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FlhC-regulated promoters analyzed by gene array and lacZ gene fusions
FEMS Microbiology Letters 197 (2001) 91^97
www.fems-microbiology.org
FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions Birgit M. Pru«M *, Xiaojin Liu, William Hendrickson, Philip Matsumura Department of Microbiology and Immunology (M/C 790), College of Medicine, University of Illinois at Chicago, E-603 Medical Sciences Building, 835 S. Wolcott, Chicago, IL 60612-7344, USA Received 3 January 2001; received in revised form 9 February 2001; accepted 10 February 2001
Abstract The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF). ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Gene array; Flagellum; Transcriptional regulation; FlhD
1. Introduction Expression of the Escherichia coli £agella transcriptional hierarchy (for reviews see [1^3]) begins with the £agellar `master' operon £hD [4^6], consisting of £hD and £hC [7]. The active complex is a heterotetramer (C2 D2 ) that binds to the upstream regions of the class II operons that are under direct control of the £hD operon [8]. FlhD itself cannot bind to the promoter regions of the class II operons. FlhD alone is involved in the regulation of the cell division rate [9]. Mutants in FlhD, but not FlhC, divide rapidly as they enter the stationary phase. The putative signalling cascade [10] starts with the depletion of serine [9] and the synthesis of acetyl phosphate [11]. OmpR, phosphorylated by acetyl phosphate, inhibits £hDC expression [12]. Using transposon mutagenesis, a gene was identi¢ed that was regulated by FlhD and not by FlhC [13]. The mutant exhibited the same cell division phenotype as the FlhD mutant. The inserted gene was cadA, the second gene in the cadBA operon, encoding lysine decar-
* Corresponding author. Tel. : +1 (312) 413 0288; Fax: +1 (312) 966 6415; E-mail : [email protected]
boxylase [14^16]. It seems that FlhD changes its DNAbinding speci¢city by either complexing with another partner or binding by itself. To identify further non-£agellar targets of FlhD, we have used the Sigma-GenoSys E. coli Gene Array (Sigma-GenoSys Biotechnologies, The Woodlands, TX, USA). Since gene arrays are a newly developed technique, there is still a great deal that can be learned. The E. coli FlhD/FlhC system is a good study subject, because a large number of regulated genes is expected and because the £agella system itself provides a good positive control with as many as 50 genes. The Sigma-GenoSys array consists of 10 ng of each of 4290 polymerase chain reaction (PCR)-ampli¢ed ORF-speci¢c DNA fragments printed in duplicate on two 12U24-cm positively charged nylon membranes. The primers have been derived from the E. coli genome project [17]. The array has been used for gene expression pro¢ling before [18,19]. The expression pattern of wild-type cells was compared to that of FlhD/FlhC mutant cells. A number of promoters were identi¢ed with the array that seemed regulated by FlhD/FlhC. Eight of these were con¢rmed using transcriptional fusions to lacZ or the activity of the enzyme. The non-£agella targets are the transporter for galactose (mglBAC), the rod-shape determination proteins (mreBCD), the tricarboxylic acid cycle enzyme malate dehydrogenase (mdh) and several enzymes involved in anaer-
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 9 2 - 1
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obic respiration (anaerobic glycerol 3-phosphate dehydrogenase, glpABC ; periplasmic nitrate reductase, napFAGHBC; nitrite reductase, nrfABCDEFG; dimethyl sulfoxide (DMSO) reductase, dmsABC; and the modulator of the hydrogenases, hydN hypF). It appears that the FlhD/FlhC heterotetrameric complex is not a £agella-speci¢c transcriptional activator, but a global regulator that is involved in many cellular processes. In addition, FlhD may be able to form complexes with di¡erent partner proteins in order to change the binding speci¢city and regulate promoters such as cadBA [13]. 2. Materials and methods 2.1. Bacterial strains and growth conditions All strains were derivatives of E. coli K-12. MC1000 (F3 araD139 v(araAB leu)7697 v(lacX74) galU galK rpsL thi) was used as the genetic background for the array experiment. MC1000 £hD: :Kan contains an insertion of the kanamycin resistance gene in £hD that inhibits the expression of £hD and £hC. YK410 [20] was used as a background for the lacZ expression studies (F3 araD139 v(lacU169) rpsL thi pyrC46 nalA thyA his). YK4131 and YK4136 contain point mutations in £hD and £hC, respectively [6]. Luria^Bertani broth (LB) contains 1% tryptone, 1% NaCl and 0.5% yeast extract. It was recommended [18] to add glucose to the growth medium, because gene expression in batch culture is constantly changing during growth. However, since £hD undergoes catabolite repression, the addition of glucose was not feasible. Cells were inoculated 1:100 from an overnight culture in LB and grown at 34³C. Both strains grew at a very similar rate, the mutant exhibiting a slightly higher growth rate than the wild-type cells [9]. For the array experiments, cultures were grown to an OD600 of 0.5. At this time, cultures are in mid-exponential phase and cells have already expressed £hDC but do not reach maximal motility. The minor di¡erences in the growth rate were compensated by collecting the wild-type sample 5^10 min later than the wild-type sample. For the lacZ expression studies, cultures were grown for 9 h. Aliquots were taken at 1-h intervals. 2.2. Isolation of RNA, cDNA synthesis and hybridization Cells in growing cultures were lysed in boiling lysis bu¡er and RNA was obtained with the hot phenol method as described [18]. cDNA synthesis and hybridization were performed as recommended by the manufacturer of the membrane (Sigma-GenoSys Biotechnologies, The Woodlands, TX, USA). These procedures are described in detail at http://www.uic.edu/depts/mcmi/home.html.
2.3. Data analysis The exposed phosphor imager screens were scanned with a pixel size of 100 Wm (10 000 dots cm312 ) on a STORM 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). The intensity of each pair of duplicate spots was determined with ImageQuant (version 4.2A, Molecular Dynamics, Sunnyvale, CA, USA) and exported into Microsoft Excel (version 5.0c). Background values were determined for each ¢eld in the array by averaging the pixel values of three empty spaces. The background values were subtracted from the gene values. The data were normalized by expressing the averaged spot signal as a fraction of the signal from the averaged pixel values of the genomic DNA standards provided on the membrane [19]. Genes whose pixel values were less than twofold di¡erent from the background values were not considered [19]. Finally, the corrected spot intensity of one strain was divided by the corresponding spot intensity of the other. Positive ratios correspond to an induction of the respective gene by £hDC, negative ratios to a repression. Induction ratios higher than 2 and repression ratios lower than 32 were considered tentatively signi¢cant [19]. 2.4. Construction of the lacZ fusions, L-galactosidase and malate dehydrogenase (Mdh) assays lacZ fusions to the ¢rst gene in the respective operons were constructed as follows : primers were designed to the promoter regions of mglB, mreB, glpA, and hydN as described at http://www.uic.edu/depts/mcmi/home.html. PCR was performed under standard conditions with 30 cycles at an annealing temperature of 55³C. Using Topo TA cloning (Invitrogen, Carlsbad, CA, USA), the PCR fragments were cloned into the BamHI and EcoRI sites of vector pRS528 [21]. This is a multi-copy plasmid that promotes transcriptional fusions of the respective promoter to the structural gene of lacZ. In addition, £iA : : lacZ, £hB: :lacZ, and £iL: :lacZ were obtained from plasmids pXL11^pXL13, a chromosomal (single-copy) napF: :lacZ was obtained from strain HW2 [22], a chromosomal nrfA: :lacZ from strain HW1 [23], and a chromosomal dmsA: :lacZ from strain PC25 [23]. All constructs were either transformed or transduced with P1 into strains YK410 (wild-type), YK4131 (£hD) and YK4136 (£hC). The activity of L-galactosidase was determined from the lacZ constructs according to the procedure of Miller [24], except that the cells were disrupted by sonication and the initial rate of the reaction was measured instead of a single time-point measurement. Activities are expressed in terms of Miller Units (OD420 /OD600 ). The activity of Mdh was determined in 150 mM Tris^ HCl pH 7.8 and 3 mM MgCl2 . 10 mM malic acid and 7.5 mM NAD were used as substrates. The formation of NADH was measured at 340 nm (O = 6.22 mM31 cm31 ).
