Universität Kaiserslautern, Fachbereich Biologie, Abteilung Mikrobiologie, PO Box 3049, D-67653 Kaiserslautern, Germany1
Author for correspondence: Bernhard Henrich. Tel: +49 631 2052347. Fax: +49 631 2053799. e-mail: henrich{at}rhrk.uni-kl.de
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ABSTRACT |
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Keywords: Lactobacillus delbrueckii, ArbZ phospho-ß-glycosidase, substrate induction, catabolite repression
Abbreviations: MßGlc, methyl-ß-glucoside; oNPGal, o-nitrophenyl ß-D-galactopyranoside; oNPGalP, oNPGal 6-phosphate; pNPGlc, p-nitrophenyl ß-D-glucopyranoside; PTS, phosphotransferase system
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INTRODUCTION |
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Utilization of ß-glucosides by bacteria occurs through pathways similar to those described for the ß-galactoside lactose. In most cases, ß-glucosides are hydrolysed by extracellular or cell-wall-associated ß-glucosidases (Gräbnitz & Staudenbauer, 1988 ; González-Candelas et al., 1989
). If uncleaved ß-glucosides are transported into the cells, this is achieved either by specific permeases (Helaszek & White, 1991
) or via the PTS with subsequent hydrolysis of the resulting P-ß-glucosides into glucose 6-phosphate and the respective aglycons (Schnetz et al., 1987
; Tobisch et al., 1997
).
Connections between the metabolism of ß-glucosides and ß-galactosides were suggested by Simons et al. (1993) , who observed that, in revertants of lacG deletion mutants of Lactococcus lactis, a P-ß-glucosidase seemed to be responsible for their slow growth on lactose. In these revertants, the P-ß-glucosidase was not only induced by cellobiose but also by lactose. Relations between ß-glucoside and ß-galactoside utilization may also be deduced from sequence similarities of P-ß-galactosidases and P-ß-glucosidases which assign most of them to family I of glycosylhydrolases (Henrissat, 1991
). Furthermore, several P-ß-glucosidases and P-ß-galactosidases cleave C6-phosphorylated derivatives of both ß-galactosides and ß-glucosides (Witt et al., 1993
; Simons et al., 1993
).
Little is known about ß-glucoside metabolism in lactobacilli. For Lactobacillus plantarum, a glucose-repressible P-ß-glucosidase gene was recently identified (Marasco et al., 1998 ). We previously achieved cloning and sequence analysis of arbZ from Lactobacillus delbrueckii subsp. lactis. ArbZ seems to be involved in ß-glucoside metabolism since it conferred the ability to utilize the ß-glucoside arbutin when expressed in Escherichia coli (Weber et al., 1998
). Here we report on the expression and control of arbZ in Lactobacillus helveticus strain 3036(62), which is used in industrial food production.
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METHODS |
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Recombinant DNA techniques.
Restriction endonucleases and nucleic acid modifying enzymes (Roche) were used as recommended by the manufacturer. Cloning techniques were performed according to Sambrook et al. (1989) . Plasmids were prepared from Lb. helveticus as described by Anderson & McKay (1983)
. Electroporation of E. coli was performed as described by Dower et al. (1988)
, and Lb. helveticus was electroporated according to Bhowmik & Steele (1993)
.
For PCR amplification of a 817 bp arbZ fragment (nt 12932109, accession number Z86115), the primers 5'-CCACAATATCGAGCTCACCGCACCCAAC-3' and 5'-GCCGGCGATATCCCATGGAGTGAAGTCG-3' were used. The cat gene (encoded by pGK12 and pBW121) was amplified with the primers 5'-AAAGCACCCATTAGTTCAACAAACG-3' and 5'-AACCTTCTTCAACTAACGGGGCAGG-3'. PCR reactions were carried out with Taq DNA polymerase as recommended by the supplier (Appligene). The temperature profile (25 cycles) used for both amplifications was 1 min at 95 °C (denaturation), 1 min at 60 °C (annealing) and 1 min at 72 °C (elongation). As templates, 100 ng total bacterial DNA isolated with the Genomix scale-up kit (Talent, Triest) or 10 ng plasmid DNA (controls) was used.
Construction of pBW121.
A 1474 bp AccIXmaIII fragment, containing arbZ and the 3'-end of the preceding arbX gene ('arbX) (Weber et al., 1998 ), was made blunt at the XmaIII site with Klenow fragment of E. coli DNA polymerase I and inserted between the HpaII and SnaBI sites of the vector pGK12 (Kok et al., 1984
). The resulting plasmid, pBW121, was initially identified after transformation of E. coli ER1562 by screening on EMB-arbutin agar and then used to transform Lb. helveticus.
