Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli

ELSEVIER

FEMS MicrobiologyLetters 131 (1995) 121-126

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli Thomas J. Zupancic a,1,Joseph D. Kittle a72,Beth D. Baker a, Courtney J. Miller a, Donna T. Palmer a7*, Yoko Asai b, Masayuki Inui b, Alain Vert& aTb, Miki Kobayashi b, Yasurou Kurusu b, Hideaki Yukawa b aBattelle Memorial Institute, 505 King Avenue, Columbus, OH 43201, USA b TsukubaResearch Center, Mitsubishi Chemical Co. 8-3-l Chuo Ami, Inashiki, Ibaraki, 300-03, Japan Received 10 April 1995; revised 16 June 1995; accepted 17 June 1995

Abstract

A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength. Keywords:Brevibacterium flauum; Escherichia coli; Coryneform

bacteria;

1. Introduction Brevibacterium flavum and related bacteria are cost-effective bio-converters that are used for the

industrial production of numerous metabolites ineluding L-glutamic acid [l] and L-aspartic acid [2]. Genetic tools for engineering of the B. flavum strain

* Corresponding

author.

Tel.:

+ 1 (614) 424 5369; Fax:

+ 1

(614) 424 3716 ’ Present address: Progenitor Inc., 1507 Chambers Rd., Columbus, OH 43212, USA. *Present address: GeneMedicine, Inc., 8080 North Stadium Drive, Suite 2110, Houston, TX 77054, USA. 0378-1097/95/$09.50 0 1995 Federation SSDI 037&X-1097(95)00240-5

of European

Microbiological

Reporter gene; Shuttle vector; Genomic library; Promoter

MJ233C, including host-vector systems, transposon mutagenesis, a partition function and highly efficient transformation and gene replacement protocols, have

been developed [3-71. The isolation and characterization of specific coryneform promoter elements will allow improved control over gene expression. The construction of several plasmid vectors useful for the detection and

analysis of promoter elements in coryneform bacteria has been reported [E&11], but relatively few promoters have been analyzed to date. Thus, the specific DNA sequence elements responsible for promoter function in coryneform bacteria have not been definitely established (see for example [12,13]). Societies. All rights reserved

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T.J. Zupancic et al. / FEMS Microbiology Letters 131 (1995) 121-126

The goal of this work was to construct a promoter probe vector and a library of promoters from B. flavum strain MJ233C and to examine the function of the promoter-containing DNA fragments in Escherichia coli and in B. flavum. The construction and preliminary characterization of coryneform promoter elements described in this paper will facilitate the development of refined production strains by providing multiple promoter elements with a wide range of strengths and unique individual properties.

2.3. Construction tor pPROBEl7

of the promoter probe shuttle vec-

Standard laboratory methods were used for plasmid isolation and restriction digestion [14]. Plasmid DNA was isolated from B. flavum MJ233C using alkaline lysis [17] after pre-treatment of the cells with 5 mg mll’ lysozyme for 30 min at 37°C. Figs. 1 and 2 show plasmid features. 2.4. Construction promoter library

and screening

of the B. flavum

2. Materials and methods

2.1. Bacterial strains and plasmids B. flavum strain MJ233C [5] was the source of genomic DNA for construction of a promoter library and a host in the assessment of promoter activity. E. coli HBlOl [14] was used as a host for vector constructions. E. coli GM119 [15] was used to prepare recombinant clones for restriction analysis and to assess the relative activities of B. ftauum promoter clones in E. co/i. Plasmids used for the construction of plasmid pPROBE17 were pBR322 1161, pKPG13 (a plasmid in which transposon Tn5 is inserted into the tetracycline resistance gene of pBR322; S.R. Jaskunas, personal communication), pJCM1, and the E. coli/B. flavum shuttle vector pCRY3 [51.

2.2. Culture media and transformations B. flavum strain MJ233C was grown on AR medium when preparing cells for electroporation or on the minimal MM medium for promoter screening experiments 151. B. frauurn MJ233C cells were transformed by electroporation [5] and transformants were selected on AR medium plates with either 25 pg ml-’ kanamycin or 5 pg ml-’ chloramphenicol. E. coli HBlOl competent cells were obtained from BRL Inc., Bethesda, MD. E. coli GM119 cells were transformed using the calcium chloride procedure, and all transformed E. coli strains were selected for resistance to 20 pg ml-’ chloramphenicol [14]. E. coli strains were generally grown on LB medium.

Genomic libraries of B. jlavum MJ233C were constructed by partial digestion of B. flavum genomic DNA with either Hue111 or A/u1 in order to produce restriction fragments (average size of 7501500 bp), followed by ligation to EcoRV-cleaved pPROBE17 plasmid DNA. Two selection methods were used to isolate promoter clones. Transformed cells were plated on AR medium containing either 25 pg ml * kanamycin (kanamycin-selected clones) (chloramphenicolor 5 Fg mll’ chloramphenicol selected clones) and subcultured in 96-well microtitre plates. Clones were subsequently replicated onto fresh MM medium agar plates supplemented with kanamycin using a microtitre plate replicator (Sigma Inc., St. Louis, MO). For the initial detection of promoter clones, kanamycin concentrations of 10, 25 and 100 pg ml-’ were used. Clones were scored as ‘promoter element containing’ if they grew on kanamycin-supplemented medium. To assess relative promoter activities, promoter clones were replicated on a series of plates containing increasing kanamycin concentrations (100-1500 pg ml-‘). After overnight incubation at 32°C the maximum kanamycin concentration which could be tolerated without causing any visible reduction in cell growth was determined. 2.5. NPTII assay To assay neomycin phosphotransferase (NPTII) expression, recombinant B. flauum cells were grown to an OD,,, of 0.5 in MM medium plus 5.0 pg ml-’ chloramphenicol. Cells were collected by centrifugation, washed in 20 mM Tris . HCl (pH 6.8) and concentrated lOO-fold in 20 mM Tris . HCl (pH 6.8).

T.J. Zupancic et al. / FEMS Microbiology Letters 13I (1995) 121-126

Protein extracts were prepared by incubating the cells with 50 pg ml-’ mutanolysin for 30 min at 37°C followed by three freeze/thaw cycles. Cell debris was removed by centrifugation; total protein concentrations in the cell extracts were measured using a BA protein assay (Pierce, Rockford, IL). Typically, cell extracts contained 500 pg ml-’ protein. NPTII protein concentrations were measured using an NPTII ELISA assay kit (5 Prime-3 Prime Inc., Boulder, CO).

3. Results The promoter probe shuttle vector pPROBE17 was constructed to detect promoter elements in coryneform bacteria and E. coli (Fig. 1). This vector

Pvu II -

123

contains the pBR322 plasmid origin of replication, the Tn9 chloramphenicol resistance gene from plasmid pACYC184, a promoter-less kanamycin resistance encoding the neo gene of transposon Tn5 as a reporter, and the coryneform origin of replication of plasmid pBY.503. The EcoRV restriction site upstream of the reporter gene provides a unique cleavage site for re-insertion of DNA fragments (Fig. 2). Genomic libraries constructed in plasmid pPROBE17 were introduced into B. flauum MJ233C by electroporation. Transformants in this first screen were selected for resistance to chloramphenicol (chloramphenicol-selected). A total of 4700 chloramphenicol-selected clones were subcultured and tested for the presence of promoter elements. 899 clones were detected which grew on media supplemented with at least 10 pg ml-’ kanamycin. These clones

