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Keywords:

  • Lactose operon;
  • Lactobacillus casei;
  • Catabolite repression;
  • Transcriptional antitermination

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References

The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 [pLZ15-] and it was considered an excellent system to study the regulation of the lactose operon. This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64. Expression of the lactose operon in different mutants of L. casei ATCC 393 [pLZ15] and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References

Lactose specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) and P-β-galactosidase (P-β-gal) has only been described in Gram-positive bacteria belonging to the genera Staphylococcus, Streptococcus, Lactococcus and Lactobacillus. Certain fundamental differences are found between genera regarding their gene order, regulatory elements, accompanying genes and genetic location [1].

Staphylococcus aureus, Streptococcus mutans and Lactococcus lactis show an identical organisation of these genes in the lactose operon. These operons include, in addition, the genes encoding the enzymes of the tagatose-6-phosphate pathway. Another common feature is that they are regulated by a galactose-6-phosphate/lacR-dependent induction mechanism.

However, data known for Lactobacillus casei indicate a different genetic structure and possibly a different regulatory mechanism of the lactose operon [2–4]. Its genetic location has not been studied in detail, although it is known that most strains carry a plasmid with the lactose operon. In other instances it is chromosomally encoded, as in the case in Lactobacillus casei ATCC 393 [5, 6].

In Lactobacillus, genetic structure and nucleotide sequence of the lactose operon has only been determined in the strain L. casei 64H [2–4, 7]. Physiological studies have been performed in the past demonstrating that lactose uptake and P-β-gal activity are repressed by glucose, although with different intensities according to the strain used [8]. In L. casei ATCC 393 the lactose operon is located on the chromosome, which could make this strain quite adequate to perform regulatory studies. However, this strain has shown some unusual features: first, it has a double lactose assimilation system, the chromosomal lactose operon in addition to a permease and β-galactosidase system encoded by the plasmid pLZ15 [6] and, second, taxonomical differences are suggested by different authors [9, 10].

Previous studies showed that glucose has a clearly inhibitory effect on the expression of the lactose operon in L. casei ATCC 393 [pLZ15] [11]. After glucose was exhausted, bacterial growth stopped for nearly 20 h, then lactose was consumed and L. casei resumed growth. It was also shown that glucose can enter the cell by two different transport systems: a proton motive-force driven permease and a EIIMan-like transporter. That work showed that EIIMan was involved in the repression of lactose utilisation in this microorganism.

Hence, the first aim of this work was to determine the structure and the nucleotide sequence of the chromosomally encoded operon of L. casei ATCC 393 [pLZ15] and the deduced amino acid homology with that of plasmid pLZ64 from L. casei 64H. Studies on the regulation of the lactose operon in L. casei ATCC 393 [pLZ15] have revealed that different mechanisms, mediated by CcpA, PTS elements and an antiterminator protein, are controlling the expression of the lactose operon.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References

2.1Bacterial strains, culture conditions and plasmids

Lactobacillus strains used in this work are listed in Table 1. These strains were grown at 37°C under static conditions on MRS (Oxoid) or Fermentation MRS medium (ADSA-MICRO, Scharlau, Barcelona, Spain) supplemented with 0.5% sugar, as indicated. Escherichia coli DH5α [F, endA1, hsdR17, gyrA96, thi1, recA1, relA1, supE44, ΔlacU169 (φ80 lacZ DM15)] was used as host in the cloning procedures. These cells were grown on Luria Bertani medium [12] and ampicillin (100 μg ml−1) or erythromycin (500 μg ml−1) were added for plasmid selection. For agar plates, 1.7% (w/v) agar was added to the medium.

Table 1. Lactobacillus strains used in this study
StrainPhenotypeSource or reference
ATCC 393 [pLZ15] B.M. Chassy
64H B.M. Chassy and J.Thompson [8]
64H [pLZ64]LacChassy et al. [5]
ATCC 4646 Chassy et al. [5]
ATCC 4646 [pLac]LacB.M. Chassy
ATCC 11578 B.M. Chassy
ATCC 11578 [pLac]LacB.M. Chassy
ATCC 334 DSM
BL71CcpaV. Monedero
BL23D2DGrVeyrat et al. [11]
BL722DGr CcpaThis work

The plasmids used for cloning in E. coli were pT7Blue-T-vector (Novagen), pUC18/SmaI (SureClone ligation Kit, Pharmacia) and pJDC9 [13].

2.2DNA manipulation

The purification of genomic DNA from Lactobacillus strains was performed according to Veyrat et al. [14]. Plasmid DNA from E. coli was isolated by standard procedures [15]. Restriction and modifying enzymes were also used according to the recommendations of the manufacturers. General cloning procedures were performed according to Sambrook et al. [15]. DNA was sequenced by the dideoxy chain-termination method [16] using Sequenase (US Biochemical). Subclones were obtained and PCR products were used directly for sequencing when it was necessary. Synthetic DNA primers used for sequencing purposes were provided by Genosys. Analysis of DNA and protein sequence data was carried out with the GCG package [17].

2.3Isolation of 2 deoxy-d-glucose (2DOG)-resistant mutant of L. casei ATCC 393 [pLZ15]

Appropriate dilutions of an overnight culture of a L. casei ATCC 393 [pLZ15] ccpA mutant, called BL71 (Monedero, V. and Pérez-Martínez, G., unpublished data) was plated on Fermentation MRS agar plates containing 0.5% lactose as carbon source and 20 mM 2DOG. Numerous colonies were obtained as spontaneous resistant mutants to this analogue. One of them (called BL72) was selected that displayed no glucose/mannose-specific PTS activity when it was grown on glucose.

2.4Construction of L. casei ATCC 393 [pLZ15] library in E. coli

L. casei ATCC 393 [pLZ15] total DNA was partially digested with Sau3A endonuclease and fragments of 2–7 kb were purified by sucrose gradient. Plasmid pJDC9 was used as cloning vector and was digested with the restriction endonuclease BamHI. A total of 5000 E. coli transformants containing plasmids with random inserts of L. casei chromosomal DNA were obtained. This genomic library was screened by direct polymerase chain reaction as described by Griffin et al. [18].

2.5Total RNA purification and primer extension experiments

L. casei ATCC 393 [pLZ15] was grown on Fermentation MRS with 0.5% of glucose, lactose or ribose to mid-logarithmic phase. Total RNA extractions were prepared as described by Veyrat et al. [14].

The Lac19 (5′-TGGTCTCTCGCCTAATTAAATAGT) and Lac33 (5′-TTTGGCAAGTTGTCATCCCCTC) oligonucleotides were labelled at the 5′-end and used for reverse transcription with 15 μg of RNA. The reverse transcriptase extension products were resolved on a denaturing 6% polyacrylamide gel, together with DNA sequencing reactions performed with the same primer.

2.6PEP-dependent PTS activity and enzymatic assays

PTS activity, estimated as the consumption of PEP in the presence of lactose, glucose and lactose plus glucose was quantified according to Chassy and Thompson [8].

P-β-galactosidase activity was estimated as previously described [8] with the chromogenic substrate o-nitrophenyl–β-galactoside-6-phosphate (ONPG-6-phosphate, Sigma Chemical Co.). Protein concentration was determined according to Bradford [19], using bovine serum albumin as standard.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References

3.1Structure of the lactose operon in different L. casei strains

Lactose–PTS and P-β-gal activities have been described in different strains of L. casei, and frequently related to the presence of plasmids [5, 6]. The genes encoding EIICBLac, P-β-gal and EIIALac (lacE, lacG and lacF, respectively) have been characterised from a 35 kb lactose plasmid [pLZ64] in the strain L. casei 64H [2–4]. The lactose operon contained four genes, including lacT, and showed a genetic order different to other known lactose operons: lacT>lacElacG>lacF [7]. Its genetic organisation was taken as reference in L. casei.

The existence of a similar system in several L. casei strains (L. casei 64H, L. casei ATCC 11578, L. casei ATCC 4646 and their plasmid-cured, Lac variants, L. casei ATCC 334 and L. casei ATCC 393 [pLZ15]) has been studied by PCR with different pairs of primers. All the lactose-proficient strains had the same pattern of amplification than strain L. casei 64H, suggesting that the lactose operon in all the strains tested had the same genetic organisation, and that all plasmid-cured strains were lacking the complete operon with the exception of L. casei ATCC 393. Also, the existence of a single copy of the lactose operon in L. casei ATCC 393 [pLZ15] was verified by Southern analysis of chromosomal DNA (data not shown). Therefore, L. casei ATCC 393 [pLZ15] could be considered a good model to study the expression of lactose genes.

3.2Cloning of PTSLac genes and sequence analysis

A genetic library of L. casei ATCC 393 [pLZ15] in E. coli DH5α was screened by PCR using Lac5 (5′-GAACATGGTCATCCTTGC) and Lac6 (5′-CTTGCTGTCTAAATAGCC) oligonucleotides as primers. One recombinant plasmid carrying a DNA fragment that could encode the whole lactose operon was isolated. This E. coli transformant showed P-β-galactosidase activity (786.5 U/mg of protein) while no activity was detected in the strain bearing the cloning vector pJDC9.

Primary structure analysis of 5.219 kb DNA fragment revealed four open reading frames (ORF) and a stem-loop like structure located at 3′-end. These ORFs showed a high degree of homology (99%) to the nucleotide sequences previous reported for L. casei 64H [2–4, 7], therefore corresponding to lacT, lacE, lacG and lacF, respectively.

At the 5′-end, a putative promoter region could be identified: −35 region TTTACA and −10 region TACAAC (Fig. 1). This putative promoter showed other typical features of the promoter elements in Gram-positive organisms [20] like a TG sequence conserved at −15 position and an adenine-rich region at −45 end. A CRE-like element (5′-ATAAAACGTTTACA) (Fig. 1) could also be recognised overlapping the −35 region with one mismatch in relation to the consensus [21]. This putative promoter is followed by a sequence that could represent a potential rho-independent terminator. A sequence (5′-GGATTGTGACTATTTAATTAGGCGAG) resembling the RNA binding site of antiterminator proteins (RAT) [22] was also found preceeding and overlapping the inverted repeats of the putative terminator structure (Fig. 1).

image

Figure 1. 5′-end nucleotide sequence of L. casei ATCC 393 [pLZ15] DNA fragment. Putative promoter region (−35 and −10) is underlined. A putative rho-independent terminator is indicated by arrows. Potential CRE and RAT sequences are indicated by shaded background. The transcriptional start sites are indicated in bold shape. Oligonucleotides used in primer extension experiments (Lac19 and Lac33) are given as heavy arrow. The accession number of 5.219 kb sequence in EMBL Data Library is Z80834.

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These findings are suggesting a complex regulatory mechanism where CcpA and an antiterminator protein could be involved.

3.3Regulatory studies in L. casei ATCC 393 [pLZ15]

3.3.1PEP-dependent PTS and P-β-gal activities

PTS–lactose phosphorylation and P-β-gal activities in L. casei ATCC 393 [pLZ15] were quantified in permeabilized cells grown on lactose, glucose or glucose plus lactose, in order to determine if both activities were regulated coordinately. Lac–PTS and P-β-galactosidase activities were only found on lactose grown cells (73 nmol of PEP consumed per minute and milligram of dry weight and 2.46 μmol of ONPG-6-phosphate hydrolyzed per minute and milligram of protein, respectively). No activity was detected in the presence of glucose, indicating a strong repression by this sugar of both activities.

3.3.2P-β-galactosidase expression in different mutants

In L. casei 64H, ATCC 4646 and ATCC 393 [pLZ15], it has been described that the lactose genes are transcribed as an operon [7]. In order to examine the mechanisms that control lactose transport and metabolism, we took P-β-galactosidase activity as a measure of lactose operon expression. P-β-gal activity was quantified in the wild-type L. casei ATCC 393 [pLZ15], an EIIMan deficient mutant (BL23D) [11], the ccpA-inactivated mutant (BL71), and also in the double mutant, ccpA, EIIMan-deficient (BL72). This last strain was obtained as a spontaneous 2DGr mutant from BL71. BL72, EIIMan-deficient mutant, showed an identical profile to BL23D [11] as they could grow on other PTS sugars, such as lactose (intact EI and Hpr) or glucose (intact glucose permease), and the PTS activity on glucose was negligible (0.77 nmol of PEP consumed per minute and milligram dry weight). All these strains were grown on lactose, glucose and lactose plus glucose to the mid-logarithmic phase (Fig. 2).

image

Figure 2. P-β-galactosidase activity from wild-type and different mutants. The activity is expressed as nanomoles of o-nitrophenyl-β-d-galactoside-6-phosphate hydrolysed per minute and milligram of dry weight.

