Transcriptome analysis of K. pneumoniae ΔgalU gene expression profiles under galactose stress

83

Many literatures have reported that galactose metabolites, especially Gal-1-P, are toxic to bacterial strains deficient in the enzymes of the Leloir pathway (GalETK). Until now, how the toxic intermediates cause the cell death and what the targets of those toxic intermediates are remain elusive (93,104). Therefore, we attempted to find the targets of toxic galactose metabolites and understrand the molecular mechanism of the galactose toxicity by performing RNA sequencing. Approximately 10.41 and 10.54 million 150-bp pair end reads from K. pneumoniae ΔgalU and K. pneumoniae ΔgalU (+Gal), respectively, were generated using Illumina HiSeq 2000 Sequencing System and analyzed by CLC-Genomics Workbench.

The transcriptomic data revealed that 44 and 144 genes were up- and down-regulated, respectively more than 2 folds in the K. pneumoniae ΔgalU mutant in the presence of galactose. These genes accounted for about 3.84% of the total genes in K. pneumoniae. It has been known that adding galactose into growth medium could induce the transcriptions of gal regulon (galETK) and galactose permease (galP) in Saccharomyces cerevisiae galE and galT mutant strains (104,105). When focus on the upregulated genes of K. pneumoniae ΔgalU mutant listed in Table 3.5.1, the expression of the gal regulon (galETKM) and galactose permease (galP) was increased in the presence of galactose consistent with the previous literature. In addition, the lacYZ and genes responsible for amino acid biosynthesis were also induced in the K. pneumoniae GalU-deficient strain upon galactose stress.

Among the downregulated genes, pduCDE, D364_17595, and D364_17590 which are responsible for glycerol metabolism (106) and some sugar transporter genes are repressed.

Unexpectedly, two iron-acquisition systems, sitABCD and feoABC, were also repressed in the presence of galactose (Table 3.5.2). These two gene clusters are regulated by ferric uptake regulator (Fur) and their putative Fur binding boxes were listed in Table 3.5.5 (107). Under iron-replete conditions, dimeric Fur in complex with ferrous ion (Fe²⁺) binds to the Fur box,

84

19 bp consensus DNA sequence (GATAAT-GATAAT-CATTATC), preventing sitABCD and

feoABC from transcription (108-111). However, M9gly medium used in this study is deficient

in ferrous ion (Fe²⁺), the repressed transcription of sitABCD and feoABC under iron-limited condition implied that other regulatory factors might participate. One of the possible regulatory factors is RyhB, a 91 nucleotide non-coding RNA which was induced by iron starvation (112). In E. coli, a microarray-based analysis revealed that overproduction of RyhB indirectly repressed the expression of fhuF, feoA and feoB (113). Besides, RyhB was found to repress the expression of sitA in K. pneumoniae presumably by direct base pairing with sitA mRNA (114). To sum up, the transcriptional repressions of sitABCD, feoABC and genes encoding iron-utilizing proteins might be due to the expression of RyhB in M9gly medium (0.5% galactose).

Interestingly, the genes subjected to galactose-mediated activation and suppression in the K. pneumoniae ΔgalU mutant is clustered, suggesting that some transcriptional regulators participate in controlling the gene expression. Therefore, three hundred base pair upstream regions of these gene clusters were analyzed and the putative regulator binding sites were predicted using the bacterial regulon analyzer Virtual Footprint (http://www.prodoric.de/vfp/) (115). Most of the promoter regions, including PgalE, PgalP, PpduC, PD364_17615, PsitA and PfeoA

were predicted to have a CRP binding box (Table 3.5.3). CRP is a global transcriptional regulator and requires the binding of cyclic AMP (cAMP) for complete activity (116). Up to now, a total of 346 promoters in E. coli K12 genome have been proposed to be under the control of cAMP-CRP (117). The cAMP-CRP complex binds to specific sites upstream of promoters, causing transcriptional activation or repression (118). The production of cAMP is reduced in the presence of glucose. When bacteria are grown in glyceol minimal medium or medium supplemented with less-preferred carbon sources, bacteria produce higher levels of

85

cAMP leading to the formation of cAMP-CRP complex (119-121). Our bacterial culture condition favors more cAMP-CRP complex formation and therefore leads to galETKM, galP, and pduCDE transcription.

In addition to genes involved in carbon metabolism and iron-acquisition, 12 regulatory genes are downregulated in the K. pneumoniae ΔgalU mutant under galactose stress. Notably, two global regulators, H-NS and CsrA, were repressed in the presence of galactose. H-NS is homologous to eukaryotic histones and has been extensively studied in E. coli and

Salmonella typhimurium (122,123). H-NS plays important roles in bacterial DNA packaging

and silencing genes involved in metabolism, flagellum synthesis, virulence and adaptation to environmental challenges (123-126). However, genes known to be repressed by H-NS were not significantly upregulated in our transcriptome data (124). We also analyzed the upstream promoter regions of P_lacZ, P_{D364_17615}, P_pduC, P_sitA, and P_feoA. Although extremely conserved H-NS nucleating high-affinity sites were found (Table 3.5.4) (127), no literature has ever reported that these genes are under regulation of H-NS. CsrA, a post-transcriptional regulator, controls a large variety of physiological processes such as central carbon metabolism, virulence, pathogenesis, c-di-GMP synthesis, biofilm formation, quorum sensing as well as bacterial motility and stress response (128-134). CsrA is an RNA-binding protein. The protein binds to a region typically nearby or overlapping with Shine-Dalgarno sequence to block the ribosome bindng, resulting in inhibition of translation initiation and rapid mRNA degradation (135-137). In some cases, CsrA served as a positive regulator through binding to the 5’ untranslated region to stabilize the mRNA, leading to the enhancement of translation efficiency (138). E. coli csrA gene was shown to be essential for growth in LB medium and minimal medium supplemented with glycolytic carbon sources (139), revealing the important role of CsrA in bacterial viability. Therefore, based on the RNA-seq results, we proposed that

86

when K. pneumoniae ΔgalU mutant was cultivated in minimal medium supplemented with galactose, the accumulation of toxic galactose metabolites might indirectly target to H-NS, CsrA and several other transcriptional regulators, causing problems on transcription and translation. Eventually, the bacterial growth becomes slow and cell death increases (Fig.

3.6.3B) because of disruption of normal bacterial physiology.

In summary, our transcriptomic results indicate that expression of carbon metabolism and iron-acquisition systems was affected in our culture medium providing galactose stresses.

Based on promoter prediction results and literature review, we hypothesized that the transcription of these genes might be under the regulation of cAMP-CRP complex. In addition, K. pneumoniae ΔgalU mutant increased the expression of galETKM in nutrient-deficient growth condition. However, because GalU deficiency in K. pneumoniae interrupts normal galactose metabolism, the higher amounts of GalP and GalETKM in cells may accelerate accumulations of toxic galactose metabolites, which in turn indirectly repress the expression of hns and csrA whose functions are important in DNA packaging and bacterial viability. Previously, Slepak and colleagues found that ribosomal protein (RP) genes and genes involved in RNA metabolism were repressed in S. cerevisiae galT mutant challenged with galactose (104). This phenomenon was due to the accumulation of Gal-1-P, the toxic galactose metabolite, in galT deficient strain. It was known that these two groups of genes were repressed via signal transduction pathways when S. cerevisiae is subjected to external stress stimulus such as hydrogen peroxide, DTT or heat shock. Thus, the authors proposed that “Gal-1-P stress” repressed the transcriptions of RP and RNA metabolism genes through signal transduction pathways responsible for responding to environmental stimulus (104,140). Similarly, the toxic galactose metabolites accumulated in K. pneumoniae ΔgalU mutant can be seen as a stress signal and might influence the gene expression via a “known”

87

signal transduction pathway or regulatory factor. Therefore, understanding which signal transduction pathway or regulatory factor participated in repressing the transcriptions of hns,

csrA and transcriptional regulator genes will bring us to find the in vivo target(s) of toxic

galactose metabolites. Finally, we hope that the transcriptome study of galactose effects on K.

pneumoniae ΔgalU mutant helps us to understand how the toxic galactose metabolites cause

cell death.