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The activity of Mdh is expressed as nmol s31 mg31 protein (nkat mg31 ).
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was compared to that of an FlhD/FlhC mutant culture (MC1000 £hD: :Kan). Since the kanamycin insertion abolishes the expression of £hD and £hC, this experiment does not distinguish whether a gene is regulated by FlhD alone or by the FlhD/FlhC complex. Cultures of both strains were grown in LB at 34³C to an OD600 of 0.5. The experiment was performed twice. For experiment 1, the averaged pixel values for the genomic DNA spots were 38418 for the wild-type membrane and 35384 for the mutant membrane. This yields a normalization factor of 38418/35384 = 1.08. For experiment 2,
3. Results 3.1. £hD regulates transporters for galactose, rod-shape determination proteins, Mdh and proteins involved in anaerobic respiration The expression pro¢le of a wild-type culture (MC1000) Table 1 Array analysis of genes a¡ected by FlhD/FlhC Gene
Gene product
Experiment 1 a
(I) Flagella genes: £gA basal-body P-ring £gB proximal rod £gD hook-cap £gF proximal rod £gG distal rod £gI basal-body P-ring £gJ rod assembly £gK HAP1; ¢rst junction £gL HAP3; second junction tsr serine receptor trg ribose/galactose receptor tap dipeptide receptor cheR receptor methylation cheB receptor demethylation cheY £agellar switch cheZ deactivation of CheY motA motor rotation motB motor rotation cheA phosphorylation £hC regulator £iC £agellin £iD ¢lament cap £iE MS-ring/rod junction £iG £agellar switch £iJ export £iK hook-length control £iN £agellar switch (II) Non-£agella genes: mglB Galactose transport mglA Galactose transport mglC Galactose transport mreB Rod-shape determination mdh Malate dehydrogenase glpA Glycerol 3-P dehydrogenase glpB Glycerol 3-P dehydrogenase glpC Glycerol 3-P dehydrogenase dmsA DMSO reductase dmsB DMSO reductase dmsC DMSO reductase hypF Hydrogenase modulation
Experiment 2
wild-type
FlhD
0.095 0.084 0.078 0.032 0.079 0.722 0.031 0.074 0.104 0.037 0.028 0.110 0.066 0.148 0.049 0.086 0.124 0.200 0.128 0.037 0.718 0.062 0.085 0.056 0.092 0.050 0.072
0.025 0.004 0.025 0.010 0.038 0.326 0.003 0.005 0.021 0.007 0.008 0.027 0.028 0.041 0.002 0.005 0.058 0.036 0.023 0.007 0.018 0.004 0.037 0.008 0.036 0.018 0.016
0.768 0.039 0.022 0.339 0.312 0.092 0.274 0.064 0.092 0.645 0.510 0.899
1.779 0.490 0.106 0.129 1.003 0.031 0.134 0.027 0.046 0.326 0.050 0.258
a
wild-typea
FlhDa
ratiob
3.7 21.2 3.1 3.3 2.1 2.2 9.6 15.2 4.9 5.4 3.6 4.1 2.3 3.6 31.8 18.3 2.1 5.5 5.6 5.4 40.9 15.9 2.3 6.7 2.6 2.8 4.5
0.188 0.456 0.505 0.123 0.406 1.551 0.184 0.758 0.595 0.284 0.212 0.423 0.808 1.160 0.489 1.333 0.663 0.942 1.535 0.284 5.593 1.016 0.167 0.254 0.259 0.338 0.100
0.052 0.003 0.002 0.062 0.108 0.813 0.079 0.007 0.012 0.023 0.060 0.038 0.089 0.036 0.012 0.040 0.009 0.083 0.079 0.023 0.015 0.000 0.033 0.061 0.040 0.015 0.021
3.6 133.3 257.7 2.0 3.8 1.9 2.3 105.1 51.1 12.4 3.6 11.1 9.1 31.8 41.7 33.5 71.3 11.4 19.3 12.4 363.4 n.a. 5.0 4.2 6.4 22.3 4.7
32.3 312.5 34.8 2.6 33.2 3.0 2.1 2.4 2.0 2.0 10.2 3.5
0.804 0.110 0.017 0.479 0.997 0.567 0.828 0.612 0.260 0.974 0.080 0.439
3.490 0.446 0.034 0.083 2.278 0.055 0.137 0.025 0.090 0.106 0.079 0.075
34.3 34.0 32.0 5.8 32.3 10.3 6.0 24.4 2.9 9.2 1.0 5.9
ratio
b
a Expression of genes in wild-type cells or FlhD/FlhC mutants, grown in LB to an OD600 of 0.5. The pixel values have been determined for each pair of duplicate spots. The background has been subtracted from the averaged pixel values. In order to compare the values of wild-type cells with the mutants, the pixel values have been normalized with the hybridization standards. b Expression ratios for genes that are regulated by FlhD/FlhC. The pixel values of wild-type cells have been divided by the pixel values of FlhD/FlhC mutants for genes that are expressed higher in the wild-type cells. For genes that are expressed higher in the mutants, the pixel values of the mutants have been divided by the pixel values of the wild-type cells. These numbers are marked with a minus.
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the averaged pixel values for the genomic DNA spots for the wild-type membrane were 10211 and 17876 for the mutant. This yields a normalization factor of 17876/ 10211 = 1.75. For this reason, a threshold expression ratio for the de¢nition of tentatively regulated genes of 2 was chosen for induced genes and 32 for repressed genes. This is consistent with published threshold ratios [19]. The data have been deposited at http://www.uic.edu/ depts/mcmi/home.html. A selection of genes that give the most consistent results is presented in Table 1. Only genes were considered where the two experiments yielded two data points. This was the case for 27 of the 46 £agella
genes that are printed on the Sigma-GenoSys E. coli gene array. A number of £agella genes yielded only one data point (for example £iA and £iL). Some genes exhibited high expression in the FlhD/FlhC mutant (for example £hB). The £agella system has been used as a positive control for the array. Overall, it shows the expected induction of the majority of these genes. However, variation in the expression is noted. Several genes were found that were regulated by either FlhD alone or FlhD/FlhC and were not previously known to be regulated by £hD. The mglBAC genes (encoding the galactose transporter) were repressed. mreB, the ¢rst gene
Fig. 1. Expression from lacZ fusions and Mdh assays. Cultures of wild-type cells (squares), £hD mutants (diamonds) and £hC mutants (triangles), each containing a lacZ fusion, were grown in LB at 34³C. Aliquots were taken every hour and the activity of L-galactosidase was determined (A^G). Mdh was determined and is expressed as nkat mg31 protein (H). The experiment was done three times and the means were determined.
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in the mreBCD operon (encoding the rod-shape determining proteins) was induced. mreC and mreD were expressed at very low level (data not shown). mdh was repressed. Several enzyme complexes that contribute to the anaerobic respiratory chain were induced. These are the glycerol 3phosphate dehydrogenase (glpABC), the DMSO reductase (dmsABC) and hypF, the ¢rst gene in the hydNhypF operon (modulator of all three E. coli hydrogenases (hya, hyb and hyc). Individual genes of the napFAGHBC (encoding nitrate reductase) and nrfABCDEFG (nitrite reductase) operons were induced as well (http://www.uic.edu/depts/ mcmi/home.html). 3.2. lacZ fusions and enzyme assays con¢rm the regulation by FlhD/FlhC lacZ promoter fusions to mglB, mreB, glpA, and hydN were constructed in multi-copy as described in Section 2. In addition, single-copy fusions to napF, nrfA, and dmsA were obtained from R. Gunsalus (University of California, Los Angeles, CA, USA). Fusions of £hB, £iA, and £iL to lacZ were obtained from the laboratory collection and used as a positive control. Cultures of strains YK410 (wild-type), YK4131 (£hD) and YK4136 (£hC), each containing lacZ fusions to these promoters, were grown at 34³C for 11 h. The L-galactosidase activity was determined at 1-h intervals. The expression pattern of the wild-type cells was compared to that of the two mutants for each promoter fusion. In addition, the activity of Mdh was measured in the three strains. Each experiment was performed three to four times and the combined data sets for these genes and proteins are presented in Fig. 1. The expression of the £agella genes £hB, £iA and £iL
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was higher in the wild-type cells than in the FlhD and FlhC mutants, indicating a regulation by both transcriptional activators (data not shown). mglB expression was repressed, mreB expression was induced, Mdh activity was repressed, and glpA, napF, nrfA, dmsA, and hydN expression were induced. The regulation of mreB, glpA, napF, nrfA, dmsA, and hydN expression and Mdh activity was dependent on the presence of both transcriptional regulators, FlhD and FlhC. mglB expression is clearly higher in the FlhD mutant than in the wild-type cells. The expression level in the FlhC mutants is intermediate. As expected, di¡erences between the three strains became more dramatic after many hours of growth (Fig. 1). This is due to the cumulative character of the assay. After 3.5 h (OD600 of 0.5), the di¡erences between the strains were not very dramatic for most promoters. However, a linear increase in expression (or enzyme activity) was observed between 2^3 and 8^10 h of growth in most cases (Fig. 1). This corresponds to a range of OD from 0.3 to 1.0 in the growth curve. In order to relate the expression data obtained from the lacZ fusions to the array, the rate of increase in L-galactosidase activity per hour, during the time where expression (or enzyme activity) increased linearly, was calculated. Expression ratios from the ¢rst gene in each operon, as obtained from the array, were averaged for the two experiments as available (Table 1). For genes that yielded only one data point (data not shown), only that one data point was considered (£iA, £iL). Fig. 2 summarizes these results. The fusions and enzyme assays give the same qualitative results as the array, although quantitative di¡erences are large for some genes and proteins (mre, Mdh, glpA). A major di¡erence is seen for £hB which was not induced in the array due to a
Fig. 2. Correlation of the lacZ fusions and enzyme assays to the array. Expression ratios of the ¢rst gene in each operon were obtained from the array and were determined twice (black bars). Expression ratios were obtained from the lacZ fusions and the Mdh assay and were determined three times. Expression ratios were calculated from the experiment in Fig. 1 and are expressed as U h31 during the linear increase of expression (OD600 of 0.3^1.0 for most promoters). For induced genes, the expression ratios of wild-type cells were divided by the expression ratios of the mutants and are expressed positive. For repressed genes, the expression ratios of the mutants were divided by the expression ratios of the wild-type cells and are expressed negative. White bars represent the expression of wild-type cells relative to the FlhD mutants. Shaded bars represent the expression of wild-type cells relative to the FlhC mutants. Standard errors are indicated.
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high expression in the mutant and was, therefore, not included in Table 1. A similarly high expression level in the two mutant strains could not be observed using lacZ fusions. This may be due to the presence of a second promoter causing FlhD/FlhC-independent expression. Overall, the correlation of the array with the lacZ fusions is reasonable. 4. Discussion 4.1. Critical evaluation of the data Given the variability in the array data (Table 1), the obtained genes that seem to be regulated by FlhD/FlhC are tentative targets at best. The inability to use glucose as a supplement because it represses the expression of £hD certainly contributes to this e¡ect. The array experiment yielded a number of additional genes that seemed to be regulated by FlhD/FlhC (http://www.uic.edu/depts/mcmi/ home.html). Some of these were tested by lacZ fusion and could not be con¢rmed as being regulated by FlhD or FlhC. All together, 11 out of 18 (61%) tested promoters were con¢rmed with lacZ fusions or enzyme assays. This indicates that gene arrays are indeed a very powerful technique for the initial screening of genes and gene expression pro¢ling. The number of FlhD-regulated genes, as presented in this study, is most likely underestimated. Only genes whose trend of expression ratios (upregulated or downregulated) was consistent in two arrays and con¢rmed by lacZ fusions were reported. Preferably, operons consisting of several genes were considered, because the larger number of data points for each promoter provided more reliable results. Moreover, a large number of genes was expressed at or below background level and could not be analyzed. Certainly, growth in LB is not the optimal condition for every gene in the cell. Experiments distinguishing between primary and secondary targets are currently in progress. 4.2. FlhD/FlhC regulates anaerobic respiration The initial screen with the Sigma-GenoSys membrane array yielded a number of genes that seemed to be regulated by FlhD or FlhD/FlhC and are involved in anaerobic respiration. Therefore, a collection of single-copy lacZ fusions to genes that encode anaerobic terminal reductases was obtained from R. Gunsalus (University of California, Los Angeles, CA, USA). These were narG (cytoplasmic nitrate reductase), napF (periplasmic nitrate reductase), nrfA (nitrite reductase), dmsA (DMSO reductase), and frdA (fumarate reductase). Of these, napF, nrfA and dmsA were regulated by FlhD/FlhC, so were glpA and hydN. An induction of these enzymes also would be expected during growth under anaerobic conditions [25].
Regulation of the aerobic enzymes, such as succinate dehydrogenase (sdh) and NADH dehydrogenase I (ndh) and II (nuoA) [26], was not observed. FlhD/FlhC appears to be involved in the transition for entering the stationary phase by inducing the anaerobic respiratory chain, which is tightly regulated in response to oxygen and nitrate [27^29]. Since several enzymes involved in anaerobic respiration are a¡ected, it is most likely that their regulation is not direct. It is more likely that some element links all the metabolic targets of FlhD/FlhC. This could be any of the known regulators of respiratory chains, such as the two-component systems ArcA/ArcB [28,30] or NarQ/ NarP [31,32]. Other proteins with a similar function but a completely di¡erent mechanism include FNR [33], a typical prokaryotic gene regulator and DNA-binding protein, and AppY [34], IHF [35], H-NS [36] and Fis [37]. A transcriptional regulation of any one of these by FlhD/ FlhC could not be observed with the array, but FlhD/ FlhC could interact with one of them in order to control respiration. mreB was induced in the two experiments of the array (Table 1). The expression level of the other two genes in this operon, mreC and mreD was too low to be analyzed. In addition, the expression from the lacZ fusion was more than 10 times higher in the wild-type cells than in the FlhD or FlhC mutant (Figs. 1 and 2). The mre genes were identi¢ed as cell-shaping proteins. Mutants showed a rounded to irregular cell shape and altered sensitivity to the antibiotic mecillinam [38]. In Bacillus subtilis, the mreBCD genes constitute a ¢ve-gene operon with minC and minD, both of which are involved in positioning the septum during cell division [39]. The mre genes could constitute a link between elongation and septation, two alternating events of the cell cycle, in order to force bacterial rods to grow to a given length [40]. Whether this relates to the previously published cell division phenotype of the FlhD mutants is unclear. FlhD mutants divide more often than wild-type cells [9], whereas MinC and MinD mutants divide at the wrong position. Also, the observed cell division phenotype of the FlhD mutants was not observed for FlhC mutants. In contrast, the expression of lacZ from the mre promoter was reduced in both mutants. It is possible that the e¡ect of FlhD upon cell division and the induction of mre by FlhD/FlhC constitute two di¡erent mechanisms, both contributing to the regulation of the bacterial cell cycle. Acknowledgements We thank R.P. Gunsalus for providing strains, J. Campbell for help setting up the cDNA arrays, Y. Zhu, S.K. Radhakrishnan, and B. Shimkos for technical assistance, and P. O'Neill for critically reading the manuscript. This work was supported by Grant GM59484 from the National Institute of Health.
