Introduction
Dairy propionic acid bacteria (PAB) include the four
species Propionibacterium acidipropionici, P. freudenreichii,
with the two subspecies freudenreichii and. shermanii, P.
jensenii and P. thoenii, that can be distinguished on the
bases of DNA homology values (Cummins and Johnson,
1986; de Carvalho, 1994). These bacteria play a posi-
tive role in the manufacture of Swiss-type cheeses, but
can cause red spotting and late blowing defects in long
ripening cheeses. For their economic importance the
development and diffusion of PAB deserve to be better
investigated especially through a correct identification
of the isolates.
Studies on the structure of ribosomal operons (rrn) have
demonstrated that the intergenic region 16S–23S is
highly polymorphic in bacteria for the different number
and type of tRNA inserts, mostly tRNA
glu
, tRNA
ala
and
tRNA
ile
, for sequence divergence in the rRNA and tRNA
genes processing sites and in the interposed nucleotide
stretches. In addition, up to ten rrn operons, that may
differ in the spacer region, can be present in a single
bacterial genome (Brosius et al., 1981; Lehner et al.,
1984; Iwami et al., 1984; Jarvis et al., 1988; Bacot and
Reeves., 1991; Garnier et al., 1991; Nour et al., 1995).
Using primer set G1–L1, of sequence complementary
to highly conserved regions flanking about 40
nucleotides upstream and downstream the 16S–23S
spacer, Jensen et al. (1993) have shown that several
bacterial species of pathogens can be identified on the
basis of the variability in size and number of the PCR
amplified fragments. In subsequent experiments, further
characterizations of the amplified products were applied
in epidemiological studies improving the recognition at
the species and serotype level (Van Lith and Aarts, 1994;
Abed et al., 1995; Drebot et al., 1996).
In this research the PCR amplification of the intergenic
region 16S–23S, followed by the restriction endo-
nuclease analysis of the amplification products, was
evaluated for the identification of PAB strains isolated
from different sources.
Materials and methods
The 67 PAB strains used in this study are listed in
Table 1.
The isolates were identified using standard biochemical
tests (Britz and Riedel, 1991). All strains were cultured
in Yeast Extract Lactate broth (YEL) at 30°C for 48 h
in anaerobic conditions.
DNA extraction
Genomic DNA was extracted from 1 ml of 48 h culture
as described by de Carvalho (1994). The DNA concen-
tration was determined spectrophotometrically at
260 nm.
PCR amplication and restriction
PCR amplification with primers G1 (5'GAAGTCG-
TAACAAGG 3') and L1 (5'CAAGGCATC-
CACCGT3') was performed as reported by Jensen et al.
(1993) in 25 l reactions.
PCR and restriction products were separated on 1.5%
agarose gel and stained with 0.5 g/ml ethidium
bromide.
The amplification products from one PCR tube were
precipitated with ethanol and resuspended in 12 l of
1:10 diluted restriction buffer containing 0.2 U/l
of Hinf I. The restriction was carried out for 2 h at
37°C.
1
1
1
1
1
1
© 1997 Chapman & Hall Biotechnology Techniques · Vol 11 · No 3 · 1997 159
16S—23S Ribosomal spacer
polymorphism in dairy propionibacteria
F. Rossi, M. Sammartino and S. Torriani *
Dipartimento di Scienze e Tecnologie Agro-Alimentari, Ambientali e Microbiologiche, Universit degli Studi del
Molise, 86100 — Campobasso, Italy
The 16S—23S spacer region was amplied from 67 strains of dairy propionibacteria using primers G1 and L1. All
species are readily distinguishable from the different molecular weights of the amplied bands and sequence poly-
morphisms among the most similar groups can be enhanced by Hinf I restriction.
24 pts min base to base from Key words to line 1 of text
Biotechnology Techniques, Vol 11, No 3, March 1997, pp. 159–161