JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 5408-5415

Vol. 172, No. 9

0021-9193/90/095408-08$02.00/0 Copyright © 1990, American Society for Microbiology

Multiple Regulatory Sites in the Bacillus subtilis citB Promoter Region AGNtS FOUET,t SHENG-FANG JIN, GLEN RAFFEL, AND ABRAHAM L. SONENSHEIN* Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus, 136 Harrison Avenue, Boston, Massachusetts 02111 Received 29 March 1990/Accepted 13 June 1990

The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source of 2-ketoglutarate. It is derepressed when either of these nutrient sources becomes limiting. Repression by rapidly metabolizable carbon sources was shown previously to depend at least in part on a DNA sequence located 67 to 84 base pairs upstream of the start point of citB transcription. In the present work, this region and surrounding DNA were mutagenized to identify more precisely the target for carbon catabolite repression. Mutations in a symmetric sequence located between positions -73 and -59 led to constitutive transcription from the citB promoter in media that normally provoke catabolite repression. By gel mobility shift assays, it was shown that at least one protein in extracts of B. subtilis binds to the symmetric sequence and that DNA of constitutive mutants binds to this protein much less effectively. A second sequence located near position -45 was also implicated in this regulation. A second form of regulation of citB was also investigated. This gene is known to be derepressed when cells are induced to sporulate by exhaustion of a nutrient broth medium or limitation of guanine nucleotide synthesis. The mutations that led to constitutivity with respect to the carbon source had no effect on citB expression in nutrient broth medium, indicating that control by catabolite repression and control by components of nutrient broth (presumably amino acids) act by different mechanisms.

In Bacillus subtilis, the citB gene, which encodes aconitase (EC 4.2.1.3; citrate [isocitrate] hydro-lyase) (4), is subject to several forms of regulation at the transcriptional level (3, 17, 20). In minimal medium containing glucose or glycerol, citB expression is much lower than in a medium containing only a poorly metabolized carbon source, such as citrate (17). Full repression by rapidly metabolizable carbon sources, however, requires a good source of 2-ketoglutarate (2-KG) (e.g., glutamine or glutamate [17]). Carbon source regulation of citB transcription is probably mediated through a negative regulator, since aconitase expression is partially derepressed in cells containing multiple copies of the promoter region (from positions -84 to +36) and since deletion of a sequence lying 67 to 84 base pairs (bp) upstream of the transcription start site leads to constitutive citB expression and loss of titration activity (7). This 17-bp region presumably defines at least part of the target of catabolite repression and may therefore correspond to part of a binding site for a negative regulator. A full understanding of citB regulation has to include the effects on this gene of conditions that induce sporulation. Thus, expression of citB in cells growing in minimal glucoseglutamine medium is dramatically increased in cells treated with decoyinine (3, 22, 23). This drug, which induces sporulation by blocking the synthesis of guanine nucleotides (11), causes a rapid decrease in the intracellular concentration of 2-KG (22). Induction of citB by decoyinine treatment may therefore be linked to the requirement for a good source of 2-KG for complete catabolite repression of this gene (6). Since deletion of the sequence between positions -84 and -68 caused the loss of both catabolite repression and *

inducibility by decoyinine, we suggested that decoyinine induces citB by antagonizing the effect of the carbon sourcedependent negative regulator (7). Expression of citB is also regulated during growth and sporulation in complex media. When cells are grown in nutrient broth sporulation medium, for example, aconitase activity is low in the early exponential phase, increases at the end of the exponential phase as sporulation begins, and disappears 2 h later (3, 7, 16). This aspect of citB regulation is maintained in a version of the promoter region that contains only positions -68 through +36 (7). This suggests that induction of citB during initiation of sporulation in minimal and complex media is mediated by distinct regulatory pathways. We describe below an attempt to locate the target for catabolite repression more precisely. To do so, we created a collection of mutants with alterations in the region between positions -84 and -45. Analysis of citB expression from these mutant promoters revealed that a dyad symmetry sequence located between positions -73 and -59 (4) is important for catabolite repression. The region just downstream from the dyad symmetry also appeared to affect this regulation. Using a gel mobility shift assay, we showed that the dyad symmetry region is necessary for binding of at least one protein to the citB regulatory region and that this binding is substantially reduced in derepressed mutants. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains used are described in Table 1. The vector pAFi (7) integrates at the amyE locus of B. subtilis and enables the construction of transcriptional fusions with the lacZ gene. Plasmids pAF13 and pAF14 are pUC8 derivatives containing, respectively, the sequences found between positions -84 and +36 and -67 and +36 of the citB promoter region (7). Plasmids pAF23 and pAF24 have the same inserts as pAF13 and

Corresponding author.

t Present address: Unitd des AntigEnes Bactdriens, Institut Pasteur, 75724 Paris, France. 5408

CATABOLITE REPRESSION OF citB

VOL. 172, 1990

5409

TABLE 1. Bacterial strains Strain

Genotype

Escherichia coli TH101 RV JM103

Source

dut ung dam Alac thi &(lac pro) supE thi strA sbcCJS endA hsdR4(F' traD36 proAB lacIqZAMJ5)

T. Henkin M. Malamy D. Chikaraishi

Bacillus subtilis SMY Wild type HS1A17 trpC2 citAl AF2 citAl amyE: :4~citBlp23-lacZa AF3 citAl amyE::,0citBp24-lacZb a citBp23 is the wild-type citB promoter sequence from positions -84 to +36. b citBp24 is the wild-type citB promoter sequence from positions -67 to +36.

pAF14, but in the pAFi vector. Plasmids pAF7O through pAF93 were obtained by randomly mutagenizing pAF13 (see below). The series pAF100 through pAF123 was constructed by purifying the EcoRI-HindIII fragments from plasmids pAF7O through pAF93 and ligating them to pAFi which had -84

-67

P. Schaeffer R. Hanson SMY x DNA HS1A17 + pAF23 SMY x DNA HS1A17 + pAF24

been digested by the same enzymes; the relevant parts of their sequences are indicated in Fig. 1. Culture media and bacterial growth. Escherichia coli strains were grown in L broth or on L agar plates (21). When appropriate, the growth medium contained 100 pLg of ampi-42

-27

1

1 ACATTTTTCTC ATAAGT CGA ACTTAT TGTATTTAATAAAAACATTGATATTTACTTATGTATGATTTT

Ia

. 0 pAF23 .......... ............ pAF100 ....A. ..................... 1 .

. pAFlol ....G. ....................

pAF1O3 pAFlOS

pAF115 Ib

pAF116 pAF119 pAF122

1-3 10 >5 >10

decoyinine inducibility + + + + + +

...........T. ..............

.......T......

1

5-

A 1 1 2 2 3 3

100 60 35 40 80 90 60 85 70 75 50 85 60 95 65 95 50

80 70 50 50 80 80 50 80 70 70

0.8 1.2 1.4 1.3 1.0 0.9 0.8 0.9 1.0 0.9 1.1 0.8 1.2 0.8 1.2 0.8 1.4

..........

.

.....TA... T.T T...... .....G....GC.TC.... A.A

. ......... .

.

... ...

.

.

.

.

.

.........

A. ...A..........G ....

...... .G ... pAFilO ..............TA.G..G 4 pAFlil .T. ..C....A... ..A ........... . 5 .. .C......A.T.. . .G. .. .A.G .... pAF112 6 .

pAF113 ... .......A.. G.G. .C..... G .. .... ... pAF114 .....A.. ................ 2 ... ........... pAF117 .... ..C . ... .T 2 ...T .............T.. pAF118 ........C. . 3 .

pAF12O pAF121

expression

1 1 1 1

G................ G .........

......... . ...... ... . ... .

A pAF24 I I]............... . ... ........... pAF1O2 ........ . C ......... ..... pAF1O4 .......... ... ........... pAF1O6 .....A.....G . . C...G........ pAF1O7 .............. ... pAF1O8 ........T.G................

pAF1O9

expression in glucose

G... .G .........C ...... .C ..... . . pAF123 ..................A.A. . . .. . T .

.T..... C.

... .A.G ....G......C...

4 5 6

Multiple regulatory sites in the Bacillus subtilis citB promoter region.

The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source...
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