Malo et al., 2010

SUPPLEMENTARY MATERIALS

Malo et al., 2010

Table of Contents

1. Materials and Methods

2. Supplementary Figure 1 Activity of calf intestinal alkaline phosphatase (cIAP) in

autoclaved tap water.

3. Supplementary Figure 2 IAP activity in the stools of mice receiving different amounts

of cIAP in the drinking water.

4. Supplementary Figure 3 Effects of cIAP on bacterial growth in vitro.

Supplementary Materials and Methods

Materials. Calf intestinal alkaline phosphatase (cIAP) and DNA restriction enzymes were

purchased from New England Biolabs (Ipswich, MA). p-Nitrophenyl phosphate disodium salt

hexahydrate (pNPP) was purchased from Sigma (St. Louis, MO). LB-broth, Brain Heart Infusion

(BHI) media, LB-agar, BHI agar, McConkey agar and Brucella agar (5% horse blood) plates

were purchased from Fisher Scientific (Pittsburgh, PA). The CO2 producing gas pack and

anaerobic condition indicators were also obtained from Fisher Scientific. The kits for isolation

of bacterial DNA from mouse stool were obtained from MoBio Laboratories (Carlsbad, CA) and

Qiagen (Valencia, CA). Taq DNA polymerase and dNTP mixes were purchased from Promega

(Madison, WI). TaqGold® DNA polymerase was purchased from Applied Biosystems (Foster

City, CA). The kit for protein assay was obtained from Bio-Rad (Hercules, CA). The primers for

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genotyping and amplification of 16S rRNA genes were synthesized by Fisher Scientific. The

TOPO TA cloning kit was purchased from Invitrogen (Carlsbad, CA).)

Animals. IAP knockout (KO) mice (Mus musculus C57BL/6) construction has been described[1]

and heterozygous mice were obtained from the Burnham Institute for Medical Research (La

Jolla, CA). These animals were subsequently bred at the MGH animal facility to create

homozygous IAP-KO, heterozygous and wild-type C57BL/6 (WT) littermates. Confirmation of

genotype was performed by PCR analysis.[1] The animal experiments were reviewed and

approved by the IACUC at MGH. Animals in this study were maintained in accordance with the

guidelines of the Committee on Animals of Harvard Medical School (Boston, MA) and those

prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of

Laboratory Resources, National Research Council (Department of Health, Education and Human

Services, publication no. 85-23 (National Institute of Health), revised 1985).

Bacterial culture. Streptomycin-resistant E. coli was grown on MacConkey agar plates

containing streptomycin (100 g/ml), and Streptomycin-resistant Salmonella typhimurium

SL1344 was grown on Hektoen plates also containing streptomycin (100 g/ml). For stool

culture, individual stool samples from different animals were collected fresh directly in BHI

media (200 l) in microfuge tubes, kept on ice, weighed and then determined the weight of stool

by subtracting the pre-determined weight of tube and media. BHI media was added to each tube

obtaining a specific weight:volume ratio (1 mg stool:10 l BHI). The stool sample was then

vortexed to homogenize followed by serial dilution and plating on MacConkey agar, LB-agar,

BHI-agar and Brucella (5% horse blood) agar plates. For the growth of aerobic bacteria, plates

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were incubated in ambient air for 24 h at 37oC; anaerobic plates were incubated in a table-top

anaerobic chamber with gas packs (Fisher Scientific) for 7 days at 37oC. Samples were plated on

the bench and then placed in the chambers. The change of color of the anaerobic indicator inside

the chamber was rigorously monitored. Colony forming units (CFU) were counted and

expressed as average CFU/gm of stool +/- SEM.

To determine the effects of calf IAP (cIAP) on bacterial growth in vitro, cultures of E.

coli, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes were grown

in LB-broth containing 20 l/ml (200 U/ml) of cIAP (New England Biolabs) or 20l/ml ‘vehicle

for cIAP’ (50 mM KCl, 10 mM Tris-HCl (pH 8.2), 1 mM MgCl2, 0.1 mM ZnCl2, and 50%

glycerol). Each culture (200 l) was grown in 5 wells of a 96-well clear-bottom plate at 37oC in a

shaking incubator, and absorbance (OD600) was determined hourly. Bacterial growth in a culture

at a definite time-point was calculated as average Absorbance (OD600) +/- SD.

Terminal restriction fragment length polymorphism (TRFLP) analyses. Fecal samples of

equal weight were collected from 8 animals in each group (WT and KO). Using the MoBio

PowerSoil® soil DNA kit (MoBio Laboratories, Carlsbad, CA) bacterial DNA was extracted

from each sample following manufacture’s protocol. Success of each extraction was determined

by measuring DNA concentration in the extraction product with a Spectramaxâ

spectrophotometer (Molecular Devices, Palo Alto CA). PCR was performed to amplify an

approximately 500 bp bacterial DNA fragment using primers homologous to conserved regions

on the bacterial 16S rRNA gene.[2] The forward primer (16s.8F: 5’-

AGAGTTTGTTCMTGGCTCAG-3’) and the reverse primer (16s.530R: 5’-

GTATTACCGCGGCTGCTGG-3’)[2] were synthesized by GenSet (La Jolla, CA) (see

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Supplementary table 1). The forward primer was fluorescently labeled with a phosphamide dye

(D4). PCR reactions were carried out in 50 l using 1 µl of undiluted DNA extract, 5 µl of 10x

Buffer, 3 µl of 10 mM dNTP, 2 µl of 20 mg/ml BSA, 7 µl of 25 mM MgCl2, 1 µl (10 pmole) of

each primer, and 0.3 µl of 5 U/l TaqGold® DNA polymerase (Applied Biosystems). Reaction

temperatures and times were 96°C for 10 min, 35 cycles of 94°C for 1 min, 46.5°C for 1 min,

72°C for 2 min followed by a final extension step of 10 min at 72°C. All reactions were

performed in triplicate and then combined using a MoBio Ultracleanâ PCR Cleanup Kit (MoBio

Laboratories) following manufacturer’s protocol. PCR products were quantified using a

fluorometer tuned to the labeling dye.

TRFLP was performed following the protocols as previously described.[2] An enzyme

digest was performed on 75 ng of cleaned PCR product using one of three restriction

endonucleases Dpn II, Hae III and Hpa II (New England Biolabs). Each 40 µl digestion used 75

ng of DNA, 1 U of enzyme, and 4 µl of buffer. The samples were digested for 4 h at 37°C and

inactivated for 20 min at 65°C or 85°C depending on the enzymes inactivation requirement. The

digestion products were precipitated by ethanol and resuspended in 20 µl of formamide

containing 0.25 µl of CEQ 600 base pair DNA standard ladder. Terminal restriction fragment

profiles were obtained using a Beckman Coulter CEQ8000 DNA analysis system.

Terminal restriction fragment (TRF) length in nucleotides and TRF peak area were

exported from the CEQ8000 into Excel (Microsoft, Seattle, WA). To standardize the data for

comparison between samples, the area under each TRF peak was normalized to total amount of

DNA analyzed and expressed as parts per million (ppm). Peaks with an area of less than 10,000

ppm (<1% of the total for that sample) were excluded from analysis to reduce noise.

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Supplementary Table 1 Primers used in this study

Group PhylumTaxonomic rank Primer Sequence (5’ – 3’)

Gene-specificity

PCR product

All Bacteria(for TRFLP and cloning)   Kingdom F. AGAGTTTGTTCMTGGCTCAG

R. GTATTACCGCGGCTGCTGG 16S rRNA 523 bp

All Bacteria (for qPCR)   Kingdom F. ACTCCTACGGGAGGCAGCAGT

R. ATTACCGCGGCTGCTGGC 16S rRNA* 175 bp

Enterobacteriaceae Proteobacteria FamilyF. GTGCCAGCAGCCGCGGTAA R. GCCTCAAGGGCACAACCTCCAAG 16S rRNA* 312 bp

Clostridiales Firmicutes OrderF. ACTCCTACGGGAGGCAGCR.GCTTCTTAGTCAGGTACCGTCAT 16S rRNA* 154 bp

Lactobacillaceae Firmicutes FamilyF. AGCAGTAGGGAATCTTCCAR. CACCGCTACACATGGAG 16S rRNA* 316 bp

