Expression of a β-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A β-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high β-galactosidase activity but utilized lactose only slightly faster than the recipient. β-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the β-galactosidase gene. Growth rates of Lac⁺ H1 and 7962 derivatives were not affected after introduction of the clostridial β-galactosidase, even though β-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-β-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-β-galactosidase activity. We suggest that β-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-β-galactosidase genes.     

Sizing the Fusobacterium nucleatum genome by pulsed-field gel electrophoresis

Abstract The β-galactosidase (β-Gal) gene from Lactobacillus plantarum C3.8 was cloned and expressed in Lactococcus lactis and Escherichia coli . Hybridization experiments indicated that the gene is located on a plasmid and is present in other strains of Lactobacillus plantarum . Its sequence is very similar to a Leuconostoc lactis β-Gal gene. Expression of the gene, both in Lactobacillus plantarum and in Lactococcus lactis , was four-fold higher in cells grown in lactose compared to those grown in glucose. The presence of the β-Gal gene in Lactococcus lactis allowed this bacterium to be efficient in clotting milk.     

Lactose metabolism in Lactobacillus curvatus and Lactobacillus sake

Abstract The lactose metabolism was investigated in five strains of Lactobacillus curvatus and 14 strains of L. sake isolated from meat or meat-derived products. Strains with the ability to ferment lactose were found in both species. They exhibited either phospho-β-galactosidase (P-β-gal) or β-galactosidase (β-gal) activity, or both. P-β-gal activity of L. curvatus and L. sake was induced and detected only in the presence of lactose or galactose. Furthermore, catabolite repression by glucose was demonstrated. The immunological properties of the P-β-gal enzymes of these organisms resemble those of Lactococcus lactis . Several strains of L. sake but none of L. curvatus exhibited β-gal activity which was constitutive. In hybridisation experiments, the β-gal genes of L. sake and L. casei ATCC393 showed over 60% DNA-homology. The presence of β-gal genes in L. sake was demonstrated in both β-gal-producing and non-producing strains. This observations is consistent with a genetic potential of lactic acid bacteria exceeding their physiological capabilities.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Nonidentity between plasmid and chromosomal copies of ISS1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes.

The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS1 from Lc. lactis subsp. lactis ML3. The IS was named ISS1RS. The chromosome of this strain contains several copies of ISS1-like IS as assessed by hybridization. One of these copies was cloned and named ISS1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.     

Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy

The metabolism of glucose was studied in Lactococcus lactis subsp. lactis CNRZ 125 by ¹³C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)^-1 s^-1]. ³¹P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by ³¹P NMR as a function of the external pH during growth. When the external pH was 6·8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7·4. After the external pH had decreased to 5·5, the internal pH of strain CNRZ 141 had declined by 0·6 unit, whereas that of strain CNRZ 125 had decreased by only 0·2 unit of pH.     

Cloning and nucleotide sequence of the β-galactosidase gene from Lactococcus lactis ssp. Lactis ATCC7962

The gene encoding -galactosidase of Lactococcus lactis ssp. lactis ATCC7962 was cloned and its nucleotide sequence was determined. The -galactosidase of L. lactis was expressed in Escherichia coli and transformants containing this gene fragment appeared as blue colonies on LB plates containing X-gal. The -galactosidase activity of E. coli transformant was thirty times higher than that of L. lactis. The gene for the 115 kDa -galactosidase has a 2991-bp open reading frame preceded by a putative ribosome binding site. The deduced amino acid sequence show a high degree of homology to the -galactosidase of E. coli, and the putative active site residues are conserved (Glu-429 and Tyr-475)     

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

Transfer of conjugative plasmid pAMβ1 from Lactococcus lactis to mouse intestinal bacteria

Conjugal transfer of plasmid pAMβ1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 × 10⁻³ and with lower frequencies to other species. In vivo , using gastric intubation with the pAMβ1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis . However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 × 10⁻³, in mice whose anus was artificially closed after gastric intubation with pAMβ1-bearing Lactococcus lactis . These results demonstrate clearly that pAMβ1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.     

Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.     

Construction and Expression of Food-Grade β-Galactosidase Gene in <Emphasis Type="Italic">Lactococcus Lactis</Emphasis>

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.     

Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32.

An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.     

Lactose Hydrolysing Enzymes in Streptococcus lactis and Streptococcus cremoris and also in some other Species of Streptococci

Two types of Streptococcus lactis could be identified: cheese starter strains, which contain β-phosphogalactosidase and ferment lactose rapidly to lactate, and non-dairy strains, which contain both β-galactosidase and β-phosphogalactosidase and ferment lactose slowly to a variety of end products. All strains had homolactic glucose fermentations and heterolactic galactose fermentations. Other species of streptococci were examined for lactose hydrolysing enzymes and found to contain β-phosphogalactosidase, except Strep, thermophilus and Strep. faecium which had high levels of β-galactosidase. Discrepancies were found in the lactose hydrolysing enzymes content when the cells were treated in different ways.     

A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period. CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.     

The production of bioluminescent lactic acid bacteria suitable for the rapid assessment of starter culture activity in milk

Promoter elements from Lactobacillus casei were isolated with an Escherichia coli promoter probe vehicle and inserted 5' to the luxA/B genes from Vibrio fischeri located within a pCK1-based shuttle vector. Three independent promoter- lux constructs were each used to transform Lactobacillus casei, Lactococcus lactis and Lactococcus lactis subsp. diacetylactis by electroporation. Transformants of all three bacteria which expressed a bioluminescent phenotype in the presence of exogenous dodecanal were obtained. By virtue of monitoring changes in light production, these recombinant micro-organisms could form the basis of a rapid monitoring system for antimicrobial substances in milk active against starter culture bacteria. In addition, the research potential of in vivo bioluminescence for monitoring gene expression in lactic acid bacteria in situ within fermentation systems can now be addressed.     

The effects of osmotic stress on survival and alkaline phosphatase activity of Aeromonas hydrophila

Abstract Lactococcus lactis MG5267 is a plasmid-free strain in which the lactose operon is integrated in the bacterial chromosome. The chromosomal lac G gene which encodes phospho-β-galactosidase was inactivated by a double cross-over integration event. Unexpectedly, the resultant mutant was shown to retain a Lac-positive phenotype. The lysin gene from Listeria monocytogenes bacteriophage LM-4 was subsequently integrated into the chromosome of this strain such that expression of the heterologous gene was mediated by the lactose operon promoter. Expression of the lysin gene was shown to be regulated by growth on lactose. This represents an important strategy for the controlled and stabilised expression of biotechnologically useful genes in L lactis .     

Antibacterial activities of nisin Z encapsulated in liposomes or produced in situ by mixed culture during cheddar cheese ripening

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7 degrees C for 6 months. In some trials, L. innocua was added to cheese milk at 10(5) to 10(6) CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 +/- 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 +/- 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.     

Conjugal transfer of the determinants for bacteriocin (lacticin 481) production and immunity in Lactococcus lactis subsp. lactis CNRZ 481

Abstract The lacticin 481-producer (Lct⁺), L. lactis subsp. lactis (L. lactis ) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb. Novobiocin treatment of L. lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid. Conjugal transfer of the lacticin 481 structural gene ( lct ) into the plasmid free strain L. lactis IL1441 yielded Lct⁺ transconjugants at a 10⁻⁴ frequency, which carried a plasmid with an apparent size of 120–130 kb. Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L. lactis 481 and on the 120–130 kb plasmid in the transconjugants. The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.     

Comparative study on cell envelope-associated proteinases in natural isolates of mesophilic lactobacilli

Lactobacilli isolated from different natural sources were screened for the presence of cell envelope-associated proteinases (Prt⁺ strains). Among them 17 of 75 tested isolates were Prt⁺. All Prt⁺ strains were producers of a serine-type proteinase, since their proteolytic activity was inhibited by phenylmethylsulfonyl fluoride. Most of the natural isolates of mesophilic lactobacilli degraded only β-casein such as Lactobacillus paracasei subsp. paracasei strains BGLI17 and BGLI18 and Lact. rhamnosus BGEN1. Only Lact. divergens BG742 cleaved all three, α-, β- and κ-caseins, even in the presence of Ca²⁺ ions. Total DNA isolated from Lact. paracasei subsp. paracasei strains BGLI17 and BGLI18 hybridized to the lactococcal proteinase gene probes originated from Lactococcus lactis subsp. cremoris Wg2. Hybridization could not be linked to the plasmid DNA, and pulse-field gel electrophoresis analysis suggested that the proteinase genes of these two strains are most probably chromosomally located.