The Lactococcus lactis nisin-sucrose conjugative transposon Tn5276

The aim of the research described in this thesis was to identify and characterize the genetic element carrying the information for nisin biosynthesis and sucrose proficiency, the nisin-sucrose element. The thesis has the following outline. Chapter 2 describes our current knowledge of nisin genetics and the recent advances in the protein engineering of nisin. The cloning and analysis of several parts of the nisin-sucrose element of L. lactis strain NIZO R5 are described in the next two chapters. Chapter 3 deals with the prenisin gene, nis A, and its surrounding region, which includes the insertion sequence IS 1068 . In Chapter 4, the organization and transcription of the sucrose ( sac ) operon and the characterization of the gene for sucrose-6-phosphate hydrolase are described. The different parts of the nisin-sucrose element were used as DNA probes to investigate the nature and location of this element (Chapter 5). It was shown that the nisin-sucrose element of L. lactis NIZO R5 is a large (70 kb), conjugative transposon, designated Tn 5276. Chapter 6 describes the isolation and analysis of the genes that encode the transposition functions of Tn 5276. The experiments leading to a model for excision and insertion of Tn 5276 are described in Chapter 7. Chapter 8 deals with the distribution and evolution of nisin-sucrose elements in L. lactis . The thesis is completed with the summary and concluding remarks (Chapter 9) and a Dutch summary (Chapter 10)

Distribution and evolution of nisin sucrose elements in Lactococcus lactis.

The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed

Distribution and evolution of nisin sucrose elements in Lactococcus lactis.

The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed