Functional Analysis and Randomization of the Nisin-Inducible Promoter for Tuning Gene Expression in Lactococcus lactis

Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the ?35 and ?10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3–5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside ?35 and ?10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.     

Isolation of sensitive nisin-sensing GFP<Subscript>uv</Subscript> bioassay <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains using FACS

Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP_uv nisin bioassay. It harbors the GFP_uv encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP_uv fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml⁻¹) for nisin than the parent strain (10 pg ml⁻¹). This showed that mutants of LAC275 could successfully be isolated using FACS.     

Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Nisin-Controlled Production of Pediocin PA-1 and Colicin V in Nisin- and Non-Nisin-Producing Lactococcus lactis Strains

The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible nisA promoter and the lactococcin A-dedicated secretion genes (lcnCD) into Lactococcus lactis strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.     

Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase

NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H₂O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD⁺ ratios in the cells.     

Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

Deletion mutants and animal models have been instrumental in the study of Helicobacter pylori pathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement during H. pylori colonization and chronic infection. To achieve this goal, we adapted the Escherichia coli Tn10-derived tetracycline-inducible expression system for use in H. pylori. The ureA promoter was modified by inserting one or two tet operators to generate tetracycline-responsive promoters, named uPtetO, and these promoters were then fused to the reporter gfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboring tetR and uPtetO-GFP was characterized by measuring GFP activity and by immunoblotting. The two tet-responsive uPtetO promoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of the uPtetO-GFP construct and the nature of the promoter driving expression of tetR influenced the strength of the uPtetO promoters upon induction. Integration of uPtetO-GFP and tetR constructs at different genomic loci was stable in vivo and did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expression in vivo during chronic infection. These results open new experimental avenues for dissecting H. pylori pathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.     

Expression and delivery of an endolysin to combat Clostridium perfringens

Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-l-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P _nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.     

Tightly Regulated Expression Vectors for Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi, a bacterial species important in bioremediation and biopesticide degradation, are hindered by the lack of suitably regulated gene expression system. A tightly regulated gene-expression system was developed for O. anthropi using the lacI^q gene and a re-engineered coliphage T5 promoter containing completely symmetrical DNA segment that binds more efficiently to the lactose repressor. The β-galactosidase activity was increased 57-fold when the expression of the re-engineered T5 promoter was induced. The degree of induction was controllable by varying the concentration of inducer isopropyl-β-d-thiogalactopyranoside.     

Molecular and Genetic Characterization of a Novel Nisin Variant Produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

Purification and Some Properties of Diplococcin from Streptococcus cremoris 346

Eleven of 150 Streptococcus cremoris strains examined produced the bacteriocin diplococcin. The diplococcin activity spectrum was restricted to S. cremoris and Streptococcus lactis strains, and none of a wide range of other gram-positive or gram-negative strains were inhibited. The diplococcin produced by S. cremoris 346 was purified by ammonium sulfate precipitation and column chromatography. Purified diplococcin was very unstable at room temperature and lost 75% of its activity after heating at 100°C for 1 min. The proteolytic enzymes trypsin, pronase, and α-chymotrypsin completely inactivated diplococcin. The amino acid composition showed a high content of acidic and neutral acids and a correspondingly low content of basic amino acids, including one residue of ornithine per mole. From the amino acid analysis a molecular weight of 5,300 was estimated. Diplococcin was readily distinguished from the S. lactis bacteriocin nisin by its restricted activity spectrum, its biological properties, and by cross-reaction experiments.     

Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any ‘genetic toolkit’ is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/tetO) regulatable system from transposon Tn10 for use in Streptomyces. The synthetic Tc controllable promoter (tcp), tcp830, was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1–100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830. The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1–1 μg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducer.