Detection, Characterization, and In Vitro and In Vivo Expression of Genes Encoding S-Proteins in Lactobacillus gallinarum Strains Isolated from Chicken Crops

Abstract

Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17 genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains (lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR, respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat.Lactobacilli are commonly detected in gut samples collected from animal species, especially those from rodents, pigs (reviewed in reference 35), and chickens (13,15,20,24,45). In the chicken gut, lactobacilli are present in the crop (15), the ileum (20, 24), and the ceca (45). Lactobacilli dominate the relatively simple microbiota of the chicken crop (13), where at least some strains adhere to the crop epithelial surface (8, 12). Lactobacillus gallinarum, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus salivarius, and Lactobacillus reuteri persist in the crop throughout the life of broilers raised under commercial farming conditions (15). The first three of these species are members of the Lactobacillus acidophilus complex, which contains six closely related species in two subgroups (11,18,22). DNA homology group A contains L. acidophilus (A1), L. crispatus (A2), Lactobacillus amylovorus (A3), and L. gallinarum (A4), whereas DNA homology group B contains Lactobacillus gasseri (B1) and L. johnsonii (B2).Surface-associated crystalline protein layers, termed S-layers, have been detected on cells of strains belonging to group A of the L. acidophilus complex (GAA) (4), with each strain having two different S-protein genes. S-proteins are the individual subunits that comprise S-layers (reviewed in references 30 and 32). It has been speculated that S-layers may ...

Resource partitioning in relation to cohabitation of Lactobacillus species in the mouse forestomach

“…Hagen et al (2005) screened 38 crop isolates of L. gallinarum for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes (lgs).…”

Comparison and Utilization of Repetitive-Element PCR Techniques for Typing Lactobacillus Isolates from the Chicken Gastrointestinal Tract

“…Preparation of the cells and PFGE plugs was performed using a protocol described previously (28). Samples and the midrange PFG marker I (New England Biolabs) were run on 1% (wt/vol) agarose gels in 0.5ϫ TBE buffer (0.045 M Tris-borate, 0.001 M EDTA, pH 8.3; Amresco, Solon, OH) at 14°C using a CHEF-DR III system (Bio-Rad, Gladesville, Australia).…”

“…For example, L. agilis (7,57,62), L. amylovorus (11), L. crispatus (27,62), L. gallinarum (27,28,62), L. johnsonii (27,57,62), L. panis (43), L. reuteri (27,43,62), L. salivarius (27,62), L. saerimneri (62), L. vaginalis (7,57,62), P. acidilactici (1,38), P. pentosaceus (53), Enterococcus cecorum (7), E. faecium (26,31), E. faecalis (46), Enterococcus villorum (61), and Veillonella spp. (69) have previously been described as inhabitants of the chicken GIT.…”

The role of the crop in poultry production

“…At least some lactobacilli are capable of binding to the crop epithelium to form biofilm layers that are relatively uniquely found on non-secretory stratified squamous epithelium (Edelman et al, 2002;Fuller and Turvey, 1971;Lebeer et al, 2011). The nature of the adherence is not fully understood, but S-layer proteins may be responsible (Hagen et al, 2005). Hagen et al (2005) speculate that the diversity of Slayer proteins among Lactobacillus gallinarum strains may provide an opportunity for different strains to live together without direct competition for attachment sites on the crop epithelium.…”

“…The nature of the adherence is not fully understood, but S-layer proteins may be responsible (Hagen et al, 2005). Hagen et al (2005) speculate that the diversity of Slayer proteins among Lactobacillus gallinarum strains may provide an opportunity for different strains to live together without direct competition for attachment sites on the crop epithelium. Adherence is vital for colonisation to prevent washout in intestinal compartments with continuous digesta passage.…”