Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

Abstract

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep 2009 and orf17. One of these genes, rep 2009 , codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori 2009 ), which was identical for all four phages. DNA fragments representing the ori 2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori 2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep 2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

“…22 to create a ⌬ltnJ mutant, from which 1,056 nucleotides has been deleted. To express ltnJ, a DNA product generated with PCR primers 5 and 6 was digested with XbaI and HinDIII; ligated with similarly digested pNZ44, an E. coli-L. lactis shuttle vector with a constitutive P44 promoter (25); and electroporated into E. coli DH5␣ to generate a transcriptional fusion between the constitutive lactococcal promoter P44 and ltnJ. Plasmid was isolated, sequenced to ensure integrity, and electroporated into ⌬ltnJ to create ⌬ltnJpNZ44ltnJ.…”

“…Furthermore, we investigate whether any alternate residues can efficiently compensate for the removal of the D-alanines from these peptides. (25), respectively. L. lactis strains were grown in M17 broth supplemented with 0.5% glucose (GM17) or GM17 agar at 30°C unless stated otherwise.…”

Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome

“…The proofreading enzyme KOD Hotstart DNA polymerase (Invitrogen) was used. PCR products were purified by using the Qiagen PCR purification kit, cut with the appropriate restriction enzymes and ligated to similarly digested plasmid pNZ44 (37). The resulting ligation mix was added to chemically competent E. coli TOP10 cells (Invitrogen) and transformants were selected on LB plates containing 10 g/ml chloramphenicol.…”

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

“…Phusion Hot Start II high fidelity DNA polymerase (Thermo Fischer Scientific), FastDigest restriction enzymes (Thermo Fischer Scientific), and T4 DNA ligase (Promega) were used as recommended by the manufacturers' instructions. The pili cassette pAC44 expresses the L. rhamnosus GG genes spaA and srtC1 under the control of the constitutive promoter P44 (47). The plasmid pAC44 was obtained as follows.…”