Characterization of <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> plasmid pCB18 and development of broad host range shuttle vector for lactic acid bacteria

Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods, and they are regarded as potential food-grade hosts for protein expression. The aim of this study was to develop a broad-host-range shuttle vector for the genetic study and biotechnological evaluation of this genus by using a Leuconostoc-derived plasmid. A cryptic plasmid, pCB18, was obtained from Leuconostoc citreum CBNU75; its nucleotide sequence was 1,821 bp long and had only 39.2% G+C content. A Leuconostoc-Escherichia coli shuttle vector, pLeuCM, was constructed by combining pCB18 and pEK104, and it was successfully replicated in both E. coli and L. citreum. The shuttle vector was replicated by following the rolling circle replication mechanism, and it showed over 80% segregational stability after 100 generations of cell division. The β-galactosidase gene of Lactobacillus plantarum was subcloned into pLeuCM, and this construct was successfully expressed in L. citreum. The pLeuCM plasmid was replicated in L. citreum, L. mesenteroides, Lb. plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. These results demonstrate that pLeuCM can be used as a potential gene-delivery tool for many lactic acid bacteria.     

Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034

Lactobacillus buchneri is probably the most beneficial microorganism for efficient preservation of animal feed silages made from grass, maize and other plant material against aerobic spoilage. Its obligatory heterofermentative nature, acid resistance and robustness have drawn attention to this species for applications as silage starter culture as well as for genetic engineering. For the first time, two cryptic plasmids present in the same L. buchneri strain, L. buchneri CD034, were isolated, sequenced and characterized. The larger plasmid, designated pCD034-1 was found to be 3424 bp in length with a G + C content of 38.36%. The smaller plasmid, designated pCD034-2 was found to be 2707 bp in length with a G + C content of 38.60%. On both plasmids we predicted three open reading frames. On pCD034-1, ORF 1 encodes a putative replication protein which shares 99% identity with the RepA protein of a Lactobacillus plantarum derived pC194/pUB110-family plasmid. ORF 2 encodes a putative protein of unknown function. ORF 1 and ORF 2 of pCD034-2 correspond to RepA and RepB proteins similar to those of plasmid pLB4 from L. plantarum. ORF 3 of both plasmids encodes a putative mobilization protein similar to that of the pediococcal plasmid pF8801. Double strand origins, putative single strand origins and typical mobilization start signals were identified. Both plasmids were shown to be maintained at relatively high plasmid copy numbers. Two shuttle vectors carrying the origins of replication of pCD034-1 and pCD034-2 were constructed and used to successfully transform two other species isolated from the same environment. Hence, we consider the two novel L. buchneri plasmids a valuable resource for the generation of shuttle and expression vectors for LAB.     

Use of a Novel Escherichia coli-Leuconostoc Shuttle Vector for Metabolic Engineering of Leuconostoc citreum To Overproduce d-Lactate

Determination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC13302 revealed that it contains 20,721 bp, a G+C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stable Escherichia coli-Leuconostoc shuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced into Leuconostoc, Lactococcus lactis, and Pediococcus. This shuttle vector was used to engineer Leuconostoc citreum 95 to overproduce d-lactate. The L. citreum 95 strain engineered using plasmid pMBLT02, which overexpresses d-lactate dehydrogenase, exhibited enhanced production of optically pure d-lactate (61 g/liter, which is 6 times greater than the amount produced by the control strain) when cultured in a reactor supplemented with 140 g/liter glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of a d-lactate overproduction system in other Leuconostoc strains and Lactococcus lactis.     

Heterologous expression and secretion of <Emphasis Type="Italic">Lactobacillus amylovorus</Emphasis> α-amylase in <Emphasis Type="Italic">Leuconostoc citreum</Emphasis>

To develop a gene expression system for Leuconostoc genus, construction of expression vector and expression of a heterologus protein in Leuconostoc was performed. α-Amylase gene from Lactobacillus amylovorus was cloned into a Leuconostoc cloning vector, pLeuCM, with its own signal peptide. pLeuCMamy was introduced into Leuconostoc citreum CB2567 and a successful expression of α-amy gene was confirmed by enzyme activity assays. About 90% of α-amylase activity was detected in the culture broth, revealing most of expressed α-amylase was secreted out cells. The signal sequence of α-amy gene is a good candidate for the secretion of heterologous protein by using Leuconostoc host-vector system.     

Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Diversity of lactic acid bacteria from Miang,a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041^T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.     

Construction of hybrid plasmid vectors for cloning inEscherichia coli,Bacillus subtilis,and the cyanobacteriumAnacystis nidulans

Two hybrid plasmids capable of acting as shuttle cloning vectors inAnacystis nidulans andBacillus subtilis were constructed by in vitro ligation. One construct, pMG202, consists of theB. subtilis vector pNN101 and the endogenous cyanobacterial plasmid pUH24. This 14.6 kb plasmid confers chloramphenicol resistance in both hosts and tetracycline resistance inB. subtilis. A second vector, pMG101, consists of pNN101 linked to theA. nidulans-Escherichia coli chimeric plasmid pCB4 and is 12.9 kb in size. The pCB4 portion of the vector enables pMG101 to replicate in the third host,E. coli, and confers ampicillin resistance in this bacterium as well as inA. nidulans. Both plasmids possess identical uniqueStu I sites which permit insertional inactivation of the chloramphenicol resistance gene; and, in addition, identical uniqueXho I sites are present on both vectors. Each vector also has a third unique site:Sma I on pMG101 andXba I on pMG202.     

New method for electroporation of Lactobacillus species grown in high salt

We here describe a new method for electroporation of Lactobacillus species, obligately homofermentative and facultatively heterofermentative, based on the cell-wall weakening resulting from growth in high-salt media. For L. casei, optimum transformation efficiency of up to 10⁵ transformants per microgram of plasmid DNA was achieved following growth in the presence of 0.9 M NaCl. Plasmids of different sizes and replication origins were also similarly transformed. These competent cells could be used either directly or stored frozen, up to 1 month, for future use, with similar efficiency. This protocol was assayed with different Lactobacillus species: L. delbrueckii subsp. lactis, L. paracasei, L. plantarum and L. acidophilus, and it was found that they were transformed with similar efficiency.     

Diversity and Probiotic Potential of Lactic Acid Bacteria Isolated from Horreh,a Traditional Iranian Fermented Food

The aim of this study was to evaluate the probiotic potential of lactic acid bacteria (LAB) strains isolated from Horreh. Some probiotic properties, e.g., resistance to acid, bile tolerance, antibacterial activity, and antibiotic susceptibility, were investigated. A total of 140 Gram-positive and catalase-negative isolates from Horreh were subjected to identification and grouping by cultural methods and the 16S rRNA sequencing. The new isolates were identified to be Lactobacillus (fermentum, plantarum, and brevis) Weissella cibaria, Enterococcus (faecium and faecalis), Leuconostoc (citreum and mesenteroides subsp. mesenteroides) and Pediococcus pentosaceus. Probiotic potential study of LAB isolates showed that Lb. plantarum and Leu. mesenteroides subsp. mesenteroides isolates were able to grow at pH 2.5 and 3.5. Lactobacillus plantarum (isolate A44) showed the highest cell hydrophobicity (84.5%). According to antibacterial activity tests, Listeria innocua and Staphylococcus aureus were the most sensitive indicators against the selected LAB strains, while Escherichia coli and Bacillus cereus were the most resistant. In addition, all the isolated LAB species were resistant to vancomycin. The results of the present study suggested that the Lactobacillus fermentum and plantarum isolated from Horreh, characterized in this study, have potential use for industrial purposes as probiotics.     

Construction,properties, and application of the pCB5 plasmid,a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1–3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

     

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Screening for potential probiotic from spontaneously fermented non-dairy foods based on in vitro probiotic and safety properties

The aim of this study was to screen potential probiotic lactic acid bacteria from Chinese spontaneously fermented non-dairy foods by evaluating their probiotic and safety properties. All lactic acid bacteria (LAB) strains were identified by 16S rRNA gene sequencing. The in vitro probiotic tests included survival under low pH and bile salts, cell surface hydrophobicity, auto-aggregation, co-aggregation, antibacterial activity, and adherence ability to cells. The safety properties were evaluated based on hemolytic activity and antibiotic resistance profile. The salt tolerance, growth in litmus milk, and acidification ability were examined on selected potential probiotic LAB strains to investigate their potential use in food fermentation. A total of 122 strains were isolated and identified at the species level by 16S rRNA gene sequencing and included 62 Lactobacillus plantarum, 40 Weissella cibaria, 12 Lactobacillus brevis, 6 Weissella confusa, and 2 Lactobacillus sakei strains. One W. cibaria and nine L. plantarum isolates were selected based on their tolerance to low pH and bile salts. The hydrophobicity, auto-aggregation, co-aggregation, and antagonistic activities of these isolates varied greatly. All of the 10 selected strains showed multiple antibiotic resistance phenotypes and no hemolytic activity. The highest adhesion capacity to SW480 cells was observed with L. plantarum SK1. The isolates L. plantarum SK1, CB9, and CB10 were the most similar strains to Lactobacillus rhamnosus GG and selected for their high salt tolerance and acidifying activity. The results revealed strain-specific probiotic properties were and potential probiotics that can be used in the food industry.     

Construction of recombinant mercury resistant Acidithiobacillus caldus

A mercury-resistant plasmid of pTMJ212 which was able to shuttle between Acidithiobacillus caldus and Escherichia coli was constructed by inserting the mercury resistant determinants, the mer operon of Acidithiobacillus ferrooxidans, into the IncQ plasmid of pJRD215. pTMJ212 was transferred from Escherichia coli into Acidithiobacillus caldus through conjugation. Furthermore, pTMJ212 was transferred back from Acidithiobacillus caldus into Escherichia coli, thereby confirming the initial transfer of pTMJ212 from Escherichia coli to Acidithiobacillus caldus. Compared to the control, the cell growth of the recombinant Acidithiobacillus caldus increased markedly under mercury (Hg²⁺) stress especially at Hg²⁺ concentrations ranging from 2.0 to 4.5 μg/ml.     

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.     

Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae

Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp.     

Display of heterologous protein on the surface of Lactobacillus plantarum by using the CspI anchor protein

The C-terminus of the putative cell surface protein CspI which contains one putative LPxTG motif region and a signal peptides fragment were amplified from L. plantarum CICC6024, and the green fluorescent protein gene gfp was amplified from the plasmid pACGFP. The three genes were ligated and the fusion gene was named SgfpL. The fusion gene SgfpL was then cloned into shuttle expression vector pMG36e and transformed into L. plantarum. SDS-PAGE identified that the fusion protein was expressed and the band of fusion protein was observed at the predicated molecular size. Fluorescence assay, western blot against GFP antibody, protease accessibility and SDS sensitivity assays were performed to determine that the GFP was successfully displayed on the surfaces of L. plantarum cells and the maximum display capacity of the GFP fusion protein was ca. 65 μg?ml^?1. The fermentation condition experiments determined that the amounts of GFP fusion protein were increased at a higher temperature and reached the peak at 2.5 h. Then, the β-galactosidase from Bifidobacterium bifidum was functionally displayed on the surface of L. plantarum cells via CspI to demonstrate the applicability of the CspI-mediated surface display system.     

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat _GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0?×?10^?7 and 4.7?×?10^?7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H.?pylori recipients, with pHel2 showing an efficiency of 2.0?×?10^?5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylorirecA ⁺ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.?pylori demonstrate the general usefulness of?this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.