Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System

S Chen; M Bagdasarian; M G Kaufman; E D Walker

doi:10.1128/AEM.01577-06

. 2006 Dec 22;73(4):1089–1100. doi: 10.1128/AEM.01577-06

Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System^▿

S Chen ^1,^*, M Bagdasarian ¹, M G Kaufman ², E D Walker ¹

PMCID: PMC1828668 PMID: 17189449

Abstract

We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (∼1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli, thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac, functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5′ rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.

Flavobacteria belong to a systematically diverse yet phylogenetically coherent group within the phylum Bacteroidetes (5, 12, 51). In the past referred to as the Cytophaga-Flavobacterium-Flexibacter-Bacteroides, Cytophaga-Flavobacterium-Bacteroides, or CFB group, this cluster represents an abundant and ubiquitous assemblage of heterotrophic bacteria, with Flavobacterium being one of many genera (5, 22). Bacteria in the Bacteroidetes are widespread in freshwater and marine ecosystems and inhabit the water column, biofilms, sediments, soils, and feces (7, 12, 22-25). An important functional role of these organisms is the transformation of high-molecular-weight, dissolved organic matter in aquatic environments (12, 24, 25). Some flavobacteria, in particular Flavobacterium psychrophilum and Flavobacterium columnare, are pathogens of fish (36, 48), while other bacteria in Bacteroidetes are facultative pathogens of immunocompromised adult humans (28).

Molecular genetic manipulation studies of bacteria in Bacteroidetes have proven to be difficult and are limited in scope. Plasmids, selectable markers, and transposons functional in proteobacteria fail to function in Bacteroidetes (2, 31, 43). Genetic techniques, including cosmid complementation and transposon-mediated mutagenesis, were used to identify several gld genes associated with gliding motility in Flavobacterium johnsoniae (1, 8, 9, 15-17, 21, 29-31). These techniques were recently extended to F. psychrophilum, leading to characterization of the tlp gene possibly involved in gliding motility and biofilm formation (3). An expression system was developed to express homologous and heterologous genes under the control of the hepA regulatory region using the Pedobacter heparinus (previously Flavobacterium heparinum) conjugation-integration plasmid pIBXF1 (6, 43). Modifications of this plasmid permitted in vivo expression of several heparinase genes as well as the gene cslA encoding the chondroitinase A enzyme as a reporter (6). Expression of Escherichia coli lacZY in F. psychrophilum was recently investigated by Alvarez et al. by inserting this reporter gene under a putative open reading frame 1 (ORF1) promoter on the pCP23 plasmid (2). A xylosidase/arabinosidase bifunctional reporter system has also been developed for Bacteroides and Porphyromonas (49). The aforementioned studies notwithstanding, genetic manipulation of this important group of bacteria is underdeveloped. Remarkably, there are only few selectable antibiotic resistance markers available (41), and very few studies of native promoters and ribosome binding sites (RBSs) of this group have been undertaken. Given the ubiquity of Bacteroidetes and their importance in ecological processes, as well as the association of the group with pathogenesis in many fish species, further development of genetic tools is warranted. In particular, there is a need to identify and characterize native promoters that will allow for the expression of gene inserts in these bacteria. Because larval mosquitoes such as Ochlerotatus triseriatus, dwelling in water-filled tree holes and similar habitats, feed upon Flavobacterium and other microorganisms (19, 20, 47; M. Kaufman, E. Walker, and S. Chen, unpublished observations), our interest is in the potential use of these highly ingestible bacteria to express genes encoding proteins toxic to mosquito larvae. Here, we report progress towards this end, with emphasis on development of new plasmid systems for genetic transformation in an environmental isolate of Flavobacterium, analysis of native promoter structure and function using the green fluorescent protein (GFP) reporter, and an investigation of properties of the RBS.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table 1. Flavobacterium strain W22, here classified as F. hibernum, was initially isolated from a water-filled tree hole in an American beech tree located near the Michigan State University campus. Flavobacterium johnsoniae UW101 (ATCC 17061) was obtained from Mark McBride of the University of Wisconsin—Milwaukee. Strains of E. coli were routinely grown in Luria-Bertani broth at 37°C. Flavobacterium strains were grown at 26°C in Casitone yeast extract (CYE) medium as previously described (31). Liquid cultures were incubated with shaking at 200 rpm. Solid CYE medium contained 20 g of agar per liter. Antibiotics were used as indicated at the following concentrations: ampicillin, 100 μg/ml; erythromycin, 100 μg/ml; tetracycline, 10 μg/ml; rifampin, 50 μg/ml; chloramphenicol, 25 μg/ml; streptomycin, 100 μg/ml; and kanamycin, 30 μg/ml.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristic(s) and/or plasmid construction^a	Source or reference
Strains
E. coli
DH5α	endA1 hsdR17 supE44 thi-1 recA1 gyrA96 (Nal^r) relA1Δ(lacIZYA-argF)U169 deoR(φ80dlacZΔM15)	Clontech
S17-1	hsdR17(r_K⁻ m_K⁻) recA RP4-2 (Tc^r::Mu-Km^r::Tn7 Str^r)	42
JM109	F′ [traD36 proAB⁺lacI^qlacZΔM15]/recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi-1 mcrA Δ(lac-proAB)	Promega
F. hibernum
W22	Wild type	This study
W22 R1	Rifampin-resistant mutant	This study
F. johnsoniae UW101	ATCC 17061	1
Plasmids
pGEM-T easy vector	Cloning vector, Ap^r	Promega
pKEN2	gfpmut3, Ap^r	11
pMMB66EH	IncQ RSF1010Δ(PstI-PvuII, 2.87 kb) Ω [lacI^qtacP rrnB] Ap^r	13
pMMB207	IncQ RSF1010 derived, Cm^r	33
pMMB503	IncQ RSF1010 derived, Sm^r	32
pCP23	ColE1 ori (pCP1 ori), E. coli-Flavobacterium shuttle plasmid, Ap^r (Tc^r)	1
pCP29	ColE1 ori, (pCP1 ori), E. coli-Flavobacterium shuttle plasmid, Ap^r (Cfx^r, Em^r)	21
pNJR5	IncQ, E. coli-Bacteroides shuttle vector, Km^r (Em^r)	41
pSCH01	gfpmut3 gene inserted in BamHI and PstI sites in pMMB603EH, Ap^r	This study
pSCH02F	Expression cassettes including lacI^q, ptac, and gfpmut3 fragment in pCP23 (ORF1, forward), Ap^r (Tc^r)	This study
pSCH02R	Expression cassettes in pCP23 (ORF1, reverse), Ap^r (Tc^r)	This study
pSCH03	Promoterless gfpmut3 including E. coli RBS in pCP23, Ap^r (Tc^r)	This study
pSCH41	HU fragment trapped in pSCH03, Ap^r (Tc^r)	This study
pSCH41.−210	D1, 375-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
pSCH41.−100	D2, 300-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
pSCH41.−52	D3, 275-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
pSCH41.−23	D4, 217-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
pSCH41.34	D5, 98-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
pSCH41.67	D6, 68-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
pSCH41.97	D7, 36-bp fragment of HU fused to gfpmut3, Ap^r (Tc^r)	This study
WT RBS	Wild-type RBS, Ap^r (Tc^r)	This study
M1	RBS mutant, TAATA→TCGTT, Ap^r (Tc^r)	This study
M2	RBS mutant, TAATA→TTGTT, Ap^r (Tc^r)	This study
M3	RBS mutant, TAATA→TATTA, Ap^r (Tc^r)	This study
M4	RBS mutant, TAATA→TTTTA, Ap^r (Tc^r)	This study
M5	RBS mutant, TAATA→TTTTT, Ap^r (Tc^r)	This study
M6	RBS mutant, TAATA→TGATA, Ap^r (Tc^r)	This study
M7	RBS mutant, TAATA→TAATG, Ap^r (Tc^r)	This study
M8	RBS mutant, TAATA→TAAAA, Ap^r (Tc^r)	This study
M9	RBS mutant, TAATA→TAGTA, Ap^r (Tc^r)	This study

