Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA1
Department of Food Science, University of Wisconsin-Madison, 1605 Linden Drive, Madison, WI 53706, USA2
Author for correspondence: James L. Steele. Tel: +1 608 262 5960. Fax: +1 608 262 6872. e-mail: jlsteele{at}facstaff.wisc.edu
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ABSTRACT |
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Keywords: Lactococcus lactis, lactic acid bacteria, aspartate aminotransferase, amino acid biosynthesis, aspartate biosynthesis
Abbreviations: Ap, ampicillin; ATase, aminotransferase; CFE, cell-free extract; Em, erythromycin; Opp, Lactococcus lactis oligopeptide transport system
The GenBank accession number for the sequence reported in this paper is AF035157
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INTRODUCTION |
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Strains of L. lactis used by the dairy industry are auxotrophic for a number of amino acids. The specific amino acid requirements are strain specific (Chopin, 1993 ) but generally include isoleucine, valine, leucine, histidine and methionine. Bovine milk is on average 3·6% protein by weight and the majority of the nitrogen exists as >11 kDa proteins called caseins (Swaisgood, 1985
). The non-protein nitrogen (free amino acids and small peptides) fraction of milk is not sufficient to support the growth of lactococci in milk to high cell densities (Juillard et al., 1995a
). Therefore, industrially important L. lactis strains contain a number of proteolytic and peptidolytic enzymes to liberate amino acids from milk caseins.
The proteolytic pathway of L. lactis is a well characterized metabolic system consisting of (at least) a cell-envelope associated proteinase (PrtP) and twelve intracellular peptidases (for a recent review see Christensen et al., 1999 ). Additionally, a broad specificity oligopeptide transport system (Opp; Tynkkynen et al., 1993
), two transport systems for di- and tripeptides (Foucaud et al., 1995
; Hagting et al., 1994
) and at least nine different amino acid transport systems (Poolman, 1993
) have been described. Mutants deficient in PrtP and/or Opp have been used to demonstrate that lactococcal strains that are unable to liberate and/or transport casein-derived oligopeptides only achieve 210% the final c.f.u. ml-1 in milk compared to PrtP+ Opp+ strains (Juillard et al., 1995a
). This suggests free amino acids and oligopeptides initially present in milk do not contribute significantly to the overall growth of L. lactis. In vitro data suggests that lactococci can obtain all amino acids through hydrolysis and transport of ß-casein-derived oligopeptides (Juillard et al., 1995b
). However, more recent in vivo studies have shown that lactococci utilize only a limited number of oligopeptides derived from the C-terminal end of ß-casein (Kunji et al., 1998
).
It is clear that PrtP and Opp are indispensable for growth of lactococci in milk to high cell densities, and that the rate of hydrolysis of caseins by PrtP is growth-rate limiting (Bruinenberg et al., 1992 ; Juillard et al., 1995a
; Helinck et al., 1997
). However, the importance of de novo amino acid biosynthesis for optimal growth cannot be excluded. In previous work, a L. lactis aspartic acid (Asp) auxotroph was isolated that acidified milk at a reduced rate compared to the wild-type strain (Wang et al., 1998
, 2000
). The molecular basis of the mutation(s) in this strain, which was derived by acriflavine mutagenesis, has not been reported. A mutant strain carrying a plasmid copy of the lactococcal pyruvate carboxylase gene, which is probably involved in Asp biosynthesis, acidified milk faster than the strain lacking this plasmid. However, the acidification rate of this plasmid-carrying mutant strain was still different to that of the parent strain containing the vector alone. Therefore, the construction of isogenic strains is necessary to clarify the importance of Asp biosynthesis during growth of L. lactis in milk.
Our laboratory has been studying amino acid aminotransferases (ATases) from L. lactis, with particular focus on their role in amino acid catabolism (Gao et al., 1997 , 1998
; Atiles et al., 2000
). ATases also catalyse the last biosynthetic step of many amino acids. Therefore, we are also interested in creating ATase mutants, identifying the amino acid biosynthetic pathways affected and determining whether the diminished ability of L. lactis to synthesize specific amino acids affects this organisms growth in milk. This paper describes the cloning and characterization of the aspartate ATase (aspC) gene of L. lactis LM0230 and the demonstration that strains lacking this gene are unable to synthesize the amino acids Asp or asparagine (Asn). Additionally, the mutant strain was unable to grow in milk, suggesting neither naturally present amino acids and peptides nor oligopeptides liberated from caseins by PrtP and transported by Opp are capable of fulfilling the Asp or Asn requirement of L. lactis.
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METHODS |
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Construction of a genomic library of L. lactis LM0230.
Chromosomal DNA was isolated from stationary phase M17-G grown L. lactis LM0230 by the method of Ausubel et al. (1989) . DNA was partially digested with Sau3AI and fragments were separated on a 1·0% agarose gel. Chromosomal fragments between approximately 6·5 and 9·5 kb were isolated using a GeneCapsule (Geno Technology). Fragments were ligated with alkaline phosphatase treated, BamHI digested pTRKL2. The products of ligation were electroporated into E. coli SURE and cells were plated onto LB agar containing Em (LBE). After incubation for 1 d at 37 °C, 2030 white colonies were picked into LBE broth, grown overnight and 125 µl aliquots from each were combined in a centrifuge tube. Plasmid DNA was isolated from the combined cultures by alkaline lysis (Sambrook et al., 1989
).
Cloning of aspC by complementation in E. coli DL39.
The plasmid pool created above was electroporated into E. coli DL39. Colonies growing on M9 plates lacking Asp were picked into LBE broth and plasmid encoded ATase activities were confirmed by enzyme assays.
DNA sequencing and sequence analysis.
DNA sequencing was performed essentially as described by Chen & Steele (1998) , using the Tn1000 kit (Gold BioTechnology) to generate nested sets of transposon insertions in pSUW419. Sequencing reactions were performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer-Applied Biosystems) and a DNA Thermal Cycler 480 (Perkin Elmer-Applied Biosystems). Sequencing reactions were analysed on an ABI 377XL Automated DNA Sequencer at the University of Wisconsin-Madison Biotechnology Center. Additional sequence information was obtained using primer walking to sequence across the SalI site of pSUW417. Sequences were analysed using the GCG sequence analysis package (Genetics Computer Group). Searches for protein sequences similar to the putative AspC sequence were performed using the BLAST network service (Altschul et al., 1997
).
Subcloning of the lactococcal aspC.
PCR subcloning of the aspC gene was accomplished using the primers 5'-AAAAAAAGATCTTCAATAAAGCGAACCAAG-3' (AspC-up) and 5'-ATATAAAGATCTCTAATTCAAAATCAGCCG-3' (AspC-down), and pSUW417 as template DNA (see Fig. 3). The primers were designed with a 6 bp recognition site for BglII (5'-AGATCT-3') at the 5' end. PCR was performed using the PCR Elongase Kit of Life Technologies. The cycling conditions were: 94 °C for 30 s, 50 °C for 30 s and 68 °C for 2 min for 30 cycles. The amplified product was digested with BglII and ligated to alkaline phosphatase treated, BglII digested pTRKL2. