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Appl Environ Microbiol, June 1998, p. 2240-2246, Vol. 64, No. 6
Department of Microbiology,
Received 5 December 1997/Accepted 29 March 1998
Use of the green fluorescent protein (Gfp) from the jellyfish
Aequorea victoria is a powerful method for nondestructive
in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter
protein, new variants have been constructed by the addition of short
peptide sequences to the C-terminal end of intact Gfp. This rendered
the Gfp susceptible to the action of indigenous housekeeping proteases,
resulting in protein variants with half-lives ranging from 40 min to a
few hours when synthesized in Escherichia coli and
Pseudomonas putida. The new Gfp variants should be useful for in situ studies of temporal gene expression.
Studies of specific gene expression
in bacteria have been greatly facilitated by the use of reporter genes.
The simple and sensitive enzymatic assays for enzymes like
It was recently shown by Keiler and coworkers (12) that
specific C-terminal oligopeptide extensions can render otherwise stable
proteins susceptible to degradation by certain intracellular tail-specific proteases. Our strategy has been to exploit this natural
protein degradation system, which is based on ssrA-mediated tagging of prematurely terminated polypeptides at the COOH end (12). The ssrA transcript of Escherichia
coli is a stable 362-nucleotide RNA molecule (5, 22)
that exhibits some tRNA-like properties and can be charged with alanine
(15). Genes homologous to ssrA have been
identified in both gram-negative (2, 25) and gram-positive (26) bacteria, implying that the ssrA-mediated
peptide-tagging system may be a conserved trait in bacteria. In the
E. coli model proposed by Keiler et al. (12) the
ssrA transcript targets proteins translated from incomplete
or damaged mRNAs (e.g., mRNAs lacking a termination codon).
Subsequently, a peptide tag with the sequence AANDENYALAA is attached
to the carboxyl terminus of the nascent polypeptide chain by
cotranslation switching of the ribosome from the damaged mRNA to the
ssrA transcript. Finally, the resulting protein, carrying a
C-terminal AANDENYALAA peptide tag, is recognized and rapidly degraded
by intracellular tail-specific proteases (12). In the
periplasm, the proteolytic degradation of AANDENYALAA-tagged proteins
has been shown to be executed by the tail-specific Tsp protease,
whereas tagged proteins located in the cytoplasm are believed to be
broken down by a currently unidentified functional Tsp homolog
(12, 23). It is important to be aware of the possibility that a highly expressed protein carrying this type of target tail may
compete with naturally occurring products from the translation process,
and the consequence could be a reduced growth rate.
Since changes in the last three residues of the AANDENYALAA
consensus sequence are known to alter protein stability
(13), we reasoned that it should also be possible to obtain
Gfp mutants of varying stability by constructing variants carrying
C-terminal peptide tags with minor alterations in the Tsp consensus
sequence.
Bacterial strains.
E. coli and Pseudomonas
putida strains used in this study are listed in Table
1.
Media.
The basic medium used was either modified
Luria-Bertani (LB) medium (1) containing 4 g of
NaCl/liter instead of the normal 10 g of NaCl/liter or ABT minimal
medium (AB minimal medium [7] containing 2.5 mg of
thiamine/liter).
Plasmids.
The plasmids used in this study are listed in
Table 1. Construction of pJBA24 (Table 1 and Fig.
1A) was done as follows. PCR
amplification with the primer set P1 and P2 (Table
2) and with pQE70 (21) as the
template produced a 1.05-kb XbaI-StuI fragment
containing a synthetic ribosome binding site (RBSII), at an optimal
distance from a SphI site, followed by a
HindIII site, translational stop codons in all three
reading frames, and two strong transcriptional terminators,
T0 (derived from phage
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
New Unstable Variants of Green Fluorescent Protein
for Studies of Transient Gene Expression in Bacteria
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
-galactosidase and luciferase have allowed detailed investigations
of gene regulation, obtained after construction of the relevant fusions
between the promoters of interest and the respective reporter genes.
Such investigations have been performed successfully in suspended
monocultures of many bacterial species (in vitro experiments), for
which the addition and spatial distribution of enzymatic substrates
represent no problem. For analysis of heterogeneous and complex
populations like surface-bound microbial communities there is a need
for gene expression reporters that (i) allow for detection at the
single-cell level and (ii) circumvent the problem with introduction and
distribution of chemical substrates for the enzymatic reporter assays.
