SpoIIAB is an anti-v factor that binds to and inhibits transcription by regulatory protein crF from Bacillus subtilis

The 0.F factor is a regulatory protein that is responsible for directing gene expression in the forespore compartment of developing cells of the spore-forming soil bacterium BacMlus subtilis. The cJF factor is encoded by the promoter-distal member of sporulation operon spollA, which consists of cistrons called spoIIAA, spollAB, and spollAC. Genetic evidence indicates that the activity of oF is negatively regulated by the product (SpoIIAB) of the spolIAB cistron. We now report that SpoIIAB is capable of binding to cF and inhibiting its capacity to direct transcription by core RNA polymerase from the promoter for a forespore-expressed gene. SpoIIAB is an anti-a factor that may be directly involved in the compartmentalization of arF-directed gene expression. Spore formation in the Gram-positive soil bacterium Bacillus subtilis involves the formation of an asymmetrically positioned septum that partitions the developing cell into two cellular compartments called the forespore and the mother cell (1, 2). Gene expression in the compartments is regulated differentially, with certain genes being expressed in the forespore and others in the mother cell (3, 4). At intermediate to late stages of development, differential gene expression is governed by two compartment-specific RNA polymerase v factors. These are oG, which directs transcription in the forespore, and acK, which directs transcription in the mother cell (5-7). Transcription of the structural genes for crG (spolIIG) and caK (sigK) is, in turn, governed by two earlyacting o factors. These are o7F, which directs the transcription of spoIIIG (8) and other forespore-expressed genes (9), and a'E, which directs the transcription of sigK (10) and other mother-cell-expressed genes (11-14). Interestingly, o.F iS produced in the predivisional sporangium (ref. 15; S. R. Partridge and J. Errington, personal communication) but does not become active in directing gene expression until after septum formation when its activity is restricted to the forespore (16). These findings indicate that a.F is subject to a regulatory mechanism that restricts its activity to one cellular compartment. A possible clue to this control mechanism comes from genetic experiments that suggest that the activity of crF iS governed by the sporulation regulatory proteins SpoIIAA and SpoIIAB (16-18). All three proteins are encoded within a three-cistron operon known as spolIA (19-21). The a-F factor is encoded by the promoter-distal member of the operon (5) spolIAC, whereas SpoIIAA and SpollAB are the products of the promoter-proximal cistrons spoIIAA and spollAB, respectively. The results of genetic experiments have shown that SpoIlAB is a negative regulator of o-Fdirected gene expression (16-18, 22) and that SpoIIAA inhibits or otherwise counteracts the inhibitory effect of SpoIIAB on aF (16, 17). In the present communication, we investigate the nature of the interaction between SpoIlAB and aF by using proteins purified from cells of Escherichia coli that have been engineered to express spoIIAB and spoIIAC at high levels. Our results indicate that SpollAB is an anti-a factor that binds to aF and blocks its capacity to direct transcription. MATERIALS AND METHODS Construction of SpoIIAB and gF Expression Vectors. An expression vector capable ofdriving transcription ofspoIIAC from a T7 RNA polymerase promoter was constructed by cloning a 1-kb Bgl II-Pst I fragment containing spoIIAC from pPM34 into BamHI/Pst I-digested pT713 (BRL), thereby creating pLD4. [pPM34 was derived from pPM24 (17) by replacing the mutant spoIIAC allele with a wild-type allele; P. Margolis and R.L., unpublished results.] An expression vector bearing spollAB downstream of a T7 RNA polymerase promoter was constructed by cloning an 820-bp Dra I-Sal I insert from pHM2 (23) into Sma I/Sal 1-digested pT713 to create pLD5. To increase SpoIIAB production, the spolIAB ribosome binding site was replaced with a stronger ribosome binding site by using the Kunkel method (24) of site-directed mutagenesis and the oligonucleotide 5'-CGATTTGAACAATCTGAGCTCAGGAGGTAAAAATCATGAAAAATG-3' to generate plasmid pLD8. This plasmid was used to produce [35S]methionine-labeled SpoIlAB for use in in vitro crosslinking studies. When unlabeled SpoIlAB was produced, however, we used pSP72IIAB-SD1 (C. Moran, personal communication), which is similar to pLD8 except that the oligonucleotide used to replace the spollAB ribosome binding site was 5'-CTGCAGAGGAGGTAAAACATTATGAAAAA-3', and the resulting gene was cloned into the Pst I site of the phage T7 RNA polymerase expression vector pSP72 (Promega). pLD4 and pLD8 were transformed into an E. coli T7 RNA polymerase expression strain, BL21(DE3)/pLysS (Novagen, Madison, WI), to create strains LDE7 and LDE15, respectively, whereas pSP72IIAB-SD1 was transformed into E. coli BL21(DE3) (Novagen) to generate strain EUD72AB. Control strains called LDE6 and LDE8 were produced by transforming the parental plasmid pT713 into BL21(DE3) and BL21(DE3)/pLysS, respectively. [3"SjMethionine Labeling of SpoIIAB and 0F. LDE15 and LDE7 were grown at 370C in 20 ml of M9 minimal medium supplemented with 0.2% glucose, vitamin B1 (1 ,ug/ml), ampicillin (75 ,ug/ml), and chloramphenicol (25 ug/ml) until OD595 was >0.2 at which time 17 RNA polymerase synthesis was induced by the addition of isopropyl f-D-thiogalactoside to 1 mM. After 30 min, rifampicin (Sigma) in methanol was added to 200 ,ug/ml (final concentration) to inhibit E. coli RNA polymerase. After host mRNA species were allowed to decay for 1 h, 1-ml aliquots of cells were labeled with 20 ,Ci Abbreviation: DSS, disuccinimidyl suberate. 2325 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2326 Biochemistry: Duncan and Losick of [35S]methionine (NEN; 1 Ci = 37 GBq) for 5 min. After the addition of unlabeled excess methionine, the reaction was allowed to proceed 5 min. The labeled cells were then collected by centrifugation, washed, and frozen at -70°C. Cell pellets containing 1 ml of [35S]methionine-labeled cells were resuspended in 500 ,ul of cell lysis buffer (50 mM Tris HCl, pH 8/2 mM EDTA/100 mM NaCl/0.1 mM dithiothreitol/0.5% Triton X-100). Cells were lysed by repeated freeze-thaw steps, and particulate matter was removed by