First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.     

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.     

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.     

Determination of aspartase activity in dairy Propionibacterium strains

Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix. Here a method for aspartase activity was established and a number of dairy propionibacteria belonging to P. freudenreichii ssp. shermanii and freudenreichii were screened for this enzyme activity. A wide range of aspartase activity could be found in PAB isolates originating from cheese. The majority, i.e. 70% of the 100 isolates tested, showed very low levels of aspartate activity.     

Production of conjugated linoleic acid by dairy starter cultures

Nineteen different strains of lactobacilli, lactococci, streptococci and propionibacteria commonly used as dairy starter cultures were tested for their ability to produce conjugated linoleic acid (CLA) from free linoleic acid in vitro. Two strains of Propionibacterium freudenreichii ssp. freudenreichii and one strain of P. freudenreichii ssp. sheramnii were found to be capable of converting free linoleic acid to extracellular CLA. The highest level of CLA formed in the media was 265 μg ml⁻¹. Of the different isomers, cis- and trans-9,11-octadecadienoic acid represented more than 70% of the total CLA formed. The inhibitory effect of linoleic acid on the growth of the bacteria and its conversion to CLA in different media by propionibacteria are discussed.     

Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii

Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium. we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B12 producer, Propionibacterium.     

Manganese-dependent ribonucleotide reductase from Propionibacterium freudenreichii subsp. Shermanii: partial purification, characteristics and role in DNA biosynthesis

Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids, P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficient P. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

Construction of a reporter vector system for in vivo analysis of promoter activity in Propionibacterium freudenreichii

A beta-galactosidase reporter system for the analysis of promoter elements in Propionibacterium freudenreichii was designed. The pTD210 in vivo reporter vector was constructed using a promoterless lacZ gene from Bifidobacterium longum cloned into the pAMT1 plasmid. The utility of the pTD210 reporter vector was demonstrated by an investigation of six predicted promoters in P. freudenreichii. The system produced accurate and reproducible measurements that facilitated both promoter identification and the quantification of promoter activities.     

Anti-stress activity of Propionibacterium freudenreichii: identification of a reactivative protein

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.     

费氏丙酸杆菌两个亚种的分离与鉴定

用亨格特（Hungate）厌氧技术从不同奶制品分离出3株丙酸杆菌Propionibacterium,编号为PTC－1,PTC－2和PTC－3。细胞呈多形态,杆状,革兰氏阳性,不形成芽孢,不运动,菌落在PYG深层洋菜中呈双凸透镜状,白至土黄色,从葡萄糖、乳糖等8种碳水化合物发酵产酸,葡萄糖发酵产物包括大量丙酸和乙酸,少量异丁酸,琥珀酸和CO₂。厌氧至耐氧。PTC－1的DNA的GC百分含量测定值为67.8mol％（Tm）。三株菌的特性很接近,只在硝酸盐还原和牛奶凝固特性有差     

Efficient transformation system for Propionibacterium freudenreichii based on a novel vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per microg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (> or =10(8) colonies per microg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.     

Induction of an outer membrane protein of 78 kDa in Vibrio vulnificus cultured in the presence of desferrioxamine B under iron-limiting conditions

Fermentation balances and growth yields were determined with various bacteria fermenting lactate to acetate plus propionate either via methylmalonyl-CoA or via acrylyl-CoA. All strains fermented lactate to acetate plus propionate at approximately a 1:2 ratio. Growth yields of Propionibacterium freudenreichii were more than twice as high as those of Clostridium homopropionicum or Veillonella parvula. Hydrogen was formed as a side product to a significant extent only by V. parvula and Pelobacter propionicus; the latter formed hydrogen preferentially when using ethanol as substrate. Acrylyl-CoA reductase of C. homopropionicum and Clostridium neopropionicum was found nearly exclusively in the cytoplasm thus confirming that this reduction step is unlikely to be involved in energy conservation. C. homopropionicum exhibited higher K(S) and higher micro(max) values, as well as higher specific substrate turnover rates than P. freudenreichii. The results allow us to conclude that C. homopropionicum using the acrylyl-CoA pathway with low growth yield obtains its specific competitive advantage compared to P. freudenreichii not through higher substrate affinity or metabolic shift toward enhanced acetate-plus-hydrogen formation but through faster specific substrate turnover.     

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.     

Structural studies on exopolysaccharides produced by three different propionibacteria strains

The exopolysaccharides produced by three propionibacteria strains, Propionibacterium freudenreichii 109, Propionibacterium freudenreichii 111, and Propionibacterium thoenii 126, grown on whey-based media, were found to be charged heteropolymers, composed of D-glucose, D-mannose, and D-glucuronic acid in molar ratios of 2:2:1. By means of methylation analysis, mass spectrometry, partial acid hydrolysis, and 1D/2D NMR (1H and 13C) studies, it was determined that all three exopolysaccharides contain the same branched, pentasaccharide repeating unit: [Formula: see text].     

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.     

Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply

The end-product profile of the glucose fermentation by Propionibacterium freudenreichii ET-3 changed on an electrochemical treatment, in which the culture vessel was filled with a carbon felt anode. Acetate and propionate were produced as final end products in a molar ratio of 2:3 without any electrochemical treatments at the point of the consumption of lactate as an intermediate of the glucose fermentation. The ratio was changed to 1:1 at the point of the lactate consumption by the electrochemical incubation at an electrode potential of 0.4 V versus Ag|AgCl for 100 h. During further electrochemical incubation, propionate was oxidized to acetate as a final end-product in the microbe-containing anode chamber. 1,4-Dihydroxy-2-naphthoic acid produced by P. freudenreichii ET-3 itself would receive electrons from the metabolic pathway and serve as an electron transfer mediator from the microbial cells to the electrode.     

Occurrence of Propionibacterium freudenreichii bacteriophages in swiss cheese.

We isolated bacteriophages active against Propionibacterium freudenreichii from 16 of 32 swiss cheese samples. Bacteriophage concentrations ranged from 14 to 7 x 10(5) PFU/g, depending on the sample and the sensitive strain used for detection. Only a few strains, 8 of the 44 strains of P. freudenreichii in our collection, were sensitive. We observed that multiplication of bacteriophages occurred in the cheese loaf during multiplication of propionibacteria in a warm curing room, but it seems that these bacteriophages have no adverse effect on the development of the propionic flora. We also found that sensitive cells, originating from either the starter or the cheese-making milk, were present at a high level (10(9) CFU/g) in the cheese.     

Improved trehalose production from biodiesel waste using parent and osmotically sensitive mutant of Propionibacterium freudenreichii subsp. shermanii under aerobic conditions

Trehalose is an important nutraceutical of wide commercial interest in the food processing industry. Recently, crude glycerol was reported to be suitable for the production of trehalose using a food microbe, Propionibacterium freudenreichii subsp. shermanii, under static flask conditions. Similarly, enhanced trehalose yield was reported in an osmotically sensitive mutant of the same strain under anaerobic conditions. In the present study, an effort was made to achieve higher production of trehalose, propionic acid, and lactic acid using the parent and an osmotically sensitive mutant of P. freudenreichii subsp. shermanii under aeration conditions. Under aeration conditions (200 rpm in shake flasks and 30 % air saturation in a batch reactor), biomass was increased and approximately 98 % of crude glycerol was consumed. In the parent strain, a trehalose titre of 361 mg/l was achieved, whereas in the mutant strain a trehalose titre of 1.3 g/l was produced in shake flask conditions (200 rpm). In the mutant strain, propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were also achieved with crude glycerol. Similarly, in controlled batch reactor culturing conditions a final trehalose titre of approximately 1.56 g/l was achieved with the mutant strain using crude glycerol as the substrate. Enhanced production of trehalose using P. freudenreichii subsp. shermanii from waste under aeration conditions is reported here. Higher production of trehalose was not due to a higher yield of trehalose but to a higher final biomass concentration.