A useful option for such cases is the green fluorescent protein (Gfp)
obtained from the jellyfish Aequorea victoria. Gfp
fluoresces green and requires only the presence of oxygen to
maturate; i.e., no external compounds need to be added to organisms
expressing Gfp in order to detect green fluorescence (4).
The gfp gene may be transferred to and expressed in a wide
range of organisms, e.g., mammals (18), fishes
(19), insects (28), plants (3), yeasts
(20), and a broad variety of bacteria (4, 6). As
Gfp normally does not interfere with the growth of the host, it is an
excellent choice for nondisruptive studies of bacterial communities or
other systems which require live cells to be studied at the single-cell level. The optimal bacterial reporter for studying real-time gene expression in individual cells should possess a species-independent instability permitting monitoring of rates of expression (in the same
way as unstable mRNA). A major drawback of Gfp is that once formed it
seems to be very stable (24), which in turn renders the
protein less valuable for studies of transient (real-time) gene
expression.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
) and T1 (derived
from the rrnB operon of E. coli). This fragment was subsequently inserted into the XbaI site and the
blunt-ended HindIII site (using the Klenow fragment of
DNA polymerase I) of pUC18Not (11). The resulting plasmid,
pJBA23, was digested with KpnI and XbaI and
ligated to a 0.27-kb KpnI-XbaI fragment carrying the LacI-repressible promoter PA1/04/03 (amplified from
pUHE24-2 [17] with the primers P3 and P4 [Table 2])
to create the cloning vector pJBA24. Construction of pJBA28 (Table 1
and Fig. 1B) was done as follows. gfpmut3* was amplified by
PCR from gfpmut3b (a kind gift of R. H. Valdivia
[8]) with the primers Pgfp(up) and Pgfp(down) (Table 2). From this PCR
product, a 0.72-kb SphI-HindIII fragment
carrying the gfpmut3* gene was isolated and subsequently
ligated to SphI-HindIII-digested pJBA24. The resulting plasmid, pJBA27, was restricted by NotI, and the
2.0-kb NotI fragment containing the
PA1/04/03-RBSII-gfpmut3*-T0-T1
cassette was finally inserted into the unique NotI site of
the pUT-miniTn5-Km vector (9) to create either
pJBA28 or pJBA29, differing only in the orientation of the
NotI insert. For the construction of gfp genes
encoding variant Gfps with differing terminal amino acids, we used
essentially the same procedure as above, substituting primers encoding
the entire C-terminal end of the variant Gfps for the down primer
(Table 2). The resulting variant genes were transferred to
pUT-miniTn5-Km to produce transposon insertion vectors with
the gfp gene oriented in either direction (for details, see
Tables 1 and 2 and Fig. 1). The novel modified genes were designated
gfp(LAA), gfp(LVA), gfp(AAV), and
gfp(ASV). Sequencing of the resulting plasmids verified that
gfpmut3*, gfp(LAA), gfp(AAV), and
gfp(ASV) had no mutations other than those derived from the customized PCR primers, whereas gfp(LVA) had a single (A 
G) point mutation in nucleotide 349, resulting in an Asp117 Gly117 (D117G) amino acid change. This point mutation does not appear to
affect the fluorescence spectrum of Gfp(LVA) compared to the other
Gfpmut3* variants (not shown).
View larger version (15K):
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FIG. 1.
Schematic drawings of cloning and transposon vectors
(not to scale). (A) Cloning vector pJBA24. (B) Transposon vector
pJBA28. (C) Transposon vectors pJBA114, pJBA116, pJBA118, and pJBA120.
In all transposon plasmid vectors only the transposon parts flanked by
the O and I ends of mini-Tn5 are shown, and the drawings
indicate only the relevant restriction sites. Abbreviations:
ori, origin of replication; lacZ', truncated
lacZ gene; bla, ampicillin resistance gene;
npt, kanamycin resistance gene; PA1/04/03,
LacI-repressible promoter; RBSII, synthetic ribosome binding site;
gfpmut3*, gene encoding Gfp(S2R, S65G, S72A) (referred to as
Gfpmut3*); T0, transcriptional terminator from phage
lambda; T1, transcriptional terminator from the
rrnB operon of E. coli; gfp(XXX), gene
encoding either Gfp(LAA), Gfp(LVA), Gfp(AAV), or Gfp(ASV) (see text
for details).
TABLE 2.
Primers used in this study
Monitoring green fluorescence from single colonies.
The
P. putida strains JB279, JB167, and JB340 [i.e., KT2442
(lacIq) carrying a transposon insertion of
gfpmut3*, gfp(LAA), or gfp(AAV), respectively, allowing expression of the gfp genes from
endogenous P. putida promoters (Table 1)] were streaked
onto semisolid agar plates of ABT medium supplemented with 10 mM
citrate, 50 µg of kanamycin/ml, 1 mM IPTG
(isopropyl--D-thiogalactoside), and 20 g of Bacto
Agar (Difco)/liter. After 18, 30, 42, 54, 73, 102, and 192 h of
incubation at 30°C, the green-fluorescence phenotypes of single
colonies were recorded with a charge-coupled device camera mounted on
an epifluorescence microscope (Zeiss Axioplan; Zeiss, Oberkochen,
Germany) equipped with a 2.5× lens. For fluorescence microscopy we
used Zeiss filter set no. 10 (excitation, 470 to 490 nm; emission, 515 to 565 nm; dichroic, 510 nm) and a Zeiss HBO-100 mercury lamp. A CH250
charge-coupled device equipped with a KAF 1400 liquid-cooled chip
(Photometrics, Tucson, Ariz.) was used for imaging.
Quantification of green fluorescence from liquid cultures.
The strains MV1190(-pir) harboring either pJBA28
(gfpmut3*), pJBA114 [gfp(LAA)], pJBA116
[gfp(LVA)], pJBA118 [gfp(AAV)], pJBA120
[gfp(ASV)], or pUT-miniTn5-Km (control strain)
(Table 1 and Fig. 1) were grown exponentially in LB medium
(1) supplemented with 50 µg of kanamycin/ml and 0.2 mM
IPTG at 37°C. At an optical density at 450 nm (OD450) of
1.0 the cultures were harvested, washed in ABT minimal medium, shifted
to the same volume of preheated (37°C) ABT minimal medium containing
50 µg of kanamycin/ml, and reincubated at 37°C. Following the
shift, culture samples were withdrawn at various time intervals and
green fluorescence was measured with a fluorometer (model RF-1501;
Shimadzu, Tokyo, Japan) set at an excitation wavelength of 475 nm and
emission detection at 515 nm. The measured values of green fluorescence
per milliliter of culture of strain MV1190(-pir)
expressing either Gfpmut3* or the C-terminal-modified Gfps were
corrected for background green fluorescence by subtracting the
corresponding measured values of green fluorescence per milliliter of
culture of MV1190(-pir) harboring the control plasmid
pUT-miniTn5-Km. Finally, the background-corrected values of
green fluorescence per milliliter of culture were converted into
relative green fluorescence and plotted as a function of postshift time
in a semilogarithmic plot (relative green fluorescence was arbitrarily
set to 100% in the time zero samples) (see Fig. 3A). Likewise, the
P. putida strains based on KT2442
(lacIq) and carrying gfpmut3* (JB279)
or the modified gfp genes {JB167 [gfp(LAA)],
JB391 [gfp(LVA)], JB340 [gfp(AAV)], and JB396
[gfp(ASV)]} or KT2442 (lacIq)
without gfp (Table 1) were grown exponentially in LB medium at 30°C. At an OD450 of 1.0 the cultures were harvested,
washed in ABT minimal medium, resuspended in the same volume of
preheated ABT minimal medium, and reincubated at 30°C. Following the
downshift, 1-ml culture samples were withdrawn after set time
intervals, and green fluorescence was measured as described above. The
measured values of green fluorescence per milliliter of culture of
strain KT2442 (lacIq) expressing either Gfp were
corrected for background green fluorescence by subtracting the
corresponding measured values of green fluorescence per milliliter of
culture of KT2442 (lacIq) and subsequently
converted into relative green fluorescence and plotted as a function of
postshift time in a semilogarithmic plot (relative green fluorescence
was arbitrarily set to 100% in the time zero samples) (see Fig. 3B).
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Construction of unstable Gfp variants. Wild-type Gfp is very stable in its mature, fluorescent form (24). The mutant protein, Gfpmut3, is approximately 20 times more fluorescent than wild-type Gfp when excited at 488 nm, and it is only weakly excited by UV light (8). This unique combination of features may allow efficient spectral separation between Gfpmut3 and other chromophores which are excited at shorter wavelengths, such as the mutant Gfp, Bfp (blue fluorescent protein) (10). We therefore chose Gfpmut3 as the template for construction of new, unstable reporter proteins aimed at applications in bacteria. We genetically modified the gfpmut3 gene in such a way that nucleotide sequences corresponding to variants of the described destabilizing peptide tail consensus sequence were added (separately) to replace the normal stop codon. This was accomplished by the use of PCR in combination with extended primers, the DNA sequences of which would encode new peptide tails of Gfpmut3 (8). The insertion of the upstream primer resulted in a change of amino acid 2 of Gfpmut3 from serine to arginine [in this study referred to as Gfpmut3*(Gfpmut3, S2R)]; we have not registered any phenotypic effects of this replacement. Several versions of the downstream primer were designed in order to introduce homologous but different C-terminal peptide tags of the encoded Gfpmut3* proteins (see Table 2 for details). We have chosen four of the resulting PCR products for further investigation: gfp(LAA), gfp(LVA), gfp(AAV), and gfp(ASV), encoding mutant Gfpmut3* proteins with the C-terminal extension sequences RPAANDENYALAA, RPAANDENYALVA, RPAANDENYAAAV, and RPAANDENYAASV, respectively. As a control we amplified the gfpmut3* gene without any C-terminal modifications in a similar way (see Materials and Methods). The selected fragments were cloned downstream of the strong LacI-repressible promoter PA1/04/03 (17), giving rise to the plasmids pJBA27 (gfpmut3*), pJBA110 [gfp(LAA)], pJBA111 [gfp(LVA)], pJBA112 [gfp(AAV)], and pJBA113 [gfp(ASV)] (Table 1). Next, fragments carrying the expression cassettes from these plasmids were transferred to mini-Tn5 transposon delivery vectors carrying the npt gene conferring resistance to kanamycin (9). Plasmids carrying the expression cassettes in both orientations were isolated (see Materials and Methods).
Expression of gfpmut3*, gfp(LAA),
gfp(LVA), gfp(AAV), and
gfp(ASV).
The 10 mini-Tn5 plasmid
derivatives were transformed to E. coli
MV1190(-pir) (11), and Gfp phenotypes of
single colonies were monitored with an epifluorescence microscope with
low magnification (see below). In this strain the
lacIq gene is located on an F' plasmid. The
clones were green fluorescent, indicating that the high copy number of
the mini-Tn5 plasmids (approximately 26 per genome compared
to 1 for the F' plasmid) may lead to titration of the LacI repressor,
resulting in partial induction of the gfp promoter.
Subsequent investigations showed that the fluorescence signals from
these clones were greatly stimulated after induction with IPTG.
Furthermore, our initial screening for fluorescence indicated that the
constructs showed the predicted differences in protein stability (see
below).
repressor carrying a C-terminal
AANDENYALAA peptide tag was significantly less stable than the
unmodified repressor in E. coli. In addition, the
suggested stability hierarchy Gfpmut3* > Gfp(AAV) > Gfp(LAA) is
similar to that observed in E. coli for unmodified
cytochrome b562, cytochrome
b562 carrying a WVAAV C-terminal tag, and
cytochrome b562 carrying a WVLAA C-terminal tag
(13). The remaining two variant gfp genes
[gfp(LVA) and gfp(ASV)] appeared by visual
inspection to give fluorescence patterns essentially the same as those
observed for gfp(LAA) and gfp(AAV),
respectively (data not shown). Similar observations of the expression
of the unstable Gfps have been made with Serratia liquefaciens
MG1 (not shown), and it is therefore likely that the unstable
Gfp reporter proteins will show similar behavior in a number of
different bacterial species. As far as we know, this is the first
demonstration that the design of unstable proteins through the addition
of this type of C-terminal tail is possible in bacterial species other
than E. coli.
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Stability of Gfpmut3*, Gfp(LAA), Gfp(LVA), Gfp(AAV), and Gfp(ASV) in E. coli and P. putida. The kinetics of induction of the different forms of the Gfp (the wild type and the four unstable variants) was investigated in an induction experiment in which IPTG was added to exponentially growing cultures of E. coli harboring the plasmids pJBA28, pJBA114, pJBA116, pJBA118, and pJBA120 (Table 1), carrying fusions between the IPTG-inducible promoter and the relevant gfp genes. The times at which fluorescence increased above the background levels for the five strains were determined, and it was found that in all cultures the initial kinetics were the same (data not shown). This strongly suggests that gene expression and maturation of the Gfp follow identical patterns for wild-type and variant genes.
