Aldolase of lactic Acid bacteria: a case history in the use of an enzyme as an evolutionary marker

[This corrects the article on p. 453 in vol. 37.].     

Use of the alr gene as a food-grade selection marker in lactic acid bacteria

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.     

Using heme as an energy boost for lactic acid bacteria

Lactic acid bacteria (LAB) are a phylogenetically diverse group named for their main attribute in food fermentations, that is, production of lactic acid. However, several LAB are genetically equipped for aerobic respiration metabolism when provided with exogenous sources of heme (and menaquinones for some species). Respiration metabolism is energetically favorable and leads to less oxidative and acid stress during growth. As a consequence, the growth and survival of several LAB can be dramatically improved under respiration-permissive conditions. Respiration metabolism already has industrial applications for the production of dairy starter cultures. In view of the growth and survival advantages conferred by respiration, and the availability of heme and menaquinones in natural environments, we recommend that respiration be accepted as a part of the natural lifestyle of numerous LAB.     

乳酸菌作为药物分子粘膜投递载体的研究进展

乳酸菌是被公认为安全的食品级微生物,广泛地应用于食品生产、保存以及作为益生菌促进人类健康.鉴于发展有效的投递药物分子策略的需要,乳酸菌成为了极有吸引力的用于口服、鼻饲及阴道进行粘膜投递药物分子的活载体.用乳酸菌作为药物分子的投递载体,安全性好,且可直接合成并投递目标蛋白,显著降低药物生产成本.到目前为止,乳酸菌作为粘膜...     

Phylogenomic reconstruction of lactic acid bacteria: an update

Background  

Lactic acid bacteria (LAB) are important in the food industry for the production of fermented food products and in human health as commensals in the gut. However, the phylogenetic relationships among LAB species remain under intensive debate owing to disagreements among different data sets.     

Carbonic acid from decarboxylation by "malic" enzyme in lactic acid bacteria

Carbonic anhydrase studies were used to determine the primary form of carbonic acid produced from decarboxylation of l-malic acid by "malic" enzyme in malolactic strains of five different species of lactic acid bacteria. Addition of carbonic anhydrase to the reaction mixture containing crude bacterial extract and l-malic acid, at pH 7, in all five cases resulted in an increase (13 to 23%) in the rate of carbon dioxide evolution over the control. The results indicated that the primary form of carbonic acid released from "malic" enzyme was not anhydrous carbon dioxide as previously supposed and as has been shown for other decarboxylating enzymes. The standard free-energy changes of the malo-lactic reaction with the various forms of carbonic acid as the primary decarboxylation product were calculated. The reaction is less exergonic when carbonic acid, bicarbonate ion, or carbonate ion is the primary decarboxylation product compared to anhydrous carbon dioxide. The free-energy of the reaction is not biologically available to the bacteria; with carbon dioxide not the primary decarboxylation product, the potential energy lost in a malo-lactic fermentation is not as great as previously considered. Endogenous carbonic anhydrase activity was not found.     

The ratio of L-form to D-form of lactic acid as a criteria for the identification of lactic acid bacteria

Stereoisomers of lactic acid produced by lactic acid bacteria were determined by HPLC by using an enantiomeric resolution column. In general, the ratios of L-form to D-form (the type of stereoisomer) obtained were compared with those in references. Values of the type of stereoisomer of lactic acid were discussed from the viewpoint of identification of lactic acid bacteria.     

Fructose-bisphosphate aldolases: an evolutionary history.

Two mechanistically distinct forms of fructose-bisphosphate aldolase are known to exist. It has been assumed that the Class II (metallo) aldolases are evolutionary more primitive than their Class I (Schiff-base) analogs since the latter had only been found in eukaryotes. With the identification of prokaryotic Class I aldolases, we present here an alternative scheme of aldolase evolution. This scheme proposes that both aldolase classes are evolutionarily ancient and rationalizes the observed highly variable expression of both enzyme types in contemporary file forms.     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Antifungal-activity-producing lactic acid bacteria as biocontrol agents in plants

Fungal infection represents a severe problem that decreases the yield and market value of fruit crops. The use of fungicides is a conventional method to control infections but it is associated with disadvantages, such as hazardous impact on public health, environmental contamination, resistance development among pathogens and high cost of agrochemicals. Biological control is an alternative approach for the treatment of fungal infections. The species of Bacillus, Pseudomonas, Enterobacter, Pantoea, Burkholderia, Lysobacter and Serratia have been successfully used in the control of fungal infections. The mechanisms involved in biocontrol are hyperparasitism or predation, production of antibiotics, lytic enzymes and induction of host resistance. Lactic acid bacteria have been used as biopreservative organisms in food and feed systems. They are a cluster of Gram-positive bacteria and include species of the genera Enterococcus, Lactobacillus, Leuconostoc, Lactococcus and Pediococcus. The ability to produce several antibacterial and antifungal substances confers biopreservation potential to lactic acid bacteria. Many have ‘generally regarded as safe’ status and are considered as safe from both human and environmental points of view. Their isolation is reported from vegetables, aerial plant surfaces, pickled cabbage, grass silage, malted cereals and also from soil. They produce antifungal substances, such as cyclic dipeptides, proteinaceous compounds, organic acids, fatty acids and reuterin. The biocontrol potential of lactic acid bacteria is demonstrated in the prevention of fungal infections of fruits, such as apples and grapes. Thus, living cells or product formulations of antifungal lactic acid bacteria may be prepared and used as an alternative biocontrol technology.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

A novel vector for lactic acid bacteria that uses a bile salt hydrolase gene as a potential food-grade selection marker

A novel vector pM4aB for lactic acid bacterial was developed using a bile salt hydrolase gene from Lactobacillus plantarum as a potential food-grade selection marker. The 3.0-kb pM4aB consisted of the replicon of Lactobacillus plasmid pM4, a multiple cloning site and the bsh gene, which was constructed by elimination of a 5.5-kb non-food-grade DNA fragment from an 8.5-kb intermediate vector pBEmpM4aB. For electroporation into Lactobacillus paracasei X9, a high transformation efficiency of 4.0±1.0×10(4) CFU/μg plasmid DNA was yielded with 0.1% (wt/vol) glycodeoxycholic acid sodium selection. A high segregation stability of the vector was also observed as only 0.1% plasmid was lost after 50 generations of growth without selection pressure. The application potential of pM4aB was further confirmed by expression of a catalase gene from Lactobacillus sakei in L. paracasei. These results revealed that the novel vector pM4aB constructed in this study would be a useful tool for genetic modification of the industrially important LAB.     

Bacteriophage development in an immobilized lactic acid bacteria system

Summary Bacteriophages were added to milk fermented byStreptococcus raffinolactis cells immobilized in calcium alginate. Beads containing the immobilized streptococci were used for five consecutive fermentations; pH, free cell and bacteriophage counts were estimated. Free cells increased from 5×10⁶ to 3×10⁸ per mL of milk, over the successive fermentations. Addition of bacteriophages reduced the free cell count by almost 1000 after 3 fermentations, but a gradual increase occurred subsequently. Bacteriophages were inoculated at 100 per mL and gradually attained 5×10⁹ per mL in the system. Rinsing of the system did not have a substantial influence on free cell or phage counts. Presence of bacteriophage reduced slightly the acidification rate in the system.Bacteriophage numeration by two layer agar method gave better results than by most probable number (MPN). MPN counts were greatly influenced byS. raffinolactis inoculation level.Contribution # 099     

Safety of industrial lactic acid bacteria.

Lactic acid bacteria (LAB) are ubiquitous in fermented and non-fermented foods and are common components of the human commensal microflora. This long history of human exposure and consumption has led to the reasonable conclusion that they are generally safe. Recent attention has also focused on their possible role as probiotic bacteria, promoting beneficial health effects. There have, however, been a number of reports of human infections caused by LAB and these are reviewed. In most cases, the source of the infection was the commensal LAB flora rather than ingested bacteria and the patient had some underlying disease or predisposing condition. Even as opportunistic pathogens, the LAB, with the notable exception of the enterococci, are much less successful than a number of other members of the commensal microflora. The use of new strains for probiotic use is likely to require more detailed evidence for their safety, particularly if the strains have been genetically modified or have been derived from animals. Procedures that have been proposed for assessing the safety of new strains are described.     

Application of the shsp gene,encoding a small heat shock protein,as a food-grade selection marker for lactic acid bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 and in the ability to grow at 52 degrees C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em(r)) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 10(4) and 1.0 x 10(4) CFU/0.5 micro g of DNA, with standard deviations of 0.54 x 10(4) and 0.32 x 10(4), for shsp and Em(r) selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10(4) and 3.8 x 10(3) CFU/0.5 micro g of DNA, with standard deviations of 0.63 x 10(4) and 3.48 x 10(3), for shsp and Em(r) selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.