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Broiler production in the United States has expanded by. 58% from 1977 to 1998 (DEFRA, 2001). Aspects of this growth call for improvement, especially with ...
IMMUNOLOGY AND MOLECULAR BIOLOGY Research Note Characterization and Electrotransformation of Lactobacillus crispatus Isolated from Chicken Crop and Intestine S. S. Beasley,* T. M. Takala,* J. Reunanen,* J. Apajalahti,† and P. E. J. Saris*,1 *Division of Microbiology, Department of Applied Chemistry and Microbiology University of Helsinki, 00014-FIN; †Enteromix Research, Danisco Innovation, FIN-02460 Kantvik, Finland ABSTRACT Lactic acid bacteria originating in the intestine have recently undergone intensive study for their potential probiotic properties. Here partial 16S rRNA gene sequencing of 8 Lactobacillus strains proved them to be Lactobacillus crispatus. Fatty acid analysis confirmed strains being closely related. These strains and type strain ATCC33820 were characterized for genetic engineering potential, thus determining aerobic growth, erythromycin sensitivity, and glycine tolerance. Out of

5 plasmids, a 2.9-kb plasmid (pLEB579) was successfully introduced into 4 chicken-originated wild-type L. crispatus strains. Transformation frequency was approximately 30 transformants per microgram of DNA, the first reported electrotransformation into chicken-originated L. crispatus. In spite of its low frequency, transformation enables bioengineering of these strains to improve the probiotic function in feed adsorption, chicken health, and food safety.

(Key words: bioengineering, lactic acid bacteria, poultry, probiotic improvement) 2004 Poultry Science 83:45–48

properties as the undefined gut content of adult poultry, because the exact mechanisms of protection and the criteria for choosing defined strains are not well documented (Stavric, 1992; van der Wielen et al., 2002). Strains of Lactobacillus crispatus, being a major component of the microflora in the chicken intestine (Sarra et al., 1985), become prime candidates for developing potential probiotic cultures. This species possesses a number of characteristics satisfying the criteria for selecting probiotic cultures, e.g., its generally recognized as safe status (Pouwels et al., 1998), its adhesion to intestinal epithelium (Edelman et al., 2002), its abundance in crop and intestine, and its capacity to decrease salmonellosis in conjunction with Clostridium lactatifermentas (Van der Wielen et al., 2002). To date, no report has been made of the transformation of DNA into poultry-originated L. crispatus, a method essential to exploit this species through genetic engineering. We have succeeded in electrotransforming a plasmid into L. crispatus, opening up the potential for engineering of this species.

INTRODUCTION Broiler production in the United States has expanded by 58% from 1977 to 1998 (DEFRA, 2001). Aspects of this growth call for improvement, especially with regard to food safety. Zoonotic pathogens, such as Salmonella (Hinton et al., 1991; Line et al., 1997; van der Wielen et al., 2002), Campylobacter (Aho et al., 1992; Line et al., 1997), and Escherichia coli O157:H7 (Stavric et al., 1993), are cited as major causes of human foodborne disease outbreaks (Bean et al., 1997). Wabeck (2002) has reported Campylobacter jejuni to be in 80% of chickens. Protective cultures have been developed to reduce this risk of zoonosis in chicken. Nurmi and Rantala (1973) investigated the effects of an undefined gut content matrix of adult poultry fed to chicks to protect them from Salmonella, a phenomenon termed competitive exclusion. Feeding undefined gut contents to chicks, however, can provide a route to deliver pathogens and parasites to newly hatched chicks (Mead and Impey, 1987). Defined Lactobacillus strains in competitive exclusion preparation have been tested for inhibiting pathogenic growth in broiler chickens. These strains have not been reported to show as high inhibition

MATERIALS AND METHODS Bacterial Strains and Plasmids Eight Lactobacillus strains,2 2 previously published (Edelman et al., 2002) (Table 1), were isolated from the intestine

2004 Poultry Science Association, Inc. Received for publication May 28, 2003. Accepted for publication September 2, 2003. 1 To whom correspondence should be addressed: per.saris@ helsinki.fi. 2 Danisco Innovation, Kantvik, Finland.

Abbreviation Key: ED = Euclidian distances; ERM = erythromycin; MRS = de Man, Rogosa, and Sharpe.

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TABLE 1. Strains used in this study and nucleotide sequence accession numbers for partial 16S rRNA gene sequence (EMBL, Germany), strain origin and transformation frequency with plasmid pLEB5791 Resistance Strain

Accession number

Origin

Transformants (per µg of DNA)

Aerobic growth

ERM (µg/mL)

Nisin (IU/mL)

Glycine (%)

Reference

28mc 145mc 29mi 81mi 101mi 119mi 134mi A33 ATCC 33820

AJ421221 —2 — — AJ421222 AJ421223 AJ421224 AJ421225 —

Cecum Cecum Ileum Ileum Ileum Ileum Crop Crop —

11 — — — 33 6 — 17 —

++ + + + + + +++ ++ +

1 1 1 1 1 1 1 1 1

10 0 10 0 0 10 10 10 10

1 0.5 0.5 0 1 2 1 2 0.5

This study This study This study This study This study This study Edelman et al. 2002 Edelman et al. 2002 Skerman et al. 1980

1

Cell growth and resistance to erythromycin (ERM), nisin and glycine in MRS broth. − = no result, +++ = good growth, ++ = moderate growth, + = poor growth.

2

of 2- to 12-d-old male Ross 208 chickens fed on a commercial wheat-based diet by enrichment on Lactobacillus selective medium (LBS) agar plates3 under anaerobic conditions (Apajalahti et al., 2001). L. crispatus ATCC338204 served as a type strain. The Escherichia coli TG-1 strain (Sambrook et al., 1989) was used for further retransformation. Plasmids pLEB579 (2.9 kb) and pLEB580 (4.2 kb) (Takala and Saris, 2002) were used for the electrotransformation. Also used were the erythromycin (ERM) resistance gene and origin of replication from plasmid pVS2 (von Wright et al., 1987) as well as plasmids pLEB599 (7.5 kb) (Reunanen and Saris, 2003) and pNZ9111 (15.4 kb) (Van der Meer et al., 1993), both with the ERM resistance gene. The plasmid pLEB590 (3.1 kb) selected with the bacteriocin nisin (Takala and Saris, 2002) was also electroporated into L. crispatus cells.

Media and Growth Conditions and DNA Techniques L. crispatus strains were cultivated in deMan, Rogosa, and Sharpe (MRS) medium5 under anaerobic conditions if needed. E. coli was grown in Luria-Bertani (LB) medium (Sambrook et al., 1989). When required, ERM was used at a final concentration of 5 µg/mL for L. crispatus and 200 µg/mL for E. coli, and nisin was at a final concentration of 100 IU/mL for L. crispatus. Plasmids from L. crispatus were isolated according to the method described by O’Sullivan and Klaenhammer (1993). For E. coli plasmid isolations, a standard method was applied (Sambrook et al., 1989).

3

Becton Dickinson Microbiology Systems, Cockeysville, MD. American Type Culture Collection, Manassas, VA. 5 Difco Laboratories, Sparks, MD. 6 Sigma Chemical Co., St. Louis, MO. 7 Merck, Darmstadt, Germany. 8 Perkin Elmer Inc., Boston, MA. 9 MIDI Inc., Newark, DE. 10 Pronadisa, Hispanlab, Spain. 11 Pharmacia, Piscataway, NJ. 12 BTX Cuvettes, San Diego, CA. 4

Identification and Characterization of L. crispatus Strains The resistance to ERM6 and to nisin6 as well as the sensitivity to glycine7 and growth in aerobic conditions of each strain was tested in MRS broth4 and measured at 600 nm on a Lambda Bio ultraviolet/visible spectrophotometer.8 Whole-cell fatty acid methyl esters were prepared and analyzed as described by Va¨isa¨nen et al. (1994) using a Sherlock Microbial Identification System with the aerobic library version 1.06.9 Strains 28mc, 101mi, 119mi, 134mi, and A33 were characterized by partial 16S rRNA gene sequencing using purified DNA (Van der Meer et al., 1993) and amplified by PCR (30 s at 94°C, 60 s at 55°C, and 90 s at 72°C, this cycle was repeated 29 times and finally for 120 s at 72°C) employing universal primers pA 5′AGA GTT TGA TCC TGG CTC AG 3′ and pE′ 5′ CCG TCA ATT CCT TTG AGT TT 3′ (Edwards et al., 1989). The amplified 900-bp fragments were isolated from low-melting gel LM-3,10 purified with a chloroform-isopropanol purification method (Sambrook et al., 1989), and then sequenced by Autoread Sequencing kit with ALF DNA Sequencer.11 Sequences were compared against the NCBI Blast Library (NCBI, 2001). Phylogenetic trees were based on partial 16S rRNA sequences with TreeTop—Phylogenetic Tree Prediction (GeneBee Group, 2003) and fatty acid data (Aerobic Library Moore Version 1.06).8

Transformation To initiate electroporation, strains were grown in MRS broth overnight at 37°C and reinoculated with a 1% culture and then grown for 8 h at 37°C. Inoculation was repeated and the culture was grown in MRS broth containing 0.8% glycine and harvested at an optical density at 600 nm of 0.3 to 0.4 by centrifugation at 9,000 × g (10 min, 4°C). Cells were washed twice in ice-cold electroporation buffer (0.5 M sucrose, 7 mM K3PO4, 1 mM MgCl2, pH 7.4), resuspended in electroporation buffer in 1/100 of the original culture volume, and stored on ice for a maximum of 1 h. Plasmids pLEB579, pLEB580, pLEB590, pLEB599, and

RESEARCH NOTE

pNZ9111 were transformed into the L. crispatus strains as follows: 50 µL of cells and 500 ng of plasmid DNA were pipeted into 2-mm interelectrode-gap cuvettes,12 and a 1.5kV pulse (200 ⍀, 25 µF)13 was then applied to the cells. Cells were incubated in 2 mL of MRS broth including 2 mM CaCl2 and 20 mM MgCl2 for 3 h at 37°C. Cells containing plasmids pLEB579, pLEB580, pLEB599, and pNZ9111 were cultivated on MRS agar containing 5 µg/mL of ERM, and cells with plasmid pLEM590 were cultivated on MRS agar containing 100 IU/mL of nisin and grown for 3 d at 37°C in aerobic conditions. Negative controls without the plasmid DNA addition were treated accordingly. Transformed colonies were picked from plates and placed into MRS broth with ERM for plasmid isolation. For analysis of the L. crispatus transformants and because of the better plasmid quality when purified from E. coli, plasmids were isolated and retransformed into CaCl2-treated E. coli TG1 cells. The transformed plasmids were again isolated for analysis and run on 1% agarose gel.14

RESULTS AND DISCUSSION To facilitate the handling of a potential health-promoting strain, the bacteria should be aero-tolerant and able to survive and grow in the anaerobic conditions of the gut (Charteris et al., 1998). For this reason, the aerobic growth of L. crispatus strains was ascertained. Strains were sensitive to ERM, nisin, and glycine. No strain tolerated more than 1 µg/mL of ERM or 10 IU/mL of nisin (Table 1). Glycine has been reported to weaken the bacterial cell wall in a dose-dependent manner (Holo and Nes, 1989; Thompson and Collins, 1996). Of the Lactobacillus strains discussed here, 5 were selected for further characterization because they were resistant to 0.5% glycine. A part of the 16S rRNA gene was sequenced to determine the species of the Lactobacillus isolates. The 5 Lactobacillus strains isolated from chicken crop and intestine were identified as L. crispatus, revealing a distinct similarity (98 to 100%) to type strain L. crispatus ATCC33820 in the NCBI Blast Library as well as when compared with each other. The sequence accession numbers are listed in Table 1. The 16S rRNA sequence dendrogram demonstrated a close relationship between strains within the L. crispatus species (Figure 1A). Comparing the fatty acid composition of strains proved the species to be the same (Figure 1B). In view of the similarity of the isolated strains, the fatty acid analysis results revealed poor matching against the aerobe library strains (Sherlock version 1.06). This finding accounts for the differences in comparison with the 16S rRNA sequence dendrogram. Results disclosed unknown fatty acid peaks and several sum features, consisting mainly of different 18:2 and 18:1 fatty acid chains. When matching the Euclidian distances (ED) to one another, tested strains were clearly similar, falling into the same species category (ED < 25).

13 Gene Pulser and Pulse Controler, Bio-Rad Laboratory, Richmond, CA. 14 BioCell Products Oy, Helsinki, Finland.

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FIGURE 1. Phylogenetic trees for Lactobacillus strains based on A) partial 16S rRNA sequences TreeTop—Phylogenetic Tree Prediction. Strains with differences of less than 0.03 belong to the same species. B) Fatty acid data with Euclidian distance (ED) referring to the same species when ED < 25, same strain when ED < 10, and same substrain when ED < 6.

This result provided further evidence that the strains were L. crispatus. Strains isolated from crop displayed a close relationship (ED < 10), whereas strains taken from cecum and ileum were less closely related but suggested the same species (10 < ED < 25). Phylogenetic trees based on partial 16S rRNA gene sequences and fatty acid results demonstrated the similarity among strains isolated from crop. Analysis of the plasmid content showed that all strains had a plasmid of approximately 11 kb (data not shown). L. crispatus strains 134mi and A33 have also been reported to have an adhesion capability to chicken epithelia (Edelman et al., 2002). The strains thus serve as potential probiotic cultures (Charteris et al., 1998; Reid, 1999; Todoriki et al., 2001). Fuller (1973) demonstrated that lactic acid bacteria adhere to intestinal epithelia after hatching and remain on the intestine throughout a chicken’s life. Electrotransformation of pLEB579 yielded ERM-resistant colonies in 4 L. crispatus strains, with a transformation efficiency of less than 102 transformants per microgram of DNA (Table 1). Transformations were repeated 5 to 7 times, depending on the strain. Transformations of strains not yielding transformants in the first electroporations were retransformed several times more using higher DNA concentrations (up to 5 µg) without success. Negative controls plated on MRS agar containing 5 µg/mL of ERM did not show any growth. Electrotransformation (1 to 7 attempts, depending on the strain) with other plasmids did not produce colonies, suggesting that size has an impact on transformation efficiency. The frequency of electroporation of most bacteria generally decreases with the increasing size of the transforming DNA (Gasson and Fitzgerald, 1994). Low frequency of transformation among wild-type lactobacilli is common (Aukrust and Blom, 1992). Plasmid isolation from the L. crispatus (pLEB579) transformants resulted in preparations in which the isolated DNA was extensively degraded. For this reason, plasmid content analysis of the transformants was performed by transformation of the isolated plasmids into E. coli TG-1 from which plasmids could be isolated without degradation problems. These plasmids were mostly plasmids of

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altered size and, as a rule, larger, compared with plasmid pLEB579. Only a portion of the plasmids originating from the transformants of strains 101mi, 119mi, and A33 were of the same size as pLEB579. These plasmids displayed an Sau3AI restriction pattern identical to that in pLEB579. L. crispatus is clearly efficient in rearranging plasmids. To avoid these stability problems, expression constructs should aim at integration into the chromosome, a more stable location than multicopy plasmids. Our results reveal that genetic engineering with L. crispatus originating from poultry is indeed successful.

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