Development of food-grade cloning and expression vectors for Lactococcus lactis.

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.

Assessment of stress response of the probiotic Lactobacillus acidophilus.

Different aspects of stress response of Lactobacillus acidophilus were investigated. First, the sublethal and lethal levels of bile, heat, and NaCl stresses were determined. They were 0.05% and 0.5% (bile), 53 degrees C and 60 degrees C (heat), and 2% and 18% (NaCl), respectively. To evaluate the effect of each stress at log phase, log-phase cultures were challenged directly with the lethal level of each stress (control) and were compared to log-phase cultures that were pre-exposed to the sublethal level prior to the exposure at the lethal level (test). Some, if not most, of the cells were killed in the control cultures against each of the three stresses. However, in the test cultures, the number of cells that had survived increased significantly. It appears that L. acidophilus is capable of displaying adaptive response to stress. The adaptive response to one stress was also shown to provide cross-protection against different stresses tested. The effect of each stress on stationary-phase cultures was also investigated. In contrast to log-phase culture, stationary-phase culture was inherently resistant to stress.

Molecular characterization of a new abortive infection system (AbiU) from Lactococcus lactis LL51-1.

This study reports on the identification and characterization of a novel abortive infection system, AbiU, from Lactococcus lactis. AbiU confers resistance to phages from the three main industrially relevant lactococcal phage species: c2, 936, and P335. The presence of AbiU reduced the efficiency of plaquing against specific phage from each species as follows: 3.7 x 10(-1), 1.0 x 10(-2), and 1.0 x 10(-1), respectively. abiU involves two open reading frames, abiU1 (1,772 bp) and abiU2 (1,019 bp). Evidence indicates that AbiU1 is responsible for phage resistance and that AbiU2 may downregulate phage resistance against 936 and P335 type phages but not c2 type phage. AbiU appeared to delay transcription of both phage 712 and c2, with the effect being more marked on phage c2.

Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

Survival response and rearrangement of plasmid DNA of Lactococcus lactis during long-term starvation.

The survival response of Lactococcus lactis during long-term starvation was investigated. The cells were cultured with different levels of glucose (the sole energy source) and either were kept in the resultant spent medium or transferred to fresh medium (without glucose) for up to 2 years. The survival of the cells during starvation was not dependent on the nature of transition phase, as expected, but on the nature of medium in which the cells were kept. The proliferation of cells, despite the apparent lack of glucose, could have been due to some cells being able to utilize the small amounts of peptides still present in the spent medium or to use energy sources provided by the breakup of dead cells. The 1- and 2-year-old cultures contained cells with vastly changed morphotypes. When these isolates were examined, it was revealed that the original plasmids present in the parent were rearranged in a certain way, and an entirely new plasmid was generated. Changes were also evident in the chromosomal DNA and in gene expression. Furthermore, all of the isolates exhibited a growth advantage relative to the parent cells when grown in energy-limiting media. When they were tested against different types of stresses, they exhibited a higher resistance against the bile salt and hydrogen peroxide stresses compared to the parent. Because of the similar changes observed in the 2-year-old isolates, a similar survival strategy may be operational in those cells that survive for that length of time.

LldI, a plasmid-encoded type I restriction and modification system in Lactococcus lactis.

A plasmid-encoded type I restriction and modification (R-M) system, designated LldI, was identified in Lactococcus lactis biovar diacetylactis LD10-1. LldI consists of three genes encoding endonuclease, methylase and specificity subunits, respectively. RT-PCR analysis revealed that the three genes are co-transcribed as a polycistronic mRNA in L. lactis. The specificity subunit of LldI differs significantly in the target recognition domains from those of other type I R-M systems, suggesting that LldI confers a novel specificity in L. lactis.

A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis.

Two regulatory genes (lcoR and lcoS) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (cat). RT-PCR analysis indicates that lcoR and lcoS are organized within an operon, controlling the transcription of cat in a copper-inducible manner. The amino acid sequences deduced from lcoR and lcoS show homology to the response and sensor proteins of known two-component regulatory systems. Deletion within either lcoS or both genes inactivated the copper-dependent activity, suggesting the presence of no trans-acting lcoR and lcoS homologs in the lactococcal host chromosome. The transcription start site involved in copper induction was mapped by primer extension.

Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis.

A plasmid-encoded phage abortive infection mechanism (AbiL) was identified from Lactococcus lactis biovar. diacetylactis LD10-1. AbiL conferred complete resistance to the small isometric-headed phage phi 712 (936 species) and partial resistance to the prolate-headed phage phi c2 (c2 species) when introduced into L. lactis LM0230. However, AbiL was not effective against the small isometric-headed phage ul36 (P335 species). The AbiL determinant was sequenced and it consists of two open reading frames, abiLi and abiLii. Their encoded proteins did not share significant homology with any known proteins in the protein databases. Transcriptional analysis indicated that abiLi and abiLii are organized as a single operon. Deletion within abiLii abolished the phage resistance. The levels of four phi c2-specific transcripts, three within the early transcribed region and one within the late transcribed region, were examined by RT-PCR, no effect of AbiL on synthesis of these transcripts was detected, suggesting that AbiL may act at a point after the transcription of phi c2 in L. lactis.

Differentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses.

Lactococcus lactis subspecies lactis (L. lactis ssp. lactis) and Lactococcus lactis subspecies cremoris (L. lactis ssp. cremoris) were investigated in respect to their response to acid, bile-salt and freezing stresses. First, the sublethal and lethal levels of each stress were determined for both subspecies. For acid stress, the levels were pH 4.5 and 2.5, respectively, for L. lactis ssp. lactis, and pH 5.0 and 3.0, respectively, for L. lactis ssp. cremoris. For bile-salt stress, the levels were 0.03 and 0.1%, respectively, for L. lactis ssp. lactis, and 0.01 and 0.04%, respectively, for L. lactis ssp. cremoris. For freezing stress, 10 degrees C was used as the sublethal temperature and -20 degrees C was used as the lethal temperature for both subspecies. To evaluate the effect of each stress at log phase, a log-phase culture was challenged directly with the appropriate lethal level (control culture) and a second log-phase culture was pre-exposed to the appropriate sublethal level prior to testing survival under normally lethal conditions (test culture). Some, if not most, of the cells were killed in the control cultures for all three stresses. However, in the test cultures, the viability was significantly improved for all of the L. lactis ssp. lactis strains tested, but not for the L. lactis ssp. cremoris strains. It appears, therefore, that L. lactis ssp. lactis is capable of displaying adaptive response to stresses, whereas L. lactis ssp. cremoris seems to lack this phenotype or the response is much weaker in this subspecies. The effect of each stress on stationary-phase cultures was also investigated. Unlike the log-phase cultures, the stationary-phase cultures of both subspecies, challenged directly with the lethal levels, were highly resistant to each of the three stresses tested.

LlaFI, a type III restriction and modification system in Lactococcus lactis.

We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactis LL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence. LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis.

A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal theta-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 degrees C and 37 degrees C, whereas derivatives that lack cisE were highly unstable at 37 degrees C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal theta-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 degrees C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 degrees C.

Conservation of the major cold shock protein in lactic acid bacteria.

Primers designed from consensus regions of the major cold shock gene of different bacterial species were used in PCR amplification of Lactic Acid Bacteria (LAB). An appropriately-sized PCR product was obtained from Lactococcus lactis subsp. lactis LL43-1 and MG1363; Lactococcus lactis subsp. cremoris LC10-1, LC11-1, and LC12-1; Streptococcus thermophilus ST1-1; Enterococcus faecalis EF1-1; Lactobacillus acidophilus LA1-1; Lactobacillus helveticus LH1-1; Pediococcus pentosaceus PP1-1; and Bifidobacterium animalis BA1-1. The PCR products were cloned and sequenced. The deduced amino acid sequences displayed high sequence similarity with the major cold shock proteins of Escherichia coli and Bacillus subtilis and the human Y-box factor. The amino acid residues of the cold shock domain implicated in nucleic acid binding in several unrelated species were also highly conserved in the LAB strains. It is possible, therefore, that this protein in LAB may also act as a transcriptional enhancer to other cold shock genes and/or act as an RNA chaperone unwinding tightly folded RNA molecules.

Effect of cold shock on protein synthesis and on cryotolerance of cells frozen for long periods in Lactococcus lactis.

Aspects of the cold-shock response in Lactococcus lactis subsp. lactis LL41-1 were investigated. First, it was determined whether new proteins were synthesized in response to cold shock. Cell-free extracts were prepared from a cold-shocked (exposed to 10 degreesC for 5 h) culture (cfe-cs) and from a non-cold-shocked (held at 30 degreesC continuously) culture (cfe-non), and were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A protein of approximately 6.3 kDa was present in the cfe-cs and appeared not to be present in the cfe-non. No other changes were evident. Second, the effect of cold shock on cryotolerance of cells that have been frozen at -20 degreesC for up to 1 year was examined. Without the cold-shock treatment prior to freezing the cell viability following freezing for 1 day was 34%, 14 days 32%, 182 days 7%, and 364 days 0.2%. However, with the cold shock treatment it was 83%, 82%, 12%, and 0.8%, respectively. It appears that cold shock significantly improves cryotolerance of the cells for short periods of freezing, but the protective effect was less marked following longer storage periods.

