Characterization of a theta-replicating plasmid from Streptococcus thermophilus

Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D). Here, we report the characterization of plasmids of group B and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B. Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode in S. thermophilus. Finally, a classification of 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.     

Characterization of a bacteriocin produced by Streptococcus thermophilus ST134

A pH-dependent adsorption/desorption technique was used to screen Streptococcus thermophilus strains for the production of bacteriocins. Agar-diffusion tests with S. thermophilus strains as targets identified 13 out of 41 strains as producers of antibacterial activity. Thermophilin A, the bacteriocin-like substance present in the culture supernatant of S.thermophilus ST134 was purified to homogeneity by ammonium sulfate precipitation and ion-exchange chromatography, followed by ultrafiltration. Thermophilin A is a relatively heat-stable and apparently glycosylated bacteriocin with a bactericidal mode of action against sensitive cells.     

Characterization of superoxide dismutase in Streptococcus thermophilus.

Streptococcus thermophilus AO54 possesses a single manganese-containing superoxide dismutase (MnSOD). The enzyme was found to be insensitive to cyanide or to a modified H2O2 treatment. The enzyme is expressed in a growth-phase-dependent fashion, increasing three- to fourfold upon entry into stationary phase. The specific activity for MnSOD was the same under anaerobic or aerobic conditions and was not induced by the presence of paraquat under aerobic conditions.     

Characterization of Streptococcus thermophilus Host Range Phage Mutants

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10⁻⁶. Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.     

Characterization of Streptococcus thermophilus host range phage mutants

To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10(-6). Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus.     

Complete genome sequence of Streptococcus thermophilus strain ND03

Streptococcus thermophilus strain ND03 is a Chinese commercial dairy starter used for the manufacture of yogurt. It was isolated from naturally fermented yak milk in Qinghai, China. We present here the complete genome sequence of ND03 and compare it to three other published genomes of Streptococcus thermophilus strains.     

Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains

The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity of CNRZ 385 was localized on the cell wall. The optimum pH for enzymatic activity at 37°C was around 7.0. Serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, inhibited the enzyme activity in both strains. The divalents cations Ca²⁺, Mg²⁺, and Mn²⁺ were activators, while Zn²⁺ and Cu²⁺ were inhibitors. β-Casein was hydrolyzed more rapidly than α_s1-casein. The results of DNA hybridization and immunoblot studies suggested that the S. thermophilus cell wall proteinase and the lactococcal proteinase are not closely related.     

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction

The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minus-origin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria. Received: 25 November 1997 / Received last revision: 13 April 1998 / Accepted: 19 April 1998     

Complete genome sequence of the pigmented Streptococcus thermophilus strain JIM8232

Streptococcus thermophilus is a dairy species commonly used in the manufacture of cheese and yogurt. Here, we report the complete sequence of S. thermophilus strain JIM8232, isolated from milk and which produces a yellow pigment, an atypical trait for this bacterium.     

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.     

Properties of a putative cambialistic superoxide dismutase from the aerotolerant bacterium Streptococcus thermophilus strain LMG 18311

The aerotolerance of the lactic - fermentative bacterium Streptococcus thermophilus is mainly based on the key antioxidant function of superoxide dismutase (StSOD). In this work, the comparison of recombinant StSOD (rStSOD) forms obtained from two different initiation triplets indicated that the enzyme from S. thermophilus strain LMG 18311 spans 201 residues. rStSOD is organised as a homodimer, even though protein aggregates are formed in concentrated solutions. The capability of binding and exchanging Fe or Mn in the active site classifies rStSOD as a putative cambialistic enzyme; the moderate preference for iron is counteracted by a 1.5-fold higher activity measured for the Mn-containing form. The enzyme is thermostable, being its half-inactivation time 10 min at 73.5°C; the energetic parameters of the heat inactivation process are regulated by the level of Mn cofactor. The effect of Mn content on the rStSOD sensitivity towards inhibitors and inactivators was also evaluated. Sodium azide acts as a weak inhibitor of rStSOD and its Mn content does not greatly affect this sensitivity. Concerning the physiological inactivator hydrogen peroxide, the Mn-enriched rStSOD displays a great resistance; a moderate sensitivity is instead observed in the presence of a low Mn content. Contrary to hydrogen peroxide, sodium peroxynitrite is a powerful inactivator, a behaviour enhanced in the Mn-enriched enzyme. All these results were compared with the corresponding data previously reported for the cambialistic SOD from the taxonomically related S. mutans. In S. thermophilus the regulation of the enzyme functions by the Mn content appears less relevant with respect to S. mutans.     

Highly bioluminescent Streptococcus thermophilus strain for the detection of dairy-relevant antibiotics in milk

Inefficient translational initiation is often the cause of poor foreign gene expression in gram-positive organisms. The expression of bacterial luciferase (lux) genes in Streptococcus thermophilus (bioluminescence) was improved by addressing this problem in two ways: by ribosome-binding site (RBS) replacement, and by enhancing lux RBS access by polymerase chain reaction modification either alone or combined with translational coupling to a truncated upstream open- reading frame (orf') having its own RBS. Lactococcal expression signals were employed for plasmid-based lux expression. The same constructs were used to monitor bioluminescence in Lactococcus lactis, as well as two non-lactic bacterial strains, for comparison. High lux expression was achieved in all four organisms with a heterodimeric thermostable enzyme. Surprisingly, where ready access to the lux RBS was predicted, translational coupling to the lactococcal orf remained a prerequisite for detectable lux expression in L. lactis. In contrast, high bioluminescence in S. thermophilus was independent of coupling. Consistent with these observations, inspection of published gene sequences suggests that RBS strength may be a more important factor in translation in S. thermophilus than in L. lactis. Using reduced light production in highly bioluminescent S. thermophilus as an indicator of biocide presence in milk, test times could be significantly shortened compared with a commercial test utilizing the related non-bioluminescent strain. lux genes appear to be sensitive, exponential-phase reporters of gene activity in S. thermophilus, an organism with molecular biology and genetics that remain largely unstudied.