Evolution of the Bacterial Species Lactobacillus delbrueckii: A Partial Genomic Study with Reflections on Prokaryotic Species Concept
* Nestlé Research Centre, Nestec Ltd., Lausanne, Switzerlands:%u-, 百拇医药
Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM. FF. NN., Università degli Studi di Verona, Italys:%u-, 百拇医药
Abstracts:%u-, 百拇医药
The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showinga high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of ß-galactosidase and of the cell wall–anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall–anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.
Key Words: species concept • evolution • Lactobacillus delbrueckiin6#4j, 百拇医药
Introductionn6#4j, 百拇医药
Lactobacillus delbrueckii is the type species of the genusLactobacillus. It includes three subspecies: delbrueckii, bulgaricus, and lactis. The main interest regarding these lactic acid bacteria comes from the importance of the subspecies bulgaricus and lactis in the dairy industry. Nevertheless, the three subspecies are also very interesting from an evolutionary point of view since they present different fermentation patterns and habitat: whereas the subspecies bulgaricus and lactis are almost exclusively present in milk, the subspecies delbrueckii colonizes vegetable sources. It is unable to ferment lactose and to degrade casein and therefore cannot grow in milk. L. delbrueckii subsp. bulgaricus is known to catabolize a much lower number of carbohydrates than L. delbrueckii subsp. lactis . These three taxa were firstly described as distinct species, but they were found to exhibit DNA-DNA homologies of 90%–100% among each other and accordingly were united under the same nomenclatural designation .
They are gram-positive, nonmotile, non–spore 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 49–51 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 . The other two subspecies, instead, are represented by a much higher number of strains.im;#, 百拇医药
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.
Material and Methods-d#5d8, 百拇医药
Bacterial Strains-d#5d8, 百拇医药
One hundred thirty isolates belonging to L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, and L. delbrueckii sp. were isolated from commercial starters, dairy fermented products, raw milk, and fresh cream, or were supplied by international collections, Universities of Milano, Piacenza, and Thessaloniki, Professor T. Sozzi, and INRA (France). They were grown overnight in MRS-broth (Difco) at 42°C for maintenance.-d#5d8, 百拇医药
ß-Galactosidase-d#5d8, 百拇医药
ß-Galactosidase activity was measured in MRS-grown cultures, while detection of growth of deficient strains in cell wall–anchored 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 Preparation78kvp, 百拇医药
Total DNA was extracted from Lactobacillus strains by a modification of the spooling method . 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 500–1000 µg/ml.
Southern Blot Analysis2pi5&, 百拇医药
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 ). 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 for ISL6: 2264 and 2283 on strain NCC 706, ISL7: 2263 and 2286 on strain NCC 2506, and pY30 : 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).2pi5&, 百拇医药
fig.ommitted2pi5&, 百拇医药
Table 1 Sequence of the Different Primers Used in This Study
Primers, PCR, and DNA Sequencing}%w%fu, 百拇医药
Custom-made primers were obtained from Microsynth (Balgach, Switzerland) . PCR was performed in a total volume of 50 µl containing 0.2 mM each deoxynucleoside triphosphate, 10 pmol of each primer, 0.5 U of the enzyme SuperTaq (Endotel, Allschwil, Switzerland), and the appropriate buffer. PCR amplification was performed in 35 cycles, each cycle consisting of denaturing step at 95°C for 1 min, a primer-annealing step at 55°C for 2 min, and a primer extension step at 72°C for 1 min per kb to be amplified with a DNA thermocycler (PerkinElmer). Sequencing was performed on PCR-amplified DNA by primer walking with the Thermo sequenase fluorescent–labeled primer cycle sequencing kit with 7-deaza dGTP (RPN2538, Amersham) and with custom-made primers, labeled with IR400 obtained from MWG-Biotech (Ebersberg, Deutschland) on a DNA sequencer LICOR, model 4000 from MWG-Biotech. DNA sequence analysis was done with the Wisconsin Sequence Analysis Package from Genetic Computer Group (University of Wisconsin). Sequence alignments of the 16S rDNA were generated by MegAlign of the Lasergene package (DNASTAR Inc, USA) and ClustalW (EMBL, Germany) and analyzed by DNAPARS from PHYLIP package .
Results')fwo4(, 百拇医药
After strain identification, different individual sets of data were collected from the analyzed strains to be evaluated: (1) 16S rDNAs sequence variations, (2) the lac operon and its genetic locus, which are responsible for the metabolism of lactose and therefore for growth in milk, (3) the cell wall–anchored protease, involved in the digestion of casein, (4) galactose metabolism, related to growth in milk since galactose is a product of lactose metabolism beside glucose, and (5) specific IS elements distribution (i.e., ISL6).')fwo4(, 百拇医药
Strain Identification')fwo4(, 百拇医药
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 designed from the probe pY85 specific for the L. delbrueckii group (data not shown). This probe was shown to be part of a nifS-like gene . 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 and an IS typing using PvuII and an IS-element specific to L. delbrueckii as probe ISL7 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 . The isolates with identical patterns were grouped and one representative of each group was chosen, totaling 61 strains selected to be further characterized . 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.
fig.ommitted|4h^{&, 百拇医药
FIG. 1. Autoradiogram of an agarose gel electrophoresis of DNA purified from different L. delbrueckii isolates, restricted with EcoRI, and hybridized with 32P-labeled 23S rRNA probe (ribotyping, pY30) (A) or restricted with PvuII and hybridized with an ISL7 probe (IS typing) (B)|4h^{&, 百拇医药
fig.ommitted|4h^{&, 百拇医药
Table 2 Genetic and Physiological Characteristics of a Selection of Lactobacillus delbrueckii Strains of the Nestlé Culture Collection (NCC) and Other Culture Collections|4h^{&, 百拇医药
fig.ommitted|4h^{&, 百拇医药
Table 2 Continued|4h^{&, 百拇医药
These typing techniques are able to reveal little genome reorganization from one strain to another but proved to be oversensitive in detecting differences for the determination of strain relatedness.|4h^{&, 百拇医药
Analysis of 16S rDNA Mutations|4h^{&, 百拇医药
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 . 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).
fig.ommittedm.x, 百拇医药
Table 3 Nucleotide Changes in the 16S rDNA Sequences at Positions According to the Sequence of L. delbrueckii subsp. lactis (GenBank )m.x, 百拇医药
In nine cases, the cytosine (guanine) is present in the 16S rDNA sequence of L. delbrueckii subsp. lactis and the thymine (adenine) in the sequence of the subspecies bulgaricus (six cases) and delbrueckii (five cases). The two mutations at positions 74 and 83 occurred in a hypervariable region, when compared with the 16S rDNA sequences of different lactobacilli. These two mutations in L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus may have occurred after the separation of the three subspecies. Among the other mutations, two of the base changes (pos 1161 and 1303, ) are found in the subspecies bulgaricus and delbrueckii, which suggests that this mutation had occurred before the separation of these two subspecies. Among these mutations occurring in the sequence of the 16S rDNA during speciation of the L. delbrueckii subspecies, the cytosine to thymine conversion at position 686 generated an EcoRI site in the middle of the 16S rDNA of L. delbrueckii subsp. bulgaricus. The 61 selected strains were screened for the presence of this EcoRI site. From each strain, the 16S rDNA was amplified using primers 9699 and 9700 . The amplification products were restricted with EcoRI. For all 23 strains of L. delbrueckii subsp. bulgaricus, including the type strain ATCC 11842T (NCC 641), the amplification product was cleaved at position 686, whereas for all other strains tested, including the type strains of the subspecies lactis ATCC 12315T (NCC 946) and delbrueckii ATCC 9649T (NCC 621), the amplification product was not cleaved. Another EcoRI site is present close to the 5'-end of the 16SrDNA gene of all L. delbrueckii subspecies. Consequently, EcoRI cuts the 16S rDNA at two positions for L. delbrueckii subsp. bulgaricus and one position for L. delbrueckii subsp. lactis and L. delbrueckii subsp. delbrueckii. The results are reported in (16S Eco).
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 .c^.+8, http://www.100md.com
Expression of ß-Galactosidasec^.+8, http://www.100md.com
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, , and (3) a repressor encoded by the lacR gene responsible for the regulation of lacS and lacZ gene expression .c^.+8, http://www.100md.com
Expression of ß-galactosidase was previously shown to be constitutive in L. delbrueckii subsp. bulgaricus ATCC 11842T . 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 .
The two available strains of L. delbrueckii subsp. delbrueckii, ATCC 9649T and NCFB 701744, do not ferment lactose; they are lac minus 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 ( and ). In this case, an amplification product was obtained using primers 2005 in lacA and 3033b in asnS1 ( 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 . 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.pgcp(1q, http://www.100md.com
fig.ommitted
FIG. 2. L. delbrueckii subsp. delbrueckii type strain ATCC 9649T lac operon deletion schematic representation (A) and nucleotide sequences (B). Deleted genes are represented as dotted boxes, orientation of the genes by arrowheads, primers used in the DNA amplifications by half arrows, and sites of deletion by broken arrows. Start and stop codons are boxed, insertion sequences (direct repeats) of IS-elements underlined and rho-independent terminators represented by inverted arrows. Amino acid sequence of part of the trans wall domain of the cell wall–anchored proteinase (C) in L. delbrueckii subsp. bulgaricus type strain ATCC 11842T (1), L. delbrueckii subsp. delbrueckii NCFB 701744 (2), and L. delbrueckii subsp. delbrueckii ATCC 9649T (3)(il[ls, 百拇医药
fig.ommitted(il[ls, 百拇医药
Table 4 GenBank Accession Numbers of the Different Sequences Involved in This Study(il[ls, 百拇医药
The lacR Gene(il[ls, 百拇医药
In L. delbrueckii subsp. lactis ATCC 12315T, the regulation of the lac operon expression was shown to be under the control of a repressor coded by a gene (lacR) belonging to the lac operon and localized downstream of the lacZ gene . In L. delbrueckii subsp. bulgaricus ATCC 11842T, the control of the lac genes expression by lactose was lost and ß-galactosidase is constitutively produced. Therefore, the lacR gene was sequenced from L. delbrueckii subsp. bulgaricus ATCC 11842T, and the sequence revealed that it was inactivated by several insertion/deletion events a deletion of one nucleotide (d1), 21 nucleotides downstream of the start codon, which leads to a premature stop 20 amino acids downstream and duplications of two nucleotides (i2) and 10 nucleotides (i10) further downstream. This region of the lacR gene was analyzed in all L. delbrueckii subsp. bulgaricus strains by T-track sequencing and DNA amplification using primers designed from the lacR sequence . The primers were designed with only the 3'-end last nucleotide different, which is sufficient to allow amplification of the mutated sequence but not of the wt sequence. The single nucleotide deletion (d1) was detected by amplification using primer B814 , and A033 as reverse primer and was found in all strains of L. delbrueckii subsp. bulgaricus. The two-nucleotide insertion (i2) was detected by amplification using primers G004 and A033 and was found in 75% of the strains. The large insertion of 10 nucleotides was found only in the type strain ATCC 11842T, which harbors the three mutations. In the L. delbrueckii subsp. bulgaricus strain NCC 512, a deletion of five nucleotides was detected upstream of the insertion i2. All the results are summarized in (Mut lacR). As the single nucleotide deletion is present in all strains of L. delbrueckii subsp. bulgaricus, it could have been the first event, which inactivated the lacR gene, allowing the constitutive expression of the lactose genes. Finally, the lacR genes of some L. delbrueckii subsp. lactis strains were also analysed by DNA amplification and revealed no mutation compared with the sequence of L. delbrueckii subsp. lactis ATCC 12315T lacR gene, except for the strain NCC 188. This strain was shown to belong to the lactis subspecies but has a constitutive expression of its ß-galactosidase. Its lacR gene was sequenced and revealed a single nucleotide deletion 80 nucleotides downstream of the start codon , as well as a conversion of the start codon to ATA, leading to the inactivation of this gene.
fig.ommittedc, http://www.100md.com
FIG. 3. Nucleotide sequence of the 5'-end region of the lac repressor in L. delbrueckii subsp. lactis. The insertion/deletion events (d1, i2, i10) identified in L. delbrueckii subsp. bulgaricus type strain ATCC 11842T are indicated in bold. The stop codon induced by the single nucleotide deletion (d1) is underlined. The sequences used for the primers B814 and G004 are underlined by an arrow. The black arrowhead is for the nucleotide deleted in the L. delbrueckii subsp. lactis strain NCC 188. Start and stop codons are boxedc, http://www.100md.com
Characterization of the Lactose Operon Locusc, http://www.100md.com
Several new IS-elements were localized and identified on both sides of the lac operon ( and ). The promoter of the lac operon, between the lacA gene and the first gene of the lac operon (lacS), was amplified from the 61 different L. delbrueckii strains using primers 2005 and 1994, respectively . Different amplification products were obtained. The sequence of a 0.2 kb product showed only the presence of the promoter of the lac operon, whereas larger sized products revealed the presence of one or more IS-elements. A product of 1.7 kb accounts for the presence of ISL6 and one of 2.9 kb for the presence of ISL6 and ISL7. The presence of a complex of two IS-elements (ISL4 in ISL5) generated a fragment of 3.5 kb. A variant of this complex structure gave a fragment of 2.7 kb, which revealed a deletion of 810 nucleotides from the 5'-end of ISL4. The insertion of one IS-element in another is known to inactivate the transposition of an element such as ISL5. The partial deletion of such IS-elements could even give a higher stability to the structure. The nature of the IS-elements was confirmed for all strains by specific amplifications using primers designed from the sequence of the IS-elements themselves (ISL4, 1647; ISL6, 2287; and ISL7, 2285), and a reverse primer taken from the sequence of lacS (1994, ). For some strains, the amplification product was of a different size and was not further characterized. In some cases, no amplification could be obtained. The characteristics of these different structures in the promoter region of the lac operon are summarized in (IS pro), and the position of IS-element insertion sequences (direct repeats) is shown in . The results show that the complex ISL4/ISL5 was found exclusively but in all L. delbrueckii subsp. bulgaricus strains, whereas the presence of ISL6, ISL7, or no IS-element was found in the subspecies lactis strains.
fig.ommitted*\, 百拇医药
FIG. 4. IS-elements found in the promoter of the lac operon (A) and downstream of the lacR gene (B) of different L. delbrueckii isolates. Nucleotide sequences indicate the sites of IS-element insertion (direct repeats). Genes and IS-elements are boxed, with their orientation represented by an empty arrowhead. Black arrowheads are for terminal inverted repeats of IS-elements and half arrows for primers used in the DNA amplifications. The shaded area marks the deletion of 810 nucleotides of the 5'-end of ISL4 in some of the isolates*\, 百拇医药
The region downstream of the lac operon was shown to also attract IS elements. ISL3 was isolated from this region during the study of spontaneous lac deletion mutants of L. delbrueckii subsp. bulgaricus N299, a derivative of the type strain ATCC 11842T The region between the lacR and asnS1 genes was amplified from the 61 L. delbrueckii strains using primers 2030 in lacZ and 3033b in asnS1 . Here too, different amplification products were obtained and sequenced. Products of 1.2 and 2.6 kb account for the presence of lacR alone and lacR with ISL3 inserted in the potential rho-independent terminator of lacR, respectively. Larger sized products ranging from 4 to 7 kb revealed a staggered insertion of three more IS-elements . A product of a 4.2 kb fragment showed the presence of ISL4 integrated in the promoter of the ISL3 transposase. In some strains, a 5.5 kb fragment was shown to have ISL7 inserted downstream of ISL4 in addition. Finally, in two strains, another IS-element was found close to the 3'-end of the ISL3 transposase in addition to ISL4 and ISL7. This IS-element, ISLdl1, has already been isolated from L. delbrueckii subsp. lactis ATCC 15808 by T. Alatossava (GenBank CAC16151). In a few strains, however, no amplification product could be obtained, indicating a structural difference of the genome in this region. The presence of these different IS-elements downstream of the lacR gene are reported in (IS lacR), and the position of ISL3 insertion sequence (direct repeat) is shown in . In this case too, the different staggered IS-elements were found exclusively but in all L. delbrueckii subsp. bulgaricus strains, whereas the absence of IS-element in this region was recorded in all L. delbrueckii subsp. lactis strains.
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.|6ae4x, 百拇医药
Expression of the Cell Wall-Anchored Protease|6ae4x, 百拇医药
Besides ß-galactosidase, which is responsible for the catabolism of lactose, another enzyme is essential for growth in milk. This enzyme, the cell wall–anchored 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 . 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 . 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, ). 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 wall–anchored 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 . 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 Metabolism35u, http://www.100md.com
L. delbrueckii subsp. bulgaricus is known to catabolize a much lower number of carbohydrates than L. delbrueckii subsp. lactis 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 . 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 (galT) and indicate that the subspecies bulgaricus and the subspecies delbrueckii strains have lost the galT gene.
ISL6 Distributionk]g, 百拇医药
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 (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.
Discussionq^, http://www.100md.com
It has been suggested that genomic approaches concerning the typing, taxonomy, and evolution of strains should includedifferent aspects: random whole-genome analysis, specific gene variation, and mobile genetic elements . Random whole-genome analysis techniques, such as RAPD-PCR and PFGE, have been extensively performed on L. delbrueckii strains, probably due to their important role in the dairy industry . Specific oligonucleotide probes or primers have been designed with the aim of identification . ARDRA, ribotyping and SDS-PAGE have also been applied to differentiate strains ( this study).q^, http://www.100md.com
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 .q^, http://www.100md.com
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 . 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 ( 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.
fig.ommitted6s, http://www.100md.com
FIG. 5. Evolutionary tree for the three subspecies of L. delbrueckii taking into account the occurrence of the oriented mutations in the 16S rDNA sequences6s, http://www.100md.com
The analysis of the genetic basis of the expression of the cell wall–anchored protease and of the galT gene for lactose metabolism complete the picture of specific gene variation in relation to habitat colonization capability. The GC content of the cell wall–anchored protease gene ( prtB) is 47 mol%, suggesting a late acquisition by horizontal transfer of this gene. Moreover, the prtB gene is still present in both L. delbrueckii subsp. delbrueckii strains but has been mutated differently by deletion/insertion at its 3'-end. Observed insertion/deletion events could be the results of strand slippage during DNA replication and should allow a modification of the length of this domain. Such length modification is essential in the phenomenon of horizontal transfer of the gene to account for differences in the thickness of the recipient bacteria cell wall, as the protease is cell wall anchored. In the case of our two L. delbrueckii subsp. delbrueckii strains, these mutations may have inactivated the gene. Other inactivating mutations cannot be excluded, as the whole genes were not sequenced.
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 . 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.sz, 百拇医药
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.l, 百拇医药
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 ( and ). 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.
fig.ommitted93@$h, 百拇医药
FIG. 6. Proposed evolution of the lacR gene sequence and IS-element insertions downstream of the lacR gene in different strains of L. delbrueckii subsp. bulgaricus. d1, i2, and i10 are for deletion/insertion events in the lacR gene and ISL3 to ISLdl1 are for IS-element insertions93@$h, 百拇医药
As a result of this proposed evolution, L. delbrueckii subsp. bulgaricus and subsp. lactis have lost a number of carbohydrate fermentation pathways, giving the bacteria an advantage for growth in an environment containing a high concentration of lactose as the only carbohydrate. The third subspecies, L. delbrueckii subsp. delbrueckii, isolated from a vegetal source, is unable to ferment lactose, to degrade casein, and to grow in milk.93@$h, 百拇医药
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 and reported by ( pp. 52–53): "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"( p. 464). In the case of L. delbrueckii, total DNA hybridization values range from 88% to 100%, as determined by filter-bound labeled DNA .
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 . 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.*kfgej1, http://www.100md.com
However, 16S rDNAs often lack resolving power at the species level . Therefore, alternative phylogenetic markers have been investigated to demarcate interspecific evolutionary relationships*kfgej1, http://www.100md.com
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 . Network topology, however, relies mainly on the chosen genes . This observation underlines the need for a phylogenomic approach , 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 . This is revealed by the analysis of genes involved in habitat adaptation and IS sequences, which are the principal driving forces behind genome flexibility.77qeh, http://www.100md.com
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.77qeh, http://www.100md.com
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.)*, 百拇医药
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!" ( p. 145).
Acknowledgementsmar$, http://www.100md.com
This work was supported by a grant of the EU Biotech II, contract nbr BIO4CT960439 (CH-OFES 96.0018). We gratefully acknowledge Professor Bernhard Hirt and Professor Sandra Torriani for critical reading of the manuscript, Dr. David Vilanova for help with bioinformatic tools and Dr. Elizabeth Prior for reviewing the manuscript.mar$, http://www.100md.com
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Accepted for publication September 10, 2002.(Jacques-Edouard Germond Luciane Lapierre Michèle Delley Beat Mollet Giovanna E. Felis and Franco De)
Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM. FF. NN., Università degli Studi di Verona, Italys:%u-, 百拇医药
Abstracts:%u-, 百拇医药
The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showinga high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of ß-galactosidase and of the cell wall–anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall–anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.
Key Words: species concept • evolution • Lactobacillus delbrueckiin6#4j, 百拇医药
Introductionn6#4j, 百拇医药
Lactobacillus delbrueckii is the type species of the genusLactobacillus. It includes three subspecies: delbrueckii, bulgaricus, and lactis. The main interest regarding these lactic acid bacteria comes from the importance of the subspecies bulgaricus and lactis in the dairy industry. Nevertheless, the three subspecies are also very interesting from an evolutionary point of view since they present different fermentation patterns and habitat: whereas the subspecies bulgaricus and lactis are almost exclusively present in milk, the subspecies delbrueckii colonizes vegetable sources. It is unable to ferment lactose and to degrade casein and therefore cannot grow in milk. L. delbrueckii subsp. bulgaricus is known to catabolize a much lower number of carbohydrates than L. delbrueckii subsp. lactis . These three taxa were firstly described as distinct species, but they were found to exhibit DNA-DNA homologies of 90%–100% among each other and accordingly were united under the same nomenclatural designation .
They are gram-positive, nonmotile, non–spore 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 49–51 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 . The other two subspecies, instead, are represented by a much higher number of strains.im;#, 百拇医药
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.
Material and Methods-d#5d8, 百拇医药
Bacterial Strains-d#5d8, 百拇医药
One hundred thirty isolates belonging to L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, and L. delbrueckii sp. were isolated from commercial starters, dairy fermented products, raw milk, and fresh cream, or were supplied by international collections, Universities of Milano, Piacenza, and Thessaloniki, Professor T. Sozzi, and INRA (France). They were grown overnight in MRS-broth (Difco) at 42°C for maintenance.-d#5d8, 百拇医药
ß-Galactosidase-d#5d8, 百拇医药
ß-Galactosidase activity was measured in MRS-grown cultures, while detection of growth of deficient strains in cell wall–anchored 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 Preparation78kvp, 百拇医药
Total DNA was extracted from Lactobacillus strains by a modification of the spooling method . 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 500–1000 µg/ml.
Southern Blot Analysis2pi5&, 百拇医药
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 ). 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 for ISL6: 2264 and 2283 on strain NCC 706, ISL7: 2263 and 2286 on strain NCC 2506, and pY30 : 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).2pi5&, 百拇医药
fig.ommitted2pi5&, 百拇医药
Table 1 Sequence of the Different Primers Used in This Study
Primers, PCR, and DNA Sequencing}%w%fu, 百拇医药
Custom-made primers were obtained from Microsynth (Balgach, Switzerland) . PCR was performed in a total volume of 50 µl containing 0.2 mM each deoxynucleoside triphosphate, 10 pmol of each primer, 0.5 U of the enzyme SuperTaq (Endotel, Allschwil, Switzerland), and the appropriate buffer. PCR amplification was performed in 35 cycles, each cycle consisting of denaturing step at 95°C for 1 min, a primer-annealing step at 55°C for 2 min, and a primer extension step at 72°C for 1 min per kb to be amplified with a DNA thermocycler (PerkinElmer). Sequencing was performed on PCR-amplified DNA by primer walking with the Thermo sequenase fluorescent–labeled primer cycle sequencing kit with 7-deaza dGTP (RPN2538, Amersham) and with custom-made primers, labeled with IR400 obtained from MWG-Biotech (Ebersberg, Deutschland) on a DNA sequencer LICOR, model 4000 from MWG-Biotech. DNA sequence analysis was done with the Wisconsin Sequence Analysis Package from Genetic Computer Group (University of Wisconsin). Sequence alignments of the 16S rDNA were generated by MegAlign of the Lasergene package (DNASTAR Inc, USA) and ClustalW (EMBL, Germany) and analyzed by DNAPARS from PHYLIP package .
Results')fwo4(, 百拇医药
After strain identification, different individual sets of data were collected from the analyzed strains to be evaluated: (1) 16S rDNAs sequence variations, (2) the lac operon and its genetic locus, which are responsible for the metabolism of lactose and therefore for growth in milk, (3) the cell wall–anchored protease, involved in the digestion of casein, (4) galactose metabolism, related to growth in milk since galactose is a product of lactose metabolism beside glucose, and (5) specific IS elements distribution (i.e., ISL6).')fwo4(, 百拇医药
Strain Identification')fwo4(, 百拇医药
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 designed from the probe pY85 specific for the L. delbrueckii group (data not shown). This probe was shown to be part of a nifS-like gene . 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 and an IS typing using PvuII and an IS-element specific to L. delbrueckii as probe ISL7 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 . The isolates with identical patterns were grouped and one representative of each group was chosen, totaling 61 strains selected to be further characterized . 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.
fig.ommitted|4h^{&, 百拇医药
FIG. 1. Autoradiogram of an agarose gel electrophoresis of DNA purified from different L. delbrueckii isolates, restricted with EcoRI, and hybridized with 32P-labeled 23S rRNA probe (ribotyping, pY30) (A) or restricted with PvuII and hybridized with an ISL7 probe (IS typing) (B)|4h^{&, 百拇医药
fig.ommitted|4h^{&, 百拇医药
Table 2 Genetic and Physiological Characteristics of a Selection of Lactobacillus delbrueckii Strains of the Nestlé Culture Collection (NCC) and Other Culture Collections|4h^{&, 百拇医药
fig.ommitted|4h^{&, 百拇医药
Table 2 Continued|4h^{&, 百拇医药
These typing techniques are able to reveal little genome reorganization from one strain to another but proved to be oversensitive in detecting differences for the determination of strain relatedness.|4h^{&, 百拇医药
Analysis of 16S rDNA Mutations|4h^{&, 百拇医药
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 . 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).
fig.ommittedm.x, 百拇医药
Table 3 Nucleotide Changes in the 16S rDNA Sequences at Positions According to the Sequence of L. delbrueckii subsp. lactis (GenBank )m.x, 百拇医药
In nine cases, the cytosine (guanine) is present in the 16S rDNA sequence of L. delbrueckii subsp. lactis and the thymine (adenine) in the sequence of the subspecies bulgaricus (six cases) and delbrueckii (five cases). The two mutations at positions 74 and 83 occurred in a hypervariable region, when compared with the 16S rDNA sequences of different lactobacilli. These two mutations in L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus may have occurred after the separation of the three subspecies. Among the other mutations, two of the base changes (pos 1161 and 1303, ) are found in the subspecies bulgaricus and delbrueckii, which suggests that this mutation had occurred before the separation of these two subspecies. Among these mutations occurring in the sequence of the 16S rDNA during speciation of the L. delbrueckii subspecies, the cytosine to thymine conversion at position 686 generated an EcoRI site in the middle of the 16S rDNA of L. delbrueckii subsp. bulgaricus. The 61 selected strains were screened for the presence of this EcoRI site. From each strain, the 16S rDNA was amplified using primers 9699 and 9700 . The amplification products were restricted with EcoRI. For all 23 strains of L. delbrueckii subsp. bulgaricus, including the type strain ATCC 11842T (NCC 641), the amplification product was cleaved at position 686, whereas for all other strains tested, including the type strains of the subspecies lactis ATCC 12315T (NCC 946) and delbrueckii ATCC 9649T (NCC 621), the amplification product was not cleaved. Another EcoRI site is present close to the 5'-end of the 16SrDNA gene of all L. delbrueckii subspecies. Consequently, EcoRI cuts the 16S rDNA at two positions for L. delbrueckii subsp. bulgaricus and one position for L. delbrueckii subsp. lactis and L. delbrueckii subsp. delbrueckii. The results are reported in (16S Eco).
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 .c^.+8, http://www.100md.com
Expression of ß-Galactosidasec^.+8, http://www.100md.com
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, , and (3) a repressor encoded by the lacR gene responsible for the regulation of lacS and lacZ gene expression .c^.+8, http://www.100md.com
Expression of ß-galactosidase was previously shown to be constitutive in L. delbrueckii subsp. bulgaricus ATCC 11842T . 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 .
The two available strains of L. delbrueckii subsp. delbrueckii, ATCC 9649T and NCFB 701744, do not ferment lactose; they are lac minus 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 ( and ). In this case, an amplification product was obtained using primers 2005 in lacA and 3033b in asnS1 ( 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 . 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.pgcp(1q, http://www.100md.com
fig.ommitted
FIG. 2. L. delbrueckii subsp. delbrueckii type strain ATCC 9649T lac operon deletion schematic representation (A) and nucleotide sequences (B). Deleted genes are represented as dotted boxes, orientation of the genes by arrowheads, primers used in the DNA amplifications by half arrows, and sites of deletion by broken arrows. Start and stop codons are boxed, insertion sequences (direct repeats) of IS-elements underlined and rho-independent terminators represented by inverted arrows. Amino acid sequence of part of the trans wall domain of the cell wall–anchored proteinase (C) in L. delbrueckii subsp. bulgaricus type strain ATCC 11842T (1), L. delbrueckii subsp. delbrueckii NCFB 701744 (2), and L. delbrueckii subsp. delbrueckii ATCC 9649T (3)(il[ls, 百拇医药
fig.ommitted(il[ls, 百拇医药
Table 4 GenBank Accession Numbers of the Different Sequences Involved in This Study(il[ls, 百拇医药
The lacR Gene(il[ls, 百拇医药
In L. delbrueckii subsp. lactis ATCC 12315T, the regulation of the lac operon expression was shown to be under the control of a repressor coded by a gene (lacR) belonging to the lac operon and localized downstream of the lacZ gene . In L. delbrueckii subsp. bulgaricus ATCC 11842T, the control of the lac genes expression by lactose was lost and ß-galactosidase is constitutively produced. Therefore, the lacR gene was sequenced from L. delbrueckii subsp. bulgaricus ATCC 11842T, and the sequence revealed that it was inactivated by several insertion/deletion events a deletion of one nucleotide (d1), 21 nucleotides downstream of the start codon, which leads to a premature stop 20 amino acids downstream and duplications of two nucleotides (i2) and 10 nucleotides (i10) further downstream. This region of the lacR gene was analyzed in all L. delbrueckii subsp. bulgaricus strains by T-track sequencing and DNA amplification using primers designed from the lacR sequence . The primers were designed with only the 3'-end last nucleotide different, which is sufficient to allow amplification of the mutated sequence but not of the wt sequence. The single nucleotide deletion (d1) was detected by amplification using primer B814 , and A033 as reverse primer and was found in all strains of L. delbrueckii subsp. bulgaricus. The two-nucleotide insertion (i2) was detected by amplification using primers G004 and A033 and was found in 75% of the strains. The large insertion of 10 nucleotides was found only in the type strain ATCC 11842T, which harbors the three mutations. In the L. delbrueckii subsp. bulgaricus strain NCC 512, a deletion of five nucleotides was detected upstream of the insertion i2. All the results are summarized in (Mut lacR). As the single nucleotide deletion is present in all strains of L. delbrueckii subsp. bulgaricus, it could have been the first event, which inactivated the lacR gene, allowing the constitutive expression of the lactose genes. Finally, the lacR genes of some L. delbrueckii subsp. lactis strains were also analysed by DNA amplification and revealed no mutation compared with the sequence of L. delbrueckii subsp. lactis ATCC 12315T lacR gene, except for the strain NCC 188. This strain was shown to belong to the lactis subspecies but has a constitutive expression of its ß-galactosidase. Its lacR gene was sequenced and revealed a single nucleotide deletion 80 nucleotides downstream of the start codon , as well as a conversion of the start codon to ATA, leading to the inactivation of this gene.
fig.ommittedc, http://www.100md.com
FIG. 3. Nucleotide sequence of the 5'-end region of the lac repressor in L. delbrueckii subsp. lactis. The insertion/deletion events (d1, i2, i10) identified in L. delbrueckii subsp. bulgaricus type strain ATCC 11842T are indicated in bold. The stop codon induced by the single nucleotide deletion (d1) is underlined. The sequences used for the primers B814 and G004 are underlined by an arrow. The black arrowhead is for the nucleotide deleted in the L. delbrueckii subsp. lactis strain NCC 188. Start and stop codons are boxedc, http://www.100md.com
Characterization of the Lactose Operon Locusc, http://www.100md.com
Several new IS-elements were localized and identified on both sides of the lac operon ( and ). The promoter of the lac operon, between the lacA gene and the first gene of the lac operon (lacS), was amplified from the 61 different L. delbrueckii strains using primers 2005 and 1994, respectively . Different amplification products were obtained. The sequence of a 0.2 kb product showed only the presence of the promoter of the lac operon, whereas larger sized products revealed the presence of one or more IS-elements. A product of 1.7 kb accounts for the presence of ISL6 and one of 2.9 kb for the presence of ISL6 and ISL7. The presence of a complex of two IS-elements (ISL4 in ISL5) generated a fragment of 3.5 kb. A variant of this complex structure gave a fragment of 2.7 kb, which revealed a deletion of 810 nucleotides from the 5'-end of ISL4. The insertion of one IS-element in another is known to inactivate the transposition of an element such as ISL5. The partial deletion of such IS-elements could even give a higher stability to the structure. The nature of the IS-elements was confirmed for all strains by specific amplifications using primers designed from the sequence of the IS-elements themselves (ISL4, 1647; ISL6, 2287; and ISL7, 2285), and a reverse primer taken from the sequence of lacS (1994, ). For some strains, the amplification product was of a different size and was not further characterized. In some cases, no amplification could be obtained. The characteristics of these different structures in the promoter region of the lac operon are summarized in (IS pro), and the position of IS-element insertion sequences (direct repeats) is shown in . The results show that the complex ISL4/ISL5 was found exclusively but in all L. delbrueckii subsp. bulgaricus strains, whereas the presence of ISL6, ISL7, or no IS-element was found in the subspecies lactis strains.
fig.ommitted*\, 百拇医药
FIG. 4. IS-elements found in the promoter of the lac operon (A) and downstream of the lacR gene (B) of different L. delbrueckii isolates. Nucleotide sequences indicate the sites of IS-element insertion (direct repeats). Genes and IS-elements are boxed, with their orientation represented by an empty arrowhead. Black arrowheads are for terminal inverted repeats of IS-elements and half arrows for primers used in the DNA amplifications. The shaded area marks the deletion of 810 nucleotides of the 5'-end of ISL4 in some of the isolates*\, 百拇医药
The region downstream of the lac operon was shown to also attract IS elements. ISL3 was isolated from this region during the study of spontaneous lac deletion mutants of L. delbrueckii subsp. bulgaricus N299, a derivative of the type strain ATCC 11842T The region between the lacR and asnS1 genes was amplified from the 61 L. delbrueckii strains using primers 2030 in lacZ and 3033b in asnS1 . Here too, different amplification products were obtained and sequenced. Products of 1.2 and 2.6 kb account for the presence of lacR alone and lacR with ISL3 inserted in the potential rho-independent terminator of lacR, respectively. Larger sized products ranging from 4 to 7 kb revealed a staggered insertion of three more IS-elements . A product of a 4.2 kb fragment showed the presence of ISL4 integrated in the promoter of the ISL3 transposase. In some strains, a 5.5 kb fragment was shown to have ISL7 inserted downstream of ISL4 in addition. Finally, in two strains, another IS-element was found close to the 3'-end of the ISL3 transposase in addition to ISL4 and ISL7. This IS-element, ISLdl1, has already been isolated from L. delbrueckii subsp. lactis ATCC 15808 by T. Alatossava (GenBank CAC16151). In a few strains, however, no amplification product could be obtained, indicating a structural difference of the genome in this region. The presence of these different IS-elements downstream of the lacR gene are reported in (IS lacR), and the position of ISL3 insertion sequence (direct repeat) is shown in . In this case too, the different staggered IS-elements were found exclusively but in all L. delbrueckii subsp. bulgaricus strains, whereas the absence of IS-element in this region was recorded in all L. delbrueckii subsp. lactis strains.
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.|6ae4x, 百拇医药
Expression of the Cell Wall-Anchored Protease|6ae4x, 百拇医药
Besides ß-galactosidase, which is responsible for the catabolism of lactose, another enzyme is essential for growth in milk. This enzyme, the cell wall–anchored 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 . 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 . 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, ). 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 wall–anchored 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 . 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 Metabolism35u, http://www.100md.com
L. delbrueckii subsp. bulgaricus is known to catabolize a much lower number of carbohydrates than L. delbrueckii subsp. lactis 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 . 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 (galT) and indicate that the subspecies bulgaricus and the subspecies delbrueckii strains have lost the galT gene.
ISL6 Distributionk]g, 百拇医药
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 (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.
Discussionq^, http://www.100md.com
It has been suggested that genomic approaches concerning the typing, taxonomy, and evolution of strains should includedifferent aspects: random whole-genome analysis, specific gene variation, and mobile genetic elements . Random whole-genome analysis techniques, such as RAPD-PCR and PFGE, have been extensively performed on L. delbrueckii strains, probably due to their important role in the dairy industry . Specific oligonucleotide probes or primers have been designed with the aim of identification . ARDRA, ribotyping and SDS-PAGE have also been applied to differentiate strains ( this study).q^, http://www.100md.com
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 .q^, http://www.100md.com
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 . 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 ( 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.
fig.ommitted6s, http://www.100md.com
FIG. 5. Evolutionary tree for the three subspecies of L. delbrueckii taking into account the occurrence of the oriented mutations in the 16S rDNA sequences6s, http://www.100md.com
The analysis of the genetic basis of the expression of the cell wall–anchored protease and of the galT gene for lactose metabolism complete the picture of specific gene variation in relation to habitat colonization capability. The GC content of the cell wall–anchored protease gene ( prtB) is 47 mol%, suggesting a late acquisition by horizontal transfer of this gene. Moreover, the prtB gene is still present in both L. delbrueckii subsp. delbrueckii strains but has been mutated differently by deletion/insertion at its 3'-end. Observed insertion/deletion events could be the results of strand slippage during DNA replication and should allow a modification of the length of this domain. Such length modification is essential in the phenomenon of horizontal transfer of the gene to account for differences in the thickness of the recipient bacteria cell wall, as the protease is cell wall anchored. In the case of our two L. delbrueckii subsp. delbrueckii strains, these mutations may have inactivated the gene. Other inactivating mutations cannot be excluded, as the whole genes were not sequenced.
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 . 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.sz, 百拇医药
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.l, 百拇医药
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 ( and ). 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.
fig.ommitted93@$h, 百拇医药
FIG. 6. Proposed evolution of the lacR gene sequence and IS-element insertions downstream of the lacR gene in different strains of L. delbrueckii subsp. bulgaricus. d1, i2, and i10 are for deletion/insertion events in the lacR gene and ISL3 to ISLdl1 are for IS-element insertions93@$h, 百拇医药
As a result of this proposed evolution, L. delbrueckii subsp. bulgaricus and subsp. lactis have lost a number of carbohydrate fermentation pathways, giving the bacteria an advantage for growth in an environment containing a high concentration of lactose as the only carbohydrate. The third subspecies, L. delbrueckii subsp. delbrueckii, isolated from a vegetal source, is unable to ferment lactose, to degrade casein, and to grow in milk.93@$h, 百拇医药
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 and reported by ( pp. 52–53): "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"( p. 464). In the case of L. delbrueckii, total DNA hybridization values range from 88% to 100%, as determined by filter-bound labeled DNA .
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 . 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.*kfgej1, http://www.100md.com
However, 16S rDNAs often lack resolving power at the species level . Therefore, alternative phylogenetic markers have been investigated to demarcate interspecific evolutionary relationships*kfgej1, http://www.100md.com
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 . Network topology, however, relies mainly on the chosen genes . This observation underlines the need for a phylogenomic approach , 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 . This is revealed by the analysis of genes involved in habitat adaptation and IS sequences, which are the principal driving forces behind genome flexibility.77qeh, http://www.100md.com
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.77qeh, http://www.100md.com
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.)*, 百拇医药
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!" ( p. 145).
Acknowledgementsmar$, http://www.100md.com
This work was supported by a grant of the EU Biotech II, contract nbr BIO4CT960439 (CH-OFES 96.0018). We gratefully acknowledge Professor Bernhard Hirt and Professor Sandra Torriani for critical reading of the manuscript, Dr. David Vilanova for help with bioinformatic tools and Dr. Elizabeth Prior for reviewing the manuscript.mar$, http://www.100md.com
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Accepted for publication September 10, 2002.(Jacques-Edouard Germond Luciane Lapierre Michèle Delley Beat Mollet Giovanna E. Felis and Franco De)