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References [1] Macnab, R.M. (1992) Genetics and biogenesis of bacterial £agella. Annu. Rev. Genet. 26, 131^158. [2] Pru«M, B.M. (2000) FlhD, a transcriptional regulator in bacteria. Recent Res. Dev. Microbiol. 4, 31^42. [3] Shapiro, L. (1995) The bacterial £agellum: from genetic network to complex architecture. Cell 80, 525^527. [4] Silverman, M. and Simon, M.I. (1974) Positioning £agella genes in Escherichia coli by deletion analysis. J. Bacteriol. 117, 73^79. [5] Komeda, Y., Kutsukake, K. and Iino, T. (1980) De¢nition of additional £agella genes. Genetics 94, 277^290. [6] Komeda, Y. (1982) Fusions of £agellar operons to lactose genes on a Mu lac bacteriophage. J. Bacteriol. 150, 16^26. [7] Bartlett, D.H., Frantz, B.B. and Matsumura, P. (1988) Flagellar transcriptional activators FlbB and FlaI: gene sequences and 5P consensus sequences of operons under FlbB and FlaI control. J. Bacteriol. 170, 1575^1581. [8] Liu, X. and Matsumura, P. (1994) The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli £agellar class II operons. J. Bacteriol. 176, 7345^7351. [9] Pru«M, B.M. and Matsumura, P. (1996) A regulator of the £agellar regulon of Escherichia coli, £hD, also a¡ects cell division. J. Bacteriol. 178, 668^674. [10] Pru«M, B.M. (1998) Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli. Arch. Microbiol. 170, 141^146. [11] Pru«M, B.M. and Wolfe, A.J. (1994) Regulation of acetyl phosphate synthesis and degradation, and the control of £agellar expression in Escherichia coli. Mol. Microbiol. 12, 973^984. [12] Shin, S. and Park, C. (1995) Modulation of £agellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J. Bacteriol. 177, 4696^4702. [13] Pru«M, B.M., Markovic, D. and Matsumura, P. (1997) The Escherichia coli £agellar transcriptional activator £hD regulates cell division through induction of the acid response gene cadA. J. Bacteriol. 179, 3818^3821. [14] Meng, S.Y. and Bennett, G.N. (1992) Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH. J. Bacteriol. 174, 2659^2669. [15] Meng, S.Y. and Bennett, G.N. (1992) Regulation of the Escherichia coli cad operon: location of a site required for acid induction. J. Bacteriol. 174, 2670^2678. [16] Watson, N., Dunyak, D.S., Rosey, E.L., Slonczewski, J.L. and Olson, E.R. (1992) Identi¢cation of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH. J. Bacteriol. 174, 530^540. [17] Blattner, F.R., Plunkett, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B. and Shao, Y. (1997) The complete genome sequence of Escherichia coli K-12. Science 277, 1453^1474. [18] Tao, H., Bausch, C., Richmond, C., Blattner, F.R. and Conway, T. (1999) Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J. Bacteriol. 181, 6425^6440. [19] Barbosa, T.M. and Levy, S.B. (2000) Di¡erential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA. J. Bacteriol. 182, 3467^3474. [20] Komeda, Y. and Iino, T. (1979) Regulation of expression of the £agellin gene (hag) in Escherichia coli K-12: analysis of hag^lac fusions. J. Bacteriol. 139, 721^729. [21] Simons, R.W., Houman, F. and Kleckner, N. (1987) Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53, 85^96.
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[22] Wang, H., Tseng, C.-P. and Gunsalus, R.P. (1999) The napF and narG nitrate reductase operons in Escherichia coli are di¡erentially expressed in response to submicromolar concentrations of nitrate but not nitrite. J. Bacteriol. 181, 5303^5308. [23] Tseng, C.-P., Albrecht, J. and Gunsalus, R.P. (1996) E¡ect of microaerophilic cell growth conditions on aerobic (cyoABCDE, cydAB) and anaerobic (narGHJI, frdABCD, dmsABC) respiratory pathway gene expression in Escherichia coli. J. Bacteriol. 178, 1094^1098. [24] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [25] Tran, Q.H., Bongaerts, J., Vlad, D. and Unden, G. (1997) Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. Eur. J. Biochem. 244, 155^160. [26] Calhoun, M.W. and Gennis, R.B. (1993) Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. J. Bacteriol. 175, 3013^3019. [27] Gunsalus, R.P. (1992) Control of electron £ow in Escherichia coli: coordinated transcription of respiratory pathway genes. J. Bacteriol. 174, 7069^7074. [28] Iuchi, S. and Lin, E.C.C. (1988) arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc. Natl. Acad. Sci. USA 85, 1888^1892. [29] Unden, G. and Bongaerts, J. (1997) Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochim. Biophys. Acta 1320, 217^234. [30] Unden, G. and Guest, J.R. (1985) Isolation and characterization of the FNR protein, the transcriptional regulator of anaerobic electron transport in Escherichia coli. Eur. J. Biochem. 146, 193^199. [31] Stewart, V. (1993) Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli. Mol. Microbiol. 9, 425^434. [32] Stewart, V. and Rabin, R.S. (1995) Dual sensors and dual response regulators interact to control nitrate- and nitrite-responsive gene expression in Escherichia coli. In: Two-Component Signal Transduction (Hoch, J.A. and Silhavy, T., Eds.), pp. 233^252. ASM Press, Washington, DC. [33] Unden, G. and Schirawski, J. (1997) The oxygen-responsive transcriptional regulator FNR of Escherichia coli: search for signals and reactions. Mol. Microbiol. 25, 205^210. [34] Brondsted, L. and Atlung, T. (1994) Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli. J. Bacteriol. 176, 5423^5428. [35] Friedman, D.I. (1988) Integration host factor: a protein for all reasons. Cell 55, 545^554. [36] Dersch, P., Schmidt, K. and Bremer, E. (1993) Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation. Mol. Microbiol. 8, 875^889. [37] Gonzalez, G.-G., Bringmann, P. and Kahmann, R. (1996) FIS is a regulator of metabolism in Escherichia coli. Mol. Microbiol. 22, 21^ 29. [38] Wachi, M., Doi, M., Tamaki, S., Park, W., Nakajima-Iijima, S. and Matsuhashi, M. (1987) Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. J. Bacteriol. 169, 4935^4940. [39] Levin, P.A., Margolis, P.S., Setlow, P., Losick, R. and Sun, D. (1992) Identi¢cation of Bacillus subtilis genes for septum placement and shape determination. J. Bacteriol. 174, 6717^6728. [40] Canepari, P., Signoretto, C., Boaretti, M. and Der Mer Lleo, M. (1997) Cell elongation and septation are two mutually exclusive processes in Escherichia coli. Arch. Microbiol. 168, 152^159.
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FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions Birgit M. Pru«M *, Xiaojin Liu, William Hendrickson, Philip Matsumura Department of Microbiology and Immunology (M/C 790), College of Medicine, University of Illinois at Chicago, E-603 Medical Sciences Building, 835 S. Wolcott, Chicago, IL 60612-7344, USA Received 3 January 2001; received in revised form 9 February 2001; accepted 10 February 2001
Abstract The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF). ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Gene array; Flagellum; Transcriptional regulation; FlhD
1. Introduction Expression of the Escherichia coli £agella transcriptional hierarchy (for reviews see [1^3]) begins with the £agellar `master' operon £hD [4^6], consisting of £hD and £hC [7]. The active complex is a heterotetramer (C2 D2 ) that binds to the upstream regions of the class II operons that are under direct control of the £hD operon [8]. FlhD itself cannot bind to the promoter regions of the class II operons. FlhD alone is involved in the regulation of the cell division rate [9]. Mutants in FlhD, but not FlhC, divide rapidly as they enter the stationary phase. The putative signalling cascade [10] starts with the depletion of serine [9] and the synthesis of acetyl phosphate [11]. OmpR, phosphorylated by acetyl phosphate, inhibits £hDC expression [12]. Using transposon mutagenesis, a gene was identi¢ed that was regulated by FlhD and not by FlhC [13]. The mutant exhibited the same cell division phenotype as the FlhD mutant. The inserted gene was cadA, the second gene in the cadBA operon, encoding lysine decar-
* Corresponding author. Tel. : +1 (312) 413 0288; Fax: +1 (312) 966 6415; E-mail : [email protected]
boxylase [14^16]. It seems that FlhD changes its DNAbinding speci¢city by either complexing with another partner or binding by itself. To identify further non-£agellar targets of FlhD, we have used the Sigma-GenoSys E. coli Gene Array (Sigma-GenoSys Biotechnologies, The Woodlands, TX, USA). Since gene arrays are a newly developed technique, there is still a great deal that can be learned. The E. coli FlhD/FlhC system is a good study subject, because a large number of regulated genes is expected and because the £agella system itself provides a good positive control with as many as 50 genes. The Sigma-GenoSys array consists of 10 ng of each of 4290 polymerase chain reaction (PCR)-ampli¢ed ORF-speci¢c DNA fragments printed in duplicate on two 12U24-cm positively charged nylon membranes. The primers have been derived from the E. coli genome project [17]. The array has been used for gene expression pro¢ling before [18,19]. The expression pattern of wild-type cells was compared to that of FlhD/FlhC mutant cells. A number of promoters were identi¢ed with the array that seemed regulated by FlhD/FlhC. Eight of these were con¢rmed using transcriptional fusions to lacZ or the activity of the enzyme. The non-£agella targets are the transporter for galactose (mglBAC), the rod-shape determination proteins (mreBCD), the tricarboxylic acid cycle enzyme malate dehydrogenase (mdh) and several enzymes involved in anaer-
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 9 2 - 1
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obic respiration (anaerobic glycerol 3-phosphate dehydrogenase, glpABC ; periplasmic nitrate reductase, napFAGHBC; nitrite reductase, nrfABCDEFG; dimethyl sulfoxide (DMSO) reductase, dmsABC; and the modulator of the hydrogenases, hydN hypF). It appears that the FlhD/FlhC heterotetrameric complex is not a £agella-speci¢c transcriptional activator, but a global regulator that is involved in many cellular processes. In addition, FlhD may be able to form complexes with di¡erent partner proteins in order to change the binding speci¢city and regulate promoters such as cadBA [13]. 2. Materials and methods 2.1. Bacterial strains and growth conditions All strains were derivatives of E. coli K-12. MC1000 (F3 araD139 v(araAB leu)7697 v(lacX74) galU galK rpsL thi) was used as the genetic background for the array experiment. MC1000 £hD: :Kan contains an insertion of the kanamycin resistance gene in £hD that inhibits the expression of £hD and £hC. YK410 [20] was used as a background for the lacZ expression studies (F3 araD139 v(lacU169) rpsL thi pyrC46 nalA thyA his). YK4131 and YK4136 contain point mutations in £hD and £hC, respectively [6]. Luria^Bertani broth (LB) contains 1% tryptone, 1% NaCl and 0.5% yeast extract. It was recommended [18] to add glucose to the growth medium, because gene expression in batch culture is constantly changing during growth. However, since £hD undergoes catabolite repression, the addition of glucose was not feasible. Cells were inoculated 1:100 from an overnight culture in LB and grown at 34³C. Both strains grew at a very similar rate, the mutant exhibiting a slightly higher growth rate than the wild-type cells [9]. For the array experiments, cultures were grown to an OD600 of 0.5. At this time, cultures are in mid-exponential phase and cells have already expressed £hDC but do not reach maximal motility. The minor di¡erences in the growth rate were compensated by collecting the wild-type sample 5^10 min later than the wild-type sample. For the lacZ expression studies, cultures were grown for 9 h. Aliquots were taken at 1-h intervals. 2.2. Isolation of RNA, cDNA synthesis and hybridization Cells in growing cultures were lysed in boiling lysis bu¡er and RNA was obtained with the hot phenol method as described [18]. cDNA synthesis and hybridization were performed as recommended by the manufacturer of the membrane (Sigma-GenoSys Biotechnologies, The Woodlands, TX, USA). These procedures are described in detail at http://www.uic.edu/depts/mcmi/home.html.
2.3. Data analysis The exposed phosphor imager screens were scanned with a pixel size of 100 Wm (10 000 dots cm312 ) on a STORM 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). The intensity of each pair of duplicate spots was determined with ImageQuant (version 4.2A, Molecular Dynamics, Sunnyvale, CA, USA) and exported into Microsoft Excel (version 5.0c). Background values were determined for each ¢eld in the array by averaging the pixel values of three empty spaces. The background values were subtracted from the gene values. The data were normalized by expressing the averaged spot signal as a fraction of the signal from the averaged pixel values of the genomic DNA standards provided on the membrane [19]. Genes whose pixel values were less than twofold di¡erent from the background values were not considered [19]. Finally, the corrected spot intensity of one strain was divided by the corresponding spot intensity of the other. Positive ratios correspond to an induction of the respective gene by £hDC, negative ratios to a repression. Induction ratios higher than 2 and repression ratios lower than 32 were considered tentatively signi¢cant [19]. 2.4. Construction of the lacZ fusions, L-galactosidase and malate dehydrogenase (Mdh) assays lacZ fusions to the ¢rst gene in the respective operons were constructed as follows : primers were designed to the promoter regions of mglB, mreB, glpA, and hydN as described at http://www.uic.edu/depts/mcmi/home.html. PCR was performed under standard conditions with 30 cycles at an annealing temperature of 55³C. Using Topo TA cloning (Invitrogen, Carlsbad, CA, USA), the PCR fragments were cloned into the BamHI and EcoRI sites of vector pRS528 [21]. This is a multi-copy plasmid that promotes transcriptional fusions of the respective promoter to the structural gene of lacZ. In addition, £iA : : lacZ, £hB: :lacZ, and £iL: :lacZ were obtained from plasmids pXL11^pXL13, a chromosomal (single-copy) napF: :lacZ was obtained from strain HW2 [22], a chromosomal nrfA: :lacZ from strain HW1 [23], and a chromosomal dmsA: :lacZ from strain PC25 [23]. All constructs were either transformed or transduced with P1 into strains YK410 (wild-type), YK4131 (£hD) and YK4136 (£hC). The activity of L-galactosidase was determined from the lacZ constructs according to the procedure of Miller [24], except that the cells were disrupted by sonication and the initial rate of the reaction was measured instead of a single time-point measurement. Activities are expressed in terms of Miller Units (OD420 /OD600 ). The activity of Mdh was determined in 150 mM Tris^ HCl pH 7.8 and 3 mM MgCl2 . 10 mM malic acid and 7.5 mM NAD were used as substrates. The formation of NADH was measured at 340 nm (O = 6.22 mM31 cm31 ).
