Structural and functional analysis of pCI65st, a 6·5 kb plasmid from Streptococcus thermophilus NDI-6

Abstract

The 6.5 kb cryptic plasmid pC165st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced. Five putative ORFs were identified. ORFl could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced 5. thermophilus plasmids, indicating that pC165st is one of the pC194 group of small Gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of Grampositive bacteria. ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis. ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modif ication systems. Variants of strain NDI-6 which lacked pC165st were readily isolated after subculture of the parent strain a t 32 OC. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 "C to 62 "C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant.

Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain The GenBank accession numbers for the sequences reported in this paper are AF153410–AF153414.

“…values, comparable to those obtained with the hsdS genes from pCIS31.1 and pIL7, were obtained when the hsdS gene encoded by pCI65st (O'Sullivan et al, 1999) was used in UC509.93 and IL1403 (E. Stanley, unpublished results).…”

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

“…2), a replication protein, a putative enolase, and HsdS, a putative type 1 restriction modification enzyme (239). These streptococcal replicons are readily lost at low temperatures but stably maintained at optimal growth temperatures of 42°C (239,298). The plasmid stability suggests that the ␣-Hsps are beneficial to lactic acid bacteria during fermentation of dairy products.…”

“…The 6.5-kb plasmid pCI65st from S. thermophilus NDI-6 codes for two almost identical ␣-Hsps (Hsp1 and Hsp2; an alignment is given in Fig. 2), a replication protein, a putative enolase, and HsdS, a putative type 1 restriction modification enzyme (239). These streptococcal replicons are readily lost at low temperatures but stably maintained at optimal growth temperatures of 42°C (239,298).…”

“…Small plasmids carry the low-molecular-mass stress proteins of Streptococcus thermophilus (239,298,299). On plasmids pER16 (4.5 kb), pER35 (10 kb), pER36 (3.7 kb), and pER341 (2.8 kb) originating from different S. thermophilus strains, the only other open reading frame encodes a rolling-circle replication protein (298,299).…”

Characterization of a Novel Integrative Element, ICE St1 , in the Lactic Acid Bacterium Streptococcus thermophilus

“…Sequence comparisons and hybridizations reveal that horizontal transfers between a large array of species of lactic acid bacteria have occurred, most likely during dairy cocultures (13,32). The most convincing evidence indicates that insertion sequences IS1191, IS981, ISS1, and IS1194 (4,5,14) and some open reading frames (ORFs) involved in exopolysaccharide synthesis (6) or in restriction-modification (24) were transferred between the lactic acid bacteria Streptococcus thermophilus and Lactococcus lactis in cocultures used during cheese manufacture. However, the mechanism of genetic exchange between these two species remains unknown, and no conjugative element has been previously characterized in S. thermophilus.…”