Genetics of Some Beneficial Traits ◾ 143
Bac + , production of some bacteriocins (also immunity to them)
Muc + , ability to produce mucin
Resistance to some antibiotics (such as erythromycin, Em r )
R/M system, restriction/modification
The same phenotype in a species can be encoded in different-size plasmids.
Cryptic Plasmids
Many plasmids of lactic acid bacteria carry genes that encode for specific phenotypes. An example
is the plasmid pSMB74 in Pediococcus acidilactici H, which encodes genes associated with the production of a bacteriocin, pediocin AcH. However, there are other plasmids in lactic acid bacteria
for which no specific phenotypes can be assigned, and they are designated as cryptic plasmids.
Some of them are able to integrate in the host chromosome and others are able to replicate in different homologous and heterologous hosts. For this reason, some of them have been used to construct vectors for cloning and expression of genes in lactic acid bacteria and some Gram-positive
and Gram-negative bacteria. Plasmids pWVO1 and pSH71 have been extensively used to develop
a series of cloning and expression vectors.
Plasmid Replication
Plasmids of lactic acid bacteria have a high degree of sequence homology in the origin of replication (repA) and the gene encoding the replication protein (repB). Depending on the size, a plasmid
replicates either by a sigma-type or by a theta-type mechanism. In the sigma (or rolling circle)
replication mode, replication is initiated by the binding of the plasmid encoding the Rep protein
to the origin (plus-origin site). This is followed by a nick in one and a fixation of Rep at the 5´
end of the nicked strand. The other strand is replicated from the nick site, causing displacement
of the nicked strand, thus forming the shape of a sigma. The single strand is then ligated, forming
a single-stranded circle and a duplex plasmid. The replication of the single strand is then followed
to produce a double-stranded plasmid. This process generally occurs in small and cryptic plasmids
(ca. 12 kb or less), which are relatively unstable.
In the theta-type replication, initiation starts with the formation of replication forks (as in the
chromosome) resulting from separation of the base pairs. Both strands then simultaneously replicate, either in the same or in the opposite direction, and two copies of the plasmids are formed at
the end of the replication. The plasmids are more than 12 kb, stable with a limited host range, and
encode for many economically important phenotypes.
Plasmid Mapping and Sequencing
Initially, the cells of a strain are lysed and plasmids are isolated, purified, and separated on the basis
of molecular weight by agarose gel electrophoresis. The plasmid of interest is purified from the gel
and used for mapping and sequencing. The purified plasmid is subjected to single and double digestions with a set of restriction endonuclease (RE) enzymes, and the number and molecular weight of
the fragments generated after each digestion are determined. These results are used to construct the
RE map of the plasmid. Individual fragments are then sequenced to construct the complete nucleotide sequence of the plasmid and to locate the open reading frames (ORFs) and regulatory regions
in the plasmid. In many studies, the complete nucleotide sequence of a plasmid, especially if it is
Bac + , production of some bacteriocins (also immunity to them)
Muc + , ability to produce mucin
Resistance to some antibiotics (such as erythromycin, Em r )
R/M system, restriction/modification
The same phenotype in a species can be encoded in different-size plasmids.
Cryptic Plasmids
Many plasmids of lactic acid bacteria carry genes that encode for specific phenotypes. An example
is the plasmid pSMB74 in Pediococcus acidilactici H, which encodes genes associated with the production of a bacteriocin, pediocin AcH. However, there are other plasmids in lactic acid bacteria
for which no specific phenotypes can be assigned, and they are designated as cryptic plasmids.
Some of them are able to integrate in the host chromosome and others are able to replicate in different homologous and heterologous hosts. For this reason, some of them have been used to construct vectors for cloning and expression of genes in lactic acid bacteria and some Gram-positive
and Gram-negative bacteria. Plasmids pWVO1 and pSH71 have been extensively used to develop
a series of cloning and expression vectors.
Plasmid Replication
Plasmids of lactic acid bacteria have a high degree of sequence homology in the origin of replication (repA) and the gene encoding the replication protein (repB). Depending on the size, a plasmid
replicates either by a sigma-type or by a theta-type mechanism. In the sigma (or rolling circle)
replication mode, replication is initiated by the binding of the plasmid encoding the Rep protein
to the origin (plus-origin site). This is followed by a nick in one and a fixation of Rep at the 5´
end of the nicked strand. The other strand is replicated from the nick site, causing displacement
of the nicked strand, thus forming the shape of a sigma. The single strand is then ligated, forming
a single-stranded circle and a duplex plasmid. The replication of the single strand is then followed
to produce a double-stranded plasmid. This process generally occurs in small and cryptic plasmids
(ca. 12 kb or less), which are relatively unstable.
In the theta-type replication, initiation starts with the formation of replication forks (as in the
chromosome) resulting from separation of the base pairs. Both strands then simultaneously replicate, either in the same or in the opposite direction, and two copies of the plasmids are formed at
the end of the replication. The plasmids are more than 12 kb, stable with a limited host range, and
encode for many economically important phenotypes.
Plasmid Mapping and Sequencing
Initially, the cells of a strain are lysed and plasmids are isolated, purified, and separated on the basis
of molecular weight by agarose gel electrophoresis. The plasmid of interest is purified from the gel
and used for mapping and sequencing. The purified plasmid is subjected to single and double digestions with a set of restriction endonuclease (RE) enzymes, and the number and molecular weight of
the fragments generated after each digestion are determined. These results are used to construct the
RE map of the plasmid. Individual fragments are then sequenced to construct the complete nucleotide sequence of the plasmid and to locate the open reading frames (ORFs) and regulatory regions
in the plasmid. In many studies, the complete nucleotide sequence of a plasmid, especially if it is