We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfsl through sfs12 (sugar ...
JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2644-2648
Vol. 173, No. 8
0021-9193/91/082644-05$02.00/0
Nucleotide Sequence and Characterization of the sfsl Gene: sfsl Is Involved in CRP*-Dependent mal Gene Expression in Escherichia coli MAKOTO KAWAMUKAI,1 RYUTARO UTSUMI,2* KAZUHIKO TAKEDA,1 AKIHISA HIGASHI,' HIDEYUKI MATSUDA,' YONG-LARK CHOI,3 AND TOHRU KOMANO3 Laboratory of Applied Microbiology, Faculty of Agriculture, Shimane University, Matsue 690,1 Laboratory of Biochemistry, Department of Agricultural Chemistry, Kinki University, 3327-204, Nakamachi, Nara 631,2 and Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto 606,3 Japan Received 29 October 1990/Accepted 22 January 1991
We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfsl through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nip, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfsl) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfsl encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfsl gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfsl could be a new regulatory factor involved in maltose metabolism.
Cyclic AMP receptor protein (CRP) binds to cAMP and regulates the expression of many genes negatively or positively in Escherichia coli (1, 9, 18). CRP* mutants which can actively affect gene expression without binding cAMP were isolated by many groups (3, 10, 11, 13-15). Most crp* mutants were isolated as a lactose-fermentative phenotypes in the absence of cAMP, but those crp* mutants act differently on the other sugar-fermentative genes. For example, crp*J, which was isolated by Aiba et al., induced the lac genes but not the mal genes in the absence of cAMP or cGMP (3). The maltose-metabolizing genes are located in two loci. The malPQ operon, encoding maltodextrin phosphorylase and amylomaltase, is located at the 75-min map position (26). malEGF and malK lamB are located at 91 min and are involved in the uptake of maltose. The expression of these genes is induced by maltose, and the induction is mediated by a malT activator. The cAMP-CRP complex positively regulates the malT gene and the malEGF operon. The action of cAMP-CRP on malPQ genes is mediated indirectly by MalT protein. This regulatory mechanism of malPQ is different from that of lacZYA, in which the expression is directly dependent on cAMP-CRP. Furthermore, the expression of malEGF is dependent on two activators of MalT and cAMPCRP. Apparently, the mal and lac regulatory systems are different even though they share the common activator of CRP. In this study, we found that maltose was fermented in the absence of cAMP when the sfsl gene was overexpressed under the tac promoter in a crp*l cya strain and have characterized the sfsl gene and analyzed its nucleotide sequence.
transducing the cya::Kmr gene of HY1025 (29) to W3110 by P1 phage. In MP1025, the crp*l gene of plasmid pHA7*1 was integrated into the chromosome of HY1025 [polA(Ts) cya::Kmr] (29) by using its inability to replicate plasmids at a high temperature. Plasmid pHA7*1 (3) codes for an altered CRP in which the Leu at position 148 is changed to Arg. Then, MK2001 was constructed by transducing crp*l of MP1025 to MK1010 by using a cotransducible selective marker of Strr (7). MP26 was constructed from MK2001 by P1 transduction of the malT::TnJO gene of ME8399. All strains were grown in minimal medium (M9) or L broth. MacConkey agar (Difco) with 1% sugar was used for the sugar fermentation test. Phage libraries were amplified in the LE392-grown liquid culture before plaque hybridization experiments were done (19). Enzymes and chemicals. The DNA sequencing kit, restriction endonuclease, DNA polymerase I, Klenow fragment, exonuclease III, mung bean nuclease, and DNA ligation kit were obtained from Takara Shuzo Co., Ltd. The reaction conditions for the enzymes were as described in the suppliers' manuals. Isopropyl-f3-D-thiogalactopyranoside, 5-bromo4-chloro-3-indolyl-p-D-galactoside, and a glucose B test kit were purchased from Wako Pure Chemical Industries Ltd. [a-32P]dCTP from New England Nuclear was used for DNA labeling. A DNA labeling kit was purchased from Nippon Gene Corp. Isolation of DNA and Southern hybridization. Plasmid DNAs were prepared by the procedure of Birnboim and Doly (4). In small-scale preparation, the alkaline-sodium dodecyl sulfate (SDS) method of Davis et al. was used (7). Chromosomal DNA was prepared by the usual method (25). Bacteriophage isolation was done as described by Maniatis et al. (22). The ordered E. coli DNA bank was supplied by Y. Kohara. To map cloned genes, plaque hybridization with the ordered DNA bank was done by the method of Southern (28). DNA sequencing. DNA sequences were determined by the dideoxy-chain termination method with vectors derived
MATERIALS AND METHODS Bacteria and plasmids. The E. coli strains and plasmids used are listed in Table 1. MK1010 is constructed by *
Corresponding author. 2644
TABLE 1. Bacterial strains and plasmids used in this study Strains and plasmids
Source or reference
Relevant genotype
Strains
W3110 MM386 HY1025 MP1025 MK1010 MK2001 MP26 TP2010 TP2O1OR1 TP2139
ME8399
Wild type polAJ2 rha lac strA MM386, cya::Kmr MM386, cya::Kmr crp*l W3110, cya::Kmr W3110, cya::Kmr crp*l strA MK2001, malT::TnJO xyl cya argH lacX74 recA ilvA srl::TnlO TP2010, crp*l xyl ilvA argH lacX74 crp recAl araD139 (argF-lac)U169 rpsL deoCI relAl thiA ptsFflbB malT::TnlO
Laboratory stock 29 29 This study This study This study This study Danchin This study Danchin
pHA7*1 pUPVC2
pPVC2Km
Ampr Ampr tacP, BamHI site (tet region) of pKK223-3 is disrupted Ampr crp*1 Ampr, EcoRI-BamHI fragment (1.3 kb) of sfsl was cloned in pUC18 so that its expression can be regulated under lac promoter Ampr, HincII fragment (Km') derived from pUC4K is inserted at ClaI site within sfsl of pPVC2
bean nuclease after recloning of the inserted fragment of pPVC2 into M13 mpl8. Enzyme assay. Cells (optical density at 600 nm, 0.5) grown in M9 were centrifuged, suspended with 25 mM sodium phosphate buffer (pH 7.5) containing 0.5% Triton X-100, and disrupted by sonication (45 s, twice, 4°C). After centrifugation, the supernatant was used as crude extract for amylomaltase. This amylomaltase activity was measured by the method of Sharp (27) by using a glucose B test kit. One unit of enzymatic activity corresponds to the formation of 1 pmol of glucose per minute. Protein was measured by the standard procedure of Lowry et al. (21). Nucleotide sequence accession number. The GenBank accession number for the sfsl gene is M60726. RESULTS
Nishimura
tonA
Plasmids pUC18 and pUC19 pKK223-4
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SEQUENCE AND CHARACTERIZATION OF THE sfsl GENE
VOL. 173, 1991
8 3 This study
This study
from bacteriophage M13 (23, 30). DNA was subcloned in the M13 mpl8 and M13 mpl9 by using restriction fragments of plasmid pPVC2. Other subcloned fragments for sequencing were constructed by the deletion method with exo III-mung
Cloning of sfs genes. Chromosomal DNA was digested with EcoRI, HindIII, PvuII, PstI, and Sau3A and ligated with the EcoRI-, HindIII-, SmaI-, PstI-, and BamHI-digested pKK223-4, respectively. The ligation mixture was used to transform strain MK2001 (crp*l cya::Km), in which maltose is not fermented, and the transformants were selected on MacConkey agar containing 1% maltose and 50 mg of ampicillin per liter. Red colonies, which can use maltose, were isolated, and the sizes of cloned fragments were checked. Table 2 shows the list of cloned genes selected in this way. Surprisingly, 12 different DNA fragments were cloned from about 140,000 recombinants. We considered these genes to be involved in sugar fermentation. Therefore, we tentatively named them sfsl through sfsl2 (sugar fermentation stimulation). The chromosomal location of sfs genes. As pHC1 (sfs8) could suppress the cya genotype (Table 2) and the restriction map was the same as already reported for the cya gene (2), we concluded that pHC1 includes the cya gene. In order to test whether other sfs genes are already known, Southern hybridization was performed with an ordered phage library of the E. coli chromosome (19). pHC2 (sfs9) hybridized with gene banks E4E4 and E5B4, which contained the malA gene locus. In another experiment, we found that pPC3 (sfs6) hybridized with the melB
TABLE 2. Cloned plasmids containing sfs genesa Plasmid
pPC1 pPC3 pPC4 pPC23 pHC1 pHC2 PHC4 pHC11 pSC2 pSC4 pPVC1 pPVC2 pKK223-4
Insert (kb)
Cloning site' (insert/vector)
Map positionC
3.5 4.7 12.3 9.2 5.3 6.5 7.0 17.0 16.0 19.0 7.0 1.3 None
P/P P/P P/P P/P H/H H/H H/H H/H S/B S/B Pv/Sm Pv/Sm None
53 (sfs4) 93 (sfs6) 45 (sfs3) 69 (sfS7)e 85 (sfs8)f 75 (sf9Yf 74 (sfs5)
(sfslO)* I (sfsJ)* (sfsl2)* 8 (sfs2) 3.5 (sfsl) None
Mal
MK101Od Lac
+ + + + + + ++ ++++ + + + + + + ++ _ -
+ + ++ _ -
Mal
MK2001d Lac
*, Experiments were not done. bEach fragment was cloned in pKK2234. P, PstI; H, HindIII; S, SaulIIA; B, BamHI; Pv, PvuII; Sm, c Map positions given in minutes. The sfs genes contained by the plasmids are in parentheses. d MK2001: crp*l, cya::Km', MK1010: crp+, cya::Km'. **, All clones show positive characteristics. e Nlp was identified by Choi et al. (6). f pHC1 and pHC2 contained cya+ and maIA+, respectively. a
SmaI.
Amylomaltase
activity (units)
8 16 11 7 *
33 * 3 * * 8 11 1
2646
94 67 43
30
*
~ ~.
KAWAMUKAI ET AL.
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J. BACTERIOL.
TABLE 3. Effect of MalT on amylomaltase activity increased by Sfsl overexpression
activity Amylomaltase (units/4tg of protein)
Strain/plasmid
MK2001/pKK223-4 .................................. MK2001/pUPVC2a .................................. MK2001/pPVC2 ..................................
MK2001/pPVC2Kmb ............. **' S FS 1
W3110/pKK223-4 .................................. W3110/pPVC2 .................................. MP26/pPVC2 ..................................
22 36 205 13 126 148