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The activity of Mdh is expressed as nmol s31 mg31 protein (nkat mg31 ).
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was compared to that of an FlhD/FlhC mutant culture (MC1000 £hD: :Kan). Since the kanamycin insertion abolishes the expression of £hD and £hC, this experiment does not distinguish whether a gene is regulated by FlhD alone or by the FlhD/FlhC complex. Cultures of both strains were grown in LB at 34³C to an OD600 of 0.5. The experiment was performed twice. For experiment 1, the averaged pixel values for the genomic DNA spots were 38418 for the wild-type membrane and 35384 for the mutant membrane. This yields a normalization factor of 38418/35384 = 1.08. For experiment 2,
3. Results 3.1. £hD regulates transporters for galactose, rod-shape determination proteins, Mdh and proteins involved in anaerobic respiration The expression pro¢le of a wild-type culture (MC1000) Table 1 Array analysis of genes a¡ected by FlhD/FlhC Gene
Gene product
Experiment 1 a
(I) Flagella genes: £gA basal-body P-ring £gB proximal rod £gD hook-cap £gF proximal rod £gG distal rod £gI basal-body P-ring £gJ rod assembly £gK HAP1; ¢rst junction £gL HAP3; second junction tsr serine receptor trg ribose/galactose receptor tap dipeptide receptor cheR receptor methylation cheB receptor demethylation cheY £agellar switch cheZ deactivation of CheY motA motor rotation motB motor rotation cheA phosphorylation £hC regulator £iC £agellin £iD ¢lament cap £iE MS-ring/rod junction £iG £agellar switch £iJ export £iK hook-length control £iN £agellar switch (II) Non-£agella genes: mglB Galactose transport mglA Galactose transport mglC Galactose transport mreB Rod-shape determination mdh Malate dehydrogenase glpA Glycerol 3-P dehydrogenase glpB Glycerol 3-P dehydrogenase glpC Glycerol 3-P dehydrogenase dmsA DMSO reductase dmsB DMSO reductase dmsC DMSO reductase hypF Hydrogenase modulation
Experiment 2
wild-type
FlhD
0.095 0.084 0.078 0.032 0.079 0.722 0.031 0.074 0.104 0.037 0.028 0.110 0.066 0.148 0.049 0.086 0.124 0.200 0.128 0.037 0.718 0.062 0.085 0.056 0.092 0.050 0.072
0.025 0.004 0.025 0.010 0.038 0.326 0.003 0.005 0.021 0.007 0.008 0.027 0.028 0.041 0.002 0.005 0.058 0.036 0.023 0.007 0.018 0.004 0.037 0.008 0.036 0.018 0.016
0.768 0.039 0.022 0.339 0.312 0.092 0.274 0.064 0.092 0.645 0.510 0.899
1.779 0.490 0.106 0.129 1.003 0.031 0.134 0.027 0.046 0.326 0.050 0.258
a
wild-typea
FlhDa
ratiob
3.7 21.2 3.1 3.3 2.1 2.2 9.6 15.2 4.9 5.4 3.6 4.1 2.3 3.6 31.8 18.3 2.1 5.5 5.6 5.4 40.9 15.9 2.3 6.7 2.6 2.8 4.5
0.188 0.456 0.505 0.123 0.406 1.551 0.184 0.758 0.595 0.284 0.212 0.423 0.808 1.160 0.489 1.333 0.663 0.942 1.535 0.284 5.593 1.016 0.167 0.254 0.259 0.338 0.100
0.052 0.003 0.002 0.062 0.108 0.813 0.079 0.007 0.012 0.023 0.060 0.038 0.089 0.036 0.012 0.040 0.009 0.083 0.079 0.023 0.015 0.000 0.033 0.061 0.040 0.015 0.021
3.6 133.3 257.7 2.0 3.8 1.9 2.3 105.1 51.1 12.4 3.6 11.1 9.1 31.8 41.7 33.5 71.3 11.4 19.3 12.4 363.4 n.a. 5.0 4.2 6.4 22.3 4.7
32.3 312.5 34.8 2.6 33.2 3.0 2.1 2.4 2.0 2.0 10.2 3.5
0.804 0.110 0.017 0.479 0.997 0.567 0.828 0.612 0.260 0.974 0.080 0.439
3.490 0.446 0.034 0.083 2.278 0.055 0.137 0.025 0.090 0.106 0.079 0.075
34.3 34.0 32.0 5.8 32.3 10.3 6.0 24.4 2.9 9.2 1.0 5.9
ratio
b
a Expression of genes in wild-type cells or FlhD/FlhC mutants, grown in LB to an OD600 of 0.5. The pixel values have been determined for each pair of duplicate spots. The background has been subtracted from the averaged pixel values. In order to compare the values of wild-type cells with the mutants, the pixel values have been normalized with the hybridization standards. b Expression ratios for genes that are regulated by FlhD/FlhC. The pixel values of wild-type cells have been divided by the pixel values of FlhD/FlhC mutants for genes that are expressed higher in the wild-type cells. For genes that are expressed higher in the mutants, the pixel values of the mutants have been divided by the pixel values of the wild-type cells. These numbers are marked with a minus.
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the averaged pixel values for the genomic DNA spots for the wild-type membrane were 10211 and 17876 for the mutant. This yields a normalization factor of 17876/ 10211 = 1.75. For this reason, a threshold expression ratio for the de¢nition of tentatively regulated genes of 2 was chosen for induced genes and 32 for repressed genes. This is consistent with published threshold ratios [19]. The data have been deposited at http://www.uic.edu/ depts/mcmi/home.html. A selection of genes that give the most consistent results is presented in Table 1. Only genes were considered where the two experiments yielded two data points. This was the case for 27 of the 46 £agella
genes that are printed on the Sigma-GenoSys E. coli gene array. A number of £agella genes yielded only one data point (for example £iA and £iL). Some genes exhibited high expression in the FlhD/FlhC mutant (for example £hB). The £agella system has been used as a positive control for the array. Overall, it shows the expected induction of the majority of these genes. However, variation in the expression is noted. Several genes were found that were regulated by either FlhD alone or FlhD/FlhC and were not previously known to be regulated by £hD. The mglBAC genes (encoding the galactose transporter) were repressed. mreB, the ¢rst gene
Fig. 1. Expression from lacZ fusions and Mdh assays. Cultures of wild-type cells (squares), £hD mutants (diamonds) and £hC mutants (triangles), each containing a lacZ fusion, were grown in LB at 34³C. Aliquots were taken every hour and the activity of L-galactosidase was determined (A^G). Mdh was determined and is expressed as nkat mg31 protein (H). The experiment was done three times and the means were determined.
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in the mreBCD operon (encoding the rod-shape determining proteins) was induced. mreC and mreD were expressed at very low level (data not shown). mdh was repressed. Several enzyme complexes that contribute to the anaerobic respiratory chain were induced. These are the glycerol 3phosphate dehydrogenase (glpABC), the DMSO reductase (dmsABC) and hypF, the ¢rst gene in the hydNhypF operon (modulator of all three E. coli hydrogenases (hya, hyb and hyc). Individual genes of the napFAGHBC (encoding nitrate reductase) and nrfABCDEFG (nitrite reductase) operons were induced as well (http://www.uic.edu/depts/ mcmi/home.html). 3.2. lacZ fusions and enzyme assays con¢rm the regulation by FlhD/FlhC lacZ promoter fusions to mglB, mreB, glpA, and hydN were constructed in multi-copy as described in Section 2. In addition, single-copy fusions to napF, nrfA, and dmsA were obtained from R. Gunsalus (University of California, Los Angeles, CA, USA). Fusions of £hB, £iA, and £iL to lacZ were obtained from the laboratory collection and used as a positive control. Cultures of strains YK410 (wild-type), YK4131 (£hD) and YK4136 (£hC), each containing lacZ fusions to these promoters, were grown at 34³C for 11 h. The L-galactosidase activity was determined at 1-h intervals. The expression pattern of the wild-type cells was compared to that of the two mutants for each promoter fusion. In addition, the activity of Mdh was measured in the three strains. Each experiment was performed three to four times and the combined data sets for these genes and proteins are presented in Fig. 1. The expression of the £agella genes £hB, £iA and £iL
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was higher in the wild-type cells than in the FlhD and FlhC mutants, indicating a regulation by both transcriptional activators (data not shown). mglB expression was repressed, mreB expression was induced, Mdh activity was repressed, and glpA, napF, nrfA, dmsA, and hydN expression were induced. The regulation of mreB, glpA, napF, nrfA, dmsA, and hydN expression and Mdh activity was dependent on the presence of both transcriptional regulators, FlhD and FlhC. mglB expression is clearly higher in the FlhD mutant than in the wild-type cells. The expression level in the FlhC mutants is intermediate. As expected, di¡erences between the three strains became more dramatic after many hours of growth (Fig. 1). This is due to the cumulative character of the assay. After 3.5 h (OD600 of 0.5), the di¡erences between the strains were not very dramatic for most promoters. However, a linear increase in expression (or enzyme activity) was observed between 2^3 and 8^10 h of growth in most cases (Fig. 1). This corresponds to a range of OD from 0.3 to 1.0 in the growth curve. In order to relate the expression data obtained from the lacZ fusions to the array, the rate of increase in L-galactosidase activity per hour, during the time where expression (or enzyme activity) increased linearly, was calculated. Expression ratios from the ¢rst gene in each operon, as obtained from the array, were averaged for the two experiments as available (Table 1). For genes that yielded only one data point (data not shown), only that one data point was considered (£iA, £iL). Fig. 2 summarizes these results. The fusions and enzyme assays give the same qualitative results as the array, although quantitative di¡erences are large for some genes and proteins (mre, Mdh, glpA). A major di¡erence is seen for £hB which was not induced in the array due to a
Fig. 2. Correlation of the lacZ fusions and enzyme assays to the array. Expression ratios of the ¢rst gene in each operon were obtained from the array and were determined twice (black bars). Expression ratios were obtained from the lacZ fusions and the Mdh assay and were determined three times. Expression ratios were calculated from the experiment in Fig. 1 and are expressed as U h31 during the linear increase of expression (OD600 of 0.3^1.0 for most promoters). For induced genes, the expression ratios of wild-type cells were divided by the expression ratios of the mutants and are expressed positive. For repressed genes, the expression ratios of the mutants were divided by the expression ratios of the wild-type cells and are expressed negative. White bars represent the expression of wild-type cells relative to the FlhD mutants. Shaded bars represent the expression of wild-type cells relative to the FlhC mutants. Standard errors are indicated.
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high expression in the mutant and was, therefore, not included in Table 1. A similarly high expression level in the two mutant strains could not be observed using lacZ fusions. This may be due to the presence of a second promoter causing FlhD/FlhC-independent expression. Overall, the correlation of the array with the lacZ fusions is reasonable. 4. Discussion 4.1. Critical evaluation of the data Given the variability in the array data (Table 1), the obtained genes that seem to be regulated by FlhD/FlhC are tentative targets at best. The inability to use glucose as a supplement because it represses the expression of £hD certainly contributes to this e¡ect. The array experiment yielded a number of additional genes that seemed to be regulated by FlhD/FlhC (http://www.uic.edu/depts/mcmi/ home.html). Some of these were tested by lacZ fusion and could not be con¢rmed as being regulated by FlhD or FlhC. All together, 11 out of 18 (61%) tested promoters were con¢rmed with lacZ fusions or enzyme assays. This indicates that gene arrays are indeed a very powerful technique for the initial screening of genes and gene expression pro¢ling. The number of FlhD-regulated genes, as presented in this study, is most likely underestimated. Only genes whose trend of expression ratios (upregulated or downregulated) was consistent in two arrays and con¢rmed by lacZ fusions were reported. Preferably, operons consisting of several genes were considered, because the larger number of data points for each promoter provided more reliable results. Moreover, a large number of genes was expressed at or below background level and could not be analyzed. Certainly, growth in LB is not the optimal condition for every gene in the cell. Experiments distinguishing between primary and secondary targets are currently in progress. 4.2. FlhD/FlhC regulates anaerobic respiration The initial screen with the Sigma-GenoSys membrane array yielded a number of genes that seemed to be regulated by FlhD or FlhD/FlhC and are involved in anaerobic respiration. Therefore, a collection of single-copy lacZ fusions to genes that encode anaerobic terminal reductases was obtained from R. Gunsalus (University of California, Los Angeles, CA, USA). These were narG (cytoplasmic nitrate reductase), napF (periplasmic nitrate reductase), nrfA (nitrite reductase), dmsA (DMSO reductase), and frdA (fumarate reductase). Of these, napF, nrfA and dmsA were regulated by FlhD/FlhC, so were glpA and hydN. An induction of these enzymes also would be expected during growth under anaerobic conditions [25].
Regulation of the aerobic enzymes, such as succinate dehydrogenase (sdh) and NADH dehydrogenase I (ndh) and II (nuoA) [26], was not observed. FlhD/FlhC appears to be involved in the transition for entering the stationary phase by inducing the anaerobic respiratory chain, which is tightly regulated in response to oxygen and nitrate [27^29]. Since several enzymes involved in anaerobic respiration are a¡ected, it is most likely that their regulation is not direct. It is more likely that some element links all the metabolic targets of FlhD/FlhC. This could be any of the known regulators of respiratory chains, such as the two-component systems ArcA/ArcB [28,30] or NarQ/ NarP [31,32]. Other proteins with a similar function but a completely di¡erent mechanism include FNR [33], a typical prokaryotic gene regulator and DNA-binding protein, and AppY [34], IHF [35], H-NS [36] and Fis [37]. A transcriptional regulation of any one of these by FlhD/ FlhC could not be observed with the array, but FlhD/ FlhC could interact with one of them in order to control respiration. mreB was induced in the two experiments of the array (Table 1). The expression level of the other two genes in this operon, mreC and mreD was too low to be analyzed. In addition, the expression from the lacZ fusion was more than 10 times higher in the wild-type cells than in the FlhD or FlhC mutant (Figs. 1 and 2). The mre genes were identi¢ed as cell-shaping proteins. Mutants showed a rounded to irregular cell shape and altered sensitivity to the antibiotic mecillinam [38]. In Bacillus subtilis, the mreBCD genes constitute a ¢ve-gene operon with minC and minD, both of which are involved in positioning the septum during cell division [39]. The mre genes could constitute a link between elongation and septation, two alternating events of the cell cycle, in order to force bacterial rods to grow to a given length [40]. Whether this relates to the previously published cell division phenotype of the FlhD mutants is unclear. FlhD mutants divide more often than wild-type cells [9], whereas MinC and MinD mutants divide at the wrong position. Also, the observed cell division phenotype of the FlhD mutants was not observed for FlhC mutants. In contrast, the expression of lacZ from the mre promoter was reduced in both mutants. It is possible that the e¡ect of FlhD upon cell division and the induction of mre by FlhD/FlhC constitute two di¡erent mechanisms, both contributing to the regulation of the bacterial cell cycle. Acknowledgements We thank R.P. Gunsalus for providing strains, J. Campbell for help setting up the cDNA arrays, Y. Zhu, S.K. Radhakrishnan, and B. Shimkos for technical assistance, and P. O'Neill for critically reading the manuscript. This work was supported by Grant GM59484 from the National Institute of Health.