In order to estimate the half-lives of the mature Gfp variants in vivo, we carried out quantitative fluorometer measurements of green fluorescence expressed by the strains derived from E. coli MV1190(-pir) or P. putida KT2442
(lacIq) encoding the variant Gfps (the same
strains used for the colony investigations). The strains were grown in
rich medium and shifted at mid-log phase to minimal medium without
inducer. After the shift there was no increase in OD450 for
4 to 5 h, which means that protein synthesis (including that of
Gfp) was negligible during this period. Fluorescence was measured at
time intervals after the shift (Fig. 3).
The stability of Gfpmut3* and the four tagged versions was estimated by
using the relative green fluorescence value as a function of the time
after the shift, represented as the slopes of the curves in Fig. 3. In
E. coli it was found that the green fluorescence of
Gfp(LAA), Gfp(LVA), Gfp(AAV), and Gfp(ASV) in the absence of Gfp
synthesis decreased linearly from T = 10 min to
T = 80 min after the downshift (Fig. 3A). The slope
constants, µ, were determined to be approximately 
0.018
min
1 for the strains encoding Gfp(LAA) and Gfp(LVA) and
0.012 and 0.0062 min1 for the strains encoding
Gfp(AAV) and Gfp(ASV), respectively. This corresponds to in vivo
half-lives of mature Gfp(LAA) and mature Gfp(LVA) of approximately 40 min (T1/2 = ln 2/µ), while Gfp(AAV) and
Gfp(ASV) appear to have half-lives of approximately 60 and 110 min,
respectively. In striking contrast, mature Gfpmut3* appears completely
stable within the time of the experiment. This is in accordance with
previous observations that both colonies and liquid cultures of
E. coli MV1190(-pir) expressing Gfpmut3* remain green fluorescent after several weeks of incubation (data not
shown). Consequently, we conservatively estimated the in vivo half-life
of mature Gfpmut3* to be more than 1 day.
|
, MV1190(
, MV1190(
, MV1190(
,
MV1190(0.0115
min1 for Gfp(LVA), indicating that the in vivo half-life
of mature Gfp(LVA) in P. putida is in the range of 60 min.
Interestingly, the other three Gfp variants, Gfp(LAA), Gfp(AAV), and
Gfp(ASV), all displayed similar degradation rates of 0.0037
min1 (corresponding to a half-life of approximately 190 min). It is possible that the increased expression of Gfp, observed as
stronger fluorescence, from the chromosomally integrated variant
gfp genes in P. putida may affect the degradation
rates of the Gfps (e.g., through titration of the protease activity),
resulting in apparently more stable proteins. Furthermore, the
proteases degrading tagged proteins in P. putida may differ
in specificity from the E. coli protease, resulting in
altered half-lives.
Consequently, the half-life estimates obtained in the experiments
presented are not to be taken as absolute, fixed values. The protease
reactions resulting in degradation of Gfp may be dependent on strains,
growth conditions, specific features of the surroundings, competing
targets in the cell, etc. The important feature, however, is that a
protein known to be very stable may be converted to an unstable variant
in a semipredictable way through the employment of a natural protease
activity, which is apparently found in many different bacteria.
Concluding remarks. We have constructed new variant gfp genes encoding Gfps with reduced half-lives compared to that of the wild-type protein. The apparent half-lives of these proteins were estimated in liquid cultures, and in all cases the novel proteins displayed half-lives markedly shorter than that of the corresponding unmodified Gfpmut3* protein. We believe that transcriptional fusions between an appropriate gene and the described novel Gfp variants will facilitate studies of real-time gene expression in situ through monitoring of the green-fluorescent phenotype. We are presently assessing the value of these new reporter proteins in connection with physiological studies of bacterial biofilms.
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ACKNOWLEDGMENTS |
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The work was supported by grants to S.M. from the Danish Research Councils under the Biotechnology Program and a contract from EU (BIO4-CT96-0181).
Brendan Cormack, Rafael H. Valdivia, and Stanley Falkow are acknowledged for the gift of the gfpmut3b gene used in this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Building 301, The Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: 45 45 25 25 13. Fax: 45 45 88 73 28. E-mail: sm{at}im.dtu.dk.
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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