* Nestlé Research Centre, Nestec Ltd., Lausanne, Switzerland
Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM. FF. NN., Università degli Studi di Verona, Italy
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Abstract |
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Key Words: species concept evolution Lactobacillus delbrueckii
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Introduction |
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They are gram-positive, nonmotile, nonspore forming, obligatory homofermentative with exclusive production of D(-)-lactic acid, with an optimal growth temperature between 40° and 44°C and the GC content of their DNA is 4951 moles %. Moreover, L. delbrueckii subsp. delbrueckii includes only two strains in major culture collections, and only recently some other strains belonging to this subspecies have been isolated from sourdough (Corsetti et al. 2001). The other two subspecies, instead, are represented by a much higher number of strains.
In this study, several genetic aspects related or not with growth in milk were investigated in order to depict an evolutionary scenario for the three subspecies. Traits considered in the analysis were 16S rDNAs heterogeneity, the lac operon, and the cell wall-anchored protease, galactose metabolism, and the presence of different insertion sequences (IS-element). To this aim, phylogenetic, genomic, and phenotypic evidences from a large number of strains were combined in order to infer infraspecific genealogical relationships without resorting to complete genome sequencing, which would have been prohibitive. Furthermore, some considerations on the prokaryotic species concept were drawn based on the presented results.
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Material and Methods |
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ß-Galactosidase
ß-Galactosidase activity was measured in MRS-grown cultures, while detection of growth of deficient strains in cell wallanchored protease and in ß-galactosidase was carried out in skimmed milk without or with 0.5% yeast extract and without or with 1% glucose at 42°C. ß-Galactosidase activity was determined by adding 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) to the bacterial culture (MRS) at a final concentration of 200 µg/ml. This analogue of lactose is cleaved by the ß-galactosidase, liberating its indol group and generating a blue color in the medium.
Chromosomal DNA Preparation
Total DNA was extracted from Lactobacillus strains by a modification of the spooling method (Delley, Mollet, and Hottinger 1990). Bacteria grown to the mid-log phase in MRS broth were collected by centrifugation, washed once with 1M NaCl, and incubated for 1 h at 37°C in the presence of proteinase K (250 µg/ml) and pronase E (500 µg/ml). Cells were washed twice in TE10 (10 mM Tris-HCl pH 8.0; 1 mM EDTA), resuspended in TE50 (50 mM Tris pH 8.0 and 10 mM EDTA) containing mutanolysin (125 µg/ml) and lysozyme (1 mg/ml), and incubated for 1 h at 37°C. One volume of 0.5% sodium dodecyl sulfate (SDS) was added to lyse the cells. Finally, proteinase K was added to 200 µg/ml and the solution further incubated for 30 min at 65°C. The DNA was extracted with phenol, precipitated with ethanol, spooled out on a sterile toothpick, washed in 70% ethanol, and drained. The DNA was then dissolved in TE50 in the presence of 200 µg/ml RnaseA, chloroform extracted, reprecipitated in ethanol, and spooled again. The purified DNA was dissolved in TE10 to give a final concentration of 5001000 µg/ml.
Southern Blot Analysis
Chromosomal DNA was digested to completion with the chosen restriction enzyme, separated on a 0.8% agarose gel in TAE buffer (40 mM Tris, 20 mM sodium acetate, 2 mM EDTA, pH 8.0) and transferred to GeneScreen (DuPont, NEN) membranes (Sambrook, Fritsch, and Maniatis 1989). Prehybridization and hybridization were performed in the presence of 1% Blotto (skimmed-milk powder), 1% SDS and 1.5x SSPE (1x SSPE : 8.76 g NaCl, 1.38 g NaH2PO4, 0.37 g EDTA for 1L). Washing was done under stringent conditions at 65°C. Probes were obtained by DNA amplification using specific primers (table 1) for ISL6: 2264 and 2283 on strain NCC 706, ISL7: 2263 and 2286 on strain NCC 2506, and pY30 (Pittet and Hottinger 1989): 2587 and 2588 on strain NCC 641 (ATCC 11842). They were then 32P labeled with a[32P]dCTP (Amersham) by the random priming method (Boehringer-Mannheim).
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Results |
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Strain Identification
The 130 bacterial isolates of the Nestlé Culture Collection (NCC) chosen for the study were first shown to belong to the L. delbrueckii species by DNA amplification using primers 1378 and 1379 (table 1) designed from the probe pY85 specific for the L. delbrueckii group (Delley, Mollet, and Hottinger 1990) (data not shown). This probe was shown to be part of a nifS-like gene (Leong-Morgenthaler et al. 1994). A selection of isolates to be further characterized was made by genetic typing based on restriction fragment length polymorphism (RFLP). Two different typing strategies were chosen: a ribotyping obtained by digestion of total DNA with EcoRI and hybridization with the 23S ribosomal probe pY30 (Pittet and Hottinger 1989) and an IS typing using PvuII and an IS-element specific to L. delbrueckii as probe ISL7 (Lapierre, Mollet, and Germond 2002) in the same way. The patterns of tagged DNA fragments obtained by these techniques allow detection of mutations and genome reorganization events linked to the restriction enzyme and the probe chosen. Some examples of such DNA patterns are shown in figure 1. The isolates with identical patterns were grouped and one representative of each group was chosen, totaling 61 strains selected to be further characterized (table 2). For L. delbrueckii subsp. lactis, 75% of the isolates could be selected, as most of them presented specific patterns, whereas for L. delbrueckii subsp. bulgaricus, only 27% of the isolates could be selected, as many identical DNA patterns were found.
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Analysis of 16S rDNA Mutations
The 16S rDNA sequences available from GenBank were aligned in order to analyze the evolutionary relationships between the three subspecies of L. delbrueckii. Most of the base differences observed between the 16S rDNAs of the three subspecies are cytosine or guanine in L. delbrueckii subsp. lactis and thymine or adenine in the subspecies bulgaricus and delbrueckii (table 3). The spontaneous deamination of cytosine is a frequent event. This deamination leads to uracil, which is efficiently excised, whereas deamination of methyl-cytosine leads to thymine. If the resulting mispaired base escapes repair, the result is an oriented mutation (i.e., a conversion of a cytosine to a thymine).
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For Lactobacillus helveticus, a close relative to L. delbrueckii, there is only one EcoRI site in the middle of the 16S rDNA gene generating different DNA fragments allowing distinction between this species and the L. delbrueckii group (Delley and Germond 2002).
Expression of ß-Galactosidase
The lac operon is composed of three genes: (1) the lactose permease (lacS) gene, involved in the uptake of lactose through the membrane, (2) the ß-galactosidase (lacZ) gene, needed for the cleavage of lactose, (Leong-Morgenthaler, Zwahlen, and Hottinger 1991), and (3) a repressor encoded by the lacR gene responsible for the regulation of lacS and lacZ gene expression (Lapierre, Mollet, and Germond 2002).
Expression of ß-galactosidase was previously shown to be constitutive in L. delbrueckii subsp. bulgaricus ATCC 11842T (Leong-Morgenthaler, Zwahlen, and Hottinger, 1991). The 61 selected strains were grown in MRS, and the ß-galactosidase activity was detected in all the 23 L. delbrueckii subsp. bulgaricus strains grown in the presence of glucose or lactose, indicating a constitutive expression of the enzyme. In L. delbrueckii subsp. lactis strains, however, ß-galactosidase activity was observed only in the presence of lactose, indicating an inducible expression of the gene. Only one exception was found: strain NCC 188 was shown to present a constitutive expression of the ß-galactosidase (table 2).
The two available strains of L. delbrueckii subsp. delbrueckii, ATCC 9649T and NCFB 701744, do not ferment lactose; they are lac minus (table 2). All attempts to amplify different parts of their lac operon failed. Finally, two primers were chosen in the genes present upstream (lacA) and downstream (asnS1) of the lac operon (fig. 2A and table 4). In this case, an amplification product was obtained using primers 2005 in lacA and 3033b in asnS1 (table 1) from the strain ATCC 9649T. The sequencing of the amplification product revealed that the entire lac operon was deleted between 6 bp downstream of the stop codon of the lacA gene and 15 bp downstream of the stop codon of the lacR gene (fig. 2B). The potential rho-independent terminator of lacR is then directly linked to the end of lacA in this strain. For the strain NCFB 701744, no amplification product could be obtained, even when using different primers located in lacA and asnS1, suggesting a larger deletion at this locus.
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The different IS elements found on both sides of the lac operon may have been involved in the establishment of the operon in the genome. They may also have been responsible for the deletion of the operon in L. delbrueckii subsp. delbrueckii.
Expression of the Cell Wall-Anchored Protease
Besides ß-galactosidase, which is responsible for the catabolism of lactose, another enzyme is essential for growth in milk. This enzyme, the cell wallanchored protease, is responsible for the digestion of casein. Its gene (prtB), located downstream of the lac operon, was isolated and characterized from L. delbrueckii subsp. bulgaricus ATCC 11842T (Gilbert et al. 1996). The expression of prtB was recently shown to be differently regulated. In L. delbrueckii subsp. bulgaricus, the protease is expressed constitutively, whereas in the subspecies lactis it is tightly repressed by the presence of peptides in the growth media (Gilbert et al. 1997). The 61 L. delbrueckii strains were grown in skimmed milk and growth was recorded by RABIT (Rapid Automated Bacterial Impedance Technique). All strains of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis exhibited growth in milk, except the two L. delbrueckii subsp. delbrueckii strains (ATCC 9649T and NCFB 701744), which do not grow in milk even when supplemented with glucose (prtB, table 2). Besides the lac minus phenotype, these strains have a protease minus phenotype. Indeed, the addition of a protein hydrolysate to the milk allows their growth. However, the cell wallanchored protease gene ( prtB) was detected by DNA amplification using several primers taken from different regions of the gene. The different amplification products were of the expected size, except in the 3'-end region of the gene. The amplification of this region of prtB using primer 1451 (located inside the gene) and 4158 (downstream of the gene) produced fragments of different size. The fragments obtained were shorter for NCFB 701744 and longer for ATCC 9649T compared with ATCC 11842T. This region, which encodes the domain spanning the cell wall, is composed of repeats of four amino acids with variations (fig. 2C). The amplification products were sequenced for the two L. delbrueckii subsp. delbrueckii strains, showing that several groups of amino acids have been inserted or deleted, with a reduction of the number of the groups for NCFB 701744 and an increase for ATCC 9649T compared with L. delbrueckii subsp. bulgaricus ATCC 11842T.
Galactose Metabolism
L. delbrueckii subsp. bulgaricus is known to catabolize a much lower number of carbohydrates than L. delbrueckii subsp. lactis (Dellaglio, Bottazzi, and Trovatelli 1973; Weiss, Schillinger, and Kandler 1983) The L. delbrueckii subsp. bulgaricus strains of the NCC were shown to metabolize only glucose, fructose, mannose, and the disaccharide lactose, whereas L. delbrueckii subsp. lactis strains can metabolize, in addition, galactose, different modified carbohydrates such as N-acetyl-glucosamine, esculin, arbutin, and salicin and disaccharides such as sucrose, maltose, and threalose. As galactose is produced from lactose by the ß-galactosidase beside glucose, the galactose operon was studied in more detail. The presence of one gene of the gal operon was tested by DNA amplification in the 61 strains. DNA was amplified using two degenerated primers (8875 and 8876) designed from the sequences of the galactosyl-1-phosphate uridyl transferase (galT) genes of different gram-positive bacteria (table 4). Amplification products were obtained for all strains of L. delbrueckii subsp. lactis, but none was obtained for the subspecies bulgaricus and delbrueckii. The results are shown in table 2 (galT) and indicate that the subspecies bulgaricus and the subspecies delbrueckii strains have lost the galT gene.
ISL6 Distribution
The IS-element ISL6 was shown to be inserted in the promoter region of some strains of L. delbrueckii subsp. lactis. The distribution of this IS-element was analyzed in the 61 L. delbrueckii strains by IS-typing. Chromosomal DNA was restricted with SalI and hybridized, after agarose gel analysis, to a labeled ISL6 probe. The results are shown in table 2 (Nbr ISL6). The number of DNA fragments detected by the probe gives a minimal estimate of the number of ISL6 present in the genomes. ISL6 was found in L. delbrueckii subsp. lactis and L. delbrueckii subsp. delbrueckii strains with more than 10 copies in several strains. It is, however, absent from most of the L. delbrueckii subsp. bulgaricus strains except for NCC 522 and 576, both of which possess only one copy of this element. It is likely that L. delbrueckii subsp. bulgaricus have evolved from an ISL6-free strain of L. delbrueckii subsp. lactis, and the single copy of this element found in two strains could have been acquired later by horizontal transfer.
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Discussion |
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However little work has been done concerning the evolutionary significance of those data, since the authors' aim was strain differentiation. Moreover, random whole-genome techniques probably provide no greater insight into genome diversity than classical phenotypic methods (Gurtler and Mayall 2001).
The most interesting characteristic of the three L. delbrueckii subspecies is that they present extremely different nutritional requirements and sources but are members of the same taxon as determined by whole-genome DNA hybridization. Therefore, gene mutations, especially of genes implicated in habitat adaptation, and mobile genetic elements could supply valuable evolutionary information, if placed side by side with the analysis of the conventional phylogenetic marker, 16S rDNA.
The 16S rDNA sequence variations (i.e., the cytosine to thymine oriented mutations) suggest that all three L. delbrueckii subspecies evolved from a common ancestor, with L. delbrueckii subsp. lactis still very close to this ancestor and L. delbrueckii subsp. bulgaricus quite different, having accumulated the maximum number of mutations (fig. 5). However, 16S rDNAs sequences show 11 overall mutations, which may be too few to support an evolutionary insight. Physical localization and organization of the lac operon were conserved, therefore, the absence of the entire operon in the two strains belonging to the subspecies delbrueckii, ATCC 9649T (NCC 621) and NCFB 701744 (NCC 801), is probably due to a deletion induced by the numerous IS-elements present upstream and downstream of the operon, rather than to an insertion event of horizontally transferred lac genes. This hypothesis is enforced by the observation that the deleted regions are different in the two strains and by the fact that spontaneous mutants are easily obtained in L. delbrueckii subsp. bulgaricus (Mollet and Delley 1990; Germond et al. 1995). Base composition of this operon is also in favor of the hypothesis of a deletion event, since the GC content of the lac operon is around 50 mol%, which corresponds to the GC content of the entire genome.
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Amplification targeted to the galT gene of the galactose operon revealed that the subspecies delbrueckii and bulgaricus had lost the gene. It is known that galactose, one moiety of lactose, plays a role in the antiport mechanism for lactose uptake (Poolman et al. 1989). In milk, which contains exclusively lactose in large amounts, the loss of the galactose metabolic pathway will favor the uptake of lactose and the exclusive use of the glucose metabolic pathway. This inactivation of the galactose metabolic pathway associated to other carbohydrate metabolic pathway loss and the constitutive expression of the ß-galactosidase and the protease probably contributed to the selection of L. delbrueckii subsp. bulgaricus for fast fermentation of milk.
In L. delbrueckii subsp. delbrueckii, galT was also lost as the lac operon. These deletions and the prtB mutations could either have caused the inability to grow in milk or could have been caused by the change of habitat, that is, mutations could have been "attracted" by useless genome regions.
Analysis of the structure and distribution of several insertion sequences revealed a complex series of events, which probably were the driving force behind the evolution of L. delbrueckii species.
In the process of the speciation of L. delbrueckii subsp. bulgaricus, the insertion of the complex ISL4/ISL5 close to the lac promoter, the single nucleotide deletion in the lacR gene, the insertion of ISL3 downstream of the lacR gene, and the loss of the galT gene from an ISL6-free strain seem to have been the starting point of a stable constitutive expression of the lac genes. From that point, L. delbrueckii subsp. bulgaricus strains continued to evolve rapidly under the pressure of their use in dairy manufacturing for the rapid fermentation of milk in yogurt production. This evolution can be followed by the sequential addition of mutation in the lacR gene and the staggered insertion of different IS-elements in ISL3 (table 2 and fig. 6). The first two events, the single nucleotide deletion in the lacR gene and the insertion of ISL3 downstream of this gene, were found in NCC 1051. From this strain, two independent events could have occurred, the insertion of ISL4 into ISL3 in strain NCC 9 and a second mutation in the lacR gene of the strain NCC 25. From the latter, two additional independent events occurred, a third mutation in lacR, which is found in the type strain ATCC 11842T (NCC 641) and the insertion of ISL7 close to ISL4 in the strain NCC 421. Finally, a last IS-element, ISLdl1, was inserted into ISL3 in the strain NCC 474.
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From these data, it is clear that the three subspecies of L. delbrueckii form quite a heterogeneous taxon, even if they are members of the same species. The prokaryotic species concept is an issue of importance in biology. There is no official definition of a species in microbiology, however at present, the most feasible one is that originally proposed by (Gordon 1978) and reported by (Rosselló-Mora and Amann 2001, pp. 5253): "a microbial species is a concept represented by a group of strains (omissis) which have in common a set or pattern of correlating stable properties that separates the group from other groups of strains." The Committee on Reconciliation of Approaches to Bacterial Systematics recommended that the boundaries for species circumscription should be described in terms of DNA-DNA binding: "a species generally would include strains with approximately 70% or greater DNA-DNA relatedness and with 5°C or less delta Tm" (Wayne, Brenner, and Colwell 1987, p. 464). In the case of L. delbrueckii, total DNA hybridization values range from 88% to 100%, as determined by filter-bound labeled DNA (Dellaglio, Bottazzi, and Trovatelli 1973; Weiss, Schillinger, and Kandler 1983).
The DNA hybridization approach does not provide information on the evolutionary history of taxa; an insight into this aspect is supplied by the phylogenetic analysis, firstly proposed by Woese (Woese 1987). A correlation between the two approaches has been observed, since organisms with genomic similarity above 70% usually share more than 97% 16S rRNA sequence similarity.
However, 16S rDNAs often lack resolving power at the species level (Fox, Wisotzkey, and Jurtshuk 1992). Therefore, alternative phylogenetic markers have been investigated to demarcate interspecific evolutionary relationships (Palys, Nakamura, and Cohan 1997).
Infraspecific analysis is a more difficult task and it has been suggested that infraspecific phylogenetic relationships could be reconstructed as networks rather than as trees (Posada and Crandall 2001). Network topology, however, relies mainly on the chosen genes (Alber et al. 2001). This observation underlines the need for a phylogenomic approach (Eisen 1998; Pennisi 1998; Gurtler and Mayall 2001), but the sequencing of complete genomes of many strains of one single species seems to be, at present, an unfeasible task.
The present partial genomic study revealed a deep internal heterogeneity of the investigated species. It was confirmed that any particular cut-off value is arbitrary and cannot reliably yield groups of bacteria corresponding to real ecological units (Vandamme et al. 1996; Palys, Nakamura, and Cohan 1997). This is revealed by the analysis of genes involved in habitat adaptation and IS sequences, which are the principal driving forces behind genome flexibility.
Other considerations concerning the concept of bacterial species as a group of strains sharing a set of stable properties could be proposed. Stability refers to maintenance of a trait over a long period of time, even if evolution occurs. However, genotypic maintenance does not necessarily imply phenotypic expression of the trait. Moreover, in flexible genomes, a loss of entire regions of the genome could happen, such as the lac operon in L. delbrueckii subsp. delbrueckii, thus loosing both phenotypic and genotypic traits.
In bacterial species delineation, characters considered unstable could and should be omitted, but the iteration of the exclusions can lead to the impossibility of fixing the set of stable characters. Therefore, the only valid methods that could be considered are total DNA hybridization, phylogenetic analysis, or both. However, phylogenetic analysis could be affected by horizontal gene transfer and DNA-DNA hybridization percentages may be influenced by large deletion or insertion events. Genomic flexibility of prokaryotes collides with a stable classification necessary from a scientific and applied point of view.
It was shown that the type species of the genus Lactobacillus, L. delbrueckii subsp. delbrueckii, is a kind of dying taxon, confined to a habitat very different from the probable native one, milk, and is represented by only two strains in the major culture collections. The fast evolution of L. delbrueckii subsp. bulgaricus and its genetic plasticity appear to be very important also from an applied point of view, since it is widely employed and exposed to a very strong selective pressure in industrial processes.
From a general point of view, the present paper suggests a new way of investigating infraspecific relationships focusing on genes involved in the colonization of ecological niches. Probably the bacterial species definition should be integrated with even partial genomic aspects, and should continue to evolve: "the adequacy of characterization of a bacterium is a reflection of time; it should be as full as modern techniques make possible. Unfortunately, one now regarded as adequate is likely, in 10 years time, to be hopelessly inadequate!" (Cowan 1965, p. 145).
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Acknowledgements |
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Footnotes |
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