88

3.5 Tables

Table 3.5.1 Upregulated genes in CG43S3 ΔgalU (+Gal) comparing with CG43S3 ΔgalU

Feature ID Gene Name Fold Change Proposed Function

D364_03825

galE

81.26 UDP-glucose 4 -epimerase

D364_03820

galT

54.15 galactose-1-phosphate uridylyltransferase

D364_03815

galK

49.74 galactokinase

galM(D364_03810) galM

29.80 galactose-1-epimerase

D364_17020

galP

15.74 D-galactose transporter

D364_07970

lacY

23.91 galactoside permease

D364_07975

lacZ

22.76

-

D-galactosidase

D364_20570

dgoR

11.91



galactonate operon transcriptional repressor

D364_20560

dgoA

6.82 2-dehydro-3-deoxy-6-phosphogalactonate aldolase

D364_20555

dgoD

13.22 galactonate dehydratase

D364_20550 5.64 galactonate transporter

89

(Continued)

Feature ID Gene Name Fold Change Proposed Function

D364_07650 19.87 di- and tricarboxylate transporters, L-tartrate/succinate antiporte

D364_21575

metE

9.73 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase

glyA(D364_14465) glyA

7.64 Ser hydroxymethyltransferase

glnA(D364_20910) glnA

7.44 glutamine synthetase

D364_17015

metK

7.25 S-adenosylmethionine synthetase

glnH(D364_04210) glnH

6.02 glutamine ABC transporter periplasmic protein D364_03465

gltI

5.70 glutamate and aspartate transporter subunit

D364_08005 5.33 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase

metQ(D364_01015) metQ

4.95 DL-methionine transporter substrate-binding subunit

D364_13485

argT

4.85 lysine-, arginine-, ornithine-binding periplasmic protein

D364_13470

hisM

4.76 histidine/lysine/arginine/ornithine ABC transporter permease HisM D364_03460

gltJ

4.60 glutamate/aspartate ABC transporter permease GltJ

90

Table 3.5.2 Downregulated genes in CG43S3 ΔgalU (+Gal) comparing with CG43S3 ΔgalU

Feature ID Gene Name fold change Proposed Function

D364_17620

dhaT

-38.34 1,3-propanediol oxidoreductase (alcohol dehydrogenase)

D364_17615 -12.23 hypothetical protein

pduC(D364_17610) pduC

-11.26 propanediol dehydratase large subunit

pduD(D364_17605) pduD

-16.59 propanediol dehydratase medium subunit

pduE(D364_17600) pduE

-14.89 propanediol dehydratase small subunit

D364_17595 -14.12 glycerol dehydratase

D364_17590 -12.61 glycerol uptake facilitator GlpF

D364_17580 -7.60 acid-resistance membrane protein

hdeB(D364_16510) hdeB

-4.96 acid-resistance protein

hdeB(D364_17585) hdeB

-4.25 acid-resistance protein

D364_23340 -7.02 sn-glycerol-3-phosphate transport system permease

glpA(D364_13220) glpA

-5.96 sn-glycerol-3-phosphate dehydrogenase subunit A

glpT(D364_13215) glpT

-4.94 sn-glycerol-3-phosphate transporter

malE(D364_22000) malE

-8.11 maltose ABC transporter periplasmic protein D364_22015

malM

-5.86 maltose regulon periplasmic protein

D364_22005 -6.75 ABC-type sugar transport systems, ATPase components

D364_15140 -8.51 PTS system glucitol/sorbitol-specific transporter subunit IIA

srlA(D364_15130) srlA

-4.08 glucitol/sorbitol-specific PTS family enzyme IIC component

91

(Continued)

