Vectors that facilitate the replacement of transcriptional lacZ fusions in Streptococcus mutans and Bacillus subtilis with fusions to gfp or gusA

Abstract

Plasmid vectors have been constructed for Streptococcus mutans and Bacillus subtilis that make possible rapid replacement of the widely used reporter gene lacZ (encoding β-galactosidase) with either gfp (encoding green fluorescent protein) or gusA (encoding β-glucuronidase). The lacZ→gfp replacement vectors greatly facilitate the analysis of the spatial location of gene expression in biofilms of S. mutans and in sporulating B. subtilis. The lacZ→gusA replacement vectors facilitate the comparison of two promoters within the same organism. A vector is also described that enables gusA to be replaced with gfp in B. subtilis.

Streptococcus mutans, Bacillus subtilis, Replacement vector

1 Introduction

Transcriptional fusions to reporter genes offer a very convenient and widely used method to monitor gene expression. Several reporter genes have been used in low-GC gram-positive species, including lacZ, encoding β-galactosidase, gusA, encoding β-glucuronidase, and gfp, encoding green fluorescent protein [[,,,,,,[]. The lacZ gene is probably the most commonly used reporter, and many promoter-lacZ transcriptional fusions have been constructed for Streptococcus mutans and Bacillus subtilis, e.g. [[,[]. Although the lacZ fusions are very useful for assessing promoter activity and for genetic screens, they are technically demanding to use for the study of the spatial location of gene expression. For the latter purpose, gfp has become the probe of choice [[]. In this paper, we describe vectors that facilitate the rapid replacement of lacZ, which is present in many existing promoter fusions, with gfp. We describe vectors that work efficiently for S. mutans, detecting gene expression within biofilms, and for B. subtilis, demonstrating compartment-specific expression during spore formation. The reporters in the vectors have strong ribosome-binding sites that should enable them to function in a range of low-GC gram-positive species. For pathogenic streptococci, there is a general reluctance to use vectors that contain a gene for ampicillin resistance, bla. Accordingly, we have designed vectors that do not contain the bla gene. We also describe vectors that facilitate replacement of lacZ with gusA. β-Glucuronidase is much less widely used than β-galactosidase as reporter, but has most of its advantages [[,[,[]; the ability to combine gusA fusions with lacZ fusions can greatly strengthen the power of a genetic screen [[], and provides an accurate way for comparing the times of expression of different promoters during the growth cycle and during development [[]. We also describe a vector for the replacement of gusA with gfp.

2 Materials and methods

2.1 Strains

The parent strains used were S. mutans UA159 [[0] and B. subtilis BR151 [[1].

2.2 Media and growth conditions

Todd Hewitt broth and defined FMC medium [[2] were used for S. mutans; L broth and modified Schaeffer's sporulation medium (MSSM) [[3] were used for B. subtilis; L broth was used for Escherichia coli. The media were solidified with 1.8% Difco Bacto agar where appropriate. Neomycin (Nm) was used for B. subtilis at 15 μg ml⁻¹ in L broth and at 3.5 μg ml⁻¹ in MSSM and for E. coli at 75 μg ml⁻¹. Ampicillin (Amp) was used at 100 μg ml⁻¹ for E. coli. Chloramphenicol (Cm) was used at 10 μg ml⁻¹ for S. mutans, at 5 μg ml⁻¹ for B. subtilis and at 50 μg ml⁻¹ for E. coli. Kanamycin (Kan) was used at 300 μg ml⁻¹ for S. mutans.

2.3 Plasmids

Plasmids were maintained in E. coli DH5α unless otherwise indicated. The replacement vectors for S. mutans and B. subtilis were derived from pDH32 [[4] via pVK56. pDH32 was digested with Sma I and Nae I, and the 6.8 kb vector fragment, devoid of cat (conferring Cm resistance) and the amyE (3′) region, was self-ligated to yield pVK56.

