An Alkaline Phosphatase Reporter for use in Clostridium difficile

Abstract

Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia.

1. INTRODUCTION

Although Clostridium difficile is a major nosocomial pathogen that causes severe gastrointestinal disease, the molecular mechanisms that control pathogenesis in this bacterium are poorly understood. The primary reasons that C. difficile remains an understudied pathogen are that the bacterium has long been difficult to cultivate and genetically manipulate. First, C. difficile is a fastidious organism and a strict anaerobe. Thus, for optimal and consistent growth, C. difficile is typically cultivated in rich growth medium within a strict anaerobic environment. Culture conditions notwithstanding, one of the greatest impediments has been the shortage of genetic tools for use in C. difficile.

Many of the molecular biology tools and techniques that are used to genetically manipulate other species have been difficult to adapt for C. difficile. However, techniques for studying C. difficile molecular biology have been developed in the past decade, including methods for gene disruption and plasmid transfer, random mutagenesis strategies and the application of next-generation sequencing technologies (1–7). Despite the advancements in molecular technologies over the past decade, genetic analysis of C. difficile remains a challenge.

The constraints of an anaerobic growth environment have been particularly problematic for the adaption of gene reporter technologies used in other bacteria. Reporter gene fusions are incredibly useful tools in the study of gene regulation and signal transduction, defining regulatory elements and promoters, and for the development of genetic screens (8). The most commonly used reporter gene fusions include colorimetric reporters, such as β-galactosidase (lacZ) and β-glucuronidase (gusA), fluorescent proteins (e.g., gfp, dsRed, mCherry), and chemiluminescent reporters (i.e., luciferase; luxAB). Unfortunately, these reporters require oxygen for proper reporter protein folding or full enzymatic activity (9–12) (data not shown), and consequently have little utility in anaerobic bacteria. Antibiotic-based reporters, such as Chloramphenicol Acetyl Transferase (cat) and neomycin (neo), are excluded because cat is one of the few selectable markers available and because C. difficile has high intrinsic resistance to aminoglycosides. A cyan fluorescent protein (CFP) reporter (13), a mCherry reporter (14), and anaerobically-functioning fluorescent reporters have been described, including SNAP (15) and LOV-based fusions (16, 17). While these fluorescent reporters are useful, visualization of activity can be difficult due to photo-bleaching and to high variability of fluorescence within a population of cells (13, 15–17). β-glucuronidase (gusA) reporters have been used in the heterologous host, Clostridium perfringens, an anaerobe that is somewhat aerotolerant, to measure C. difficile-specific gene expression (18). In addition, gusA reporters have been used in C. difficile (19); however, these assays exhibit low sensitivity and are cost-prohibitive.

While other methods are available for quantifying C. difficile gene transcription, including qRT-PCR, microarray and RNA-seq analyses, these techniques are expensive and require an investment in specialized equipment and enzymes. Since these methods are expensive and time-consuming, it is not practical to employ these techniques in large-scale genetic screens. In contrast, colorimetric reporter assays are relatively inexpensive and require a low investment in materials and reagents. In this study, we evaluate the alkaline phosphatase gene, phoZ, from Enterococcus faecalis as a transcriptional reporter for use in C. difficile. We report that C. difficile can express an active phoZ transcriptional fusion, which functions as a sensitive and quantifiable reporter of gene expression. This alkaline phosphatase reporter provides C. difficile researchers with a versatile new tool for assessing gene expression.

2. MATERIALS AND METHODS

2.1. Bacterial strains and growth conditions

The strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were cultured at 37°C in LB (20) or BHIS me dium (21) aerobically or anaerobically, and supplemented with 20 µg chloramphenicol ml⁻¹ or 100 µg ampicillin ml⁻¹, as needed. Enterococcus faecalis was cultured on BHI or BHIS medium as previously described (22). Clostridium difficile strains were cultured at 37°C in an anaerobic chamber as previously detailed (23, 24). C. difficile strains were grown in BHIS medium supplemented with 2–10 µg thiamphenicol ml⁻¹, 5 µg erythromycin ml⁻¹, 0.5–5 µg nisin ml⁻¹ or 100–400 µg XP (BCIP, 5-Bromo-4-chloro-3-indolyl phosphate; Sigma-Aldrich) ml⁻¹ as indicated.

Table 1.