Feature ID Gene Name fold change Proposed Function

D364_15335

sitA

-40.33 iron ABC transporter substrate-binding protein

D364_15340

sitB

-23.93 manganese/iron transport system ATP-binding protein D364_15345

sitC

-23.24 iron transport system inner membrane permease component

D364_15350

sitD

-12.23 manganese ABC transporter, inner membrane permease protein SitD

feoA(D364_18960) feoA

-9.36 ferrous iron transport protein A

feoB(D364_18965) feoB

-7.14 ferrous iron transport protein B D364_18970

feoC

-10.99 ferrous iron transport protein FeoC D364_18675

bfd

-30.03 bacterioferritin-associated ferredoxin

D364_05240 -19.35 hypothetical protein;iron permease

D364_05245 -18.90 hypothetical protein; iron uptake system component EfeO D364_24070

fhuF

-13.22 ferric iron reductase involved in ferric hydroximate transport

D364_10960

tonB

-12.62 transport protein TonB

D364_13700

mntH

-7.93 manganese transport protein MntH

D364_11490 -5.78 ferrichrome-iron receptor

D364_00795 -4.25 iron-hydroxamate transporter substrate-binding subunit

D364_00785 -4.10 ferrichrome outer membrane transporter

92

(Continued)

Feature ID Gene Name fold change Proposed Function

D364_10085 -8.51 LysR family transcriptional regulator

D364_12135 -7.44 LysR family transcriptional regulator

D364_17675 -6.38 PadR family transcriptional regulator

D364_06175 -5.32 DNA-binding transcriptional activator OsmE

D364_13730

yfeR

-5.05 LysR family transcriptional regulator D364_01955

bolA

-5.00 transcriptional regulator BolA

D364_11035

hns

-4.78 global DNA-binding transcriptional dual regulator H-NS D364_04125

ybiH

-4.73 DNA-binding transcriptional regulator

D364_07595 -4.41 DNA-binding transcriptional regulator

D364_18425

envR

-4.08 DNA-binding transcriptional regulator EnvR D364_23815

cstA

-6.25 carbon starvation protein CstA

D364_15080

csrA

-4.06 carbon storage regulator

93

Table 3.5.3 Predicted CRP binding site in the upstream region of galactose regulated genes

Gene CRP binding site

5′-TG(T/C)GA:N6:TC(A/G)CA-3′

PgalE -75 CTTGT:GCTATG:TCACA -60

PgalP -81 CGTGA:TTATAG:TCACG -66

PD364_17615 -209 TGTGA:TCGCCC:GCAAT -194

P_pduC -223 ATTGC:GGGCGA:TCACA -202

P_pduC -127 ATTTA:TTTTTT:TCACC -106

P_sitA -68 CTTGT:GCTATA:TAACA -53

PfeoA -156 CCTGC:AGCGCA:TCATA -141

Numbers are relative to the position of the translational start codon (Details are found in Appendix II). Nucleobase identical to the consensus CRP binding sequences are underlined.

94

Table 3.5.4 Predicted H-NS binding site in the upstream region of the galactose regulated genes

Gene H-NS binding site 5′-TCGATAAATT-3′

PlacZ -105 TCTGTTAATT -96

PD364_17615 -283 TCGTTTCAAT -274

PD364_17615 -185 TCAATTAATA -176

P_pduC -10 CCGATGAACA -1

P_sitA -79 TTTATAAATA -70

P_sitA -44 GCTATAAACG -35

PfeoA -189 TCAATAAAAA -180

PfeoA -20 GCGATAGACA -11

Numbers are relative to the position of the translational start codon (Details are found in Appendix II). Nucleobase identical to the consensus H-NS binding sequences are underlined.

95

Table 3.5.5 Predicted Fur binding site in the upstream region of the galactose regulated genes

Gene Fur binding box

5′-GATAAT:GATAAT:CATTATC-3′

PsitA -99 GCAAAT:AAGAAT:TATTTTC -81

P_feoA -134 GATGAT:AAAAAC:CATTCTC -116

Numbers are relative to the position of the translational start codon (Details are found in Appendix II). Nucleobase identical to the consensus Fur binding sequences are underlined.

96

3.6 Figures

Fig. 3.6.1 Effects of glucose or galactose on K. pneumoniae CG43 S3 and ΔgalU mutant using the disk diffusion assay

The bacterial cultures were applied onto LB agar plates and the paper disks impregnated with 3

l of 50% galactose or glucose were placed on the top of the

bacterial lawn. After an overnight incubation at 37^oC, the plates were examined and the pictures were taken by a Canon camera (PowerShot G10).