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References [1] Macnab, R.M. (1992) Genetics and biogenesis of bacterial £agella. Annu. Rev. Genet. 26, 131^158. [2] Pru«M, B.M. (2000) FlhD, a transcriptional regulator in bacteria. Recent Res. Dev. Microbiol. 4, 31^42. [3] Shapiro, L. (1995) The bacterial £agellum: from genetic network to complex architecture. Cell 80, 525^527. [4] Silverman, M. and Simon, M.I. (1974) Positioning £agella genes in Escherichia coli by deletion analysis. J. Bacteriol. 117, 73^79. [5] Komeda, Y., Kutsukake, K. and Iino, T. (1980) De¢nition of additional £agella genes. Genetics 94, 277^290. [6] Komeda, Y. (1982) Fusions of £agellar operons to lactose genes on a Mu lac bacteriophage. J. Bacteriol. 150, 16^26. [7] Bartlett, D.H., Frantz, B.B. and Matsumura, P. (1988) Flagellar transcriptional activators FlbB and FlaI: gene sequences and 5P consensus sequences of operons under FlbB and FlaI control. J. Bacteriol. 170, 1575^1581. [8] Liu, X. and Matsumura, P. (1994) The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli £agellar class II operons. J. Bacteriol. 176, 7345^7351. [9] Pru«M, B.M. and Matsumura, P. (1996) A regulator of the £agellar regulon of Escherichia coli, £hD, also a¡ects cell division. J. Bacteriol. 178, 668^674. [10] Pru«M, B.M. (1998) Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli. Arch. Microbiol. 170, 141^146. [11] Pru«M, B.M. and Wolfe, A.J. (1994) Regulation of acetyl phosphate synthesis and degradation, and the control of £agellar expression in Escherichia coli. Mol. Microbiol. 12, 973^984. [12] Shin, S. and Park, C. (1995) Modulation of £agellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J. Bacteriol. 177, 4696^4702. [13] Pru«M, B.M., Markovic, D. and Matsumura, P. (1997) The Escherichia coli £agellar transcriptional activator £hD regulates cell division through induction of the acid response gene cadA. J. Bacteriol. 179, 3818^3821. [14] Meng, S.Y. and Bennett, G.N. (1992) Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH. J. Bacteriol. 174, 2659^2669. [15] Meng, S.Y. and Bennett, G.N. (1992) Regulation of the Escherichia coli cad operon: location of a site required for acid induction. J. Bacteriol. 174, 2670^2678. [16] Watson, N., Dunyak, D.S., Rosey, E.L., Slonczewski, J.L. and Olson, E.R. (1992) Identi¢cation of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH. J. Bacteriol. 174, 530^540. [17] Blattner, F.R., Plunkett, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B. and Shao, Y. (1997) The complete genome sequence of Escherichia coli K-12. Science 277, 1453^1474. [18] Tao, H., Bausch, C., Richmond, C., Blattner, F.R. and Conway, T. (1999) Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J. Bacteriol. 181, 6425^6440. [19] Barbosa, T.M. and Levy, S.B. (2000) Di¡erential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA. J. Bacteriol. 182, 3467^3474. [20] Komeda, Y. and Iino, T. (1979) Regulation of expression of the £agellin gene (hag) in Escherichia coli K-12: analysis of hag^lac fusions. J. Bacteriol. 139, 721^729. [21] Simons, R.W., Houman, F. and Kleckner, N. (1987) Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53, 85^96.
97
[22] Wang, H., Tseng, C.-P. and Gunsalus, R.P. (1999) The napF and narG nitrate reductase operons in Escherichia coli are di¡erentially expressed in response to submicromolar concentrations of nitrate but not nitrite. J. Bacteriol. 181, 5303^5308. [23] Tseng, C.-P., Albrecht, J. and Gunsalus, R.P. (1996) E¡ect of microaerophilic cell growth conditions on aerobic (cyoABCDE, cydAB) and anaerobic (narGHJI, frdABCD, dmsABC) respiratory pathway gene expression in Escherichia coli. J. Bacteriol. 178, 1094^1098. [24] Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [25] Tran, Q.H., Bongaerts, J., Vlad, D. and Unden, G. (1997) Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. Eur. J. Biochem. 244, 155^160. [26] Calhoun, M.W. and Gennis, R.B. (1993) Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. J. Bacteriol. 175, 3013^3019. [27] Gunsalus, R.P. (1992) Control of electron £ow in Escherichia coli: coordinated transcription of respiratory pathway genes. J. Bacteriol. 174, 7069^7074. [28] Iuchi, S. and Lin, E.C.C. (1988) arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc. Natl. Acad. Sci. USA 85, 1888^1892. [29] Unden, G. and Bongaerts, J. (1997) Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochim. Biophys. Acta 1320, 217^234. [30] Unden, G. and Guest, J.R. (1985) Isolation and characterization of the FNR protein, the transcriptional regulator of anaerobic electron transport in Escherichia coli. Eur. J. Biochem. 146, 193^199. [31] Stewart, V. (1993) Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli. Mol. Microbiol. 9, 425^434. [32] Stewart, V. and Rabin, R.S. (1995) Dual sensors and dual response regulators interact to control nitrate- and nitrite-responsive gene expression in Escherichia coli. In: Two-Component Signal Transduction (Hoch, J.A. and Silhavy, T., Eds.), pp. 233^252. ASM Press, Washington, DC. [33] Unden, G. and Schirawski, J. (1997) The oxygen-responsive transcriptional regulator FNR of Escherichia coli: search for signals and reactions. Mol. Microbiol. 25, 205^210. [34] Brondsted, L. and Atlung, T. (1994) Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli. J. Bacteriol. 176, 5423^5428. [35] Friedman, D.I. (1988) Integration host factor: a protein for all reasons. Cell 55, 545^554. [36] Dersch, P., Schmidt, K. and Bremer, E. (1993) Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation. Mol. Microbiol. 8, 875^889. [37] Gonzalez, G.-G., Bringmann, P. and Kahmann, R. (1996) FIS is a regulator of metabolism in Escherichia coli. Mol. Microbiol. 22, 21^ 29. [38] Wachi, M., Doi, M., Tamaki, S., Park, W., Nakajima-Iijima, S. and Matsuhashi, M. (1987) Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. J. Bacteriol. 169, 4935^4940. [39] Levin, P.A., Margolis, P.S., Setlow, P., Losick, R. and Sun, D. (1992) Identi¢cation of Bacillus subtilis genes for septum placement and shape determination. J. Bacteriol. 174, 6717^6728. [40] Canepari, P., Signoretto, C., Boaretti, M. and Der Mer Lleo, M. (1997) Cell elongation and septation are two mutually exclusive processes in Escherichia coli. Arch. Microbiol. 168, 152^159.
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