Bacterial Strains and plasmids

Plasmid or Strain		Relevant genotype or features	Source, construction or reference
Strains
E. faecalis
	V583	clinical isolate	(40, 41)
E. coli
	HB101	F-mcrB mrr hsdS20(rB -mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20	B. Dupuy
	MC101	HB101 pRK24	B. Dupuy
	MC445	HB101 containing pRK24 and pMC358	This study
	MC461	HB101 containing pRK24 and pMC368	This study
	MC468	HB101 containing pRK24 and pMC375	This study
B. subtilis
	BS49	CU2189::Tn916	P. Mullany
	MC472	BS49 Tn916::pMC370	This study
	MC473	BS49 Tn916::pMC371	This study
C. difficile
	R20291	Clinical isolate
	630	Clinical isolate	(42)
	630Δerm	ErmS derivative of strain 630	N. Minton (43)
	MC324	630Δerm pMC123	(25)
	MC448	630Δerm pMC358	This study
	MC486	630Δerm pMC368	This study
	MC488	630Δerm Tn916::phoZ	This study
	MC489	630Δerm Tn916::PcprA::phoZ	This study
	MC499	630Δerm pMC375	This study
Plasmids
	pRK24	Tra+, Mob+; bla, tet
	pUC19	Cloning vector; bla	(44)
	pSMB47	Tn916 integrational vector; CmR, ErmR	(45) (GenBank U69267)
	pMC123	E. coli-C. difficile shuttle vector; bla, catP	(37)
	pMC211	pMC123 PcprA	(25)
	pMC358	pMC123 phoZ	This study
	pMC368	pMC123 PcprA::phoZ	This study
	pMC370	pSMB47 phoZ	This study
	pMC371	pSMB47 PcprA::phoZ	This study
	pMC375	pMC123 Pdlt::phoZ	This study

2.2. Strain and plasmid construction

E. faecalis strain V583 (GenBank accession AE016830) was used as a template for phoZ amplification. Oligonucleotides used for PCR and qRT-PCR are listed in Table 2. Isolation of plasmid DNA, PCR and cloning were performed using standard protocols. The 1477 bp phoZ coding sequence and upstream ribosomal binding site were amplified using primers oMC829/oMC830. This fragment was cloned into the BamHI and SphI sites of pMC123 (4) to create pMC358 (Figure 1A). pMC368 was created by cloning the PcprA fragment from pMC211 (25) as EcoRI/BamHI into pMC358. pMC375 was created by amplifying a 600 bp fragment upstream of dltD with oMC850/oMC823 and cloning this fragment as EcoRI/BamHI into pMC358. pMC370 was created by cloning the phoZ fragment from pMC358 as BamHI/SphI into pSMB47 (Figure 1B). To construct pMC371, the PcprA::phoZ insert was amplified from pMC368 using primers oMC909/oMC830, followed by digestion and cloning as a SalI/SphI fragment into pSMB47. Note that the cat cassette of pSMB47 is only functional as a selectable marker in E. coli. Sequencing of cloned DNA fragments was performed by Eurofins MWG Operon. Plasmid maps were created using SnapGene Viewer software.

Table 2.

Oligonucleotides

Primer	Sequence (5’ → 3’)	Use/location
oMC44	5’-CTAGCTGCTCCTATGTCTCACATC-3’	rpoC qPCR (CD0067) (4)
oMC45	5'-CCAGTCTCTCCTGGATCAACTA-3’	rpoC qPCR (CD0067) (4)
oMC96	5'-CGTTCAGGTCAATTCTCTCTAGGC -3'	cprA qPCR (CD1349) (4)
oMC97	5'-GGTCAAGACCATTTGTAGGCTC -3'	cprA qPCR (CD1349) (4)
oMC823	5'-gccggatccATTTTCTCTCCTCTAAAAATATTCAAA -3'	PdltD cloning (CD2854)
oMC829	5'-gccggatccGTCAATGTATGGGTAGATATGAAGG -3'	phoZ cloning (EF2973)
oMC830	5'-gcgcatgcCGTTCTGCTTTTTCTTCATTTTG -3'	phoZ cloning (EF2973)
oMC850	5'-gcggaattcTTCTTATATACCATCTGAAATACAGG -3'	PdltD cloning (CD2854)
oMC901	5'-CTGAAGCGAAGGCAACTGAA-3'	phoZ qPCR (EF2973)
oMC902	5'-GCTTGCTGTCCGACCAAATA-3'	phoZ qPCR (EF2973)
oMC909	5'-GCCgtcgacGACATGGAAGTAGAAGTTAAGG-3'	PcprA cloning

Figure 1. Overview of alkaline phosphatase reporter constructs.

