臺灣博碩士論文加值系統

在枯草桿菌體內的ycgMNO operon分別可以轉譯出proline dehydrogenase、??-pyrroline-5-carboxylate dehydrogenase和proline permease，做為proline的吸收和proline代謝成glutamate所需。然而對於參與ycgMNO operon表現的調控仍尚未被研究。藉由搜尋枯草桿菌的基因體序列，顯示在ycgO基因下游有一個功能未知的ycgP基因。ycgP所轉譯出的蛋白質與目前資料庫中已知功能的蛋白質在胺基酸序列上皆無有意義的相似性，但在YcgP 蛋白質的C端可比對出可能的DNA結合區域。在本篇論文研究中，我們已證實不論是在Luria-Bertani培養液或是M9 minimal medium中的proline誘導作用，ycgM基因都必須有ycgP基因的存在才可表現。由膠體遲滯的實驗亦顯示，純化出的His-tagged YcgP 蛋白質可專一地與ycgM基因啟動子上游inverted repeat序列結合，並且藉由序列刪除及定點突變的分析更加確認此段inverted repeat對於ycgM基因表現的重要性。而由細胞生長曲線分析也顯示，ycgM及ycgP基因對於枯草桿菌代謝proline作為唯一碳源或氮源都是必需的。因此，上述結果皆可支持YcgP蛋白質為一個新型的轉錄活化子，可在in vivo中藉由結合到inverted repeat序列上來活化ycgMNO的轉錄。此外，我們發現ycgP基因在M9 minimal medium中可持續微量的表現，並且ycgP的表現並不會受到自身調控及proline的誘導。由破壞可轉譯出與YcgM相似物的yusM基因並不會影響ycgM的表現，及yusM在野生型和ycgM突變菌株中表現都相當低的結果，推測YcgM是枯草桿菌體內唯一有功能的proline dehydrogenase。另一方面，透過產孢效率的分析，可發現菌體正常產孢需要ycgM而非ycgP的參與，並且ycgM突變株的產孢缺陷也不會因添加glutamate而改善。最後，我們發現casamino acid抑制ycgM 基因表現的作用是一種CodY非依賴型的方式，而casamino acid所造成之抑制現象的詳細機制仍有待釐清。

The Bacillus subtilis ycgMNO operon, which encodes proline dehydrogenase, ??-pyrroline-5-carboxylate dehydrogenase, and proline permease, is known to be responsible for proline uptake and subsequent conversion of proline to glutamate. However, regulation of expression of the ycgMNO operon has not been extensively studied. A search in the genome of B. subtilis revealed that the ycgP gene, whose function is unknown, is located immediately downstream of the ycgO gene. The ycgP-encoded putative protein shows no significant overall amino acid sequence homology to other proteins with known functions in the database. Nevertheless, YcgP contains a putative DNA-binding motif at its C-terminal region. In this study, we have demonstrated that the ycgP gene is not only essential for ycgM expression in Luria-Bertani medium, but is also required for proline induction of ycgM expression in M9 minimal medium. Electrophoretic mobility shift assays showed that purified His-tagged YcgP protein could specifically interact with an inverted repeat sequence located upstream of the ycgM promoter. Deletion analysis and site-directed mutagenesis confirmed the importance of the inverted repeat sequence in ycgM expression. Cell growth assays have also established that both ycgP and ycgM are essential for metabolism of proline as a sole carbon or nitrogen source in B. subtilis. Taken together, these results support that the novel transcriptional activator YcgP can activate transcription of ycgMNO in vivo through binding to the inverted repeat sequence. In addition, we found that the ycgP gene was constitutively expressed at a low level in M9 minimal medium and that ycgP expression was not subject to negative autoregulation and proline induction. Disruption of the yusM gene, which encodes an YcgM homolog, did not affect ycgM expression. The expression level of yusM was low both in wild-type cells and in the ycgM mutant. These results are consistent with the idea that YcgM is the only functional proline dehydrogenase in B. subtilis. Moreover, sporulation efficiency analysis showed that ycgM but not ycgP is required for normal sporulation and that the sporulation defect of the ycgM mutant could not be suppressed by the addition of glutamate. Finally, we found that casamino acid could repress ycgM expression in a CodY-independent manner. The precise mechanism underlying casamino acid repression remains to be clarified.

