Common and distinguishing regulatory and expression characteristics of the highly related KorB proteins of streptomycete plasmids pIJ101 and pSB24.2

Abstract

The conjugative plasmid pIJ101 of the spore-forming bacterium Streptomyces lividans contains a regulatory gene, korB, whose product is required to repress potentially lethal expression of the pIJ101 kilB gene. The KorB protein also autoregulates korB gene expression and may be involved in control of pIJ101 copy number. KorB (pIJ101) is expressed as a 10-kDa protein in S. lividans that is immediately processed to a mature 6-kDa repressor molecule. The conjugative Streptomyces cyanogenus plasmid pSB24.1 is deleted upon entry into S. lividans to form pSB24.2, a nonconjugative derivative that contains a korB gene nearly identical to that of pIJ101. Previous evidence that korB of pSB24.2 is capable of overriding pIJ101 kilB-associated lethality supported the notion that pIJ101 and pSB24.2 encode highly related, perhaps even identical conjugation systems. Here we show that KorB (pIJ101) and KorB (pSB24.2) repress transcription from the pIJ101 kilB promoter equally well, although differences exist with respect to their interactions with kilB promoter sequences. Despite high sequence and functional similarities, KorB (pSB24.2) was found to exist as multiple stable forms ranging in size from 10 to 6 kDa both in S. lividans and S. cyanogenus. Immediate processing of KorB (pIJ101) exclusively to the 6-kDa repressor form meanwhile was conserved between the two species. A feature common to both proteins was a marked increase in expression or accumulation upon sporulation, an occurrence that may indicate a particular need for increased quantities of this regulatory protein upon spore germination and resumption of active growth of plasmid-containing cells.

FIG. 1.

KorB proteins encoded by plasmids pIJ101 and pSB24.2. (A) KorB encoded by pIJ101 as represented by open horizontal arrows in both unprocessed full-length 10-kDa and mature 6-kDa forms. A region previously identified (11) as a putative DNA-binding domain is indicated, as is the approximate position of posttranslational cleavage toward the C-terminal end. Amino acid positions Q3 (glutamine) and E30 (glutamic acid) are indicated on both the unprocessed and mature forms. (B) The full-length 10-kDa form of KorB encoded by pSB24.2. The putative DNA-binding domain, identical in sequence to the analogous region from KorB (pIJ101) with the exception of a lysine at amino acid position 30 (K30), is indicated. Amino acid position H3 (histidine) designates the only other sequence difference between the two KorB proteins. kDa, kilodaltons.

FIG. 2.

The kilB-xylE fusion vector pSCON2. Plasmid pSCON2 contains the 351-bp PstI-BstEII kilB-promoter-containing pIJ101 region inserted upstream of the xylE structural gene in the orientation shown on the E. coli-Streptomyces shuttle vector pXE4 (10). The Streptomyces SCP2* replicon, tsr (thiostrepton resistance gene), bla (beta-lactamase gene), and pBR322 ori (origin of replication) regions of pXE4 are indicated, as is the position of inverted repeat sequences between the putative −35 and −10 determinants of the korB-regulated kilB promoter. Upstream of the kilB promoter within the inserted fragment is a pIJ101 open reading frame (orf66) of undetermined function (11). The line drawing for the 351-bp PstI-BstEII sequence of pIJ101 is not to scale.

FIG. 3.

EMSA analysis of the korB-regulated pIJ101 kilB promoter conducted by using the KorB proteins of pIJ101 and pSB24.2. (A) Approximately 17 μg of each relevant KorB-containing S. lividans TK23 extract (prepared as described in Materials and Methods) was incubated with 30,000 counts of a radiolabeled 0.1-kb fragment per min, which includes the 74-bp FspI-BstEII kilB-promoter-containing region of pIJ101 (Fig. 2), and reactions were then subjected to electrophoresis on a 5% nondenaturing polyacrylamide gel followed by autoradiography. Lane 1, no extract added; lane 2, TK23 (pHYG1) extract added; lane 3, KorB (pIJ101)-containing TK23 (pHYG1:D8) extract added; and lane 4, KorB (pSB24.2)-containing TK23 (pSCON22) extract added. Two separate shifted bands (bands I and II) for KorB (pSB24.2) are indicated, as is the single KorB (pIJ101) shifted band. (B) Concentration-dependent characteristics of KorB (pSB24.2) binding to the pIJ101 kilB promoter. EMSA analysis was performed as described above, except that various amounts of KorB (pSB24.2)-containing TK23 (pSCON22) cell extracts were added to reactions. Total extract amount added: lane 1, none; lane 2, 0.53 μg; lane 3, 1.1 μg; lane 4, 2.1 μg; lane 5, 4.2 μg; lane 6, 8.4 μg; and lane 7, 17 μg.

FIG. 4.

Comparative DNase I footprinting of the korB-regulated kilB promoter by the KorB proteins of pIJ101 and pSB24.2. A fragment containing the 191-bp SalI-BstEII region of pIJ101 that includes the kilB promoter (Fig. 2) was radiolabeled on the coding strand, was incubated with 17 μg of each relevant S. lividans cell extract, and was then assayed by DNase I footprinting as described in Materials and Methods. Lane 1, Maxam-Gilbert A+G reactions of the same fragment; lanes 2, 4, and 6, TK23 (pHYG1) extract added; lane 3, KorB (pIJ101)-containing TK23 (pHYG1:D8) extract added; and lane 5, KorB (pSB24.2)-containing TK23 (pSCON22) extract added. The putative −35, −10, and +1 elements of the kilB promoter as described previously (23) are indicated to the left of the lanes. Protected regions from −40 to +21 relative to the kilB transcription start site for KorB (pIJ101) and from −40 to +3 for KorB (pSB24.2) are based on the data presented here. Two independent trials of the DNase I footprinting analysis shown produced identical results.

FIG. 5.

Appearance and processing of KorB (pIJ101) and KorB (pSB24.2) throughout differentiation of S. lividans. Spores of S. lividans TK23 containing either the pIJ101 derivative pIJ303 (14) or the pSB24.2 derivative pSB24.202 (18) were used to inoculate either R5 agar plates (A) or YEME broth (B), and cells were harvested at the indicated times (in hours). Protein extracts were separated on SDS-PAGE gels, transferred to nitrocellulose, and probed with antibodies raised against the His₆-tagged, unprocessed (10-kDa) form of KorB (pSB24.2). The sizes of KorB (pSB24.2) larger than 6 kDa are given on the right, while the positions of 15- and 6-kDa molecular mass standards (A) and 16- and 6-kDa molecular mass standards (B) are indicated on the left.

FIG. 6.

Processing of KorB (pIJ101) and KorB (pSB24.2) in S. cyanogenus. Spores of S. cyanogenus NRRL-B12354 containing either pIJ303 or pSB24.202 were used to inoculate YEME broth, and cells were harvested at the indicated times (in hours). Protein extracts were analyzed by Western blotting as described in the legend to Fig. 5. The sizes of KorB (pSB24.2) forms greater than 6 kDa are indicated on the right, while the positions of 16- and 6-kDa molecular mass standards are shown on the left.