extracts and immunodetection 28
2-13 Circular dichroism spectra of Fis 29
3 Results and Discussion 30
3-1 Validation of EcLonS679D as trap for substrate identification 30
3-2 Identification of EcLonS679D-trapped Proteins 31
Figure 2. Principle of the EcLonS679D-based substrate identification approach 33
Figure 4. Workflow of identification potential substrates of EcLon 35
Figure 5. Scheme of the identified proteins in BW25113 and JW0429 39
3-3 Validation of the Potential Substrates in EcLon 40
Figure 6. in vitro and in vivo putative substrates validation 41
Table 5. Summary of in vitro and in vivo degradation of 10 selected candidates 42
3-3.1 Validation of five novel substrates 43
3-3.1.1 Fis 43
Figure 7. Fis is subjected to Lon-dependent proteolysis both in vitro and in vivo 46
Figure 8. Cleavage sites of Fis and respective positions on tertiary structure. 47
3-3.1.2 BssR and ArnA 48
Figure 9. Interaction between BssR and ArnA 52
Figure 10. Validation of BssR and ArnA in vitro and in vivo 53
Figure 11. Cleavage sites of BssR 54
3-3.1.3 XerC/D 55
Figure 12. XerC was subjected to Lon-mediated proteolysis in vivo 58
Figure 13. Position of His6-tag influenced the tendency for degradation in XerD 59
Figure 14. Cleavage sites of XerD 60
Figure 15. Respective positions of cleavage sites in XerD on tertiary structure. 61
Figure 16. Possible regulation mechanisms for XerC/D proteolysis 62
3-3.1.4 UspG and its truncated form (UspG△136) 63
Figure 17. Validation of UspG in vitro and in vivo 65
Figure 18. UspG△136 might be subjected to Lon-mediated proteolysis in vivo 66
3-3.2 Not all proteins co-purified with LonS679D are protease substrates 67
3-3.2.1 FabR 67
3-3.2.2 MukE 68
3-3.2.3 BasR 68
3-3.2.4 CbpA 69
Figure 19. Validation of FabR in vivo 70
Figure 20. Validation of MukE in vitro and in vivo 71
Figure 21. Validation of BasR in vitro and in vivo 72
Figure 22. Validation of CbpA in vitro and in vivo 73

References 74

1.Iyer LM, Leipe DD, Koonin EV, & Aravind L (2004) Evolutionary history and higher order classification of AAA+ ATPases. Journal of structural biology 146(1-2):11-31.
2.Snider J, Thibault G, & Houry WA (2008) The AAA+ superfamily of functionally diverse proteins. Genome biology 9(4):216.
3.Striebel F, Kress W, & Weber-Ban E (2009) Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Current opinion in structural biology 19(2):209-217.
4.Wickner S, Maurizi MR, & Gottesman S (1999) Posttranslational quality control: folding, refolding, and degrading proteins. Science 286(5446):1888-1893.
5.Gottesman S, Wickner S, & Maurizi MR (1997) Protein quality control: triage by chaperones and proteases. Genes & development 11(7):815-823.
6.Gottesman S & Maurizi MR (1992) Regulation by proteolysis: energy-dependent proteases and their targets. Microbiological reviews 56(4):592-621.
7.Hershko A & Ciechanover A (1992) The ubiquitin system for protein degradation. Annual review of biochemistry 61:761-807.
8.Hochstrasser M (1996) Ubiquitin-dependent protein degradation. Annual review of genetics 30:405-439.
9.Keiler KC, Waller PR, & Sauer RT (1996) Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271(5251):990-993.
10.Sauer RT & Baker TA (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annual review of biochemistry 80:587-612.
11.Maurizi MR, Trisler P, & Gottesman S (1985) Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable. Journal of bacteriology 164(3):1124-1135.
12.Swamy KH & Goldberg AL (1981) E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature 292(5824):652-654.
13.Tsilibaris V, Maenhaut-Michel G, & Van Melderen L (2006) Biological roles of the Lon ATP-dependent protease. Research in microbiology 157(8):701-713.
14.Melnikov EE, et al. (2008) Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments. Acta biochimica Polonica 55(2):281-296.
15.Li M, et al. (2005) Crystal structure of the N-terminal domain of E. coli Lon protease. Protein science : a publication of the Protein Society 14(11):2895-2900.
16.Rotanova TV, et al. (2006) Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein science : a publication of the Protein Society 15(8):1815-1828.
17.Lee I & Suzuki CK (2008) Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates. Biochimica et biophysica acta 1784(5):727-735.
18.Van Melderen L & Aertsen A (2009) Regulation and quality control by Lon-dependent proteolysis. Research in microbiology 160(9):645-651.
19.Amerik A, et al. (1991) Site-directed mutagenesis of La protease. A catalytically active serine residue. FEBS letters 287(1-2):211-214.
20.Duman RE & Lowe J (2010) Crystal structures of Bacillus subtilis Lon protease. Journal of molecular biology 401(4):653-670.
21.Neuwald AF, Aravind L, Spouge JL, & Koonin EV (1999) AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome research 9(1):27-43.
22.Lupas AN & Martin J (2002) AAA proteins. Current opinion in structural biology 12(6):746-753.
23.Smith CK, Baker TA, & Sauer RT (1999) Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proceedings of the National Academy of Sciences of the United States of America 96(12):6678-6682.
24.Hattendorf DA & Lindquist SL (2002) Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding. Proceedings of the National Academy of Sciences of the United States of America 99(5):2732-2737.
25.Ogura T & Wilkinson AJ (2001) AAA+ superfamily ATPases: common structure--diverse function. Genes to cells : devoted to molecular & cellular mechanisms 6(7):575-597.
26.Charette MF, Henderson GW, & Markovitz A (1981) ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proceedings of the National Academy of Sciences of the United States of America 78(8):4728-4732.
27.Chin DT, Goff SA, Webster T, Smith T, & Goldberg AL (1988) Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. The Journal of biological chemistry 263(24):11718-11728.
28.Lin YC, et al. (2009) DNA-binding specificity of the Lon protease alpha-domain from Brevibacillus thermoruber WR-249. Biochemical and biophysical research communications 388(1):62-66.
29.Chung CH & Goldberg AL (1982) DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 79(3):795-799.
30.Charette MF, Henderson GW, Doane LL, & Markovitz A (1984) DNA-stimulated ATPase activity on the lon (CapR) protein. Journal of bacteriology 158(1):195-201.
31.Fu GK, Smith MJ, & Markovitz DM (1997) Bacterial protease Lon is a site-specific DNA-binding protein. The Journal of biological chemistry 272(1):534-538.
32.Goldberg AL, Moerschell RP, Chung CH, & Maurizi MR (1994) ATP-dependent protease La (lon) from Escherichia coli. Methods in enzymology 244:350-375.
33.Park SC, et al. (2006) Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli. Molecules and cells 21(1):129-134.
34.Rudyak SG, Brenowitz M, & Shrader TE (2001) Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis. Biochemistry 40(31):9317-9323.
35.Botos I, et al. (2004) Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution. Journal of structural biology 146(1-2):113-122.
36.Schmidt R, Bukau B, & Mogk A (2009) Principles of general and regulatory proteolysis by AAA+ proteases in Escherichia coli. Research in microbiology 160(9):629-636.
37.Baker TA & Sauer RT (2006) ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends in biochemical sciences 31(12):647-653.
38.Dougan DA, Truscott KN, & Zeth K (2010) The bacterial N-end rule pathway: expect the unexpected. Molecular microbiology 76(3):545-558.
39.Dulebohn D, Choy J, Sundermeier T, Okan N, & Karzai AW (2007) Trans-translation: the tmRNA-mediated surveillance mechanism for ribosome rescue, directed protein degradation, and nonstop mRNA decay. Biochemistry 46(16):4681-4693.
40.Bonnefoy E, Almeida A, & Rouviere-Yaniv J (1989) Lon-dependent regulation of the DNA binding protein HU in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 86(20):7691-7695.
41.Kuroda A, et al. (2001) Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli. Science 293(5530):705-708.
42.Raju RM, Goldberg AL, & Rubin EJ (2012) Bacterial proteolytic complexes as therapeutic targets. Nature reviews. Drug discovery 11(10):777-789.
43.Pruteanu M & Baker TA (2009) Proteolysis in the SOS response and metal homeostasis in Escherichia coli. Research in microbiology 160(9):677-683.
44.Heuveling J, Possling A, & Hengge R (2008) A role for Lon protease in the control of the acid resistance genes of Escherichia coli. Molecular microbiology 69(2):534-547.
45.Torres-Cabassa AS & Gottesman S (1987) Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. Journal of bacteriology 169(3):981-989.
46.Leffers GG, Jr. & Gottesman S (1998) Lambda Xis degradation in vivo by Lon and FtsH. Journal of bacteriology 180(6):1573-1577.
47.Gottesman S, Gottesman M, Shaw JE, & Pearson ML (1981) Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability. Cell 24(1):225-233.
48.Shah IM & Wolf RE, Jr. (2006) Inhibition of Lon-dependent degradation of the Escherichia coli transcription activator SoxS by interaction with ''soxbox'' DNA or RNA polymerase. Molecular microbiology 60(1):199-208.
49.Griffith KL, Shah IM, & Wolf RE, Jr. (2004) Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons. Molecular microbiology 51(6):1801-1816.
50.Chung CH & Goldberg AL (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proceedings of the National Academy of Sciences of the United States of America 78(8):4931-4935.
51.Gur E & Sauer RT (2008) Recognition of misfolded proteins by Lon, a AAA(+) protease. Genes & development 22(16):2267-2277.
52.Gur E & Sauer RT (2009) Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine. Proceedings of the National Academy of Sciences of the United States of America 106(44):18503-18508.
53.Bissonnette SA, Rivera-Rivera I, Sauer RT, & Baker TA (2010) The IbpA and IbpB small heat-shock proteins are substrates of the AAA+ Lon protease. Molecular microbiology 75(6):1539-1549.
54.Katz C, et al. (2009) Temperature-dependent proteolysis as a control element in Escherichia coli metabolism. Research in microbiology 160(9):684-686.
55.Biran D, Gur E, Gollan L, & Ron EZ (2000) Control of methionine biosynthesis in Escherichia coli by proteolysis. Molecular microbiology 37(6):1436-1443.
56.Little JW (1984) Autodigestion of lexA and phage lambda repressors. Proceedings of the National Academy of Sciences of the United States of America 81(5):1375-1379.
57.Neher SB, Flynn JM, Sauer RT, & Baker TA (2003) Latent ClpX-recognition signals ensure LexA destruction after DNA damage. Genes & development 17(9):1084-1089.
58.Neher SB, et al. (2006) Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon. Molecular cell 22(2):193-204.
59.Frank EG, Ennis DG, Gonzalez M, Levine AS, & Woodgate R (1996) Regulation of SOS mutagenesis by proteolysis. Proceedings of the National Academy of Sciences of the United States of America 93(19):10291-10296.
60.Ishii Y & Amano F (2001) Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine. The Biochemical journal 358(Pt 2):473-480.
61.Mizusawa S & Gottesman S (1983) Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proceedings of the National Academy of Sciences of the United States of America 80(2):358-362.
62.Kuroda A, Murphy H, Cashel M, & Kornberg A (1997) Guanosine tetra- and pentaphosphate promote accumulation of inorganic polyphosphate in Escherichia coli. The Journal of biological chemistry 272(34):21240-21243.
63.Kuroda A, Nomura K, Takiguchi N, Kato J, & Ohtake H (2006) Inorganic polyphosphate stimulates lon-mediated proteolysis of nucleoid proteins in Escherichia coli. Cellular and molecular biology 52(4):23-29.
64.Goldberg AL & St John AC (1976) Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annual review of biochemistry 45:747-803.
65.Pruteanu M, Neher SB, & Baker TA (2007) Ligand-controlled proteolysis of the Escherichia coli transcriptional regulator ZntR. Journal of bacteriology 189(8):3017-3025.
66.Van Melderen L & Saavedra De Bast M (2009) Bacterial toxin-antitoxin systems: more than selfish entities? PLoS genetics 5(3):e1000437.
67.Koga M, Otsuka Y, Lemire S, & Yonesaki T (2011) Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system. Genetics 187(1):123-130.
68.Kawano M, Aravind L, & Storz G (2007) An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin. Molecular microbiology 64(3):738-754.
69.Christensen SK, et al. (2004) Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system. Molecular microbiology 51(6):1705-1717.
70.Christensen-Dalsgaard M, Jorgensen MG, & Gerdes K (2010) Three new RelE-homologous mRNA interferases of Escherichia coli differentially induced by environmental stresses. Molecular microbiology 75(2):333-348.
71.Van Melderen L, Bernard P, & Couturier M (1994) Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria. Molecular microbiology 11(6):1151-1157.
72.Shah D, et al. (2006) Persisters: a distinct physiological state of E. coli. BMC microbiology 6:53.
73.Keren I, Shah D, Spoering A, Kaldalu N, & Lewis K (2004) Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. Journal of bacteriology 186(24):8172-8180.
74.Shah IM & Wolf RE, Jr. (2006) Sequence requirements for Lon-dependent degradation of the Escherichia coli transcription activator SoxS: identification of the SoxS residues critical to proteolysis and specific inhibition of in vitro degradation by a peptide comprised of the N-terminal 21 amino acid residues. Journal of molecular biology 357(3):718-731.
75.Gonzalez M, Frank EG, Levine AS, & Woodgate R (1998) Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis. Genes & development 12(24):3889-3899.
76.Higashitani A, Ishii Y, Kato Y, & Koriuchi K (1997) Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon. Molecular & general genetics : MGG 254(4):351-357.
77.Schoemaker JM, Gayda RC, & Markovitz A (1984) Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death. Journal of bacteriology 158(2):551-561.
78.Christensen SK, Mikkelsen M, Pedersen K, & Gerdes K (2001) RelE, a global inhibitor of translation, is activated during nutritional stress. Proceedings of the National Academy of Sciences of the United States of America 98(25):14328-14333.
79.Van Melderen L, et al. (1996) ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions. The Journal of biological chemistry 271(44):27730-27738.
80.Langklotz S & Narberhaus F (2011) The Escherichia coli replication inhibitor CspD is subject to growth-regulated degradation by the Lon protease. Molecular microbiology 80(5):1313-1325.
81.Flynn JM, Neher SB, Kim YI, Sauer RT, & Baker TA (2003) Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Molecular cell 11(3):671-683.
82.Datsenko KA & Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America 97(12):6640-6645.
83.Wang TF & Wang AH (2006) Preparation of sticky-end PCR products and ligation into expression vectors for high-throughput screening of soluble recombinant proteins. CSH protocols 2006(1).
84.Wang TF & Wang AH (2006) Preparation of vectors for high-throughput screening of soluble recombinant proteins. CSH protocols 2006(1).
85.Sambrook J (2001) Molecular cloning : a laboratory manual / Joseph Sambrook, David W. Russell (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y).
86.Kelly SM & Price NC (1997) The application of circular dichroism to studies of protein folding and unfolding. Biochimica et biophysica acta 1338(2):161-185.
87.Liao JH, et al. (2010) Binding and cleavage of E. coli HUbeta by the E. coli Lon protease. Biophysical journal 98(1):129-137.
88.Ishii Y, et al. (2000) Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli. Journal of biochemistry 127(5):837-844.
89.Finkel SE & Johnson RC (1992) The Fis protein: it''s not just for DNA inversion anymore. Molecular microbiology 6(22):3257-3265.
90.Travers A, Schneider R, & Muskhelishvili G (2001) DNA supercoiling and transcription in Escherichia coli: The FIS connection. Biochimie 83(2):213-217.
91.Nishii W, et al. (2005) Cleavage mechanism of ATP-dependent Lon protease toward ribosomal S2 protein. FEBS letters 579(30):6846-6850.
92.Nishii W, Maruyama T, Matsuoka R, Muramatsu T, & Takahashi K (2002) The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli. European journal of biochemistry / FEBS 269(2):451-457.
93.Ali Azam T, Iwata A, Nishimura A, Ueda S, & Ishihama A (1999) Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid. Journal of bacteriology 181(20):6361-6370.
94.Domka J, Lee J, & Wood TK (2006) YliH (BssR) and YceP (BssS) regulate Escherichia coli K-12 biofilm formation by influencing cell signaling. Applied and environmental microbiology 72(4):2449-2459.
95.Breazeale SD, Ribeiro AA, & Raetz CR (2002) Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose. The Journal of biological chemistry 277(4):2886-2896.
96.Arciszewska LK & Sherratt DJ (1995) Xer site-specific recombination in vitro. The EMBO journal 14(9):2112-2120.
97.Subramanya HS, et al. (1997) Crystal structure of the site-specific recombinase, XerD. The EMBO journal 16(17):5178-5187.
98.Blakely G, Colloms S, May G, Burke M, & Sherratt D (1991) Escherichia coli XerC recombinase is required for chromosomal segregation at cell division. The New biologist 3(8):789-798.
99.Kuempel PL, Henson JM, Dircks L, Tecklenburg M, & Lim DF (1991) dif, a recA-independent recombination site in the terminus region of the chromosome of Escherichia coli. The New biologist 3(8):799-811.
100.Blakely G, et al. (1993) Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12. Cell 75(2):351-361.
101.Kvint K, Nachin L, Diez A, & Nystrom T (2003) The bacterial universal stress protein: function and regulation. Current opinion in microbiology 6(2):140-145.
102.Weber A & Jung K (2006) Biochemical properties of UspG, a universal stress protein of Escherichia coli. Biochemistry 45(6):1620-1628.
103.Bochkareva ES, Girshovich AS, & Bibi E (2002) Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL. European journal of biochemistry / FEBS 269(12):3032-3040.
104.Kopp J & Schwede T (2004) The SWISS-MODEL Repository of annotated three-dimensional protein structure homology models. Nucleic acids research 32(Database issue):D230-234.
105.Schwede T, Kopp J, Guex N, & Peitsch MC (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic acids research 31(13):3381-3385.
106.Zhang YM, Marrakchi H, & Rock CO (2002) The FabR (YijC) transcription factor regulates unsaturated fatty acid biosynthesis in Escherichia coli. The Journal of biological chemistry 277(18):15558-15565.
107.Yamanaka K, Ogura T, Niki H, & Hiraga S (1996) Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli. Molecular & general genetics : MGG 250(3):241-251.
108.Feng J, Yamanaka K, Niki H, Ogura T, & Hiraga S (1994) New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli. Molecular & general genetics : MGG 243(2):136-147.
109.Sawitzke JA & Austin S (2000) Suppression of chromosome segregation defects of Escherichia coli muk mutants by mutations in topoisomerase I. Proceedings of the National Academy of Sciences of the United States of America 97(4):1671-1676.
110.Nagasawa S, Ishige K, & Mizuno T (1993) Novel members of the two-component signal transduction genes in Escherichia coli. Journal of biochemistry 114(3):350-357.
111.Hagiwara D, Yamashino T, & Mizuno T (2004) A Genome-wide view of the Escherichia coli BasS-BasR two-component system implicated in iron-responses. Bioscience, biotechnology, and biochemistry 68(8):1758-1767.
112.Ueguchi C, Kakeda M, Yamada H, & Mizuno T (1994) An analogue of the DnaJ molecular chaperone in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 91(3):1054-1058.