目錄 i
表目錄 iii
圖目錄 iii
中文摘要 iv
Abstract v
壹、緒論 1
一、 枯草桿菌 (Bacillus subtilis)之簡介 1
二、 Proline的合成與分解 2
三、 細菌中合成及代謝基因的調控 4
四、 細菌的壓力反應 (stress response) 7
五、 滲透壓力 (osmostress)之簡介 8
六、 細菌產孢 (sporulation)及Spo0A (Stage 0 sporulation protein A)之簡介 10
貳、實驗儀器、材料與方法 12
一、 實驗儀器 12
二、 實驗材料 14
(一) 菌種、質體 14
(二) 實驗所用之培養液及培養基 14
(三) 緩衝液 15
(四) 實驗試劑 16
三、 實驗方法 17
(一) 染色體的萃取 17
(二) 質體DNA萃取 17
(三) 質體DNA片段回收 18
(四) Total RNA萃取 18
(五) 大腸桿菌勝任細胞製備 18
(六) 大腸桿菌之轉形作用 19
(七) 枯草桿菌轉形作用 19
(八) Site-directed mutagenesis 20
(九) Single cross-over方式製備突變株 20
(十) 蛋白質的表現與純化 21
(十一) 不含 α-amylases (AmyE)之菌株篩選 21
(十二) BgaB (β-galactosidase)酵素活性分析 22
(十三) 孢子形成效率(sporulation efficiency) 23
(十四) 引子延伸分析 (primer extension) 23
(十五) DNA定序 (DNA sequencing) 23
(十六) Reverse transcription-PCR (RT-PCR) 24
(十七) Real-time RT-PCR 24
(十八) 膠體遲滯分析 (EMSA) 24
參、實驗結果 26
一、 破壞ycgM基因下游的未知功能基因ycgP會降低ycgM基因的表現 26
二、 YcgP蛋白質與ycgM基因啟動子區域有專一性的結合 27
三、 YcgP蛋白質可直接與ycgM基因啟動子上游inverted repeat序列結合 28
四、 ycgM基因啟動子上游inverted repeat序列對ycgM基因的表現是必需的 29
五、 Proline誘導ycgM基因的表現需透過ycgP基因的幫助 29
六、 破壞ycgM基因會提升本身ycgM基因啟動子之報導基因的表現 30
七、 ycgP基因的表現不受到proline的誘導及自身的調控 (autoregulation) 30
八、 利用引子延伸的方法找到ycgM基因及ycgP基因轉錄起始點 31
九、 yusM基因並不影響和代償ycgM基因的功能 32
十、 ycgM和ycgP基因對枯草桿菌利用proline做為唯一碳源或氮源是必需的 33
十一、 破壞ycgP基因不影響孢子形成 (sporulation)的過程 33
十二、 Casamino acid並非透過CodY來達到抑制ycgM基因表現的作用 34
十三、 ycgM基因的表現會受到高滲透壓力的刺激而有降低趨勢 35
肆、討論 36
伍、圖表 45
陸、參考文獻 72
柒、附錄 82

1. Waldburger, C., D. Gonzalez, and G.H. Chambliss, Characterization of a new sporulation factor in Bacillus subtilis. J Bacteriol, 1993. 175(19): p. 6321-7.
2. Grossman, A.D., Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu Rev Genet, 1995. 29: p. 477-508.
3. Moir, A., Bacterial spore germination and protein mobility. Trends Microbiol, 2003. 11(10): p. 452-4.
4. Kunst, F., et al., The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 1997. 390(6657): p. 249-56.
5. Kobayashi, K., et al., Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A, 2003. 100(8): p. 4678-83.
6. Tanner, J.J., Structural biology of proline catabolism. Amino Acids, 2008. 35(4): p. 719-30.
7. Buxton, R.S., Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage. J Gen Virol, 1980. 46(2): p. 427-37.
8. Ogura, M., et al., Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis. J Bacteriol, 1994. 176(18): p. 5673-80.
9. Belitsky, B.R., et al., Multiple genes for the last step of proline biosynthesis in Bacillus subtilis. J Bacteriol, 2001. 183(14): p. 4389-92.