97

Fig. 3.6.2 Growth curve of K. pneumoniae CG43 S3 and the ΔgalU mutant

(A)K. pneumoniae CG43 S3 (B)K. pneumoniae CG43 ΔgalU mutant. The overnight bacterial cultures were sub-cultured in fresh LB medium containing various concentrations of galactose. The optical density of each bacterial culture was measured every hour at the wavelength of 595 nm by a spectrophotometer (Jasco V-530).

98

Fig. 3.6.3 The growth curve and Live/Dead cell staining of the K. pneumoniae CG43 ΔgalU mutant

(A) Growth curve of K. pneumoniae CG43 S3 ΔgalU mutant in M9gly medium containing 0.5% galactose. The green solid arrow indicates the time point at which the galactose is added into bacterial cultures. The numbers represent the time point at which the bacterial cells are collected for Live/Dead cell staining. (B)When the OD595

reached to 0.1 - 0.2 AU, galactose was added into the bacterial cultures at the final concentration of 0.5% (w/v). After being treated with galactose for 3 h, the bacterial cells were mixed with the fluorescence dye for 15 min in dark and examined under a fluorescence microscope with a 100x objective lens.

99

References

1. Cleasby, A., Wonacott, A., Skarzynski, T., Hubbard, R. E., Davies, G. J., Proudfoot, A. E., Bernard, A. R., Payton, M. A., and Wells, T. N. (1996) The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution. Nature structural biology 3, 470-479

2. Wu, B., Zhang, Y., Zheng, R., Guo, C., and Wang, P. G. (2002) Bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase is the point of control for GDP-D-mannose biosynthesis in Helicobacter pylori. FEBS

letters 519, 87-92

3. Rocchetta, H. L., Pacan, J. C., and Lam, J. S. (1998) Synthesis of the A-band polysaccharide sugar D-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa.

Molecular microbiology 29, 1419-1434

4. Proudfoot, A. E., Payton, M. A., and Wells, T. N. (1994) Purification and characterization of fungal and mammalian phosphomannose isomerases.

Journal of protein chemistry 13, 619-627

5. Proudfoot, A. E., Turcatti, G., Wells, T. N., Payton, M. A., and Smith, D. J.

(1994) Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase. European journal of biochemistry / FEBS 219, 415-423

6. Jensen, S. O., and Reeves, P. R. (1998) Domain organisation in phosphomannose isomerases (types I and II). Biochimica et biophysica acta

1382, 5-7

7. Shinabarger, D., Berry, A., May, T. B., Rothmel, R., Fialho, A., and Chakrabarty, A. M. (1991) Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa. The Journal of biological chemistry 266, 2080-2088

8. Friedman, L., and Kolter, R. (2004) Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. Journal of bacteriology 186, 4457-4465

9. Jackson, K. D., Starkey, M., Kremer, S., Parsek, M. R., and Wozniak, D. J.

(2004) Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation. Journal

of bacteriology 186, 4466-4475

10. Duggleby, R. G., Peng, H. L., and Chang, H. Y. (1996) An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products. Experientia 52, 568-572

100

11. Sousa, S. A., Moreira, L. M., Wopperer, J., Eberl, L., Sa-Correia, I., and Leitao, J. H. (2007) The Burkholderia cepacia bceA gene encodes a protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities. Biochemical and biophysical research communications 353, 200-206

12. Makinen, K. K., Makinen, P. L., Wilkes, S. H., Bayliss, M. E., and Prescott, J.

M. (1982) Photochemical inactivation of Aeromonas aminopeptidase by 2,3-butanedione. The Journal of biological chemistry 257, 1765-1772

13. Tawfik, D. S. (2002) Modification of Arginine Side Chains with p-Hydroxyphenylglyoxal. in The Protein Protocols Handbook (Walker, J. M.

ed.), second Ed., Humana Press, Totowa, New Jersey. pp 475-476

14. Koplin, R., Arnold, W., Hotte, B., Simon, R., Wang, G., and Puhler, A. (1992) Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis.

