Construction of vectors for inducible and constitutive gene expression in Lactobacillus.
ABSTRACT Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.
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ABSTRACT: The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 megadaltons) was genetically marked with the chloramphenicol resistance (Cmr) gene from pC194. The recombinant plasmid (pGK1, 2.4 megadaltons) replicated and expressed Cmr in Bacillus subtilis. From this plasmid an insertion-inactivation vector was constructed by inserting the erythromycin resistance (Emr) gene from pE194 cop-6. This plasmid (pGK12, 2.9 megadaltons) contained a unique BclI site in the Emr gene and unique ClaI and HpaII sites outside both resistance genes. It was stably maintained in B. subtilis at a copy number of approximately 5. pGK12 also transformed Escherichia coli competent cells to Cmr and Emr. The copy number in E. coli was about 60. Moreover, pGK12 transformed protoplasts of Streptococcus lactis. In this host both resistance genes are expressed. pGK12 is stably maintained in S. lactis at a copy number of 3.Applied and Environmental Microbiology 11/1984; 48(4):726-31. · 3.68 Impact Factor
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ABSTRACT: Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription. Expression of the lac genes in the absence of the inducer arabinose indicates that transcription originates within the inserted fragment; non-expression of lac with arabinose present indicates that transcription is terminated by the fragment. Using different cloning vectors, DNA fragments generated by a wide variety of restriction endonucleases can be inserted between ara and lac. This procedure has been used to identify and isolate endonuclease-generated DNA fragments from theEscherichia coli chromosome, various R plasmids, bacteriophage T5 and Drosophila melanogaster that contain nucleotide sequences capable of functioning as promoters in E.coli. A characteristic level of lac expression is determined by the amount of transcription that proceeds to the lac genes from a promoter located within each fragment. The effects of genetic regulatory mechanisms acting on a promoter can be assayed by alterations in the level of lac expression.These cloning vectors were also used to bring structural genes located within an inserted DNA fragment under the control of the ara promoter. Insertion of HindIII endonuclease-generated fragments carrying the tetracycline-resistance determinant of pSC101 or the sulfonamide-resistance determinant of the R6-5 plasmid into such vectors resulted in arabinose-induced resistance to tetracycline or sulfonamide.Journal of Molecular Biology 05/1980; 138(2):179-207. · 3.91 Impact Factor
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ABSTRACT: Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.Proceedings of the National Academy of Sciences 04/2005; 102(11):3906-12. · 9.74 Impact Factor