Plasmid maps of pMC358 (for construction of plasmid phoZ reporters) and pMC370 (for construction of chromosomal phoZ reporters).

pMC358, pMC368 and pMC375 were introduced into E. coli strain MC101 by transformation, resulting in strains MC445, MC461 and MC468, respectively. MC445, MC461 and MC468 were conjugated independently to C. difficile strain 630Δerm as previously described (26, 27) using 10 µg thiamphenicol ml⁻¹ to select for the plasmids in C. difficile and 50 µg kanamycin ml⁻¹ to counterselect against E. coli after conjugation.

pMC370 and pMC371 were integrated into the Tn916 region within the chromosome of B. subtilis strain BS49 and selected for on BHIS plates containing 5 µg erythromycin ml⁻¹ as previously described (28, 29), except that 50 µg kanamycin ml⁻¹ was added to counterselect against B. subtilis after conjugation.

2.3 Alkaline Phosphatase Assays

Assays of alkaline phosphatase (AP) activity in C. difficile were adapted from previously described assays with some modifications (30, 31). To begin, overnight C. difficile cultures were back-diluted to an OD₆₀₀ of approximately 0.05 in BHIS medium (21) with or without addition of nisin, as indicated. Duplicate (2 ml) samples of each culture were taken when the cells reached an OD₆₀₀ of approximately 0.5. Samples were pelleted and the supernatant discarded, then stored at −20°C. To prepare samples for assay, cell pellets were first washed with 0.5 ml of cold Wash buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgSO₄), pelleted and resuspended in 1 ml Assay buffer (1 M Tris-HCl, pH 8.0, 0.1 mM ZnCl₂). 500 µl of cell suspension was transferred to a separate tube for alkaline phosphatase tests. 300 µl of additional Assay buffer, 50 µl of 0.1% SDS and 50 µl of chloroform were added to the suspension, which was then vortexed for 1 min and incubated at 37°C for 5 min. Sample tubes were then placed on ice for 5 min to cool. To begin the assay, samples were pre-warmed to 37°C, then 100 µl of 0.4% pNP (p-nitrophenyl phosphate in 1M Tris-HCl, pH 8.0; Sigma-Aldrich) was added to each sample at 10 second intervals. A blank without cells was prepared as a negative control in all experiments. Samples were inverted to mix and placed in a 37°C water bath. Upon development of a light yellow color, 100 µl of stop solution (1 M KH₂PO₄) was added and tubes placed in an ice bath to stop the phosphatase reaction. Time elapsed (min) for the assay was recorded. Assay samples were then centrifuged at max speed for 5 minutes. Absorbance for each sample was read at OD₄₂₀ and OD₅₅₀. Units of activity were calculated and normalized to cell volume as follows: $\frac{[\mathrm{O}\mathrm{D}420-(1.75\times \mathrm{O}\mathrm{D}550)]1000}{t(\text{min})\times \mathrm{O}\mathrm{D}600\times \text{vol}.\text{cells}(\mathrm{m}\mathrm{l})}$. Technical duplicates were performed and averaged for each AP assay set. A minimum of three biological replicates were performed for each experiment. The results are presented as the means and standard error of the experimental replicates. The two-tailed Student's t test was used to compare the results of the variable and control sets as indicated.

2.4 Quantitative reverse transcription PCR analysis (qRT-PCR)

Cultures for qRT-PCR analysis were harvested in parallel with the alkaline phosphatase assay samples described above. Cultures were harvested into cold 1:1 ethanol:acetone prior to RNA purification as previously described (4, 32). Total RNA was DNase I treated and cDNA synthesized as previously described (33). qRT-PCR primers were designed using the IDT PrimerQuest tool and primer efficiencies were determined for each primer set (http://www.idtdna.com/Scitools/Applications/Primerquest). Technical triplicates were performed for each qPCR reaction set for a minimum of three biological replicates. Relative quantification of transcription was performed using the internal control transcript, rpoC, for normalization. The transcriptional ratios of variable and control sets are presented as the means and standard error of the means for the data obtained. The two-tailed Student’s t test was used to compare the ratios of the variable and control sets as indicated.