Journal of bacteriology 174, 191-199

15. Griffin, A. M., Poelwijk, E. S., Morris, V. J., and Gasson, M. J. (1997) Cloning of the aceF gene encoding the phosphomannose isomerase and GDP-mannose pyrophosphorylase activities involved in acetan biosynthesis in Acetobacter xylinum. FEMS microbiology letters 154, 389-396

16. Pain, R. (2004) Determining the CD Spectrum of a Protein. in Current

Protocols in Protein Science (John E. Coligan, B. M. D., David W. Speicher,

Paul T. Wingfield ed.), Jonh Wiley & Sons, Hoboken, New Jersey. pp Unit 7.6 17. Magliery, T. J., Wilson, C. G., Pan, W., Mishler, D., Ghosh, I., Hamilton, A. D.,

and Regan, L. (2005) Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. Journal

of the American Chemical Society 127, 146-157

18. Hung, R. J., Chien, H. S., Lin, R. Z., Lin, C. T., Vatsyayan, J., Peng, H. L., and Chang, H. Y. (2007) Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1. The Journal of biological

chemistry 282, 17738-17748

19. Papoutsopoulou, S. V., and Kyriakidis, D. A. (1997) Phosphomannose isomerase of Xanthomonas campestris: a zinc activated enzyme. Molecular

and cellular biochemistry 177, 183-191

20. Hurwitz, C., and Rosano, C. L. (1967) The intracellular concentration of bound and unbound magnesium ions in Escherichia coli. The Journal of

biological chemistry 242, 3719-3722

21. Wells, T. N., Scully, P., and Magnenat, E. (1994) Arginine 304 is an active site residue in phosphomannose isomerase from Candida albicans. Biochemistry

33, 5777-5782

22. Rogers, T. B., Borresen, T., and Feeney, R. E. (1978) Chemical modification of

101

the arginines in transferrins. Biochemistry 17, 1105-1109

23. Swan, M. K., Hansen, T., Schonheit, P., and Davies, C. (2004) Structural basis for phosphomannose isomerase activity in phosphoglucose isomerase from Pyrobaculum aerophilum: a subtle difference between distantly related enzymes. Biochemistry 43, 14088-14095

24. May, T. B., Shinabarger, D., Boyd, A., and Chakrabarty, A. M. (1994) Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa. The Journal of biological chemistry 269, 4872-4877

25. Sousa, S. A., Moreira, L. M., and Leitao, J. H. (2008) Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities. Applied

microbiology and biotechnology 80, 1015-1022

26. Barends, T. R., Dunn, M. F., and Schlichting, I. (2008) Tryptophan synthase, an allosteric molecular factory. Current opinion in chemical biology 12, 593-600

27. Sa-Correia, I., Darzins, A., Wang, S. K., Berry, A., and Chakrabarty, A. M.

(1987) Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene. Journal of

bacteriology 169, 3224-3231

28. Ghosh, I., Hamilton, A. D., and Regan, L. (2000) Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. Journal of the American Chemical Society 122, 5658-5659

29. Wilson, C. G., Magliery, T. J., and Regan, L. (2004) Detecting protein-protein interactions with GFP-fragment reassembly. Nature methods 1, 255-262

30. Rodrigue, A., Quentin, Y., Lazdunski, A., Mejean, V., and Foglino, M. (2000) Two-component systems in Pseudomonas aeruginosa: why so many? Trends in

microbiology 8, 498-504

31. Stock, A. M., Robinson, V. L., and Goudreau, P. N. (2000) Two-component signal transduction. Annual review of biochemistry 69, 183-215

32. Akerley, B. J., Cotter, P. A., and Miller, J. F. (1995) Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell 80, 611-620

33. Lux, R., and Shi, W. (2005) A novel bacterial signalling system with a combination of a Ser/Thr kinase cascade and a His/Asp two-component system. Molecular microbiology 58, 345-348

102

34. Hsu, J. L., Chen, H. C., Peng, H. L., and Chang, H. Y. (2008) Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1. The Journal of

biological chemistry 283, 9933-9944

35. Lin, C. T., Huang, Y. J., Chu, P. H., Hsu, J. L., Huang, C. H., and Peng, H. L.

(2006) Identification of an HptB-mediated multi-step phosphorelay in Pseudomonas aeruginosa PAO1. Research in microbiology 157, 169-175 36. Garsin, D. A., Duncan, L., Paskowitz, D. M., and Losick, R. (1998) The kinase

activity of the antisigma factor SpoIIAB is required for activation as well as inhibition of transcription factor sigmaF during sporulation in Bacillus subtilis.

Journal of molecular biology 284, 569-578

37. Mattoo, S., Yuk, M. H., Huang, L. L., and Miller, J. F. (2004) Regulation of type III secretion in Bordetella. Molecular microbiology 52, 1201-1214

38. Mougous, J. D., Gifford, C. A., Ramsdell, T. L., and Mekalanos, J. J. (2007) Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nature cell biology 9, 797-803

39. Mukhopadhyay, S., Kapatral, V., Xu, W., and Chakrabarty, A. M. (1999) Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa. Journal of

bacteriology 181, 6615-6622

40. Motley, S. T., and Lory, S. (1999) Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa. Infection and

immunity 67, 5386-5394

41. Deziel, E., Comeau, Y., and Villemur, R. (2001) Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. Journal of bacteriology 183, 1195-1204

42. Kohler, T., Curty, L. K., Barja, F., van Delden, C., and Pechere, J. C. (2000) Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. Journal of bacteriology 182, 5990-5996

43. Tremblay, J., and Deziel, E. (2008) Improving the reproducibility of Pseudomonas aeruginosa swarming motility assays. Journal of basic

microbiology 48, 509-515

44. Campbell, E. A., Masuda, S., Sun, J. L., Muzzin, O., Olson, C. A., Wang, S., and Darst, S. A. (2002) Crystal structure of the Bacillus stearothermophilus anti-sigma factor SpoIIAB with the sporulation sigma factor sigmaF. Cell 108, 795-807

45. Min, K. T., Hilditch, C. M., Diederich, B., Errington, J., and Yudkin, M. D.

(1993) Sigma F, the first compartment-specific transcription factor of B.

subtilis, is regulated by an anti-sigma factor that is also a protein kinase. Cell

103

74, 735-742

46. Yang, X., Kang, C. M., Brody, M. S., and Price, C. W. (1996) Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. Genes & development 10, 2265-2275

47. Kozak, N. A., Mattoo, S., Foreman-Wykert, A. K., Whitelegge, J. P., and Miller, J. F. (2005) Interactions between partner switcher orthologs BtrW and BtrV regulate type III secretion in Bordetella. Journal of bacteriology 187, 5665-5676

48. (2000) Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose. in Molecular Cloinig, A Laboratory Manual (Joseph Sambrook, D. W. R. ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. pp 15.36-15.39

49. Schulenberg, B., Aggeler, R., Beechem, J. M., Capaldi, R. A., and Patton, W. F.

(2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. The Journal of biological chemistry

278, 27251-27255

50. Schulenberg, B., Goodman, T. N., Aggeler, R., Capaldi, R. A., and Patton, W. F.

(2004) Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry.

Electrophoresis 25, 2526-2532

51. Schwyn, B., and Neilands, J. B. (1987) Universal chemical assay for the detection and determination of siderophores. Analytical biochemistry 160, 47-56

52. Louden, B. C., Haarmann, D., and Lynne, A. M. (2011) Use of Blue Agar CAS Assay for Siderophore Detection. Journal of microbiology & biology

education : JMBE 12, 51-53

53. Latifi, A., Winson, M. K., Foglino, M., Bycroft, B. W., Stewart, G. S., Lazdunski, A., and Williams, P. (1995) Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites

53. Latifi, A., Winson, M. K., Foglino, M., Bycroft, B. W., Stewart, G. S., Lazdunski, A., and Williams, P. (1995) Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites

在文檔中 綠膿桿菌PAO1中兩個雙功能酵素PslB和PA3346功能特性之研究與克雷白氏肺炎桿菌CG43 galU突變株在半乳糖壓力下其轉錄體之分析 (頁 101-142)