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Antibiotic resistance phenotype: Ap^r, ampicillin; Tc^r, tetracycline; Em^r, erythromycin; Cfx^r, cefoxitin; Cm^r, chloramphenicol; Km^r, kanamycin; and Sm^r, streptomycin. Unless indicated otherwise, antibiotic resistance phenotypes are those expressed in E. coli. Antibiotic resistance phenotypes listed in parentheses are those expressed in Flavobacterium strains but not in E. coli.

Conjugal transfer of plasmid DNA from E. coli to F. hibernum strain W22.

Rifampin resistance was used as a counterselectable marker for conjugation. Rifampin-resistant F. hibernum strain W22 mutants were obtained by plating more than 10⁹ cells on CYE medium supplemented with 50 μg of rifampin per ml. Equal amounts of F. hibernum strain W22 cells and E. coli cells (approximately 10⁸ cells of each strain) at mid-log phase were harvested by centrifugation, washed with CYE medium, mixed, spotted onto CYE agar containing 5 mM CaCl₂, and incubated at 26°C for 12 h. After conjugation, cells were scraped off the plates, diluted, and plated on CYE agar containing the appropriate antibiotic. For conjugal transfer of plasmids pCP23 and pCP29, counterselection was unnecessary because the tetracycline resistance and erythromycin resistance were not expressed in E. coli.

Electroporation.

Flavobacterium cells were harvested during exponential growth, washed three times in 10% (vol/vol) glycerol at 4°C, and resuspended to a cell density of approximately 10¹¹/ml in GYT medium (10% [vol/vol] glycerol, 0.125% [wt/vol] yeast extract, and 0.25% [wt/vol] tryptone, pH 7.0). Approximately 80 ng of plasmid DNA was added to 100 μl of cells. Electroporation was conducted by using a Bio-Rad pulser in a 2-mm cuvette according to the manufacturer's instructions. Following electroporation, 1 ml of CYE medium was immediately added to each cuvette. Cells were incubated at 26°C for 1.5 h to allow expression of antibiotic resistance and plated on CYE agar with the appropriate antibiotic. Colonies were counted after 2 to 3 days of incubation at 26°C.

Plasmid constructions.

An expression vector using tac promoter was constructed as follows. A 750-bp, promoterless version of gfpmut3 was amplified by PCR from plasmid pKEN2 containing the E. coli ribosome binding site by using the primers gfpF1 and gfpR1 (Table 2). The gfpmut3 fragment was digested with BamHI and PstI and inserted into pMMB66EH at the same restriction sites. The resulting plasmid was named pSCH01. The expression cassette (including the lacI^q, tac promoter, gfpmut3, and transcriptional terminator regions) from plasmid pSCH01 was amplified by PCR with primers 66EHF and 66EHR. The resultant fragment was ligated into pCP23, which had been digested with KpnI and SphI, blunt ended with T4 DNA polymerase, dephosphorylated, and gel purified. The constructs were transformed into E. coli DH5α, and the inserts in both orientations were screened, yielding plasmids pSCH02F and pSCH02R. These plasmids were extracted from E. coli and transformed into F. hibernum strain W22 and F. johnsoniae by electroporation.

TABLE 2.

Primers used in this study

Primer	Sequence^a
63f	5′-CAGGCCTAACACATGCAAGTC-3′
1387r	5′-GGGCGGWGTGTACAAGGC-3′
gfpF1	5′-CGCGGATCCTTTAAGAAGGAGATATACATATGAGTAAAGGAGAAG-3′
gfpR1	5′-AAACTGCAGGAATTCTTATTTGTATAGTTC-3′
66EHf	5′-CCTGCTAATTGGTAATACC-3′
66EHr	5′-CGGAAATGTTGAATACTCATAC-3′
D1	5′-CGGGGTACCTTACCAGCAGATGCGG-3′
D2	5′-CGGGGTACCCAGGATCGACAAGCGAC-3′
D3	5′-CGGGGTACCCTAAATTTAAAGAAAACACTTGC-3′
D4	5′-CGGGGTACCCGGATTTCCTATTAAATTTGTG-3′
D5	5′-CGGGGTACCCTAATTATTATGAACAAATCAG-3′
D6	5′-CGGGGTACCGCTATCGCTGCTGATGCAGG-3′
D7	5′-CGGGGTACCGCTGCAGCTAAATTAGC-3′
gfpSphIR	5′-ACATGCATGCGAATTCTTATTTGTATAGTTC-3′
RBSM1	5′-CGCGGATCCAATTAATAATTCGTTTTTATGAGTAAAGGAGAAG-3′
RBSM2	5′-CGCGGATCCAATTAATAATTTGTTTTTATGAGTAAAGGAGAAG-3′
RBSM3	5′-CGCGGATCCAATTAATAATTATTATTTATGAGTAAAGGAGAAG-3′
RBSM4	5′-CGCGGATCCAATTAATAATTTTTATTTATGAGTAAAGGAGAAG-3′
RBSM5	5′-CGCGGATCCAATTAATAATTTTTTTTTATGAGTAAAGGAGAAG-3′
RBSM6	5′-CGCGGATCCAATTAATAATTGATATTTATGAGTAAAGGAGAAG-3′
RBSM7	5′-CGCGGATCCAATTAATAATTAATGTTTATGAGTAAAGGAGAAG-3′
RBSM8	5′-CGCGGATCCAATTAATAATTAAAATTTATGAGTAAAGGAGAAG-3′
RBSM9	5′-CGCGGATCCAATTAATAATTAGTATTTATGAGTAAAGGAGAAG-3′
WT	5′-CGCGGATCCAATTAATAATTAATATTTATGAGTAAAGGAGAAG-3′
SCH08race	5′-CCGCTCTGCCTCTTGCTCCAGGTCTTG-3′
SCH13race	5′-CCACATTGAAGTGTAAGCTGCCTGAACAGCTGC-3′
SCH17race	5′-GTCAATAAGTTCGTCTTTGTTAGCAGGCC-3′
SCH36race	5′-TGGCATTTGGATTGGATTACCCGTTCC-3′
SCH40race	5′-GTCTACTTCTCCCCAAACCGGTTTCCATG-3′
SCH41race	5′-GTAAGTAGCATCACCTTCACCTTCACCGG-3′
SCH52race	5′-CAACGCTTCGTACCCGTCAACACCTTTGG-3′

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Restriction sites on the primers are underlined.

A promoter-probe vector was constructed as follows. The promoterless gfpmut3 construct was released from pSCH01 by BamHI and PstI and ligated into the same sites of pCP23, resulting in the promoter-probe vector herein designated pSCH03.

In order to analyze promoter structure in detail, promoter deletion derivatives for the strongest promoter found in the analysis shown below (on plasmid pSCH41) were constructed. The positions of N-terminal primers on the putative pSCH41 promoter region were as follows, with the TSP assigned as +1: D1, −210 bp; D2, −100 bp; D3, −52 bp; D4, −23 bp; D5, +34 bp; D6, +67 bp; and D7, +97 bp. Amplification of the deletion derivatives and gfpmut3 was performed with the above N-terminal primers (containing an N-terminal KpnI site) and C-terminal primer gfpSphIR (with an SphI site) complementary to the 3′ end of gfpmut3. The PCR products were inserted into the T-easy vector and sequenced. The inserts were released from this vector by KpnI and SphI and cloned in the same sites of pCP23 to create the following deletion vector series: pSCH41.−210, pSCH41.−100, pSCH41.−52, pSCH41.−23, pSCH41.34, pSCH41.67, and pSCH41.97.

In order to elucidate properties of the ribosome binding site, site-specific mutagenesis was performed by using the oligonucleotide-directed PCR method. The sequences of the nine mutagenic oligonucleotides used for mutagenesis of the RBS, labeled RBSM1 through RBSM9, are shown in Table 2. These N-terminal oligonucleotides were designed to mutagenize the 4-bp putative RBS region upstream of the translation start codon bearing a BamHI site, whereas the C-terminal primer gfpSphIR was designed to have a SphI site. The product of the PCR with the mutagenic primer and C-terminal primer was purified, inserted into T-easy vector, and sequenced. The inserts were released by BamHI and SphI and cloned into the same sites on pSCH41, resulting in the desired RBS mutagenesis series (RBSM1 to RBSM9).

Promoter-trapping and native promoter analysis.

Chromosomal DNA of F. hibernum W22, extracted with the genomic DNA extraction kit (Promega), was partially digested with Sau3AI and size fractionated by agarose gel electrophoresis. DNA fragments ranging from 0.3 to 2.0 kb were purified and ligated into the BamHI site of pSCH03 (extracted from F. hibernum strain W22). The ligation mixture was electroporated into electrocompetent Flavobacterium cells. Qualitative screening of transformants for expression of the gfpmut3 gene was performed by fluorescence microscopy (excitation wavelength of 485 nm and emission length of 510 nm). Quantitative analysis of GFP production was performed using a microtiter fluorescence spectrometer (Dynex, Chantilly, VA). Strains were grown overnight in CYE medium supplemented with 10 μg/ml tetracycline. Two hundred microliters of each culture was centrifuged, washed with phosphate-buffered saline, and diluted to an optical density at 600 nm of 0.4. The samples were analyzed under the following conditions: excitation wavelength of 485 nm, emission wavelength of 510 nm, and 0.2-s interval at 25°C. To ensure that the values recorded were due to GFP, cultures of untransformed strains were used as the appropriate blanks for calculation of the relative units of fluorescence. Colonies showing high fluorescence were subcultured and stored in CYE-glycerol medium at −80°C.

RNA isolation.

Flavobacterium cells (2 ml) from exponentially growing cultures (turbidity at 650 nm of 0.3 to 0.4) were stabilized using 2 volumes of RNAprotect bacterial reagent (QIAGEN) for 5 to 10 min. Total RNA was extracted by using the RNeasy kit (QIAGEN) according to the protocol of the manufacturer. Following extraction, total RNA was treated with DNase I. The DNase I was later heat inactivated at 70°C for 15 min. To concentrate the samples, total RNA was precipitated with ethanol and resuspended in 30 μl of RNase-free water. Samples were stored at −80°C.

Transcriptional start site.

The transcriptional start site was determined by using the method 5′ rapid amplification of cDNA ends based on the switching mechanism at 5′ end of RNA transcript (SMART-RACE system), as recommended by the supplier (Clontech, Mountain View, CA) with 3 μg of total RNA (DNA free). A gfpmut3 gene-specific primer (gfpSphIR) was used to initiate the first strand of cDNA synthesis for 1.5 h at 42°C. Small aliquots of the above cDNA as a template were amplified using SMART PCR primer UP and gene-specific primers (Table 2). The PCR products were cloned into the T-easy vector according to standard procedures and sequenced.

DNA sequencing.

Plasmid DNA was prepared using QIAGEN Mini-Prep spin columns. The insert fragment was sequenced by the dideoxy termination method using an automated sequencing system (Applied Biosystem). GenBank database searches were carried out using the National Center for Biotechnology Information BLAST web server (http://www.ncbi.nlm.nih.gov/BLAST). Multiple sequence alignments were carried out with the ClustalW program and later adjusted manually.

Nucleotide sequence accession number.

The inserter sequences of the promoter clones reported in this paper have been deposited in the GenBank database under the following accession numbers: SCH08, DQ834946; SCH13, DQ834947; SCH14, DQ834948; SCH15, DQ834949; SCH16, DQ834950; SCH17, DQ834951; SCH19, DQ834952; SCH24, DQ834953; SCH25, DQ834954; SCH28, DQ834955; SCH29, DQ834956; SCH35, DQ834957; SCH36, DQ834958; SCH40, DQ834959; SCH41, DQ834960; SCH42, DQ834961; SCH45, DQ834962; SCH47, DQ834963; SCH51, DQ834964; and SCH52, DQ834965.

RESULTS

Phylogenetic placement of F. hibernum strain W22.

Genomic DNA was extracted from strain W22, and the 16S rRNA gene was amplified using primers 63f and 1387r (Table 2). Both strands of the amplified fragment were sequenced. BLAST analysis of the 16S rRNA gene indicated that the strain was a Flavobacterium sp. Sequence analysis showed that the sequence was 99% identical to the 16S rRNA gene of the Antarctic psychrotroph F. hibernum (GenBank accession no. L39067.1). Placement of the sequence into a phylogenetic tree using ARB (26) revealed a close relationship to other F. hibernum sequences (Fig. 1).

FIG. 1. — Placement of the *Flavobacterium* 16S rRNA sequence into a phylogenetic tree using ARB, revealing affinity with *F. hibernum*. An approximately 1.3-kb region of the 16S rRNA gene was amplified using forward primer 63f and reverse primer 1387r and sequenced. The phylogenetic tree was constructed by neighbor-joining analysis of partial 16S rRNA sequences according to the ARB manual (27). The bar represents 10% sequence divergence.

Transfer of plasmids into F. hibernum strain W22 by conjugation and electroporation.

Attempts to transfer broad-host-range plasmids pMMB66EH, pMMB207, and pMMB503, derived from IncQ plasmid RSF1010, or the E. coli-Bacteroides shuttle plasmid pNJR5, by conjugation into F. hibernum strain W22 did not result in any transconjugants (frequency of <10⁻⁹). On the other hand, plasmids pCP23 and pCP29, derived from cryptic F. psychrophilum D12 plasmid pCP1, were transferred by conjugation from E. coli to F. hibernum strain W22 at frequencies of 2.8 × 10⁻⁵ per recipient cell. A procedure was developed to allow for the direct introduction of DNA into F. hibernum strain W22 cells by electroporation. Plasmid pCP23, isolated from F. hibernum strain W22, was used to optimize this procedure. The optimal parameters for transfer by electroporation were determined to be 10.0 kV/cm and 400 Ω. Under these conditions, 2.5 × 10⁵ tetracycline-resistant transformants per μg of DNA were obtained. Only ∼200 colonies per μg of DNA were obtained when pCP23 isolated from E. coli was used. This indicated that a restriction barrier existed between E. coli and Flavobacterium.

Lack of E. coli lac promoter function in Flavobacterium strains.

Plasmids pSCH02F and pSCH02R, both constructed in this study and containing the expression cassette including the lacI^q, tac promoter, gfpmut3, and transcriptional terminator regions, resulted in intensive green fluorescence of the colonies when introduced into E. coli DH5α grown on LB agar plates supplemented with isopropyl-β-d-thiogalactopyranoside (IPTG). However, no detectable fluorescence was recorded in F. hibernum strain W22 or F. johnsoniae, into which pSCH02F and pSCH02R had been transferred after 48 h on the CYE agar plates with IPTG as an inducer (data not shown).

Construction and screening of an F. hibernum strain W22 promoter library.

Fragments of chromosomal DNA with an average length of 1,200 bp, obtained by partial digestion with Sau3AI, were inserted into the unique BamHI site of the plasmid pSCH03, and the ligation mixture was introduced into F. hibernum strain W22. Out of approximately 9,000 colonies screened by fluorescence microscopy under the conditions described in Methods and Materials, 48 colonies with strong GFP fluorescence were isolated and liquid cultures prepared from them were screened by quantitative fluorimetry (Fig. 2). Nucleotide sequence determination and restriction enzyme analysis indicated that the inserts in the 20 isolates with a high level of gfp expression ranged from 300 to 1,800 bp. The nucleotide and predicted amino acid sequences of these inserts were used to search the GenBank databases, and putative identification of the genes encoded was made based on homology data (Fig. 3). Most of the inserts had annotated ORFs and showed high homology (74% to ∼100%) with coding sequences from the recently sequenced genome of F. johnsoniae UW101. Some of them functioned as bacterial cell maintenance proteins such as transcription termination factor rho (SCH16), enolase (SCH47), haloacid dehalogenase-like (HAD) hydrolase (SCH08), ABC transporter protein (SCH25), histone-like bacterial DNA-binding protein (SCH41), and ribosomal proteins (SCH45 and SCH52). High homology with some important enzymes involved in amino acid or sugar metabolism, e.g., carboxylate dehydrogenase (SCH13), amino acid aminotransferase (SCH14), alpha-glucosidase (SCH28 and SCH40), citrate synthase (SCH29), biotin synthase (SCH35), and ATPase (SCH36) and some conserved proteins (SCH15, SCH17, and SCH28) were found in the trapped fragments.

FIG. 2. — Comparison of levels of GFP fluorescence emitted by representative *F. hibernum* strain W22 library isolates. (Top panel) Cultures were incubated overnight in CYE medium and quantified with fluorescence spectrometry as described in Materials and Methods. (Lower panels) Demonstration of GFP expression in *F. hibernum* strain W22. (A) SCH03 clone (promoterless *gfpmut3*). (B) SCH41 clone. (C) SCH03 cells. (D) SCH41 cells.

FIG. 3. — Description of clones containing putative promoter regions from *F. hibernum* strain W22. The diagrams show the *F. hibernum* strain W22 chromosomal DNA inserts containing the gene or protein sequence identity, as determined by BLASTN or BLASTX searches. ORFs were identified with Gene Finder. The solid arrows represent the direction of putative ORFs with the putative promoter(s), the single lines represent coding regions without promoter(s), and the dashed lines represent noncoding fragments. The solid thick lines represent promoterless *gfpmut3*. DNA inserts are not drawn to scale relative to each other. The insert size was determined by restriction digestion analysis with KpnI and BamHI. The database homology is only given to the ORFs with the putative promoter. The abbreviations of annotated genes from promoter clones were assigned like those previously published. The GenBank accession numbers for the annotated genes are as follows: *had*, EAS57413; *p5cd*, EAS59076.1; *bcat*, EAS58378; *cp1*, EAS58170; *rho*, EAS57797; *cp2*, EAS57669; *2ogfo*, EAS57503; *abt*, EAS59161; *malt1*, EAS61144; *cp3*, EAS57757; *cs1*, EAS59912; *bts3r*, EAS60446; *iop*, EAS60447; *atp*, EAS58692; HU, EAS59688; *rpl2*, EAS59936; *esp*, EAS58086; *eno*, EAS59913; *alcd*, EAS61717; *rps10*, EAS59940; and *rpl3*, EAS59939. N/A, data not available.

Comparisons of GFP production in E. coli and F. hibernum strain W22.

Plasmids harboring putative promoters were extracted and transformed into E. coli DH5α in order to determine the relative strength of each promoter in both F. hibernum strain W22 and E. coli. Flavobacterium and E. coli strains harboring plasmid pSCH03, which did not produce green fluorescent protein, were used as negative controls. Strong expression was observed, ranging from 20- to 80-fold increases in F. hibernum strain W22 compared with the control strain SCH03 (Fig. 4). Very little GFP was detected in E. coli transformants, although plasmid DNA isolated from E. coli indicated that their copy number was up to 10-fold higher than that in F. hibernum strain W22 when the same concentration of cells was used (data not shown). Reintroduction of these plasmids into F. hibernum strain W22 resulted in intensive green fluorescent colonies, showing that weak GFP production in E. coli was not due to mutation.

FIG. 4. — Alignment of experimentally determined or proposed promoters functional in *Flavobacterium* and determination of GFP fluorescence from different promoter clones in *E. coli* and *F. hibernum* strain W22. The consensus sequence derived from this alignment is given at the bottom. It was defined as nucleotides that are present at any given position in more than 50% of the sequences. The promoters marked with an “a” were identified in this study; the other letters and associated reference numbers are as follows: b, Chen et al., unpublished data; c, reference 17; d and g, reference 8; e, reference 34; f, reference 35; h, reference 6; i, reference 37; j, reference 4. The −7 and −33 consensus regions are capitalized and boldface. The relative GFP production level of each promoter clone is given as relative fluorescence values. Triplicate samples were used, and the standard deviations are shown. The SCH03 clone is used as a negative control. N/A, data not available.

Identification of putative TSPs and consensus promoters.

The following criteria were used for selection of “hot” promoter clones in this study: (i) clones that exhibited the highest fluorescence, (ii) sequences upstream of the reporter having homology to known bacterial genes, and (iii) fragments where putative regulatory sequences were close enough to the reporter gene (gfp) to facilitate the transcriptional start point (TSP) determination. In order to determine the TSP, SMART-5′ RACE PCR analysis was performed in seven putative promoter clones. For each clone, we obtained a single dominant 5′RACE PCR product (data was not shown), indicating there was no alternative transcriptional start site. To ensure there was no contaminating DNA present in the RNA preparation, a negative control was included using the RNA that had not been reverse transcribed. The amplified 5′-RACE products were cloned into T-easy vectors and sequenced as described previously. Nucleotide sequences adjacent to the TSP were aligned with 10 promoters functional in Flavobacterium which were either experimentally identified or proposed in other studies, as shown in Fig. 4. The consensus sequence determined from this alignment consists of nucleotides that occur in more than 50% of the clones at any position. The mapped TSP was A, C, or G, with A present in 9 of 17 promoters examined. A putative −7 region matching the consensus Bacteroides sequence (TAnnTTTG) (4) was located from 2 to ∼16 nucleotides upstream from the TSP, and a putative −33 region matching the Bacteroides conserved sequence (TTG) (4) was centered on −33 region from the TSP. The space between −7 and −33 regions was 17 to ∼23 bases.

Deletion analysis of a strong promoter.

In order to determine which portion at the 5′ end of the promoter was critical for maximum promoter activity, we chose the strongest promoter clone (SCH41) for the following experiment. Deletion derivatives of the trapped fragment encoding histone-like bacterial DNA-binding protein (HU) were generated by PCR, inserted into the T-easy vector, and sequenced. The individual deletion promoter fragments were released from T-easy vectors and fused to the same position on pCP23. These plasmids were transformed into F. hibernum strain W22 and F. johnsoniae, and cells harboring these constructs were grown to log phase and subjected to quantitative fluorescence analysis. Results from sequential deletion of the strongest promoter clone (SCH41; Fig. 5) showed that there was no significant difference in the level of gfpmut3 expression in plasmids pSCH41.−210 through pSCH41.−52 in both F. hibernum strain W22 and F. johnsoniae. Deletion plasmid pSCH41.−23 exhibited an eightfold lower level of fluorescence than pSCH41.−210 in F. hibernum strain W22 and a fivefold lower level in F. johnsoniae. These data indicate that the 52-bp fragment upstream of the TSP in the HU promoter is required for maximal expression of gfpmut3 in log-phase F. hibernum strain W22 and F. johnsoniae under our testing conditions.

FIG. 5. — Serial deletions of HU promoter. (A) The N-terminal primer D1 to D7 with a KpnI site and C-terminal primer gfpSphIR with an SphI site were used to amplify the deletion derivatives. Left dotted arrows represent the N-terminal primers (D1 to D7). The right dotted arrow represents the C-terminal primer gfpSphIR. The solid box represents *gfpmut3*. P.C., the positive control (SCH41); N.C., the negative control (SCH03). (B) Quantitative analysis of GFP production of the deletion series in *F. hibernum* strain W22. (C) Quantitative analysis of GFP production of the deletion series in *F. johnsoniae*.

Putative RBS in F. hibernum strain W22.

Alignment of 12 putative RBSs from this study with 13 RBSs from various published Flavobacterium strains revealed that TAAAA was the consensus sequence (Fig. 6), with some variations. The putative RBS was followed by a possible initiation codon ATG located after a spacer of 2 to 12 bp. The RBS consensus sequence of the 16S rRNA of our F. hibernum strain W22 was predicted to be TAGAAA. The majority of the nucleotide sequences upstream of ATG in the putative RBS regions we analyzed were A-T rich (63 to ∼90% in 30-mer, 69 to ∼100% in 16-mer) (Fig. 6). The native RBS for ribosomal protein L3 gene from F. hibernum strain W22 (10) was chosen for site-directed mutagenesis in order to determine which base(s) in the putative RBS were critical for translation. The engineered RBS from E. coli (10) was used with the same promoter as a control. Individual or multiple base changes at four positions in this putative RBS AATA region (positions −4, −5, −6, and −7) were assessed for their effects on GFP production in F. hibernum strain W22 (Fig. 7). If these nucleotides constituted part of a recognition site for ribosomes, substitutions would severely affect GFP production levels. Six of the mutations resulted in reduced GFP production (Fig. 7). Five of those were reduced by 50% or more (M1 through M5; see mutations as indicated in Fig. 7). For mutant M9, a single change (−6A→G) decreased the GFP level by 25% compared to the wild type. At the same position, the −6A→T nearly eliminated GFP production (mutant M3). The individual point change at position −7 (−7A→G) in mutant M6 had no significant effect on the expression level compared to the wild type. The dual mutations at positions −6 and −7 (−6A→G and −7A→C) in mutant M4 resulted in 40% of the expression observed in the wild-type gene. The triple mutations (−4A-6A-7A→TTT) in mutant M5 decreased GFP production dramatically to about 5% of the wild type. In contrast, in mutant M8, the T→A change at position −5 of the predicted consensus region, resulting in the sequence TAAAA, increased the GFP production by 50% compared to the wild-type RBS. Similarly, a single change in mutant M7 (−6A→G) resulted in a 25% increase in GFP production. The same GFP production pattern was recorded in F. johnsoniae when these constructs were transformed (data not shown).

FIG. 6. — Alignment of putative *Flavobacterium* RBS regions. The 30-bp nucleotide sequences upstream of the start codon were chosen for comparison. The sources of the selected genes are as follows: a, *F. hibernum* strain W22; b, *F. johnsoniae*; c, *F. columnare*; d, *P. heparinus*; and e, *F. psychrophilum*. The putative consensus RBSs are indicated as boldface and capitalized. These were defined as nucleotides that are present at any given position in more than 50% of the sequences. The AT contents within 30 bp and 16 bp upstream of the start codon are given as percentages.

FIG. 7. — Effects of various mutations in the putative RBS region on GFP production level. Oligonucleotide-directed mutagenesis in the putative RBS was created by PCR, and products were transcriptionally fused to HU promoter in SCH41. Replacement bases are underlined. The GFP level was expressed relative to that measured with the wild type.

DISCUSSION

Optimization of gene transfer techniques using electroporation and modified plasmid constructs allowed us to develop a promoter-trap system (herein named pSCH03), isolate and analyze several strong promoters, and use GFPmut3 as a reporter in F. hibernum strain W22. The gfp construct is well established as a marker for gene expression in other bacteria (14, 27, 38), but had not been used previously with species of Bacteroidetes. Promoter trapping has been used frequently to isolate and characterize native promoters (14, 27) and is dependent on functional reporter systems such as GFPmut3. GFPmut3 has several advantages as a reporter (11): it is nontoxic to its host, allows the in situ study of gene expression in environmental samples without the additional exogenous substrates, and is compatible with high-throughput screening techniques such as fluorescence-activated cell sorting. Sequence analysis shows that most of the trapped fragments contain annotated ORFs that show high homology with other Flavobacterium strains. In this study, GFP production in F. hibernum strain W22 was often associated with sequences that resembled those of authentic promoters. Notably, expression of gfp in E. coli was nil for all of these promoter constructs, even though plasmid copy number was demonstrably higher in that host compared to F. hibernum strain W22 (Fig. 4).