Construction of the lacZ→gfp replacement vector for S. mutans, pMC4, involved several steps. The gfp-mut3b* allele [[,[5] was used in S. mutans. It was amplified by PCR using oligonucleotides designed so that it had the enhanced ribosome-binding site of pGreenTIR [[6] in order to increase gfp expression, and cloned in pTRKL2 [[7], yielding pJAR2. A 1 kb Cla I–Sma I cat cassette from pR326 [[8] was cloned in pVK56, which had been digested with Cla I and Eco RV; the resulting plasmid, pMC2, contained the cat cassette replacing a 287 bp internal fragment of lacZ. The gfp-mut3b* allele with the enhanced RBS was isolated from pJAR2 as a 0.7 kb blunt endNru I–Eco RV fragment and cloned upstream of the cat marker at the Cla I site (ends filled in) in pMC2 to yield pMC3 (with gfp in the same orientation as lacZ). In the final step, pMC3 was digested with Nsi I and Ahd I in order to delete the amp gene; the 7.1 kb vector fragment was gel purified, its ends made blunt, and then it was self-ligated to generate pMC4 (Fig. 1). The structures of these and all plasmids were confirmed by PCR and/or by restriction digestion as appropriate.

1

Vectors that facilitate the replacement of lacZ with gfp. pMC4 is for S. mutans and pVK209 for B. subtilis. Here, and in Figs. 2 and 3, the ColE1 replication determinant is indicated, as are the antibiotic-resistance determinants and the reporter genes. The pMC plasmids are designed for S. mutans, but also work with B. subtilis. The pVK plasmids are designed for B. subtilis; they contain an amp gene, making them unsuitable for streptococci. The DNA sequences of these plasmids are available on request.

The lacZ→gfp replacement vector for B. subtilis, pVK209, was constructed in two steps. First, the gfp-mut1 gene was obtained from pGreenTIR [[6] as a 0.7 kb Eco RI fragment, and cloned at the Xba I site of pVK59 [[9] (the ends of both made blunt with Klenow polymerase), to yield an intermediate pVK199R1. In the second step, the gfp-neo (neo confers resistance to Nm and Kan) cassette was isolated as a 2.0 kb Eco RV–Not I fragment from pVK199R, its ends were filled in, and it was ligated at the Eco RV site in the lacZ gene of pVK56 to yield pVK209 (Fig. 1).

The lacZ→gusA replacement vector for S. mutans, pMC5, was derived from pMC4. pMC4 was digested with Hin dIII, and the ends were made blunt; it was then digested with Bgl II. The resulting 6.3 kb Hin dIII(blunt)–Bgl II fragment (with gfp deleted) was gel purified and ligated to a 2 kb Bam HI–Xmn I fragment from pMLK113 that contained gusA [[] to yield pMC5 (Fig. 2).

2

Vectors that facilitate the replacement of lacZ with gusA. pMC5 is for S. mutans and pVK266 for B. subtilis.

The lacZ→gusA replacement vector for B. subtilis, pVK266, was constructed in a single step. The gusA-neo cassette was isolated as a 3.1 kb Hin dIII–Bam HI fragment from pMLK117 [[], its ends were made blunt with Klenow polymerase and it was cloned into the Eco RV site of pVK56 to yield pVK266 (Fig. 2).

The gusA→gfp replacement vector for B. subtilis, pVK268, was constructed in two steps. First, the gusA vector pMLK72 [[] was digested with Not I–Sal I, and a 72 bp oligomer containing the MCS was removed. The ends were made blunt with Klenow polymerase, and the fragment was self-ligated to yield an intermediate plasmid pVK265. The 2.0 kb blunt-end gfp-neo cassette obtained from pVK196R1 was cloned between the Hin cII sites of pVK265 (deleting a 524 bp internal region of gusA) to yield pVK268 (Fig. 3).

3

Vector pVK268 that enables gusA to be replaced with gfp in B. subtilis.

Transformation of B. subtilis and S. mutans was as described [[3,[0]. In general, the replacement vectors were linearized with a restriction enzyme at an appropriate site so as to favor double-crossover recombination. However, this step was found to be unnecessary, as >90% of the transformants obtained with non-linearized vectors resulted from double-crossover recombination in both B. subtilis and S. mutans. Transformants of E. coli with pMC4 and pMC5 were selected with 50 μg Cm ml⁻¹. Transformants of E. coli with pVK209, pVK266 and pVK268 were selected using Amp (100 μg ml⁻¹), or Nm (75 μg ml⁻¹) or both (each at 75 μg ml⁻¹).