10. Hahn, J., et al., Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA. Mol Microbiol, 1993. 10(1): p. 99-111.
11. Kempf, B. and E. Bremer, Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch Microbiol, 1998. 170(5): p. 319-30.
12. Measures, J.C., Role of amino acids in osmoregulation of non-halophilic bacteria. Nature, 1975. 257(5525): p. 398-400.
13. Sans, N., U. Schindler, and J. Schroder, Ornithine cyclodeaminase from Ti plasmid C58: DNA sequence, enzyme properties and regulation of activity by arginine. Eur J Biochem, 1988. 173(1): p. 123-30.
14. Baumberg, S. and C.R. Harwood, Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis. J Bacteriol, 1979. 137(1): p. 189-96.
15. Gardan, R., G. Rapoport, and M. Debarbouille, Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. J Mol Biol, 1995. 249(5): p. 843-56.
16. Atkinson, M.R., L.V. Wray, Jr., and S.H. Fisher, Regulation of histidine and proline degradation enzymes by amino acid availability in Bacillus subtilis. J Bacteriol, 1990. 172(9): p. 4758-65.
17. Adams, E., Metabolism of proline and of hydroxyproline. Int Rev Connect Tissue Res, 1970. 5: p. 1-91.
18. Bearne, S.L. and R. Wolfenden, Glutamate gamma-semialdehyde as a natural transition state analogue inhibitor of Escherichia coli glucosamine-6-phosphate synthase. Biochemistry, 1995. 34(36): p. 11515-20.
19. Tsuge, H., et al., Crystal structure of a novel FAD-, FMN-, and ATP-containing L-proline dehydrogenase complex from Pyrococcus horikoshii. J Biol Chem, 2005. 280(35): p. 31045-9.
20. Becker, D.F. and E.A. Thomas, Redox properties of the PutA protein from Escherichia coli and the influence of the flavin redox state on PutA-DNA interactions. Biochemistry, 2001. 40(15): p. 4714-21.
21. Menzel, R. and J. Roth, Regulation of the genes for proline utilization in Salmonella typhimurium: autogenous repression by the putA gene product. J Mol Biol, 1981. 148(1): p. 21-44.
22. Vilchez, S., M. Manzanera, and J.L. Ramos, Control of expression of divergent Pseudomonas putida put promoters for proline catabolism. Appl Environ Microbiol, 2000. 66(12): p. 5221-5.
23. Muro-Pastor, A.M., P. Ostrovsky, and S. Maloy, Regulation of gene expression by repressor localization: biochemical evidence that membrane and DNA binding by the PutA protein are mutually exclusive. J Bacteriol, 1997. 179(8): p. 2788-91.
24. Brown, E.D. and J.M. Wood, Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli. J Biol Chem, 1992. 267(18): p. 13086-92.
25. Fisher, S.H. and A.L. Sonenshein, Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression. J Bacteriol, 1984. 157(2): p. 612-21.
26. Belitsky, B.R. and A.L. Sonenshein, Modulation of activity of Bacillus subtilis regulatory proteins GltC and TnrA by glutamate dehydrogenase. J Bacteriol, 2004. 186(11): p. 3399-407.
27. Matsuoka, H., K. Hirooka, and Y. Fujita, Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation. J Biol Chem, 2007. 282(8): p. 5180-94.
28. Yamane, K., M. Kumano, and K. Kurita, The 25 degrees-36 degrees region of the Bacillus subtilis chromosome: determination of the sequence of a 146 kb segment and identification of 113 genes. Microbiology, 1996. 142 ( Pt 11): p. 3047-56.
29. Glaser, P., et al., Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees. Mol Microbiol, 1993. 10(2): p. 371-84.
30. Sonenshein, A.L., Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol, 2007. 5(12): p. 917-27.
31. Gutowski, J.C. and H.J. Schreier, Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo. J Bacteriol, 1992. 174(3): p. 671-81.
32. Wray, L.V., Jr., et al., TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis. Proc Natl Acad Sci U S A, 1996. 93(17): p. 8841-5.
33. Fisher, S.H., Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference! Mol Microbiol, 1999. 32(2): p. 223-32.
34. Belitsky, B.R., P.J. Janssen, and A.L. Sonenshein, Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression. J Bacteriol, 1995. 177(19): p. 5686-95.
35. Bohannon, D.E., M.S. Rosenkrantz, and A.L. Sonenshein, Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source. J Bacteriol, 1985. 163(3): p. 957-64.
36. Henkin, T.M., The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis. FEMS Microbiol Lett, 1996. 135(1): p. 9-15.
37. Moreno, M.S., et al., Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Mol Microbiol, 2001. 39(5): p. 1366-81.
38. Fujita, Y., et al., Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr. Mol Microbiol, 1995. 17(5): p. 953-60.
39. Galinier, A., et al., The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression. Proc Natl Acad Sci U S A, 1997. 94(16): p. 8439-44.
40. Ramseier, T.M., et al., In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of gram-positive bacteria. FEMS Microbiol Lett, 1995. 129(2-3): p. 207-13.
41. Shivers, R.P. and A.L. Sonenshein, Bacillus subtilis ilvB operon: an intersection of global regulons. Mol Microbiol, 2005. 56(6): p. 1549-59.
42. Lulko, A.T., et al., Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 2007. 12(1-2): p. 82-95.
43. Shivers, R.P. and A.L. Sonenshein, Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids. Mol Microbiol, 2004. 53(2): p. 599-611.
44. Molle, V., et al., Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis. J Bacteriol, 2003. 185(6): p. 1911-22.
45. Serror, P. and A.L. Sonenshein, CodY is required for nutritional repression of Bacillus subtilis genetic competence. J Bacteriol, 1996. 178(20): p. 5910-5.
46. Inaoka, T. and K. Ochi, RelA protein is involved in induction of genetic competence in certain Bacillus subtilis strains by moderating the level of intracellular GTP. J Bacteriol, 2002. 184(14): p. 3923-30.
47. Lopez, J.M., A. Dromerick, and E. Freese, Response of guanosine 5'-triphosphate concentration to nutritional changes and its significance for Bacillus subtilis sporulation. J Bacteriol, 1981. 146(2): p. 605-13.
48. Ratnayake-Lecamwasam, M., et al., Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev, 2001. 15(9): p. 1093-103.
49. Guedon, E., et al., Pleiotropic transcriptional repressor CodY senses the intracellular pool of branched-chain amino acids in Lactococcus lactis. Mol Microbiol, 2001. 40(5): p. 1227-39.
50. Jarmer, H., et al., Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions. FEMS Microbiol Lett, 2002. 206(2): p. 197-200.
51. Slack, F.J., et al., A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol, 1995. 15(4): p. 689-702.
52. Fisher, S.H., K. Rohrer, and A.E. Ferson, Role of CodY in regulation of the Bacillus subtilis hut operon. J Bacteriol, 1996. 178(13): p. 3779-84.
53. Liebs, P., et al., Formation of some extracellular enzymes during the exponential growth of Bacillus subtilis. Folia Microbiol (Praha), 1988. 33(2): p. 88-95.
54. Hightower, L.E., Stress Proteins In Biology and Medicine. Richard I. Morimoto, Alfred Tissieres, and Costa Georgopoulos, Eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1990. x, 450 pp., illus. $97. Cold Spring Harbor Monograph Series 19. Science, 1990. 249(4968): p. 572-573.
55. Volker, U., et al., Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology, 1994. 140 ( Pt 4): p. 741-52.
56. Hecker, M., W. Schumann, and U. Volker, Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol, 1996. 19(3): p. 417-28.
57. Price, C.W., et al., Genome-wide analysis of the general stress response in Bacillus subtilis. Mol Microbiol, 2001. 41(4): p. 757-74.
58. Boylan, S.A., et al., Stress-induced activation of the sigma B transcription factor of Bacillus subtilis. J Bacteriol, 1993. 175(24): p. 7931-7.
59. Benson, A.K. and W.G. Haldenwang, The sigma B-dependent promoter of the Bacillus subtilis sigB operon is induced by heat shock. J Bacteriol, 1993. 175(7): p. 1929-35.
60. Volker, U., B. Maul, and M. Hecker, Expression of the sigmaB-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis. J Bacteriol, 1999. 181(13): p. 3942-8.
61. Csonka, L.N. and A.D. Hanson, Prokaryotic osmoregulation: genetics and physiology. Annu Rev Microbiol, 1991. 45: p. 569-606.
62. Hoffmann, T., et al., Responses of Bacillus subtilis to hypotonic challenges: physiological contributions of mechanosensitive channels to cellular survival. Appl Environ Microbiol, 2008. 74(8): p. 2454-60.
63. Chen, M., et al., Expression of Bacillus subtilis proBA genes and reduction of feedback inhibition of proline synthesis increases proline production and confers osmotolerance in transgenic Arabidopsis. J Biochem Mol Biol, 2007. 40(3): p. 396-403.
64. Burg, M.B., E.D. Kwon, and D. Kultz, Regulation of gene expression by hypertonicity. Annu Rev Physiol, 1997. 59: p. 437-55.
65. Miller, K.J. and J.M. Wood, Osmoadaptation by rhizosphere bacteria. Annu Rev Microbiol, 1996. 50: p. 101-36.
66. Whatmore, A.M. and R.H. Reed, Determination of turgor pressure in Bacillus subtilis: a possible role for K+ in turgor regulation. J Gen Microbiol, 1990. 136(12): p. 2521-6.
67. Whatmore, A.M., J.A. Chudek, and R.H. Reed, The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis. J Gen Microbiol, 1990. 136(12): p. 2527-35.
68. Kappes, R.M., B. Kempf, and E. Bremer, Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD. J Bacteriol, 1996. 178(17): p. 5071-9.
69. Kempf, B. and E. Bremer, OpuA, an osmotically regulated binding protein-dependent transport system for the osmoprotectant glycine betaine in Bacillus subtilis. J Biol Chem, 1995. 270(28): p. 16701-13.
70. Lin, Y. and J.N. Hansen, Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168. J Bacteriol, 1995. 177(23): p. 6874-80.
71. Boch, J., B. Kempf, and E. Bremer, Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J Bacteriol, 1994. 176(17): p. 5364-71.
72. Peng, Z., Q. Lu, and D.P. Verma, Reciprocal regulation of delta 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants. Mol Gen Genet, 1996. 253(3): p. 334-41.
73. Wengender, P.A. and K.J. Miller, Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli. Appl Environ Microbiol, 1995. 61(1): p. 252-9.
74. Wood, J.M., Proline porters effect the utilization of proline as nutrient or osmoprotectant for bacteria. J Membr Biol, 1988. 106(3): p. 183-202.
75. Jung, H., Towards the molecular mechanism of Na(+)/solute symport in prokaryotes. Biochim Biophys Acta, 2001. 1505(1): p. 131-43.
76. von Blohn, C., et al., Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B. Mol Microbiol, 1997. 25(1): p. 175-87.
77. Spiegelhalter, F. and E. Bremer, Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A- and sigma B-dependent stress-responsive promoters. Mol Microbiol, 1998. 29(1): p. 285-96.
78. Hahne, H., et al., A comprehensive proteomics and transcriptomics analysis of Bacillus subtilis salt stress adaptation. J Bacteriol, 2010. 192(3): p. 870-82.
79. Errington, J., Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev, 1993. 57(1): p. 1-33.
80. Piggot, P.J. and D.W. Hilbert, Sporulation of Bacillus subtilis. Curr Opin Microbiol, 2004. 7(6): p. 579-86.
81. Predich, M., G. Nair, and I. Smith, Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H. J Bacteriol, 1992. 174(9): p. 2771-8.
82. Zhao, H., et al., DNA complexed structure of the key transcription factor initiating development in sporulating bacteria. Structure, 2002. 10(8): p. 1041-50.
83. Baldus, J.M., et al., Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. J Bacteriol, 1994. 176(2): p. 296-306.
84. Sonenshein, A.L., Control of sporulation initiation in Bacillus subtilis. Curr Opin Microbiol, 2000. 3(6): p. 561-6.
85. Rossignol, D.P. and J.C. Vary, L-Proline site for triggering Bacillus megaterium spore germination. Biochem Biophys Res Commun, 1979. 89(2): p. 547-51.
86. Boland, F.M., et al., Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB. Microbiology, 2000. 146 ( Pt 1): p. 57-64.
