**4. Materials and methods**

*Synthetic Biology - New Interdisciplinary Science*

*GenBank deposited nucleotide sequence L77091.1 [26].*

tip fibrillum and joins it to the rod [24].

*prs* (*pap*-related sequence) operon [18].

of the P blood group and on uroepithelial cell fimbriae [18]. Further receptors for P-fimbriae are present on erythrocytes from pigs, pigeon, fowl, goats and dogs but not on those from cows, guinea pigs or horses [19]. These fimbriae are encoded in the *pap* operon, consisting of 11 different genes (see **Figure 2A**): *papA* (558 bp), *papB* (315 bp), *papC* (2511 bp), *papD* (720 bp), *papE* (522 bp), *papF* (504 bp), *papG* (1008 bp), *papH* (588 bp), *papI* (234 bp), *papJ* (582 bp) and *papK* (537 bp) [20]. The product of the *papA* gene is the major subunit protein A (19.5 kDa) [19]. In *papB* a regulatory protein (13 kDa) is encoded. PapB is necessary for the activation of the *papA* expression [21]. PapC (80 kDa) is located in the outer membrane and forms the assembly platform for fimbrial growth. PapD (27.5 kDa) is present in the periplasmic space and is involved in the translocation of fimbrial subunits across the periplasmic space to the outer membrane prior to assembly. PapE (16.5 kDa), PapF (15 kDa) and PapG (35 kDa) are minor fimbrial components. PapG is the adhesin molecule conferring the binding specificity [19]. PapH (20 kDa) terminates fimbrial assembly and helps anchor the fully grown fimbriae to the cell surface [22]. PapI (12 kDa) is another regulatory protein involved in *papA* expression due to activation of *papB* promoter [21]. PapJ (18 kDa) is a periplasmic protein required to maintain the integrity of P-fimbriae [23]. PapK (20 kDa) regulates the length of the

*Scheme of pap and F17 operon and annealing sites of the used primers. Genes in the operon are presented as* 

*(A) Scheme of pap operon. The scheme of pap operon was drawn based on the GenBank deposited nucleotide sequence X61239.1 [20] and (B) scheme of F17 operon. The scheme of F17 operon was drawn based on the* 

*boxes. The positions of used primers to amplify the studied genes are marked with arrows.* 

Many variants of P-fimbriae exist. PapA molecules from different P-fimbrial serovariants have a high degree of similarity at the N and C termini, while the central portions of PapA exhibit a great variation in the primary structure. This central part of PapA is hydrophilic and exposed and hence under selective pressure from the host immune system. Substantial heterogeneity is also between different minor fimbrial subunits (PapE, PapF and PapG) [19]. In addition also P-fimbriarelated fimbriae, the so-called Prs-fimbriae, exist. Prs-fimbriae are encoded in the

F17-fimbriae are found on pathogenic *E. coli* strains, isolated from infections in domestic animals. They are mainly detected on bovine and ovine *E. coli* associated with diarrhoea or septicaemia but also on *E. coli* from other hosts, including humans. The F17 adhesin binds to N-acetyl-d-glucosamin receptors of bovine intestinal cells;

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**3.2 F17-fimbriae**

**Figure 2.**

#### **4.1 Bacterial strains, growth media and conditions**

The analysed 24 clinical haemolytic *E. coli* strains [5] originated from dogs with diarrhoea and were isolated at the Veterinary Microbiological Diagnostics Centre of Utrecht University, the Netherlands. Some more details about the strains are given in **Table 1**. As positive control strains, a dog uropathogenic *E. coli* strain (strain 1473) and a cattle mastitis *E. coli* strain (strain E5) from Wim Gaastra's *E. coli* collection were used [31].

All used bacterial strains were stored at −80°C as a suspension in a 1:1 mixture of L-broth and glycerol as published by Garcia et al. [32]. The strains were grown overnight on LB plates and in liquid LB medium at 37°C. When grown in liquid LB medium, the flasks with the bacterial culture were incubated with aeration.

#### **4.2 Isolation of chromosomal DNA**

Chromosomal DNA was isolated from all 24 clinical haemolytic *E. coli* strains [5] and strains used for positive controls [31] using a slightly modified protocol based on the protocol of miniprep of bacterial genomic DNA published by Ausubel et al. [33]. To summarise, 2 ml of an overnight bacterial culture was centrifuged for 2 min at 14,000 rpm at room temperature. The obtained bacterial pellet was resuspended in 567 μl of buffer TE and 6 μl of 0.5 M EDTA. The suspension was incubated for 15 min at −80°C. Following the incubation at −80°C, the suspension was thawed, and 10 μl of 25 mg/ml proteinase K solution was added. The suspension was mixed thoroughly, and 30 μl of 10% SDS was added to the suspension and mixed thoroughly again. A 2-hour incubation at 37°C followed, and then 100 μl of 5 M NaCl was added to the suspension and mixed thoroughly. Next 80 μl of CTAB/NaCl was added, mixed thoroughly again and incubated at 65°C for 10 min. After the incubation the suspension was treated with 200 μl of chloroform/isoamyl alcohol and centrifuged for 5 min at 14,000 rpm at room temperature. The aqueous supernatant was transferred to a fresh microcentrifuge tube and treated with 100 μl of phenol/ chloroform/isoamyl alcohol and centrifuged for 5 min at 14,000 rpm at room temperature. The aqueous supernatant was transferred to a fresh microcentrifuge tube, and the DNA in the aqueous supernatant was precipitated with addition of 0.6 volume of isopropanol. The precipitated chromosomal DNA was transferred to a fresh microcentrifuge tube containing 100 μl of 70% ethanol. The precipitated


*MSHA is an abbreviation of mannose-sensitive haemagglutination, and MRHA is an abbreviation of mannoseresistant haemagglutination. The erythrocytes are abbreviated as follows: B, bovine erythrocytes; E, equine erythrocytes; C, canine; O, ovine; P, porcine. NT, non-typable.*

#### **Table 1.**

*Characteristics of the 24 studied E. coli strains [5, 31].*

DNA in 70% ethanol was pelleted with centrifugation (10 min at 14,000 rpm at room temperature). The 70% ethanol was then removed and the chromosomal DNA pellet air-dried at 37°C. Finally the chromosomal DNA pellet was dissolved in 100 μl of sterile distilled water.

#### **4.3 PCR mixtures for PCR amplification of P- and F17-fimbrial genes**

One μl of the isolated chromosomal DNA was used in 50 μl PCR mixtures consisting of 20 pmol of each primer, 0.2 mM dNTP mixture and 0.625 U of *Taq*-polymerase in PCR buffer [5]. In PCRs for P-fimbrial genes for positive control samples, the isolated chromosomal DNA of the dog uropathogenic *E. coli* strain (strain 1473) was used. In PCRs for F17-fimbrial gene for positive control samples, the isolated chromosomal DNA of the cattle mastitis *E. coli* strain (strain E5) was used. In all PCRs for the negative control, sterile distilled water was used [31].

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shown in **Figure 2**.

**Table 2.**

**PCR for gene(s)**

final extension for 10 min at 72°C.

**4.5 Agarose gel electrophoresis**

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions*

Primers used in the PCRs to amplify the studied genes are listed in **Table 2**.

*papA* 22 84.1

*papEF* POP 63.8

*papG* GOD1 65.5

*F17G* F17G-1 72.4

**Primers (name, sequence of the primer 5′→ 3′) Tm (°C) of primers**

23 68.3

BAD 72.9

GOD2 62.4

F17G-2 76.4

Predicted primer annealing sites of the used primers on the target operons are

The expected sizes of PCR products were determined with the 'Primer-BLAST' online tool (data set nr organism *Escherichia coli*) on the Internet page of the National Center for Biotechnology Information, US National Library of Medicine (http://www. ncbi.nlm.nih.gov) as follows: *papA*—552 bp (GenBank deposited nucleotide sequence LR134092.1), 555 bp (GenBank deposited nucleotide sequence CP025703.1), 534 bp (GenBank deposited nucleotide sequence CP018957.1), 564 bp (GenBank deposited nucleotide sequence CP029579.1) and 561 bp (GenBank deposited nucleotide sequence CP024886.1); *papEF*—1372 bp (GenBank deposited nucleotide sequence CP027701.1), 1373 bp (GenBank deposited nucleotide sequence CP028304.1), 1367 bp (GenBank deposited nucleotide sequence CP026853.1) and 1371 bp (GenBank deposited nucleotide sequence LR134238.1); *papG*—1000 bp (GenBank deposited nucleotide sequence CP026853.1) and 1003 bp (GenBank deposited nucleotide sequence M20181.1); and *F17G*—888 bp (GenBank deposited nucleotide sequence AF055313.1)

Samples of isolated chromosomal DNA (5 μl of isolated chromosomal DNA and 1 μl of 6 × loading dye) were subjected to analysis with agarose gel electrophoresis using

and 885 bp (GenBank deposited nucleotide sequence CP001162.1).

The PCR amplification in all the reactions for all studied genes was carried out in the following steps: heating at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and the

**4.4 Primers and PCRs used to amplify P- and F17-fimbrial genes**

ATGATGAATTCGGTTATTGCCGGTGCGG

CTGAGAATTCAGGTTGAAATTCGC

ATGGTACCCAGCTTTGTTATTTTCC

TGGCAATATCATGAGAAGCTTT

CAGGCGGCAGTTTCATTTATTGGC

CCGGACTGAGGGTGACGTTTCCGT

*Primers and their melting temperatures (Tm) used to amplify the studied genes.*

CCACGTTTGAATTGACATATCG

CCGTAGCAATGCCCGGGC

*DOI: http://dx.doi.org/10.5772/intechopen.85164*