2.4 Enzyme assays

β-Galactosidase and β-glucuronidase activities at pH 7.0 were assayed essentially as described [[]. Reactions were stopped by the addition of Na₂CO₃ to 0.25 M. Specific activity is expressed as nmole o-nitrophenyl-β-d-galactoside or p-nitrophenyl-β-d-glucuronide hydrolyzed min⁻¹ (mg bacterial dry wt)⁻¹, using the extinction coefficients at pH 10.2 of 21,300 at 420 nm for o-nitrophenol and of 16,800 at 410 nm for p-nitrophenol.

3 Results and discussion

3.1 Vectors that replace lacZ with gfp

S. mutans colonizes the oral cavity, as a part of the dental plaque. We have been studying formation and subsequent survival in biofilms of S. mutans under sugar-limiting conditions [[1]. We have utilized a lacZ promoter-probe vector to identify genes that are expressed during stationary phase [[] (Renye, Buttaro and Piggot, unpublished), and wished to convert lacZ fusions to gfp in order to study the spatial distribution of gene expression in biofilms. The plasmid pMC4 (Fig. 1) was constructed specifically for this purpose. It lacks the amp gene, which is present in many E. coli plasmids. It contains the cat gene from pR326 [[8], which provides a selectable marker for both E. coli and S. mutans. It contains the gfp-mut3b* gene, which encodes a derivative of GFP that functions in streptococci [[,[5], under the control of the enhanced RBS from pGreenTIR [[6], which we have found to greatly increase GFP expression in S. mutans and B. subtilis (unpublished observations).

S. mutans strain SL12471 is a derivative of UA159 containing a clpE–lacZ transcriptional fusion linked to neo within the vector pFP1 [[] and integrated by single crossover into the chromosome at clpE; this fusion was expressed during stationary phase (Renye, Buttaro and Piggot, unpublished observations). The strain was transformed with linearized pMC4, selecting for resistance to Cm (Table 1). Cm^R transformants were analyzed by fluorescent microscopy and revealed a GFP signal in batch cultures and in biofilms, with no detectable β-galactosidase activity (data not shown). Thus, pMC4 provides a rapid way to replace lacZ with gfp.

1

Transformation of B. subtilis and S. mutans strains with the replacement vectors

Recipient	Relevant genotype	Donor plasmida	Transformants (μg DNA)	Replacement efficiency (%)b	Gfp signalc
					FS	MC	WC
SL12471 (S. mutans)	clpE–lacZ	pMC4 lacZ→gfp	5.6 × 104	100	−	−	−
pMC5 lacZ→gusA	4.2 × 103	93	−	−	−
SL10614 (B. subtilis)	spoIIR–lacZ	pVK209 lacZ→gfp	2.4 × 103	98	49	0	1
pMC3 lacZ→gfp	2.2 × 103	100	48	0	2
pVK266 lacZ→gusA	1.4 × 103	94	−	−	−
SL12626 (B. subtilis)	spoIID–gusA	pVK268 gusA→gfp	2.4 × 103	100	0	48	2

^aPlasmids were linearized with a restriction enzyme at a site in the vector region so as to favor double cross over.

^bBased on blue/white colony phenotype on agar medium containing X-gal or X-gluc.

^cExcitation at 488 nm, emission peak at 509 nm.

Spore formation by B. subtilis has become a paradigm for the study of bacterial differentiation. There are extensive studies of the compartmentalization of gene expression that accompanies spore formation [[2], and GFP has become a major tool for such studies. For B. subtilis, we constructed the replacement vector pVK209 in which the selectable marker is neo (Fig. 1). The gfp gene in pVK209 is the mut1 allele associated with the enhanced ribosome-binding site from pGreenTIR [[6]. Plasmid pVK209 was used to insert gfp within lacZ of a transcriptional fusion to spoIIR (Table 1). The great majority (98%) of Nm-resistant transformants were white on X-gal-supplemented sporulation agar, consistent with disruption of lacZ by double crossover. Expression of gfp from the spoIIR promoter was largely confined to the prespore during spore formation (Table 1), as would be expected for such a σ^F-directed promoter [[2]; similar results were obtained for the spoIIIG promoter (data not shown). The vectors pMC3 and pMC4 can also be used for B. subtilis and potentially other low-GC gram-positive species to convert lacZ to gfp (pMC3, Table 1); their selectable marker is cat, making them useful when the lacZ fusion in B. subtilis is not linked to a cat gene.