Northern and Southern hybridizations.
For Northern blot analyses, total RNA (4 µg), isolated with the RNeasy total RNA kit (Qiagen), was electrophoretically separated and blotted as described previously (Henrich et al., 1990 ). A 817 bp PCR-amplified arbZ probe was non-radioactively labelled, hybridized to the blotted RNA, and detected by using a digoxigenin DNA labelling and detection kit (Roche) as recommended by the supplier.
For Southern blot analyses, total DNA from Lb. helveticus 3036(62) was isolated using the Genomix scale-up kit. DNA fragments (2 µg), obtained by restriction with HindIII or ClaI, were separated on 0·7% agarose gels and blotted to nylon membranes (Sambrook et al., 1989 ). The 817 bp arbZ probe was fluorescently labelled, hybridized to the blotted DNA fragments, and detected as recommended by the manufacturer (Amersham) of the ECL random prime labelling and detection system used.
In vivo assay of ß-glycosidase activity.
Strains of Lb. helveticus were grown in SFB supplemented with appropriate sugars. At OD600 0·60·8, cells from 2 ml aliquots of the cultures were collected by centrifugation, washed twice with 1 ml Z-buffer (Miller, 1972 ), and resuspended in 1·5 ml of the same buffer. When appropriate, cells were permeabilized by the addition of chloroform to a final concentration of 14% (v/v). Three hundred microlitres of these suspensions was mixed with 10 µl of the respective substrates (10 mg ml-1 in Z-buffer) and incubated at 37 °C. When using the chromogenic substrates p-nitrophenyl ß-D-glucopyranoside (pNPGlc) (Fluka) and o-nitrophenyl ß-D-galactopyranoside (oNPGal) (Sigma), the reactions were stopped by the addition of 300 µl 2 M Na2CO3 as soon as a light-yellow colour had developed. Cells were subsequently removed by centrifugation, and the amounts of liberated p- or o-nitrophenol were determined photometrically at 410 nm or 420 nm, respectively. Cleavage of salicin or arbutin was assayed as described by Schaefler (1967)
. Each reaction was repeated at least four times. Units of ArbZ activity were expressed as nmol of the aglycons liberated from the respective substrates min-1. Specific activities were calculated as units per OD600 unit of the cell suspensions used in the assays.
Assay of (phospho) ß-glycosidase activity in cell-free extracts.
To prepare crude cell extracts, Lb. helveticus strains were cultivated in SFB supplemented with appropriate sugars. At OD600 0·40·7, cells were pelleted from 20 ml aliquots by centrifugation, washed twice with Z-buffer, and resuspended in 1·7 ml Z-buffer (mg cell wet wt)-1. After addition of 2·5 ml glass beads (/ 0·18 mm) (mg cell wet wt)-1, the bacteria were disrupted by shaking in a Vibrogen cell mill (Bachhofer) for 20 min at 4 °C at a frequency of 70 Hz. The samples were subsequently diluted by the addition of Z-buffer [1·5 ml (mg cell wet wt)-1] and thoroughly mixed. Cell debris and glass beads were removed by centrifugation for 10 min at 14800g and 4 °C, and the protein concentration in the supernatants (cell-free extracts) was determined with Coomassie blue (Spector, 1978
). To determine the activity of ß-glycosidases and P-ß-glycosidases in cell extracts (Weber et al., 1998
), the chromogenic substrates oNPGal, oNPGal 6-phosphate (oNPGalP) (Sigma) and pNPGlc (Fluka) were used. Each reaction was repeated at least three times. Enzyme units were defined as nmol nitrophenol liberated min-1, and specific activities were expressed in units (mg protein)-1.
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RESULTS AND DISCUSSION |
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Lb. helveticus 3036(62) was transformed with the arbZ-containing plasmid pBW121, or with the vector pGK12 as a control. Of about 100 chloramphenicol-resistant clones obtained from two independent transformations with pBW121, 24 were tested for utilization of the ß-glucoside arbutin in SFB-arbutin. Of these transformants, only four (two from each transformation) were stably positive for arbutin fermentation. No arbutin-fermenting clones were detected among a similar number of pGK12 transformants, even after prolonged incubation.
Attempts to prepare plasmids from pBW121 transformants and pGK12 transformants (Anderson & McKay, 1983 ) did not yield electrophoretically detectable amounts of plasmid DNA. In a more sensitive assay, we used the material obtained from these preparations to retransform the restriction/modification-deficient E. coli strain ER1562. As estimated from control experiments with known concentrations of pBW121 and pGK12, fresh transformants of Lb. helveticus, after growth in chloramphenicol-containing MRS broth for 12 h, contained less than one plasmid copy per cell. More than 50% of these plasmids were clearly reduced in size as compared with pBW121 and pGK12, respectively. These effects pointed to structural instability of pBW121 and pGK12 in the Lb. helveticus host, since both plasmids were structurally stable during propagation in E. coli. After repeated subculturing of Lb. helveticus 3036(62)(pBW121) and Lb. helveticus 3036(62)(pGK12), no plasmid DNA at all was detectable in the E. coli retransformation assay.
When total DNA prepared from subcultures of each of the four arbutin-positive pBW121 transformants was used as template for PCR, arbZ as well as the plasmid-encoded cat gene could be amplified, whereas in pGK12 transformants only cat was detectable. The presence of arbZ in the arbutin-utilizing Lb. helveticus 3036(62)(pBW121) clones was also verified by Southern blot analysis (Fig. 1). An arbZ probe gave a single hybridization signal with total DNA from each of the four clones, digested with restriction enzymes (HindIII or ClaI) which do not cut within arbZ. No signals were obtained with DNA from pGK12 transformants. When ClaI- or HindIII-restricted plasmid pBW121 (one ClaI site, no HindIII site) was probed, the positions of arbZ-specific signals were clearly different from those observed with total DNA from the arbutin-positive pBW121 transformants (Fig. 1
). This indicated that arbZ had been integrated into the chromosome of the transformants.
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As Lb. helveticus 3036(62) is not able to ferment ß-glucosides and has no native (P)-ß-glucosidase (see Table 1), the presence of a functional arbZ homologue in this strain can be excluded. So, even if weak homology between the 'arbX-arbZ region in pBW121 and the Lb. helveticus chromosome was assumed, integration into a non-functional arbZ-like sequence of the host would most probably lead to inactivation and disruption of the plasmid-encoded arbZ gene. This was excluded by measurements of ArbZ activity in the transformants (see Table 1
) and by demonstrating the integrity of arbZ in PCR and Northern blot (Fig. 2b
) analyses.
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Fermentation of ß-glucosides by Lb. helveticus 3036(62)(pBW121)
Our previous studies showed that arbZ, when expressed in E. coli, allowed for utilization of arbutin, whereas fermentation of other ß-glucosides could not be detected (Weber et al., 1998 ). With the arbutin-positive Lb. helveticus 3036(62)(pBW121) clones a different substrate pattern was observed. In SFB, not only arbutin, but also the ß-glucosides salicin and cellobiose, and, with a lower efficiency, methyl-ß-glucoside (MßGlc) (Sigma), were utilized as carbon sources and converted to acidic fermentation products. The growth rates ranged between 0·003 and 0·004 min-1 for cellobiose, salicin and arbutin and was 0·0012 min-1 for MßGlc. Cleavage of arbutin and salicin was verified by assaying the hydrolytic activity of the transformants towards these substrates. The arbutin-positive Lb. helveticus 3036(62)(pBW121) clones, in addition, were able to cleave aesculin, as indicated by a positive reaction on aesculin-iron agar. The transformants with pGK12, in contrast, were not able to grow in the presence of arbutin, salicin, cellobiose or MßGlc or to hydrolyse arbutin, salicin or aesculin. When glucose or the ß-galactoside lactose was used as carbon source, no differences in the growth rates (0·0056 min-1 for glucose, 0·0053 min-1 for lactose) were observed between transformants carrying pBW121 or pGK12, respectively. This indicated that the ability of Lb. helveticus to utilize ß-glucosides depended on the presence of the cloned arbZ gene.
arbZ confers P-ß-glycosidase activity
Expression of arbZ was clearly detectable in the in vivo assay. After growth with glucose, lactose or xylose, each of the four arbutin-positive Lb. helveticus 3036(62)(pBW121) clones showed 1030-fold higher activities towards the ß-glucoside pNPGlc than the respective pGK12 transformants (Table 1). Permeabilization of the cytoplasmic membrane by chloroform prior to the assay resulted in a decrease of ArbZ activity, which was most pronounced (45-fold) after growing the cells in the presence of salicin. This suggested that ArbZ activity towards ß-glycosides required the integrity of the cytoplasmic membrane. As possible explanations, membrane association of ArbZ or its requirement for phosphorylated substrates were considered. Since membrane location of ArbZ was unlikely (Weber et al., 1998
), we chose the C6-phosphorylated ß-galactoside oNPGalP for activity tests with cell-free extracts. This substrate seemed appropriate, as chromogenic derivatives of phosphorylated ß-glucosides were not available and cleavage of oNPGalP had previously been reported for P-ß-glucosidases of E. coli (Witt et al., 1993
) and Lc. lactis (Simons et al., 1993
). Cell-free extracts prepared from the arbutin-positive Lb. helveticus 3036(62)(pBW121) clones, after growth with glucose or xylose, showed 5- or 1·5-fold higher activities towards oNPGalP than those from pGK12 transformants, which demonstrated that ArbZ contributed to cleavage of this substrate. Notably, extracts from salicin-grown cells were more than 20-fold less active towards pNPGlc or oNPGal than towards oNPGalP (Table 1
). This clearly indicated that ArbZ was able to efficiently hydrolyse the phosphorylated substrate in the absence of a functional cytoplasmic membrane. We therefore classified ArbZ as a P-ß-glycosidase.
After growth with lactose, extracts of transformants with both pBW121 or pGK12 contained more than 100-fold higher oNPGalP-hydrolysing activities, as compared with extracts of glucose-grown cells (Table 1). This suggested that, under these conditions, the activity of ArbZ towards oNPGalP was concealed by a native, lactose-inducible P-ß-galactosidase of Lb. helveticus 3036(62). As deduced from in vitro assays (Table 1
) and in vivo assays (not shown) with oNPGal after growth on lactose, this strain in addition has a native ß-galactosidase. Coincidence of both activities in the same strain has previously been reported for other lactic acid bacteria (Premi et al., 1972
; Okamoto & Morichi, 1979
).
Since Lb. helveticus, in the absence of cloned arbZ, did not grow at all with salicin as carbon source, we could not exclude that high oNPGalP-hydrolysing activities in extracts of salicin-grown pBW121 transformants were partly due to induction of the native P-ß-galactosidase by salicin. This, however, seemed unlikely, since induction of oNPGalP cleavage by another ß-glucoside, MßGlc, also required the expression of arbZ, and no induction of the native P-ß-galactosidase by MßGlc was observed in this case (not shown).
Induction of ArbZ activity by ß-glucosides
ArbZ activity of the arbutin-positive Lb. helveticus 3036(62)(pBW121) clones depended on the carbon source in the growth medium (Table 1). When salicin or arbutin was used, the in vivo activities were about 10-fold higher than in the presence of xylose and more than 25-fold higher than in the presence of lactose. This indicated that ArbZ is subject to substrate induction, which is a common feature in bacterial ß-glucoside catabolism (Schnetz & Rak, 1988
; Krüger et al., 1996
; Woodward & Wiseman, 1982
).
Two independently isolated arbutin-positive Lb. helveticus 3036(62)(pBW121) clones were grown in SFB-xylose, and the time courses of ArbZ induction were followed after addition of various ß-glucosides. Since both clones showed very similar induction kinetics, only one set of curves is presented in Fig. 2(a). Cellobiose, although being a substrate for ArbZ, was only a poor inducer. A similar observation, previously reported for a ß-glucosidase from Botryodiplodia theobromae (Woodward & Wiseman, 1982
), has been ascribed to repression by glucose, which is liberated by cleavage of cellobiose in twofold higher amounts than from other ß-glucosides. Arbutin, salicin and MßGlc led to strong increase of activity within 60 min after their addition (Fig. 2a
). Arbutin was the most potent inducer, while induction by MßGlc occurred more slowly. The activities induced by arbutin and salicin slowly declined after reaching a maximum, probably due to cleavage of these ß-glucosides. The inducing effect of MßGlc persisted longer, which is in accordance with low ArbZ activity towards MßGlc, as concluded from growth experiments.
Total RNA, prepared from arbutin-positive Lb. helveticus 3036(62)(pBW121) transformants after induction with MßGlc, was analysed in Northern hybridizations with an arbZ-specific probe. Increase of ArbZ activity after induction was found to be accompanied by simultaneous increase of a 960 nt arbZ transcript (Fig. 2b). A similar transcript was not found in pGK12 transformants. This demonstrated that ß-glucoside-dependent control of ArbZ occurs at the transcriptional level. Antitermination may be considered as a possible mechanism to enhance arbZ transcription, since several other prokaryotic systems for ß-glucoside metabolism are subject to this type of control (Schnetz & Rak, 1988
; Krüger et al., 1996
). The arbZ upstream sequence, however, does not contain any stemloop structures or RAT motifs (Aymerich & Steinmetz, 1992
) typical of other transcriptional antiterminators.
We previously demonstrated that the 1474 bp fragment of chromosomal DNA present in pBW121 contains a functional promoter between 'arbX and arbZ and a terminator downstream of arbZ. These signals determined the ends of a 960 nt arbZ transcript which we detected in Lb. delbrueckii subsp. lactis DSM 7290 after growth with arbutin or salicin (Weber et al., 1998 ). It should be noted that the arbZ transcripts, detected after MßGlc induction of Lb. helveticus 3036(62)(pBW121) transformants (Fig. 2b
), had exactly the same size. This clearly indicated (i) that arbZ had retained its physical integrity during integration into the Lb. helveticus genome and (ii) that substrate induction actually occurred at the native arbZ promoter and not at a promoter which might be located adjacent to the arbZ integration site.
Catabolite control of ArbZ
After growth with glucose, whole cells or extracts of the four arbutin-positive Lb. helveticus 3036(62)(pBW121) clones showed 5- or 3·6-fold lower activities towards pNPGlc or oNPGalP, respectively, than after growth with xylose (Table 1). ArbZ therefore seemed to be subject to catabolite repression in these transformants. To verify this, different hexoses were added to cultures of two independently isolated arbutin-positive Lb. helveticus 3036(62)(pBW121) clones grown in SFB-xylose, and simultaneously arbZ was induced with MßGlc. As shown in Fig. 3
, each of the sugars tested (glucose, fructose, mannose, galactose) had an inhibitory effect on induction of ArbZ activity. There were, however, significant differences in the time courses of ArbZ induction, which allowed for classifying the sugars into two groups. (i) Inhibition by glucose and mannose was very effective. In the presence of 0·3% of these sugars no induction of ArbZ was observed within 7 h. The repressive effect was detectable immediately after addition of the sugars and resulted in a time delay of induction. The length of this delay was proportional to the concentration of the respective sugars whereas the final ArbZ activities were inversely related. This effect was more pronounced for glucose than for mannose (Fig. 3a
, b
). The immediate repression effect of glucose and mannose may be explained by a transport-dependent control mechanism such as inducer exclusion (Ye & Saier, 1996
) or involvement of a transcriptional regulator with a PTS regulation domain (PRD) (Stülke et al., 1998)
. It is also reminiscent of transient repression, previously described for the lac operon of E. coli (Tyler & Magasanik, 1969
). (ii) Inhibition by fructose and galactose was more moderate; distinct ArbZ induction was still observed in the presence of 0·3% of the sugars (Fig. 3c
, d
). In contrast to glucose and mannose, the inhibitory effect of galactose and fructose only became visible about 60 min after addition of the sugars and resulted in flattening of the induction curves. Such effects would be expected for a repression mechanism that depends on formation of specific intermediates during catabolism of the respective sugars. The maximum ArbZ activities after addition of fructose or galactose showed a similar dependence on sugar concentrations as observed for glucose and mannose.
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CcpA, through its interaction with intermediates of carbohydrate catabolism, is able to respond not only to glucose but also to a number of other sugars (Saier et al., 1996 ). Therefore, repression of ArbZ by each of the four sugars tested might be a reflection of a CcpA-related control mechanism. This option is further supported by the presence of a palindromic sequence (5'-TGAGAACGATGACT-3') upstream of arbZ (Weber et al., 1998
), which overlaps with the first nucleotide of the putative -35 region and matches the consensus sequence of catabolite responsive elements (cre) (Weickert & Chambliss, 1990
) in 13 out of 14 positions. The cre sequence is known to interact with CcpA in a number of catabolite-controlled operons, and CcpA usually has a repressive effect if (as in the case of arbZ) cre overlaps the respective promoters or is located downstream of them (Weickert & Chambliss, 1990
; Hueck et al., 1994
; Saier et al., 1996
). It is therefore tempting to speculate that cre and CcpA homologues are involved in catabolite repression of arbZ.
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ACKNOWLEDGEMENTS |
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Received 29 March 2000;
accepted 28 April 2000.
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