Enterococcus Firmicutes FamilyF. CCCTTATTGTTAGTTGCCATCATTR. ACTCGTTGTACTTCCCATTGT 16S rRNA* 165 bp

Bacteroidetes Bacteroidetes ClassF. AGCAGCCGCGGTAATR. GCATTTCACCGCTA 16S rRNA* 184 bp

E. coli Proteobacteria GenusF. GACGTCTCGTTGCTGCATAAACCR. CAGCAGCAGACCATTTTCAATCC

LacZ (external) 402 bp

E. coli Proteobacteria GenusF. ATGATGATTTCAGCCGCGCTR. GCTTCAATCAGCGTGCCGTC

LacZ (internal) 280 bp

Note: F, forward primer; R, reverse primer; *, see Barman et al. [3].

Statistical analyses. Normalized TRFLP data sets were transformed by taking the square root of

the area under each TRF peak to de-emphasize large TRF peaks while still taking relative

abundance into account.[4] Transformed data were analyzed by Bray-Curtis similarity and

ordinated by multi-dimensional scaling (MDS) using the PRIMER5 statistics package (Primer-E

Ltd., Ivybridge, United Kingdom). Significance testing was performed using analysis of

similarity (ANOSIM) with PRIMER5.

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Construction of the library of bacterial 16S small-subunit ribosomal RNA genes. Stool

samples of equal weight from 10 individual wild-type (WT) mice were pooled and used for

isolation of bacterial DNA using the DNA isolation kit from Qiagen. Similarly, DNA was

isolated from pooled stool samples of 10 IAP-KO animals. The forward and reverse primers[2]

that were used in PCR amplification of the 16S rRNA gene fragments for TRFLP analyses (see

above) were also used to amplify the same 500 bp 16S rRNA gene fragments for cloning. PCR

was performed using Taq DNA polymerase in a thermocycler (PTC-200, MJ Research,

Waltham, MA). The PCR conditions were: initial denaturation for 2 min, then 32 cycles of

denaturation (94oC for 30 sec), annealing (45oC for 30 sec) and extension (72oC for 60 sec)

followed by a final extension step of 5 min at 72oC. The PCR products were verified by

electrophoresis through a 2% agarose gel. Using TOPO TA cloning kit (Invitrogen) fresh PCR

products were cloned into pCR2.1 TA cloning vector following the manufacturer’s protocol. The

transformants were plated on LB-agar plates containing ampicillin (100 g/ml) and X-gal (40

mg/ml) and incubated at 37oC overnight. Approximately one thousand white transformant

colonies from each group (WT and KO) were grown at 37oC overnight in 96-well plates, each

well containing 150 l of LB-broth with 100 g/ml ampicillin.

Sequencing. Using M13 forward and reverse primers bidirectional sequencing was performed on

all clones, 1,000 from each library (WT and KO), using dye-labeled dideoxynucleotide chain

terminators at the Molecular Cloning Laboratory (South San Francisco, CA).

Phylogenetic analyses. Cloned 16S rRNA gene sequences were analyzed with the ‘Classifier’

program developed by Michigan State University (http://rdp.cme.msu.edu/). The program

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produced the name and number of 16S rRNA gene sequences, and arranged them in taxonomical

hierarchy. Percentage of each sequence was calculated using the Microsoft Excel program. Chi

squared analysis was performed to determine statistical significance in the distribution of clones

in WT and KO libraries, and p<0.05 was considered significant.

Quantitative real-time PCR (qPCR)

Semi-quantitative limited-cycle PCR (<20 cycles) was performed on WT and KO stool DNA

using Taq DNA polymerase with E. coli LacZ and subphylum-specific 16S rRNA gene primers

(see Supplementary table 1) in a thermocycler (MJ Research). Quantitative real-time PCR was

performed in an IQ5 Thermocycler (Bio-Rad, Hercules, CA) using SYBR Green PCR kit from

New England Biolabs (Ipswich, MA). Primers were synthesized by the MGH Core DNA

Synthesis Facility.

For absolute quantitation of bacterial DNA, serial dilutions of a known amount of E. coli

DH5 genomic DNA were subjected to qPCR amplification with the 16S rRNA gene universal

primers (synthesizing 175 bp fragment, see Supplementary table 1), and a standard curve was

generated by plotting CT’s against the known amounts of DNA. DNA isolated from WT and KO

stools were subjected to qPCR using universal as well as subphylum-specific 16S rRNA gene

primers. Quantitation of Eubacterial DNA as well as subphylum-specific bacterial DNA was

calculated by comparing the known CT values against the standard values. The fold change for

Eubacteria was determined by dividing the total amount of IAP-KO stool DNA with the total

amount of WT stool DNA, whereas the fold change of a specific bacterial group was calculated

by dividing the percentage of the specific bacterial group in IAP-KO mice stool with the

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percentage of the respective bacterial group in WT mice stool. Each PCR was repeated at least 3

times.

Intestinal alkaline phosphatase (IAP) assay. IAP activity was determined following the

protocol as described in Baykov et al.[5] Briefly, individual stool samples were homogenized in

water (10 mg/100 l), centrifuged, and supernatant was collected. Then 25 l of the stool

supernatant (or aqueous cIAP solution as a control) were mixed with 175 l of phosphatase assay

reagent containing 5 mM of p-nitrophenyl phosphate (pNPP) followed by determination of

optical density at 405 nm after a specific time period when the samples usually turned yellow

due to release of p-nitrophenol. Protein concentrations of stool supernatants were measured using

a kit from Bio-Rad (Hercules, CA). The specific activity of the enzyme is expressed as average

pmole pNPP hydrolyzed per min per g of protein +/- SEM.

Colonization assay. For studying colonization of E. coli, a commensal E. coli was isolated from

the stool of a WT mouse, and a spontaneous streptomycin-resistant mutant was isolated by

culturing the bacterial sample in a MacConkey plate containing streptomycin (100 g/ml).

Spontaneous streptomycin resistant mutants are frequently a result of mutations in the rpsL gene

and are phenotypically stable.[6] Salmonella typhimurium SL1344 was grown in LB-broth and

CFU was determined by plating on Hektoen plates. Doses of bacteria for oral gavage varied from

20,000 to 500,000 CFU depending on the experiments. After oral gavage, the presence as well as

quantity of bacteria was determined by stool culture on selective media.

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Oral supplementation of IAP. For oral administration of IAP we determined the efficacy and

stability of calf IAP (cIAP) in drinking water and observed that cIAP was reasonably functional

and stable in autoclaved tap water (see below, Supplementary fig. S1). We also fed 5 WT mice

different doses of cIAP in the drinking water (autoclaved tap water) and observed that IAP

activity was dose-dependent (Supplementary fig. S2). If not indicated otherwise, for in vivo

studies, the dose of cIAP was 200 U/ml (20 l/ml) drinking water. The control animals received

‘vehicle for cIAP’ (see above, 20 l/ml) in the drinking water.

Antibiotic treatment. If required, mice were fed streptomycin (5 mg/ml) and/or ciprofloxacin

(0.5 mg/ml) in their drinking water for 3 days.

Determination of pH. Individual mouse stool samples were suspended in water (pH 7.0) at a

specific weight:volume ratio (1 mg stool:10 l water). The stool samples were then vortexed to

homogenize and centrifuged to collect supernatant. The pH of the supernatant was then

determined using pH paper strips. The pH of luminal fluids (duodenum and ileum) was

determined by directly applying the fluid on pH paper strips.

Restoration of gut flora after antibiotic treatment. Two groups of wild-type (C57BL/6) mice

(n = 5 for each group) were allowed to drink autoclaved tap water containing 5 mg/ml

streptomycin for 3 days. One group (cIAP+ Group) also received 200 U/ml cIAP (20 l/ml)

(New England Biolabs) along with streptomycin, and cIAP was continued until normal gut flora

was reestablished (usually by Day 7). The other group (cIAP Group) received equal amounts (20

l/ml) of ‘vehicle for cIAP’ for the total duration of the experiment. Water containing cIAP and

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‘vehicle for cIAP’ was replaced daily. The fecal sample from each animal was plated everyday

on MacConkey agar plates to determine the restoration of Gram-negative bacteria, especially E.

coli.

The experiment was repeated 6 times and duration of experiments varied from 7-35 days.

Data on the day of first appearance of E. coli in an animal’s stool were compiled for 39 animals

in each group. Statistical significance of the difference in the number of animals with E. coli on a

specific point of time (Day) was determined by two-tailed Fisher’s Exact Test, and p<0.05

was considered significant.

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Supplementary Figure 1 Activity of calf intestinal alkaline phosphatase (cIAP) in autoclaved

tap water. Two units of cIAP (New England Biolabs) were dissolved in 1 ml autoclaved tap

water in the presence or absence of cIAP buffer, and cIAP activity was determined at specific

time intervals following the protocol as described.[5] The results show that cIAP retains more

than 80% of its activity in the tap water without any added cIAP buffer. We wanted not to use

any buffer in the drinking water because cIAP buffer has high pH (7.9) as well as higher

concentrations of salts (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT), which

might affect the background bacterial growth and mask the effects of cIAP. Note: Animals

received cIAP in autoclaved drinking water, which was replaced everyday.

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Supplementary Figure 2 IAP activity in the stools of mice receiving different amounts of

cIAP in the drinking water. A group of mice (n = 5) was allowed to drink water containing a

specific amount of cIAP for 24 h followed by determination of IAP activity in their stools.

Specific activity (S.A.) of IAP was expressed as average pmole pNPP hydrolyzed/min/g

protein +/- SEM. The results show that the IAP activity in a stool sample is dose-dependent;

the higher the amount in the drinking water, the higher the activity in stool, indicating that

oral administration of IAP in the drinking water could be an effective route of delivery to

increase intestinal luminal concentration of IAP.

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Supplementary Figure 3 Effects of cIAP on bacterial growth in vitro. Overnight bacterial

culture was diluted 20-fold in LB-broth +/- cIAP (200 U/ml = 20 l/ml). The control culture

received 20 l/ml ‘vehicle for cIAP’ (see above) instead of cIAP. Each culture was grown in 5

wells of a 96-well plate at 37oC in a shaking incubator, and absorbance (OD600) was determined

hourly. Bacterial growth in a culture at a definite time-point was calculated as average

Absorbance (OD600) +/- SD. (A) Growth of Gram-negative bacterium E. coli in the presence of

cIAP (cIAP) or absence of cIAP (cIAP). (B) Growth of Gram-negative bacterium Salmonella

typhimurium in the presence of cIAP (cIAP) or absence of cIAP (cIAP). (C) Growth of Gram-

positive bacterium Staphylococcus aureus in the presence of cIAP (cIAP) or absence of cIAP

(cIAP). (D) Growth of Gram-positive bacterium Listeria monocytogenes in the presence of cIAP

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(cIAP) or absence of cIAP (cIAP). The experiment was repeated more than 5 times and similar

results were obtained. Note: The results show no significant effect of cIAP on bacterial growth in

vitro.

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Supplementary References

1. Narisawa S, Huang L, Iwasaki A, et al. Accelerated fat absorption in intestinal alkaline

phosphatase knockout mice. Mol Cell Biol 2003; 23: 7525-30.

2. Kaplan CW, Astaire JC, Sanders ME, et al. 16S ribosomal DNA terminal restriction

fragment pattern analysis of bacterial communities in feces of rats fed Lactobacillus

acidophilus NCFM. Appl Environ Microbiol 2001; 67: 1935-9.

3. Barman M, Unold D, Shifley K, et al. Enteric salmonellosis disrupts the microbial

ecology of the murine gastrointestinal tract. Infect Immun 2008; 76:907-15. Epub 2007

Dec 26.

4. Blackwood CB, Marsh T, Kim SH, et al. Terminal restriction fragment length

polymorphism data analysis for quantitative comparison of microbial communities. Appl

Environ Microbiol 2003; 69: 926-32.

5. Baykov AA, Evtushenko OA, Avaeva SM. A malachite green procedure for

orthophosphate determination and its use in alkaline phosphatase-based enzyme

immunoassay. Anal Biochem 1988; 171: 266-70.

6. Lagatolla C, Dolzani L, Monti-Bragadin C. Characterization of the phenotype conferred

by two different rpsL alleles coding for streptomycin dependence. New Microbiol 1998;

21: 105-11.

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