3.2 Vectors that replace lacZ with gusA

The gusA gene, encoding β-glucuronidase has proved a very useful second probe in a range of gram-positive and gram-negative species, e.g. [[,[]. It has recently been shown to function as a probe in S. mutans [[] (our unpublished observations). The vector, pMC5 (Fig. 2), was constructed so as to make possible replacement of lacZ with gusA in S. mutans. It is derived from pMC4 and has cat as selectable marker. Nearly all chloramphenicol-resistant transformants (93%) exhibited β-glucuronidase gusA activity and no β-galactosidase activity, as detected with X-gluc (the substrate for β-glucuronidase) and X-gal (the substrate for β-galactosidase). Thus, pMC5 efficiently replaced lacZ with gusA in S. mutans (Table 1). Enzyme assays of liquid cultures confirmed that β-glucuronidase had completely replaced β-galactosidase (data not shown).

Plasmid pVK266 (Fig. 2) was designed to replace the lacZ gene in B. subtilis with gusA. It has neo as selectable marker. The plasmid was used to replace sporulation-specific lacZ transcriptional fusions with gusA (Table 1). Neomycin-resistant transformants of all the strains tested exhibited β-glucuronidase gusA activity and no β-galactosidase activity, as detected with X-gluc and X-gal. Measurement of β-glucuronidase activity of liquid cultures of strain SL11913, showed a similar temporal pattern of gene expression to that of the lacZ containing parent strain, SL10614, exhibiting β-galactosidase activity (Fig. 4). The difference in time of induction between the strains in this experiment reflects variability of behavior between cultures rather than a significant difference caused by the change in reporter (unpublished observations). The results confirm that when gusA replaces lacZ, β-galactosidase activity is totally abolished in the strain. Starting with lacZ fusions to two distinct promoters, the vector pVK266 enabled us to construct a strain that made possible simultaneous analysis of the expression of two sporulation-specific gene fusions in the same strain, one with lacZ and one with gusA.

4

Effect of replacing lacZ with gusA in a B. subtilis strain containing a spoIIR–lacZ transcriptional fusion. The strains used also contain a spoIIIE36 mutation, which enhances spoIIR expression [[]. Circles, strain SL10614, containing a spoIIR–lacZ transcriptional fusion inserted at the amyE locus. Triangles, strain SL11913, a derivative of SL10614 in which the gusA gene has been inserted within lacZ by double crossover using pVK266 as donor. Open symbols, specific β-galactosidase activity. Filled symbols, specific β-glucuronidase activity. Specific activity is expressed as nmoles o-nitrophenyl-β-d-galactoside or nmoles p-nitrophenyl-β-d-glucuronide hydrolyzed min⁻¹ (mg bacterial dry wt)⁻¹.

3.3 A vector that replaces gusA with gfp

There are a number of existing promoter-gusA fusions for B. subtilis [[]. Plasmid pVK268 (Fig. 3) was constructed as a vector that makes it possible to replace gusA with gfp in B. subtilis using neo as selectable marker; we did not attempt a comparable construction for S. mutans. Plasmid pVK268 was used to replace gusA with gfp in a strain containing a spoIID–gusA transcriptional fusion. Analysis of transformants by fluorescence microscopy revealed that the strain exhibited mother cell-specific GFP expression, as expected for spoIID expression (SL12626, Table 1).

Acknowledgements

We are very grateful to Drs. Andersen, Hansen, Klaenhammer and Miller for providing strains. We thank Dr. Bettina Buttaro for helpful discussions. This work was supported by Public Health Service Grants DE014604 and GM43577 from the National Institutes of Health.