Involvement of antisense RNA in replication control of the lactococcal plasmid pND324.

pND324 belongs to a family of closely related theta-type plasmids from Lactococcus lactis. An antisense RNA, termed countertranscript (ctRNA), was identified which is complementary to the leader sequence of the mRNA that encodes RepB, a protein essential for plasmid replication. When the synthesis of ctRNA was abolished by site-directed mutagenesis within its promoter region, the mutant replicon showed a 1.8-fold increase in copy number. Similar ctRNA promoter sequences are readily identifiable in 12 other published lactococcal theta-type plasmids, suggesting that they all encode a similar ctRNA-mediated regulatory mechanism.

The effect of nisin concentration and nutrient depletion on nisin production of Lactococcus lactis.

The kinetics of nisin production was studied in batch cultures using a construct of Lactococcus lactis subsp. lactis C2SmPrt-, containing a transposon (TnNip) that encodes nisin production. The introduction of TnNip into C2SmPrt- significantly lowered the specific growth rate and the maximum A620 reached was reduced from 15.2 to 11.0. The effect of nisin concentration and nutrient depletion on nisin production of the construct, C2SmPrt-(TnNip), was examined. Nisin production was found to be inhibited by high concentrations of nisin, when grown in excess nutrient, even though growth of the culture continued because nutrient limitation was not operating. However, in low nutrient concentrations nisin production was limited by nutrient depletion. The specific growth rate of C2SmPrt-(TnNip) was altered, by using different nutrient concentrations and different sugars, in order to examine the relationship between nisin production and growth. Nisin production was shown to be growth-associated for most of growth, but near the end of growth, when the specific growth rate was 0.05 h-1 or less, the production ceased.

Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance.

When Lactic Acid Bacterial cultures were frozen at -20 degrees C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10 degrees C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25-37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested.

Isolation, cloning and characterisation of the abiI gene from Lactococcus lactis subsp. lactis M138 encoding abortive phage infection.

Plasmid pND852 (56 kb) encodes nisin resistance and was isolated from Lactococcus lactis ssp lactis (L. lactis) M138 by conjugation to L. lactis LM0230. It conferred strong resistance to the isometric-headed phage phi 712 and partial resistance to the prolate-headed phage phi c2. A 2.6 kb HpaII fragment encoding phage resistance was cloned into the streptococcal/Bacillus hybrid vector pGB301 to generate pND817. The mechanism of phage resistance encoded by pND817 involved abortive infection and this was illustrated by a reduction in burst size from 166 to 6 at 30 degrees C and from 160 to 90 at 37 degrees C. Partial resistance was therefore retained at 37 degrees C. DNA sequencing revealed that the abortive infection was encoded by a single open reading frame (ORF), designated abiI, encoding a 332 amino acid protein. Neither abiI nor the predicted product showed significant homology to any existing sequence in the GenBank database. Frame shift mutation at the unique EcoRI site within the ORF resulted in loss of the Abi+ phenotype, confirming that the ORF is responsible for the encoded phage resistance.

A novel plasmid-encoded phage abortive infection system from Lactococcus lactis biovar. diacetylactis.

A 16-kb plasmid (pND859) was identified from Lactococcus lactis biovar. diacetylactis UK12922 which encodes phage resistance to the small isometric phage 712 when tested in L. lactis LM0230. The gene encoding phage abortive infection, designated abi-859, was localized on a 1.2-kb region which consists of an open reading frame (ORF) of 846 bp preceded by a potential ribosome-binding site and a putative promoter region. A helix-turn-helix region typical of DNA-binding motifs was identified near the N-terminal of the abi-859 product, suggesting a possible interaction with the phage DNA.

Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis.

The 8.8-kb Lactococcus lactis plasmid pND302 encodes resistance to cadmium (CdR). Regions of pND302 involved in replication and CdR were subcloned and sequenced. The replication region is localized on a 1.5-kb region and consists of an open reading frame (repB) preceded by a noncoding AT-rich sequence (ori) which is highly homologous to lactococcal theta-type replicons. The CdR determinant is localized on a 2.9-kb region and encodes putative proteins similar to the Cd(2+)-specific P-type efflux ATPase (CadA) and the transcriptional regulatory repressor (CadC) identified in Staphylococcus aureus, Bacillus firmus, and Listeria monocytogenes. Similar CdR determinants were also detected by PCR in other CdR plasmids isolated from different L. lactis strains.

Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant.

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.