Promoters in F. hibernum strain W22 harbor RNA polymerase (sigma factor) binding sequences very similar to those of B. fragilis (4). This sigma factor, named σ^ABfr, with binding properties to upstream regions of the promoter was found to be widespread in the Bacteroidetes phylum as well as in the phylum Chlorobi (46). The TSPs observed in Flavobacterium strains indicated that the preferred +1 base is A. A conserved consensus sequence, TAnnTTTG, was found 2 to 16 nucleotides upstream of the TSP, and a shorter, less conserved sequence (TTG) was found centered at the −27 to −41 region upstream of the TSP. Deletion of the −33 region (pSCH.−23) did not completely abolish expression of gfp, but dramatically decreased it, indicating that the −33 region is necessary for the maximum expression of the reporter gene (Fig. 5). This result is unlike that found in Campylobacter jejuni, where only the −10 region is required (50). The space between the two conserved regions ranged from 17 to 23 bases. This space between the −7 motif and the −33 region seems to be unusually large, particularly since eubacterial RNA polymerase works within the short interval of 17 ± 1 bp (44). However, the two conserved regions and the distance between them in Flavobacterium promoters are similar to most known promoter structures found in B. fragilis (4). In addition, the TAnnTTTG motif within 20 nucleotides of TSP could also be found in many promoters in Porphyromonas gingivalis, a member of the phylum Bacteroidetes (18). In B. fragilis, mutagenesis of the −7 region completely disrupted expression of the reporter gene. Deletion of the −33 TTTG did not abolish expression as it did in the −7 element but resulted in a sharp reduction of expression (4). We have further investigated the spacer length between the −7 and −33 regions in Flavobacterium by site-directed mutagenesis and found the optimal length to be 19 bp (Chen et al., unpublished data). The differences in promoter consensus sequences found upstream of several “housekeeping” genes in our Flavobacterum strain, compared to those found in E. coli and other gram-negative bacteria (44), suggest differences in the respective sigma factors. Recently, the putative gene of a sigma factor in F. johnsoniae was cloned and the sequence showed high homology with the primary sigma factor σ^ABfr in B. fragilis. σ^ABfr in B. fragilis has been biochemically identified and exhibits several unusual features compared with those in other prokaryotes (46). The primary structure analysis shows that the protein completely lacks region 1.1, a highly acidic N-terminal domain present exclusively in primary sigma factors. In E. coli, region 1.1 has been proven to have several modulatory effects on RNA polymerase (RNAP) function and to constitute an autoinhibitory domain that prevents DNA binding and promoter recognition. σ^ABfr did not support transcription from any promoter of B. fragilis or E. coli in association with the E. coli RNAP core. In contrast, it formed an active holoenzyme only with its cognate core RNAP and recognized only Bacteroidetes promoters.

The signal for initiation of protein synthesis in bacteria consists primarily, but not exclusively, of an AUG codon and an rRNA-complementary sequence, the Shine-Dalgarno sequence (39). This sequence is usually located 4 to 9 nucleotides upstream of the initiator AUG in many mRNAs, where it is complementary to the 3′ end of 16S rRNA. When constructing the promoter-trap vector pSCH03, we originally assumed that the E. coli RBS sequence could be recognized by Flavobacterium strains. We examined the 30-bp regions upstream of the start codon from 25 ORFs from different Flavobacterium strains (Fig. 6). One of the striking features observed in most of the genes is that the majority of the nucleotide sequences upstream of AUG were A+T rich (63 to ∼90% in 30-mer, 68 to ∼100% in 16-mer). This finding contrasts with the well-characterized RBS consensus (AGGAGG) in other prokaryotes, where the RBS is typically centered in a purine-rich region (45). A consensus sequence, “TAAAA,” is proposed herein as the novel putative RBS in Flavobacterium strains (Fig. 6). In contrast, no typical “AGGAGG” RBS-like sequence existed within the 16 bp upstream of any of the putative ORFs isolated from F. hibernum strain W22. The 3′ end of the 16S rRNA of Flavobacterium strains is 5′-CUGGAUCACCUCCUUUCUA-3′. This does include a sequence complementary to the typical RBS sequence “AGGAGG” of gram-negative bacteria (40). It includes, however, an extra sequence partially complementary to the sequence “TAAAA” found upstream of the 25 open reading frames from various Flavobacterium strains. In addition, our site-directed mutagenesis analysis of the TAAAA sequence rendered further support for the proposed consensus RBS motif in Flavobacterium strains. Why the putative RBS sequences found in this work do not have a perfect complement in the 3′ end of Flavobacterium 16S rRNA requires further study.

This study is the first to characterize the consensus promoter structures and putative RBS motifs for general housekeeping genes in a Flavobacterium species using the GFP-based reporter system. This will lead to an understanding of specific gene regulation for both transcriptional and translational analysis in Bacteroidetes. Results of this study can also be extended to achieve stable protein production in Flavobacterium and related bacteria, especially when inducible systems are not applicable. The evolutionary advantage of the novel promoter and RBS motifs for modulating gene expression in F. hibernum strain W22 and related bacteria remains unclear, but it is reasonable to conclude that the departure from typical transcription and translation start signals reflects an early divergence of the Bacteroidetes group from the main bacterial branch.

Acknowledgments

We gratefully acknowledge Mark McBride for his generous advice and supply of plasmids pCP23 and pCP29 and Flavobacterium strains. We also thank Angela Sosin and Blair Bullard for their assistance.

This project was funded by NIH grant AI21884.

Footnotes

^▿

Published ahead of print on 22 December 2006.

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[r1] 1.Agarwal, S., D. W. Hunnicutt, and M. J. McBride. 1997. Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc. Natl. Acad. Sci. USA 94:12139-12144. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r2] 2.Alvarez, B., P. Secades, M. J. McBride, and J. A. Guijarro. 2004. Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Appl. Environ. Microbiol. 70:581-587. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System▿

S Chen

M Bagdasarian

M G Kaufman

E D Walker