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Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor.

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.

Acetates↗

Application of in vivo bioluminescence to the study of ionophoretic action.

Ionophores (carbonyl cyanide m-chlorophenylhydrazone; valinomycin; and the hop-derived compounds colupulone, trans-isohumulone and trans-humulinic acid) reduced the rate of dodecanal-dependent light emission from IuxA/B-transformed cells of Lactococcus lactis subsp. diacetylactis F712 when the cells were suspended in a buffered medium (pH 6.4) containing glucose. This allowed an assay for ionophores to be devised and permitted measurement of the effects of such compounds on the test organism by the use of a non-destructive technique in real time.

Anti-Bacterial Agents↗

Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin.

A new ATP bioluminescence-based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic-nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin-luciferase. Nisin-induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quantification of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN(tot):AN(ext) ratio = 1. For an initial concentration of 1.4 x 10(7) bacteria/mL, the nisin effectiveness threshold is 3.4 +/- 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells.

Adenine Nucleotides↗

DNA-based gels for oral delivery of probiotic bacteria.

A single-stranded DNA, readily extracted from industrial discarded salmon milt, was used to prepare hydrogels and complex gels by cross-linking with gelatin and kappa-carrageenan, for the oral delivery of probiotic bacteria. The complex gels showed a higher protective capability over the hydrogels for approximately one log scale. However, the hydrogels were more stable during storage at 4 degrees C. The Lactobacillus and Lactococcus due to protection of the hydrogels could better tolerate to acid than the Bifidobacterium. Furthermore, food-graded hydrogels were prepared and optimized to a similar protective capability for future applications.

Administration, Oral↗

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at </=15 degrees C and yields of active tetramer increased as the concentration of unfolded subunits decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0 degrees C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 microM subunit, respectively) when incubated at 15 degrees C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl(2), and KCl.

Amino Acid Oxidoreductases↗

Molecular analysis of lipid macroamphiphiles by hydrophobic interaction chromatography, exemplified with lipoteichoic acids.

Analyzed were (I) the oligoglucosyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Enterococcus hirae containing Glc(alpha 1-2)Glc(alpha 1-3)acyl2-Gro and a phosphatidyl derivative thereof as lipid anchor, (II) the poly(digalactosyl, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing the same glycolipid and an acyl derivative of it, and (III) the N-acetylglucosaminyl-, alanyl-substituted poly(glycerophosphate)lipoteichoic acid of Staphylococcus aureus with Glc(beta 1-6)Glc(beta 1-3)acyl2Gro as the sole lipid moiety. Hydrophobic interaction chromatography on octyl-Sepharose separated lipoteichoic acids I and II into two peaks according to the number of fatty acids. Within each peak further fractionation occurred in the order of decreasing length of the hydrophilic chain. A similar fractionation was observed within the single peak of lipoteichoic acid III. With lipoteichoic acid I and III the extent of glycosylation decreased with decreasing length of the hydrophilic chain whereas the content of alanine ester remained either constant or increased. Variations in the oligosaccharide pattern of lipoteichoic acid I and in the fatty acid composition of all lipoteichoic acids could also be observed. Collectively, the data provide for the first time a detailed picture of the complex polydispersity of lipoteichoic acids comprising the number of fatty acids, the length of the hydrophilic chain, the kind and extent of chain substitution, and the fatty acid composition. The procedure will also be applicable to the molecular analysis of lipopolysaccharides and bacterial lipoglycans. Moreover, the results are of physicochemical interest because they demonstrate for lipid macroamphiphiles an inverse relationship between hydrophobicity and the size of the hydrophilic headgroup.

Animals↗

Preparation of bacterial X-prolyl dipeptidyl aminopeptidase and its stabilization by organic cosolvents.

To obtain large amounts of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp lactis (PepX, E.C. 3.4.14.5), PepX was purified from a commercial L. lactis cell extract. The enzyme was purified in only three steps and the last one was performed by HPLC on a C4 reverse-phase column using acetonitrile as an eluent. Despite its high molecular mass (175 kDa), the enzyme was recovered with a good activity yield (75%). Advantages and drawbacks of this technique compared to the classical ones are discussed. The stability of the enzyme in aqueous solutions and in the presence of 10 water-miscible solvents was also investigated. PepX was found to be stabilized by dimethyl sulfoxide, triglyme, and glycerol.

Aminopeptidases↗

Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference.

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

Adenine↗

Use of continuous culture for the selection of plasmids with improved segregational stability.

In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described. The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemostat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched. We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAM beta 1 as selection vectors. The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties. The first class involved mutations in the pAM beta 1-derived selection plasmid. These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable. The other two classes were based on the insertion of additional, stability promoting, sequences in the selection plasmids. We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance. The site-specific resolution function of pAM beta 1 was used as an example of the latter functions. We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria.

Bacteriological Techniques↗

Positive selection, cloning vectors for gram-positive bacteria based on a restriction endonuclease cassette.

Lactococcus lactis contains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by the LlaI operon has previously been characterized from the L. lactis conjugative plasmid pTR2030. The LlaI operon is composed of six genes: First, a small regulatory gene llaIC precedes the methylase gene llaIM. The following three genes, llaI.1, llaI.2, llaI.3, are all essential for restriction endonuclease activity and are designed as the restriction cassette llaIR. The forth open reading frame of unknown function follows the llaIR gene cassette. We have successfully subcloned the three llaIR genes, llaI.1, llaI.2, and llaI.3, without llaIM, as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) from Lactobacillus acidophilus ATCC4356, which is functional in E. coli, lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated in Escherichia coli and several gram-positive bacteria. The llaIR restriction cassette was not functional in E. coli, but its presence was lethal to L. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis, and Leuconostoc gelidum. Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within the llaIR cassette. Insertions in llaI.1 resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of the llaIR genes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.

Cloning, Molecular↗

Nucleotide sequence analysis of the lactococcal EPS plasmid pNZ4000.

The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and three origin of transfer (oriT) sequences. Sequences identical to these oriT sequences were also found on two other lactococcal plasmids and a plasmid from Lactobacillus helveticus. Several complete and partial IS elements were identified on pNZ4000, including iso-ISS1, iso-IS946, and iso-IS982 sequences. Furthermore, pNZ4000 contains a gene cluster that may encode a cobalt transport system and a gene that encodes a CorA homologue which may function as a magnesium transporter.

Amino Acid Sequence↗

Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli.

A new collection of shuttle cloning vectors has been constructed that can be used in a broad host range, because they carry replication origins which are functional in Escherichia coli (p15A, pWV01, ColE1), Lactococcus lactis, lactobacilli, and Bacillus subtilis (pAMbeta1, pWV01). These plasmids contain the lacZ-T1T2 cassette from pJDC9, which allows the X-gal selection and cloning of DNA fragments that could cause plasmid instability in E. coli. In addition, they have been proved to be structurally and segregationally stable in Lactobacillus casei, in which their copy number has been determined by real-time quantitative PCR. Furthermore, the antibiotic resistance markers (beta-lactamase, chloramphenicol acetyl transferase, and erythromycin transacetylase) and the theta and rolling circle replicating origins have been combined to obtain this set of compatible plasmids (pIA family) that can be cotransformed, both in lactic acid bacteria and in E. coli.

Bacillus subtilis↗

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.

Animals↗

The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria.

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA of Lactobacillus spp. has been identified. Two orfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). The orf adjacent to attP predicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The other orf encodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region with attP. The attB site is located at the 3' end of the transfer RNALeu gene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such as Lactococcus lactis and even into Escherichia coli. In Lc. lactis, integration occurs in a previously unknown intergenic region, whereas in E. coli, it maps within the rrnD operon, 5' of rrsD gene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in the attB sequence, since Lc. lactis and E. coli only share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.

Amino Acid Sequence↗

Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain.

Genetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics. Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes. Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages. In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L. lactis host. They occurred by homologous recombination with phage-related sequences present in the host chromosome. Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication. We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages.

Bacterial Proteins↗

Mutational analysis of two structural genes of the temperate lactococcal bacteriophage TP901-1 involved in tail length determination and baseplate assembly.

Two putative structural genes, orf tmp (tape measure protein) and orf bpp (baseplate protein), of the temperate lactococcal phage TP901-1 were examined by introduction of specific mutations in the prophage strain Lactococcus lactic ssp. cremoris 901-1. The adsorption efficiencies of the mutated phages to the indicator strain L. lactic ssp. cremoris 3107 were determined and electron micrographs were obtained. Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages. Furthermore, construction of an inframe deletion or duplication of 29% in orf tmp was shown to shorten or lengthen the phage tail by approximately 30%, respectively. The orf tmp is proposed to function as a tape measure protein, TMP, important for assembly of the TP901-1 phage tail and involved in tail length determination. Specific mutations in orf bpp produced phages which were unable to adsorb to the indicator strain and electron microscopy revealed particles lacking the baseplate structure. The orf bpp is proposed to encode a highly immunogenic structural baseplate protein, BPP, important for assembly of the baseplate. Finally, an assembly pathway of the TP901-1 tail and baseplate structure is presented.

Adsorption↗

Comparative genomics reveals close genetic relationships between phages from dairy bacteria and pathogenic Streptococci: evolutionary implications for prophage-host interactions.

The genome of the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 contains eight prophage elements. Only prophage SF370.1 could be induced by mitomycin C treatment. Prophage SF370.3 showed a 33.5-kb-long genome that closely resembled the genome organization of the cos-site temperate Siphovirus r1t infecting the dairy bacterium Lactococcus lactis. The two-phage genomes shared between 60 and 70% nucleotide sequence identity over the DNA packaging, head and tail genes. Analysis of the SF370.3 genome revealed mutations in the replisome organizer gene that may prevent the induction of the prophage. The mutated phage replication gene was closely related to a virulence marker identified in recently emerged M3 serotype S. pyogenes strains in Japan. This observation suggests that prophage genes confer selective advantage to the lysogenic host. SF370.3 encodes a hyaluronidase and a DNase that may facilitate the spreading of S. pyogenes through tissue planes of its human host. Prophage SF370.2 showed a 43-kb-long genome that closely resembled the genome organization of pac-site temperate Siphoviridae infecting the dairy bacteria S. thermophilus and L. lactis. Over part of the structural genes, the similarity between SF370.2 and S. thermophilus phage O1205 extended to the nucleotide sequence level. SF370.2 showed two probable inactivating mutations: one in the replisome organizer gene and another in the gene encoding the portal protein. Prophage SF370.2 also encodes a hyaluronidase and in addition two very likely virulence factors: prophage-encoded toxins acting as superantigens that may contribute to the immune deregulation observed during invasive streptococcal infections. The superantigens are encoded between the phage lysin and the right attachment site of the prophage genome. The genes were nearly sequence identical with a DNA segment in S. equi, suggesting horizontal gene transfer. The trend for prophage genome inactivation was even more evident for the remaining five prophage sequences that showed massive losses of prophage DNA. In these prophage remnants only 13-0.3 kb of putative prophage DNA was detected. We discuss the genomics data from S. pyogenes strain SF370 within the framework of Darwinian coevolution of prophages and lysogenic bacteria and suggest elements of genetic cooperation and elements of an arms race in this host-parasite relationship.

Bacteriophages↗

Complete genomic sequence of bacteriophage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages.

The complete genomic sequence of the Lactococcus lactis virulent phage ul36 belonging to P335 lactococcal phage species was determined and analyzed. The genomic sequence of this lactococcal phage contained 36,798 bp with an overall G+C content of 35.8 mol %. Fifty-nine open reading frames (ORFs) of more than 40 codons were found. N-terminal sequencing of phage structural proteins as well as bioinformatic analysis led to the attribution of a function to 24 ORFs (41%). A lysogeny module was found within the genome of this virulent phage. The putative integrase gene seems to be the product of a horizontal transfer because it is more closely related to Streptococcus pyogenes phages than it is to L. lactis phages. Comparative genome analysis with six complete genomes of temperate P335-like phages confirmed the heterogeneity among phages of P335 species. A dUTPase gene is the only conserved gene among all P335 phages analyzed as well as the phage BK5-T. A genetic relationship between P335 phages and the phage-type of the BK5-T species was established. Thus, we proposed that phage BK5-T be included within the P335 species and thereby reducing the number of lactococcal phage species to 11.

Bacteriophages↗