3. RESULTS AND DISCUSSION

3.1. Evaluation of alkaline phosphatase activity and construction of vectors for alkaline phosphatase reporter fusions

Before evaluating the use of phoZ as a reporter, we first assessed the potential for inherent alkaline phosphatase (AP) activity in C. difficile. A protein BLAST search revealed no proteins with significant homology to phoZ within the genome of any C. difficile strain. For good measure, C. difficile strains 630Δerm and R20291 were grown overnight on BHIS agar medium with and without the chromogenic substrate XP as described in the Materials and Methods. No apparent blue color development of the colonies was detected following overnight anaerobic growth or aerobic incubation of the plates, indicating that C. difficile does not produce significant levels of alkaline phosphatase under the tested conditions (data not shown).

In order for phoZ to be useful as an anaerobic reporter, the PhoZ produced under anaerobic conditions needs to be enzymatically active. To determine whether the E. faecalis phoZ gene product was functional when produced under anaerobic conditions, E. faecalis was grown on BHIS agar with and without XP, anaerobically and aerobically. E. faecalis grown aerobically exhibited the blue colony phenotype indicative of AP activity, while the bacteria that were incubated anaerobically appeared white until the plates were allowed to develop in the presence of oxygen (data not shown). However, when cells expressing phoZ were assayed anaerobically using the AP substrate pNP, significant color change was observed, indicating that PhoZ is enzymatically active anaerobically (data not shown). Thus, the AP protein folds properly anaerobically, but oxidation is required for the XP cleavage product to produce the color-forming precipitate as previously described (34). Accordingly, AP reactions using the pNP substrate can be performed under anaerobic conditions, but reactions using the XP substrate will not produce a colored precipitate without further oxidation.

To investigate phoZ as a transcriptional reporter in C. difficile, two independent reporter constructs were created. The first was an extrachromosomally-replicating plasmid containing the phoZ reporter gene. The phoZ gene was amplified from E. faecalis and cloned into the C. difficile-E.coli shuttle vector pMC123, creating pMC358 (Figure 1A). This vector does not have a promoter to drive the expression of phoZ, thus no alkaline phosphatase activity should be produced in strains harboring this plasmid. To enable expression of phoZ in C. difficile the strong promoter, PdltD, or the nisin-inducible promoter, Pcpr, was cloned into pMC358 upstream of phoZ to give pMC375 and pMC368, respectively (4, 35, 36). All three plasmids were conjugated into C. difficile strain 630Δerm as described in the Materials and Methods.

A second phoZ vector was created to permit single-copy chromosomal-based reporter fusions. To this end, phoZ and Pcpr::phoZ were amplified and cloned independently into the plasmid pSMB47 to create pMC370 (Figure 1B) and pMC371, respectively. pSMB47 can replicate in E. coli, but integrates into the chromosome of B.subtilis strain BS49 at the Tn916 locus and can be conjugated via Tn916 onto the chromosome of C. difficile (37–39).

3.2. Alkaline phosphatase is expressed and functional in C. difficile

To investigate the activity of alkaline phosphatase expressed in C. difficile, strains carrying the plasmid-based Pdlt::phoZ (MC499), the promoterless phoZ control (MC448) or the empty vector (MC324) were grown on BHIS plates containing XP and thiamphenicol. After overnight anaerobic growth, plates were removed from the anaerobic chamber and alkaline phosphatase activity was allowed to develop aerobically. As shown in Figure 2, C. difficile containing the Pdlt::phoZ fusion turn blue on XP plates. The development of color in colonies was visible within 20 minutes of removing the plate from the anaerobic chamber. Cells carrying the empty vector or the promoterless phoZ vector remained white on XP plates. These results demonstrate that the E. faecalis PhoZ is active when produced by C. difficile.

Figure 2. Visualization of alkaline phosphatase activity from C. difficile grown on agar plates.

C. difficile strains A) MC324 (empty vector), B) MC448 (promoterless phoZ) and C) MC499 (Pdlt::phoZ) were grown anaerobically overnight on BHIS agar plates containing 400 µg XP substrate ml⁻¹ and 2 µg thiamphenicol ml⁻¹. Plates were removed from the anaerobic chamber and development of color was monitored over 24 hours (“H” indicates the number of hours after removal from the anaerobic chamber). Visible color development was observed within 20 minutes of aerobic atmospheric exposure.

3.3. Alkaline phosphatase transcriptional reporter activity correlates directly with gene-specific transcription

To determine the amount of alkaline phosphatase activity in C. difficile cells expressing phoZ, quantitative AP assays were performed. This AP assay was based on previously published assays with minor modifications (30, 31) (see Materials and Methods). C. difficile strains expressing the promoterless phoZ (MC448) and the Pdlt::phoZ fusion (MC499) from a plasmid were used to assess the expression and activity of alkaline phosphatase. Each strain was grown in BHIS broth, and culture samples were taken during mid-logarithmic growth (OD₆₀₀ = ~0.5) for AP assays. The strain expressing the Pdlt::phoZ fusion exhibited a 144-fold increase in AP activity compared to the promoterless phoZ construct, indicating that a C. difficile-specific promoter can robustly drive measurable phoZ expression (Figure 3A).

Figure 3. Alkaline phosphatase activity from the cprA::phoZ constructs is increased in the presence of nisin.

(A) C. difficile strains MC448 (promoterless phoZ on a plasmid) and MC499 (Pdlt::phoZ on a plasmid) were grown to an OD₆₀₀ = ~0.5 in BHIS medium. (B) C. difficile strains MC488 (promoterless phoZ on the chromosome), MC489 (PcprA::phoZ on the chromosome), MC448 (promoterless phoZ on a plasmid) and MC486 (PcprA::phoZ on a plasmid) were grown to an OD₆₀₀ = ~0.5 in BHIS medium in the presence or absence of nisin. Alkaline phosphatase activity was determined as described in the Materials and Methods. The means and standard errors of the means of three biological replicates are shown.

To further determine the sensitivity and linear range of the phoZ promoter, we used the inducible cpr promoter (4). C. difficile strains expressing either the promoterless phoZ integrated on the chromosome (MC488) or from a plasmid (MC448), or the Pcpr::phoZ fusion on the chromosome (MC489) or from a plasmid (MC486) were grown in BHIS broth alone or with the addition of 0.5 µg or 5 µg ml⁻¹ nisin. Culture samples were later taken during mid-logarithmic growth (OD₆₀₀ = ~0.5) for AP assays and RNA preparation. Shown in Figure 3B, strains expressing the promoterless phoZ from the chromosome or from a plasmid exhibit very low alkaline phosphatase activity. As expected, strains expressing either Pcpr::phoZ fusion had higher AP activity when grown in nisin (~10–20-fold in the presence of 0.5 µg ml⁻¹ nisin and ~30–50-fold in the presence of 5 µg ml⁻¹ nisin), demonstrating that the cprA promoter is induced in a dose-dependent manner in the presence of nisin, as observed previously (4, 35). In addition, AP activity is significantly lower from the PcprA::phoZ fusion (MC486) in BHIS medium (1.3 AP units) compared to the constitutive, stronger Pdlt::phoZ fusion (MC499; 26 AP units; compare Figure 3B to 3A). Altogether, these results indicate that the phoZ reporter can be used to accurately quantify gene expression in a wide dynamic range with little basal expression.

To determine if AP activity from the PcprA::phoZ constructs correlates with native gene expression, qRT-PCR analysis was performed to measure chromosomal cprA gene transcription. Expression of native cprA was increased ~100-fold in the presence of 0.5 µg ml⁻¹ nisin and ~170–200-fold in the presence of 5 µg ml⁻¹ nisin (Figure 4). To compare chromosomal cprA expression to the AP activity measured from the PcprA::phoZ fusion in the absence and presence of nisin, we determined the fold change in AP activity and transcript levels in various conditions (Table 3). The fold change in PcrpA::phoZ fusion AP activity and in native cprA transcription is similar between the chromosomal and plasmid reporter constructs, although the plasmid reporter constructs are more sensitive (Table 3; bolded numbers). In addition, this data demonstrates that the presence of an additional cprA promoter in the chromosome or on a plasmid does not affect native gene expression or its regulation as cprA transcription is equal between all strains in the absence or presence of nisin (qRT-PCR data in Table 3; Figure 4).

Figure 4. Native cprA transcription correlates with PcprA::phoZ alkaline phosphatase activity.

qRT-PCR analysis of cprA expression in C. difficile strains MC488 (promoterless phoZ on the chromosome), MC489 (PcprA::phoZ on the chromosome), MC448 (promoterless phoZ on a plasmid) and MC486 (PcprA::phoZ on a plasmid) grown to an OD600 = ~0.5 in BHIS medium in the presence or absence of nisin. The means and standard errors of the means of three biological replicates are shown.

Table 3.

Fold change of cprA gene expression analyzed by alkaline phosphatase activity and qRT-PCR in C. difficile grown with and without nisin.

Strain	Relevant Genotype	Nisin 0.5 µg ml−1/ BHISa,d		Nisin 5 µg ml−1/ BHISb,d		Nisin 5 µg ml−1/ Nisin 0.5 µg ml−1c,d
		AP	qRT-PCR	AP	qRT-PCR	AP	qRT-PCR
MC488	Tn916:phoZ	1.1±0.1	89.3±10.5	1.0±0.1	162.7±11.7	1.0±0.0	1.8±0.1
MC489	Tn916::PcprA::phoZ	17.3±1.1	84.7±2.6	32.5±7.9	193.0±27.1	2.7±0.3	2.3±0.4
MC448	pphoZ	1.2±0.1	110.0±8.3	1.5±0.2	201.3±18.0	1.2±0.1	1.8±0.1
MC486	pPcprA::phoZ	23.3±2.9	78.5±5.4	48.3±1.4	152.0±19.0	2.2±0.3	1.9±0.2

^aFold change of alkaline phosphatase (AP) activity or transcript levels (qRT-PCR) in cells grown in 0.5 µg ml⁻¹ nisin to BHIS medium with no supplement.

^bFold change of alkaline phosphatase (AP) activity or transcript levels (qRT-PCR) in cells grown in 5 µg ml⁻¹ nisin to BHIS medium with no supplement.

^cFold change of alkaline phosphatase (AP) activity or transcript levels (qRT-PCR) in cells grown in 5 µg ml⁻¹ nisin to 0.5 µg ml⁻¹ nisin.

^dThe means and standard errors of the means of three biological replicates are shown.

Our data demonstrate that phoZ can function as an alkaline phosphatase reporter to provide qualitative or quantitative analysis of gene expression in C. difficile. This phoZ reporter will be amenable to multiple uses, including the analysis of promoter function, identifying transcriptional regulatory effects, and genetic screens. For genetic screens performed using XP substrate, it is critical to replica plate the bacteria anaerobically prior to the removal,of cells from the chamber, as brief exposure to oxygen can kill vegetative C. difficile. In addition measuring phoZ reporter activity in sporulating cells may require additional extraction techniques, such as the use of French pressure cell press, for complete cell lysis and accurate measurement of alkaline phosphatase activity. Notably, the use of the reporter on a multi-copy plasmid will facilitate increased expression for analyzing low levels of gene expression or screening for a range of regulatory effects in a variety of conditions. As with any construct expressed on a plasmid, additional considerations must be acknowledged when interpreting the results (e.g. titration of regulatory factors and plasmid burden).

It is important to note that we normalized alkaline phosphatase activity to the optical density of cultures at the time cells were harvested. Because of cell lysis, the optical density of C. difficile cells after resuspension in assay buffer was inconsistent and unreliable. However, some strains or mutants could exhibit aberrant cellular morphology that would result in an inaccurate correlation between optical density readings and the number of cells present. In this case, alkaline phosphatase activity can be normalized to total cellular protein (mg ml⁻¹), which can be determined by precipitating cells with trichloroacetic acid, to remove cross-reacting or interfering substances, and performing a standard protein quantification assay (e.g. bicinchoninic acid assay).

CONCLUSIONS

The use of a standardized reporter for analysis of gene expression and regulatory effects as well as the development of genetic screens is an effective molecular biology tool for studying prokaryotes. Thus, the development of a colorimetric and quantifiable reporter system that functions well in an anaerobe will be useful for the study of C. difficile molecular genetics. The phoZ reporter is a simple, critically-needed system that will allow cost-effective and time-efficient genetic analysis in C. difficile and other anaerobic organisms.

HIGHLIGHTS.

• We describe an effective transcriptional reporter assay for use in C. difficile.

• Promoter::phoZ fusion activity correlates with native gene expression in vivo.

• The phoZ reporter is a simple method for quantifying gene expression.