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None of the strains showed P-β-gal activity when grown on glucose but high activity was detected on lactose-grown cells, indicating that lactose was necessary for the expression of the operon. However, when the culture medium contained glucose plus lactose, neither the wild-type nor BL71 (ccpA) displayed any activity, and the BL23D (EIIMan−) mutant had only 24% of the activity found when grown on lactose. Only the double mutant showed full derepression of P-β-gal activity.

It has been proposed that the CcpA/HPr signal transduction pathway is the main mechanism of catabolite repression in Gram-positives [23]. However, these results show that despite the presence of a CRE element in the promoter region of the lactose operon of L. casei (Fig. 1), the absence of a functional CcpA protein is not enough to override glucose repression. Full expression of P-β-gal could only be found in the double mutant BL72, suggesting the existence of another mechanism of repression of the lactose operon which is also related to the PTS or the phosphorylated intermediates that are generated.

Furthermore, total P-β-gal activity in BL71 strain and the double mutant was twice as much as that of the wild-type, indicating that, somehow, metabolites derived from lactose — possibly glucose derivatives — can also repress the expression of the operon through interaction with CcpA.

3.3.3Transcription analysis of the lactose genes

Primer extension experiments were performed in order to map the transcriptional start point of the mRNA and to prove if the observed repression by glucose of the lactose genes was due to some of the putative regulatory elements found at the 5′-end of the operon. Total RNA used in the primer extension experiments was extracted from L. casei ATCC 393 [pLZ15] grown on Fermentation MRS with 0.5% glucose, lactose or ribose.

Two primers were used to determine the relative amounts of mRNA of the different cultures and the transcription initiation site (TIS); one partially overlapped the RAT sequence and the beginning of the terminator-like structure (Lac19) and the other one was downstream to these sequences (Lac33) (Fig. 1).

The first oligonucleotide (Lac19) was used to detect all the transcripts synthesised from the promoter of the lactose operon, which included transcripts that could stop at the terminator-like structure and those that extended further in the operon (Fig. 3A). The highest amount of reverse transcript detected corresponded to cells with ribose in the medium, and progressively decreased in the lactose and glucose lanes. This strongly suggests a repressive effect played by glucose from the medium and possibly by glucose released from lactose hydrolysis. That effect could take place through the binding of CcpA protein to the CRE element found in the promoter region (Fig. 1).

image

Figure 3. Reverse transcriptase mapping using Lac19 (A) and Lac33 (B) oligonucleotides. RNA was obtained from cultures grown on 0.5% glucose (G), lactose (L) or ribose (R). Sequence reactions were performed with primers Lac19 and Lac33 and are displaying the complementary strand to the mRNA. The initiation sites are indicated by asterisks.

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With oligonucleotide Lac33, there was a difference of one nucleotide in the TIS mapped from lactose-grown cells and ribose-grown cells, where transcription was apparently initiated at nucleotide 103 and 104, respectively, from the ATG start codon (Fig. 3B). These TISs did not coincide with that determined with primer Lac19. A false mapping of the start point has been described in similar systems [24] and could be due to the presence of the stem-loop structure.

With this primer, only transcription products that proceeded through the stem-loop structure could be detected. This long mRNA was present in greater amounts on lactose than on ribose-grown cells, while on the glucose lane bands were very faint, suggesting that transcription could not overcome the terminator-like structure. As described before (Fig. 3A), a higher transcription rate occurs from the lactose promoter when cells are grown on ribose. The reverse transcript found on ribose could be explained by some escape to the control by the terminator (Fig. 3B). These findings support the hypothesis of a lactose induction mechanism by which RNA polymerase could overcame the terminator structure with the aid of lacT gene product.

4Conclusion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References

Data from enzyme activity measurements as well as molecular assays strongly suggest that, in the presence of lactose, there is an activation of the lactose operon through an antiterminator mechanism.

Glucose clearly repressed enzyme activity possibly through the PTS/CcpA signal transduction pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References

This work was financed by the EU contract number BIO2-CT92-0137. Work in the laboratory of C.A.A. was supported by Grant SFB171C17 from the Deutsche Forschungsgemeinschaft. We thank Prof. B.M. Chassy for providing Lactobacillus casei strains. We are grateful to Francisco Rico for his assistance with the computer drawings.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgements
  8. References
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