pPROBE17

Fig. 1. The promoter probe shuttle vector, pPROBE17, was constructed in E. coli HBlOl 111 by following a series of steps. The pBR322 plasmid [16] origin of replication region, present on a l.l-kb PuuII-BstYI fragment, was ligated to a synthetic DNA linker (GATCTCAAGAAGATCCPITGATCITTI’CTACGGATCCAG) designed to restore the plasmid pBR322 RNAII promoter. Ligation products were cleaved with BarnHI and ligated to a BarnHI fragment from plasmid pJCM1 which contained the transposon Tn9 chloramphenicol resistance gene. pJCM1 contains the pACYC184 HaeII fragment encoding the transposon Tn9 chloramphenicol resistance gene; this HaeII fragment had been cloned into the BamHI site of plasmid pBR322 using synthetic linkers. Following transformation, a recombinant plasmid, pBRCM102, was isolated. In order to prevent readthrough of the kanamycin resistance gene from plasmid promoters, a transcription terminator based on the sequence of the E. coli trpA gene terminator [18] was ligated to pBRCM102 plasmid DNA partially cleaved with PouII. A recombinant clone containing the terminator inserted counter-clockwise was isolated and the subsequent plasmid was designated pCMT44. The NPTII encoding gene of transposon Tn5 without its promoter element was isolated from plasmid pKPG13 on a 1.4-kb BglII-EamHI fragment and ligated to pCMT44 plasmid DNA which had been partially cleaved with BarnHI in order to generate plasmid pCKT11. The B. frauurn plasmid origin of replication was isolated on the 4.0-kb X/r01 fragment from the E. coZi/B. flavum shuttle vector pCRY3 [S] and ligated to XhoI-cleaved pCKTl1 plasmid DNA resulting in plasmid pPROBE17.

124

T.J. Zupancic et al./FEMS

m

AATT]

(CT-

EC0R”

ATGAGGATCGllTCGCfi

Barn HI&l

Kanamycin

-I

AlTGAACAA...

Fig. 2. The DNA sequence upstream of the NPTII encoding reporter gene. The EcoRV restriction site upstream of the neomycin resistance gene provides a unique cleavage site for re-insertion of DNA fragments.

were assembled into microtitre plates to create a B. flavum promoter library. Further analysis of relative promoter activities was accomplished by screening recombinant clones for their abilities to confer resistance to increasing concentrations of kanamycin. Promoter clones which conferred resistance to at least 100 pg ml -I kanamycin were further tested by replicating onto MM plates supplemented with 100-1.500 pg ml-’ kanamycin. The distribution of relative promoter ac-

Table 2 Expression

of the reporter

Promoter clone

neo gene by promoter Insert size (bp)

clones in B. flawm Kanamycin MJ233C

Letters 131 (1995) 121-126

Table 1 Distribution of relative promoter activities in B. flaauum cells of chloramphenicol-selected clones isolated in B. flauum

Tn 5 DNA ---> GATCAAGAGACAOG

On lknker GGG CTT l-i-T Tll

Microbiology

( pg ml-’ 1

41.5 25.2 20.4 6.1 3.4 1.0 1.0 1.4

Distribution is expressed as a percentage, the maximum kanamycin concentration significant growth inhibition.

which is a function of that did not result in

tivities of these chloramphenicol-selected clones is summarized in Table 1. For purposes of comparison, the level of resistance to kanamycin conferred to B. flavum MJ233C cells by the E. coli tat promoter in plasmid pPROBE17 was found to be 500 pg ml-‘. Restriction analyses that were performed on a large

strain MJ233C and E. cofi strain GM119

resistance ( pg mlGM119

’)

NPTII expression (% total protein) MJ233C

B. jlavum selected 1Ktl 2Kn 6Kn 12Kn 13Kn 5Cm 51Cm 54Cm 1OlCm 130Cm Vector and Ptac pPROBEl7 pTAC E. coli selected AlCm H2Cm ElKn a E4Kn E93Kn

125 900 500 1000 450 350 450 500 1100 500

1500 1000 1000 1000 500 1500 1500 1000 1000 750

10 250 50 10 10 500 100 500 100 100

0.25 0.12 0.09 0.09 0.08 0.58 0.17 0.22 0.14 0.16

_ 97

<5 500

<5 500

0.0 0.07

350 700 350 600 150

25 < 10 > 1000 < 10 10

100 100 500 500 500

a Appears to be identical to 5Cm (data not shown). nd: not determined.

%

10 25 100 259 500 750 1000 1500

nd

nd nd nd nd

T.J. Zupancic et al./FEMS Microbiology Letters 131 (1995) 121-126

number of promoter clones spanning the range of relative promoter activities observed confirmed that numerous different promoter elements had been isolated. A second selection of transformants was accomplished by directly selecting for kanamycin resistance (kanamycin-selected). While selection of transformed cells on chloramphenicol generally yielded approximately lo3 transformants pg-’ plasmid DNA, kanamycin selection yielded approximately lo-50 transformants pg-’ plasmid DNA. The kanamycin-selected promoter clones were found by restriction analysis to contain a small number of different DNA fragments which were recovered multiple times. The kanamycin-selected clones all contained relatively strong promoter elements as demonstrated by kanamycin resistance levels greater than 100 pg ml-’ kanamycin, in contrast to the chloramphenicol-selected clones which constituted a more diverse collection of promoters with a wider range of relative activities (Table 2). In order to test whether the sequence determinants which define promoter strength are similar in B. flauum and E. coli, several promoter clones were used in both hosts. The collection of clones used in this test contained five unique kanamycin-selected clones isolated in B. flavum which conferred resistance to at least 500 pg ml-’ kanamycin (designated clones lKn, 2Kn, etc.), and ten chloramphenicol-selected clones (designated 5Cm, 51Cm, etc.> also isolated in B. j7uvum, which conferred resistance to at least 250 pg ml - * kanamycin. The levels of expression of NPTII protein in B. flavum mediated by these 15 strong promoter clones were measured by an ELISA assay and typical results are shown in Table 2. B. jlavum cells with no plasmid vector or with plasmid pPROBE17 alone were tested to determine the cellular background reaction. These samples typically showed a limited reaction in the ELISA assay (about 0.05% of total cell protein; this background is subtracted from the values in Table 2). The measured levels of NPTII protein were consistent with the levels of resistance to kanamycin observed for each promoter clone tested. Data are also shown for several strong promoter clones isolated in E. coli (chloramphenicol-selected clones AlCm, and H2Cm and kanamycin-selected clones Elkn, E4kn, and E93kn). Results shown in Table 2 indicate that,

12.5

in contrast to their function in B. jlavum, 12 of the 15 promoters isolated in B. fZavum were relatively weak or moderate promoters in E. coli. Similarily, the activities of the promoter clones isolated in E. coli were generally less in B. jlavum. On the other hand, the tat promoter in plasmid pPROBE17 conferred resistance to 500 pg ml- ’ kanamycin in both E. coli and B. flavum.

4. Discussion A promoter probe vector, pPROBE17, useful for the isolation and characterization of coryneform promoter elements was constructed using a coryneform plasmid origin of replication and a kanamycin resistance reporter gene. Testing for the expression of the kanamycin resistance trait using the E. coli tat promoter and genomic DNA fragments from B. flavum demonstrated that this vector could be used to detect promoter activity in both coryneform bacteria and E. coli. Analysis of kanamycin resistance levels conferred by a library of 899 independently isolated B. flavum genomic promoter clones indicated that individual promoter elements with a wide range of relative activities were isolated. The most active of these promoter clones conferred resistance to at least 1500 pg ml-’ kanamycin, a level significantly higher than the level of resistance conferred by the E. coli tat promoter (500 pg ml-’ 1. Comparison of the reporter gene expression directed by a set of promoter clones in E. coli and B. flavum showed that the relative activities of the clones differed in these two species. The observed differences in promoter activities of the cloned fragments could be the result of gene regulation in B. flavum which does not function when these B. jlauum promoters are introduced into E. coli. Alternatively, these discrepancies could occur because the DNA sequence determinants of promoter activity are different in E. coli and B. flavum. Many factors contribute to the level of protein expression from a given gene construct. It is nevertheless reasonable to assume that the plasmids conferring the very highest levels of kanamycin resistance must contain, at a minimum, a strong promoter. In addition, some of these clones may contain small genes or portions of open reading frames,

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Microbiology Letters 1.31 (1995) 121-126

whose translation could contribute to the efficiency of transcription and RNA stability in the cell. On the other hand, the relatively short inserts in some of the strongest clones may be highly active in part because gene-specific repression functions have been eliminated. These sequences should represent very strong, near consensus promoters. However, most of these potentially deregulated B. jhumselected promoters are only weakly active in E. coli. Moreover, while the E. coli consensus tat promoter is functional in B. flauum, it does not appear stronger than natural promoters, as it is in E. coli. These observations therefore suggest that the genetic signals which determine promoter strength in B. flavum are different from those in E. coli. As a result, selection of promoters in 8. j7auum enabled the isolation of promoters that optimally function in a native background and that are therefore more suitable for engineering coryneform bacteria-mediated biotechnological processes.

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