Genes In Lactococcus Lactis Research Articles

Antimicrobial Impact forLactococcus lactissubsp.lactisA15 andEnterococcus faeciumA15 Isolated from Some Traditional Egyptian Dairy Products on Some Pathogenic Bacteria

Lactococcus lactis subsp. lactis A15 and Enterococcus faecium A15 had antimicrobial activity against Listeria monocytogenes EGDEe 107776. Bacteriocins produced from isolated strains are stable between pH 5 and 8 and stable also until 100C for 10 min. The amount of bacteriocin produced by Lactococcus lactis subsp. lactis A15 reached to the maximum (2560 AU/mL) at the end of logarithmic phase while Enterococcus faecium A15 was observed in logarithmic phase. PCR results revealed the presence of nisin Z structural gene in Lactococcus lactis subsp. lactis A15 and enterocin B gene for Enterococcus faecium A15. Lactococcus lactis subsp. lactis A15 was free from genes of asa1, gelE, espfm, cylA, efaAfs. Otherwise, Enterococcus faecium A15 did not contain an asal, espfm and cylA genes. Tested strains were sensitive to kanamycin, vancomycin and gentamycin. Tested strains were not able to hydrolyze red blood cells. Practical Applications Nowadays, the addition of bacteriocins and/or bacteriocin-producing lactic acid bacteria (LAB) as natural food preservatives to food products (i.e. cheese and yoghurt) has been considered with the aim of increasing the food safety of these products. Bacteriocins have successfully been used to control Listeria monocytogenes. Lactococcus lactis subsp. lactis A15 could be used as starter culture or co-culture in food system for preventing the growth of Listeria monocytogenes and increasing the safety and shelf-life of dairy products. Enterococcus faecium A15 was able to produce gelatinase but not able to hydrolyze red blood cells. On the other hand, the bacteriocin produced from Enterococcus faecium A15 may find application as bio-preservatives for increasing the shelf-life of Egyptian Dairy products.

Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

Group B Streptococcus pili mediate adherence to salivary glycoproteins

Group B Streptococcus (GBS) is a leading cause of neonatal sepsis, pneumonia and meningitis, and is responsible for a rising number of severe invasive infections in adults. For all disease manifestations, colonisation is a critical first step. GBS has frequently been isolated from the oropharynx of neonates and adults. However, little is understood about the mechanisms of GBS colonisation at this site. In this study it is shown that three GBS strains (COH1, NEM316, 515) have capacity to adhere to human salivary pellicle. Heterologous expression of GBS pilus island (PI) genes in Lactococcus lactis to form surface-expressed pili demonstrated that GBS PI-2a and PI-1 pili bound glycoprotein-340 (gp340), a component of salivary pellicle. By contrast, PI-2b pili did not interact with gp340. The variation was attributable to differences in capacities for backbone and ancillary protein subunits of each pilus to bind gp340. Furthermore, while GBS strains were aggregated by fluid-phase gp340, this mechanism was not mediated by pili, which displayed specificity for immobilised gp340. Thus pili may enable GBS to colonise the soft and hard tissues of the oropharynx, while evading an innate mucosal defence, with implications for risk of progression to severe diseases such as meningitis and sepsis.

Molecular characterisation of ABC-type multidrug efflux systems in Bifidobacterium longum

Administration of probiotic bacteria such as Bifidobacterium spp. can prevent antibiotic associated diarrhoea since they can survive the often harsh conditions of the gut. In Bifidobacterium longum subsp. longumT NCIMB 702259, two gene clusters, with homology to the ATP-binding cassette (ABC) family of efflux transporters, were identified and studied to assess their functional contribution to antibiotic resistance. Both gene clusters contained two genes encoding putative efflux transporters and a regulator gene, upstream of the structural genes. Reverse transcriptase analysis indicated that the genes in each cluster were transcribed as operons, one where all three genes, including a putative MarR-type regulator were transcribed together (BLLJ_1496/1495/1494), and the other where the two ABC-type transporter genes (BLLJ_1837/1836) were co-transcribed, but excluded the putative regulator (BLLJ_1838). Heterologous expression of the cloned BLLJ_1837/1836 transporter genes in Lactococcus lactis conferred resistance to erythromycin and tetracycline by increasing the minimum inhibitory concentration between 1.5 and 3 fold. The presence of these genes also allowed a 16% increase in the efflux of Hoechst 33342 from L. lactis cells containing the two transporter genes, BLLJ_1837-6. In B. longum, an increase in the levels of transcription of 3.3 fold was observed for BLLJ_1837 in the presence of erythromycin, as measured by multiplex quantitative PCR. In contrast to this, the expression of the genes of the BLLJ_1495/1494 operon in L. lactis did not show significant drug resistance functionality. Gel shift experiments showed that in the BLLJ_1495/1494 operon, the putative MarR-type regulator protein (BLLJ_1496) bound with high affinity to the DNA sequence upstream of the operon in which it was located but this was not erythromycin dependent. This study demonstrated the occurrence of a drug inducible, ABC-type transporter system (BLLJ_1837/1836) in B. longum as well as a putative MarR-type DNA binding protein (BLLJ_1496).

Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds

Hydroxyacid dehydrogenases limit the conversion of α-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the α-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953ΔpanE was an efficient strain producing volatile compounds related to cheese aroma.

Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis

The goal of this study was to construct a single-tube multiplex molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for the detection of 17 microbial pathogens commonly associated with septicaemia. The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene-specific sequences. The pathogens and their targeted genes were: Klebsiella spp. (phoE); Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp. (thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf), Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified gene in Lactococcus lactis, served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence-specific low-temperature fluorescent probe derived from molecular beacons. All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA. This assay used new technology to achieve an advance in the field of molecular diagnostics: a single-tube assay for detection of pathogens commonly responsible for septicaemia.

Inactivation of the ybdD Gene in Lactococcus lactis Increases the Amounts of Exported Proteins

Random insertional mutagenesis performed on a Lactococcus lactis reporter strain led us to identify L. lactis ybdD as a protein-overproducing mutant. In different expression contexts, the ybdD mutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknown regulatory processes that play a role during protein secretion.

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis.

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD + Cofactor Recycling

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.

Characterization of the tyramine-producing pathway in Sporolactobacillus sp. P3J

A sporulated lactic acid bacterium (LAB) isolated from cider must was shown to harbour the tdc gene encoding tyrosine decarboxylase. The isolate belonged to the Sporolactobacillus genus and may correspond to a novel species. The ability of the tdc-positive strain, Sporolactobacillus sp. strain P3J, to produce tyramine in vitro was demonstrated by using HPLC. A 7535 bp nucleotide sequence harbouring the putative tdc gene was determined. Analysis of the obtained sequence showed that four tyramine production-associated genes [tyrosyl-tRNA synthetase (tyrS), tyrosine decarboxylase (tdc), tyrosine permease (tyrP) and Na(+)/H(+) antiporter (nhaC)] were present and were organized as already described in other tyramine-producing LAB. This operon was surrounded by genes showing the highest identities with mobile elements: a putative phage terminase and a putative transposase (downstream and upstream, respectively), suggesting that the tyramine-forming trait was acquired through horizontal gene transfer. Transcription analyses of the tdc gene cluster suggested that tyrS and nhaC are expressed as monocistronic genes while tdc would be part of a polycistronic mRNA together with tyrP. The presence of tyrosine in the culture medium induced the expression of all genes except for tyrS. A clear correlation was observed between initial tyrosine concentration and tyramine production combined with an increase in the final pH reached by the culture. Finally, cloning and expression of the tyrP gene in Lactococcus lactis demonstrated that its product catalyses the exchange of tyrosine and tyramine.

Construction and Expression of Food-Grade β-Galactosidase Gene in Lactococcus Lactis

Recombinant Lactococcus lactis MG1363/pMG36e-lacZ exhibiting high β-galactosidase activities were constructed by us in the previous study. However, erythromycin resistance present in these recombinants restricted their practical application in food preparation. This study was conducted to delete the gene coding for erythromycin resistance present in recombinant L. lactis, as a result of which these bacteria express food-grade β-galactosidase. In this study, the recombinant plasmid pMG36e-lacZ was digested with restriction enzymes BclI and HpaI and the food-grade plasmid FGZW was rebuilt. FGZW was transformed into Escherichia coli JM109 and L. lactis MG1363. Erythromycin resistance, enzyme activity determination, gene sequencing and SDS-PAGE analysis indicated that these new recombinant bacteria lost erythromycin resistance and its relevant gene but still expressed β-galactosidase activities, although a decrease in the expression of β-galactosidase of these new strains was observed. The β-galactosidase food-grade expression system was successfully constructed and it could provide a new solution for the management of lactose intolerance. These results might promote the usage of gene-modified microorganisms and related technology in the food sector, which has the highest priority for food safety.

Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants

The facultative anaerobic bacterium Lactococcus lactis has been used as a host for expression of a gene isolated from the anaerobic rumen fungus Neocallimastix sp. The coding region of the cellulase gene was obtained from the fungus with the aid of polymerase chain reaction amplification. The gene was then transformed into pCT vector system and the constructed recombinant plasmid was introduced into two L. lactis strains (IL403 and MG1363) by electroporation. The gene encoding the fungal originated cellulase was expressed in both strains successfully although the expression level was relatively lower in comparison with the original enzyme activity. Genetically modified L. lactis strains were used as silage inoculants for pre-biodegradation of the plant biomass during ensiling. That treatment resulted in a notable reduction of the acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents of the plant biomass used as silage material. Inoculation with recombinant strain IL1043 resulted in 4.8 and 9.7 % decrease in NDF and ADF contents, respectively while the inoculation of silage with strain MG1363 decreased the ADF content by >5 %.

Functional Expression of Bacterial Zymobacter palmae Pyruvate Decarboxylase Gene in Lactococcus lactis

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.

Identification of Lactococcus lactis genes required for bacteriophage adsorption.

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.

Casitone-mediated expression of the prtP and prtM genes in Lactococcus lactis subsp. lactis BGIS29

Casitone added to chemically defined medium (CDM) specifically influenced the regulation of the proteinase activity in the natural isolate Lactococcus lactis subsp. lactis BGIS29. Comparative analysis of the influence of casitone present in CDM on the proteolytic activity of strain BGIS29 and of L. lactis subsp. cremoris strains SK11 and Wg2 indicated that the proteolytic activity of strains BGIS29 and SK11 is casitone-dependent, whereas that of strain Wg2 is not. The regulatory region of the prt genes of strain BGIS29 was cloned and sequenced. The nucleotide sequence of the prt regulatory region of strain BGIS29 was distinctly different from that of L. lactis subsp. cremoris strains SK11 and Wg2. Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene ( gusA) were used to study medium-dependent expression of two divergent prtP and prtM promoters of strain BGIS29 (designated P (prtP) and P (prtM), respectively). beta-Glucuronidase assays and Northern blot analysis showed that the activities of P (prtP) and P (prtM) are controlled by casitone at the transcriptional level.

Transcriptional and translational regulation of alpha-acetolactate decarboxylase of Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of alpha-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.

Identification of a Gene Cluster Encoding Krebs Cycle Oxidative Enzymes Linked to the Pyruvate Carboxylase Gene in Lactococcus lactis ssp. lactis C21,2

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.

Improving nisin production by increasing nisin immunity/resistance genes in the producer organism Lactococcus lactis

The effect on nisin production of increasing nisin immunity/resistance genes in Lactococcus lactis subsp. lactis MG1363 was investigated. The 60-kb nisin immunity/resistance plasmid pND300, which was isolated from a non-nisin-producing strain, encodes five genes involved in nisin immunity/resistance, which are very similar to those of the immunity/resistance system encoded by the nisin-production transposon. The introduction of pND300 into MG1363(TnNip) resulted in the construct being able to produce significantly more nisin than the parent MG1363(TnNip). The introduction of pND314, which contains the nisin immunity/resistance genes subcloned into pSA3, into MG1363(TnNip) allowed the strain to grow more rapidly than the parent MG1363(TnNip) with a concomitant increase in the rate of nisin production. This work illustrates that introduction of pND300 and a derivative containing the nisin immunity/resistance system of pND300 into MG1363 (TnNip) can result in significant alterations to the kinetics of nisin production. These observations indicate approaches that may be used successfully to improve the economics of nisin production.

Use of luciferase genes as biosensors to study bacterial physiology in the digestive tract.

A method based on the use of the bacterial luciferase genes was developed in order to investigate Lactococcus lactis gene expression in the mouse digestive tract. Germfree mice were monoassociated with different strains containing transcriptional fusions of promoters with the luciferase genes. Our results demonstrate that this method is readily applicable to the study of promoter strength and physiology of bacteria in the digestive tract.

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene.

Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed beta-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.