Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

Adaptation of lactic acid bacteria to unfavorable growth conditions

The adaptation of lactic acid bacteria (LAB) to unfavorable growth conditions, e.g., depletion of nutrient sources, overthreshold cell density of a population, or antibiotic impact, was shown to include: (1) formation of cyst-like dormant cells (CDC) providing for survival and species preservation and (2) realization of intra-population phenotypic variability, which is demonstrated by development of non-dominant colonies on plates inoculated with CDC suspensions. In Lactobacillus plantarum, the dormant cells, which retained viability and heat resistance for a long time, were formed in 10- and 20-fold concentrated suspensions of the stationary phase cells. In 4-month cell suspensions, two types of cells were present, CDC and L-forms. The CDC of Lactococcus lactis were formed in (1) post-stationary cultures grown under glucose limitation and (2) in stationary phase cultures resuspended in starvation medium (without glucose). Populations of CDC stored for different periods of time varied in the ability for phase variation; as a result, both variants exhibited a shift of the population’s CDC spectrum to the transition of the dominant S-colony type to the R-type up to complete substitution (by day 25). In Lactobacillus acidophilus AT-41, CDC appeared in (1) post-stationary cultures grown on a nitrogen-limited medium; (2) autolyzing cultures treated with ampicillin or erythromycin; and (3) concentrated (10- and 20-fold) suspensions of stationary-phase cells. At plating of L. acidophilus CDC, the substitution of the S-type for the dominant R-type in variants (1) (day 30), (2) (100 μg/ml ampicillin, day 10), and (3) (day 25) was 68.6%, 30.1%, and 61.2%, respectively. The S-variant of L. acidophilus was used for development of a novel lactofermented product based on vegetable (beet) juice fermentation, which sustained high titer of viable cells (2 × 10⁶ cells/ml).     

Metabolism of lactic acid bacteria studied by nuclear magnetic resonance

The complexity of metabolic and regulatory networks presents a great scientific challenge to an integrated view of how individual components contribute to the overall function. Nuclear magnetic resonance (NMR) spectroscopy is undoubtedly a suitable technique for global investigations of microbial metabolism, since it allows a view into living cells without disturbing the cellular organisation. Therefore, metabolic processes can be monitored in real time under physiological conditions. In the present paper, examples of the application of NMR to study the metabolism of lactic acid bacteria will be given. These include the analysis of labelling patterns in end-products using ¹³C as a tracer, thereby establishing metabolic pathways, the detection and quantification of intermediates in the pathway of exopolysaccharide biosynthesis, and on line monitoring of glycolytic kinetics to assess the effect of metabolic engineering strategies.     

Incorporation of nisI-mediated nisin immunity improves vector-based nisin-controlled gene expression in lactic acid bacteria

Lactic acid bacteria (LAB) have been used successfully to express a wide variety of recombinant proteins, ranging from flavor-active proteins to antibiotic peptides and oral vaccines. The nisin-controlled expression (NICE) system is the most prevalent of the systems for production of heterologous proteins in LAB. Previous optimization of the NICE system has revealed a strong limit on the concentration of the inducer nisin that can be tolerated by the culture of host cells. In this work, the nisin immunity gene, nisI, has been inserted into the recently reported pMSP3535H2 vector that contains the complete NICE system on a high-copy Escherichia coli-LAB shuttle vector. Fed-batch fermentation data show that Lactococcus lactis IL1403 cells transformed with the new vector, pMSP3535H3, tolerate a 5-fold increase in the concentration of the inducer nisin, and, at this elevated concentration, produce a 1.8-fold increased level of green fluorescent protein (GFP), a model recombinant protein. Therefore, the incorporation of nisI in the pMSP3535H3 NICE system described here unveils new ranges of induction parameters to be studied in the course of optimizing recombinant protein expression in LAB.     

Metabolic engineering of sugar catabolism in lactic acid bacteria

Lactic acid bacteria are characterized by a relatively simple sugar fermentation pathway that, by definition, results in the formation of lactic acid. The extensive knowledge of traditional pathways and the accumulating genetic information on these and novel ones, allows for the rerouting of metabolic processes in lactic acid bacteria by physiological approaches, genetic methods, or a combination of these two. This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

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).     

大曲来源淀粉利用型乳酸菌的筛选及其淀粉利用特性

【背景】乳酸菌是面包、馒头等发酵食品中的重要功能微生物,对改善质地和风味均具有重要作用。淀粉利用能力高的乳酸菌,因其能够在生面粉中更好地定殖而具有重要的应用价值。【目的】筛选获得淀粉水解型乳酸菌并研究其淀粉利用特性。【方法】以浓香型白酒大曲为筛选源,采用淀粉基质碳源对大曲中乳酸菌进行定向富集,结合淀粉发酵能力筛选高淀粉利用能力菌株,并对筛选得到的优良菌株展开淀粉酶表达及其酶活力研究。【结果】以贮存3-6个月的大曲为优秀筛选源,以生面糊传代富集方法可较快筛选出具有良好淀粉利用能力的乳杆菌,主要物种为植物乳杆菌和类食品乳杆菌。对其中一株具有淀粉利用能力的类食品乳杆菌LBM12001的淀粉水解特征和淀粉酶活力展开研究,该菌株淀粉水解能力达10 g/L,并且其在面糊中具有良好的定殖能力;酶活力测定表明,其α-淀粉酶和麦芽糖淀粉酶为胞外酶;麦芽糖淀粉酶水解淀粉的最适pH值为3.5,比酶活为1 240 U/mg。【结论】建立起从我国传统白酒发酵大曲中高效筛选淀粉水解型乳酸菌的富集筛选方法,以及菌株的水解能力评价方法,获得的胞外麦芽糖淀粉酶分泌型乳杆菌在酸面团、馒头等需进行生面粉发酵食品的生产中具有重要应用前景。     

Lactic acid bacteria as prime candidates for codon optimization

In species having a strong correlation of expressivity and codon bias it has been shown that heterologous expression can be optimized by changing codons of the introduced gene towards the set of codons that the host organism naturally uses in its highly expressed genes. Even though two lactic acid bacteria are fully sequenced, there are no reports on attempts of codon optimization in the literature. In this report it is demonstrated that codons used in highly expressed genes tend to differ from the codons in lowly expressed genes, and that there is a strong correlation of codon bias and empirical expressivity (codon adaptation index) in Lactococcus lactis and Lactobacillus plantarum. This strongly suggests that codon optimization strategies could be applied to expression systems with lactic acid bacteria as producer strains. A good example of a candidate for codon optimization is the mouse interleukin-2 gene, which in its natural form has an extremely low codon adaptation index for expression in Lc. lactis.     

The impact of lactic acid bacteria on cheese flavor

Abstract Chesse flavor is a manifestation of complex interactions of volatile and non-volatile flavor-active compounds plus tactual perception. Numerous agents, including lactic acid bacteria, procece the flavou sensations. The effect of lactic acid bacteria is more dominant in cheese varieties with limited growth of secondary flora. This review describes the indirect and direct impacts of lactic acid bacteria in cheese with emphasis on carbohydrate fermentation, changes in oxidation-reduction potential, interactions with non-starter bacteria, autolysis, proteolytic and peptidolytic activities, transport of metabolites and flavor production.     

alpha-Chitinase activity among lactic acid bacteria

Chitin is a polysaccharide widely distributed in nature. Among 115 strains from 29 species of lactic acid bacteria only strains belonging to Carnobacterium divergens and Carnobacterium maltaromaticum hydrolyzed alpha-chitin. This activity was not affected by temperature (10 degrees C versus 30 degrees C) and in most cases not subject to glucose catabolite repression.     

Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains

Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of -ketoglutarate since this -ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous -ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing -ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to -ketoglutarate in several natural LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Metabolic engineering of lactic acid bacteria for the production of nutraceuticals

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.     

Evolution of cell division in bacteria

Molecular evolution in bacteria is examined with an emphasis on cell division. For a bacterial cell to assemble and then divide required an immense amount of integrated cell and molecular biology structures/functions to be present, such as a stable cellular structure, enzyme catalysis, minimal genome, septum formation at mid-cell and mechanisms to take up nutrients and produce and use energy, as well as store it. The first bacterial cell(s) capable of division must have had complex cell and molecular biology functions. At this stage of evolution, they would not have been primitive cells but would have reached a threshold in evolution where cell division occurred in a regulated manner.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Cell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicum

Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.     

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.