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Journal of Bacteriology, March 1999, p. 1924-1926, Vol. 181, No. 6
Microbial Ingredients Section, NIZO Food
Research, 6710 BA Ede, The Netherlands
Received 17 June 1998/Accepted 13 January 1999
The divergently transcribed sacBK and sacAR
operons, which are involved in the utilization of sucrose by
Lactococcus lactis NZ9800, were examined by transcriptional
and gene inactivation studies. Northern analyses of RNA isolated from
cells grown at the expense of different carbon sources revealed three
sucrose-inducible transcripts: one of 3.2 kb containing
sacB and sacK, a second of 3.4 kb containing
sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by
replacement recombination resulted in the constitutive transcription of
the sacBK and sacAR operons in the presence of
different carbon sources, indicating that SacR acts as a repressor
of transcription.
Sucrose can be utilized as a sole
carbon source by many bacteria, and the vast majority of bacteria take
up this disaccharide via the sucrose-specific
phosphoenolpyruvate-dependent phosphotransferase system (PTS) (1,
3, 8, 12, 15, 16). The PTS catalyzes the transport of sucrose
across the cytoplasmic membrane concomitant with its phosphorylation by
a sucrose-specific enzyme, enzyme II. The product of this
translocation, sucrose-6-phosphate, is then hydrolyzed to
glucose-6-phosphate and fructose by a sucrose-6-phosphate hydrolase
(EC 3.2.1.26). The glucose-6-phosphate can readily be used, while
the fructose has to be phosphorylated by an ATP-dependent fructokinase
(EC 2.7.1.4) before it can be metabolized via the glycolytic pathway.
Previously, a sucrose-6-phosphate hydrolase and a fructokinase had been
purified from Lactococcus lactis and characterized in detail
(13, 14). To investigate the utilization of sucrose by
L. lactis, we have cloned and analyzed the sacB, sacK, and the sacR genes, which encode
sucrose-specific enzyme II, a fructokinase, and a regulatory
protein, respectively. In order to analyze the role of the SacR protein
in the transcriptional control of the sucrose genes, the
sacR gene was disrupted, and the effects of this disruption
on the transcription of the sucrose genes were studied.
Sequence analysis of plasmids pNZ755 and pNZ9250 containing DNA
fragments from the nisin-sucrose conjugative transposon
Tn5276 (9), isolated from L. lactis
NZ9800 (6), revealed the presence of three new genes,
i.e., sacR, sacB, and sacK (Fig.
1) (see below), in addition to the
previously described sacA gene encoding a
sucrose-6-phosphate hydrolase (10). The
sacA stop codon partly overlaps the putative SacR start
codon (GTG). If functional, the sacR gene encodes a 318-residue protein showing significant sequence similarity to proteins
of the LacI-GalR family of bacterial regulator proteins (17). A transcription initiation start site was mapped
upstream of the sacR gene, which is preceded by a sequence
corresponding to those of consensus L. lactis promoters
(data not shown [2a]). Strikingly, the sacR
promoter contains an inverted repeat similar to those identified
in the sacB and sacA promoters
(10), which could represent the binding site of a
factor involved in sucrose-specific regulation. The
sacB gene, which is in the opposite orientation to the
sacA gene, encodes a protein that contains the enzyme II ABC
domains expected for an enzyme II protein of the PTS (8). The disruption of the sacB gene by a single-crossover
recombination (7) using plasmid pNZ9251 containing an
internal PstI-BamHI fragment (Fig. 1) resulted in
a strain that was no longer able to grow at the expense of sucrose,
indicating that the sacB gene is essential for the
utilization of sucrose. The sacB gene is immediately
followed by another gene, sacK, which encodes a 290-residue protein. The NH2-terminal amino acids 2 to 26 of the
deduced SacK sequence are identical to those determined for the
purified fructokinase I from L. lactis KI (14).
Moreover, the total amino acid composition and the calculated molecular
mass of the deduced protein (31,626 Da) are highly similar to those of
the purified lactococcal fructokinase (14).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of the Divergent sacBK
and sacAR Operons, Involved in Sucrose Utilization by
Lactococcus lactis
ABSTRACT
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FIG. 1.
Genetic and transcriptional organization of the
Tn5276-located sucrose gene cluster of L. lactis NZ9800. The genes (open arrows) are shown with their
products and mapped promoters (arrowheads) as well as the mapped
transcripts (arrows). Cloned chromosomal DNA fragments relevant to this
study are illustrated below the arrows, as are the names of the derived
plasmids on which they are carried. The putative terminator downstream
of the sacK gene is indicated (T1). Relevant
restriction sites are shown as follows: B, BamHI; K,
KpnI; M, MunI; P, PstI; and X,
XbaI.
The expression of sucrose catabolic genes is in most cases regulated at the transcriptional level (2, 4, 5). Northern analyses were performed in order to investigate the transcription of the sacBK and sacAR genes. RNA was isolated from cells of strain NZ9800 grown at the expense of glucose or sucrose as the sole carbon source as described previously (6). RNA was denatured and size fractionated on a 1% agarose gel containing formaldehyde according to standard procedures (11). The blot was probed with internal DNA fragments of all individual sac genes (Fig. 2). After hybridization with either a sacB- or sacK-specific probe, a transcript of approximately 3.2 kb was observed with RNA isolated from sucrose-grown cells, but not with RNA isolated from glucose-grown cells, indicating that the sacBK genes are located on a single sucrose-inducible transcript. The size of this transcript, in conjunction with its mapped transcription initiation site (10), suggests that the transcription terminates at a putative rho-independent terminator structure that was identified immediately downstream of the sacK gene (Fig. 1). Another sucrose-inducible transcript of approximately 3.4 kb was observed when RNA from sucrose-grown cells was hybridized with either a sacA or sacR probe. This transcript was absent in RNA isolated from glucose-grown cells. When L. lactis NZ9800 was grown on a mixture of sucrose and glucose, a severe reduction of the sacBK and sacAR transcription compared to that in cells grown on sucrose could be observed, indicating a form of glucose repression (Fig. 2). A third sucrose-inducible transcript of 1.8 kb was shown to hybridize with a sacR-specific probe, confirming the existence of a second regulated promoter driving transcription of the sacR gene. This transcript is likely to end at the same transcriptional terminator as the 3.4-kb transcript that initiates from the sacA promoter.
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To investigate the role of SacR in transcriptional control of the
sucrose genes, the sacR gene was disrupted. A
double-crossover recombination (7) using plasmid
pNZ9254, in which the MunI site in the sacR open
reading frame was filled in by using Klenow polymerase, resulted in the
introduction of a frameshift mutation (Fig. 1). No differences in
growth rate could be observed between strain NZ9860
(
sacR) and the wild-type strain, NZ9800, when the cells
were grown at the expense of either glucose or sucrose. The results of
Northern analysis of RNA isolated from strain NZ9860 (sacR) grown at the expense of glucose showed that
sacBK and sacAR transcription had become
constitutive at a level comparable to that of the wild-type strain
grown at the expense of sucrose (Fig. 2). These results
demonstrate that SacR acts as a repressor of both
sacBK transcription and sacAR
transcription. The levels of sacBK and sacAR
transcription in strain NZ9860 (sacR) grown on
glucose or sucrose were found to be similar, indicating that SacR not
only is involved in substrate induction but also mediates glucose
repression. The observed substrate induction and negative autoregulation of sacR by its gene product result in
efficient transcriptional control of the sac genes in
response to variations in extracellular sucrose concentrations.
Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under accession no. Z97015.
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ACKNOWLEDGMENTS |
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This work was partly supported by the BIOTECH Programme of the European Community (contract BIO2-CT92-0137).
We are grateful to Ger Rutten and Marke Beerthuyzen for technical assistance, Jack Thompson for helpful suggestions, and Michiel Kleerebezem and Roland Siezen for critically reading the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbial Ingredients Section, NIZO Food Research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-318-659525. Fax: 31-318-650400. E-mail: kuipers{at}nizo.nl.
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Journal of Bacteriology, March 1999, p. 1924-1926, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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