87. Hudson, K.D., et al., Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J Bacteriol, 2001. 183(14): p. 4317-22.
88. Molle, V., et al., The Spo0A regulon of Bacillus subtilis. Mol Microbiol, 2003. 50(5): p. 1683-701.
89. Eichenberger, P., et al., The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol, 2004. 2(10): p. e328.
90. Rossignol, D.P. and J.C. Vary, Biochemistry of L-proline-triggered germination of Bacillus megaterium spores. J Bacteriol, 1979. 138(2): p. 431-41.
91. Fulco, A.J. and R.T. Ruettinger, Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli. Life Sci, 1987. 40(18): p. 1769-75.
92. Birnboim, H.C. and J. Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res, 1979. 7(6): p. 1513-23.
93. Igo, M.M. and R. Losick, Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J Mol Biol, 1986. 191(4): p. 615-24.
94. Chang, S. and S.N. Cohen, High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet, 1979. 168(1): p. 111-5.
95. Kunst, F. and G. Rapoport, Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol, 1995. 177(9): p. 2403-7.
96. Janknecht, R., et al., Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A, 1991. 88(20): p. 8972-6.
97. Wang, S.T., et al., The forespore line of gene expression in Bacillus subtilis. J Mol Biol, 2006. 358(1): p. 16-37.
98. Tseng, C.L., H.J. Chen, and G.C. Shaw, Identification and characterization of the Bacillus thuringiensis phaZ gene, encoding new intracellular poly-3-hydroxybutyrate depolymerase. J Bacteriol, 2006. 188(21): p. 7592-9.
99. Lin, J.S. and G.C. Shaw, Regulation of the kduID operon of Bacillus subtilis by the KdgR repressor and the ccpA gene: identification of two KdgR-binding sites within the kdgR-kduI intergenic region. Microbiology, 2007. 153(Pt 3): p. 701-10.
100. Haldenwang, W.G., The sigma factors of Bacillus subtilis. Microbiol Rev, 1995. 59(1): p. 1-30.
101. Commichau, F.M., et al., Glutamate metabolism in Bacillus subtilis: gene expression and enzyme activities evolved to avoid futile cycles and to allow rapid responses to perturbations of the system. J Bacteriol, 2008. 190(10): p. 3557-64.
102. Muro-Pastor, A.M. and S. Maloy, Proline dehydrogenase activity of the transcriptional repressor PutA is required for induction of the put operon by proline. J Biol Chem, 1995. 270(17): p. 9819-27.
103. Commichau, F.M. and J. Stulke, Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression. Mol Microbiol, 2008. 67(4): p. 692-702.
104. Vilchez, S., et al., Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates. J Bacteriol, 2000. 182(1): p. 91-9.
105. Cho, K. and S.C. Winans, The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated. Mol Microbiol, 1996. 22(5): p. 1025-33.
106. Keuntje, B., B. Masepohl, and W. Klipp, Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein. J Bacteriol, 1995. 177(22): p. 6432-9.
107. Jafri, S., et al., An Lrp-type transcriptional regulator from Agrobacterium tumefaciens condenses more than 100 nucleotides of DNA into globular nucleoprotein complexes. J Mol Biol, 1999. 288(5): p. 811-24.
108. Macaluso, A., E.A. Best, and R.A. Bender, Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes. J Bacteriol, 1990. 172(12): p. 7249-55.
109. Suhr, M. and D. Kleiner, Genetic analysis of the regulatory putP region (coding for proline permease) in Klebsiella pneumoniae M5a1: evidence for regulation by the nac system. FEMS Microbiol Lett, 1993. 114(2): p. 191-4.
110. Madhusudhan, K.T., N. Huang, and J.R. Sokatch, Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida. J Bacteriol, 1995. 177(3): p. 636-41.
111. Yuan, G. and S.L. Wong, Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE). J Bacteriol, 1995. 177(19): p. 5427-33.
112. Lereclus, D., et al., Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant. Biotechnology (N Y), 1995. 13(1): p. 67-71.
113. White, T.A., et al., Structure and kinetics of monofunctional proline dehydrogenase from Thermus thermophilus. J Biol Chem, 2007. 282(19): p. 14316-27.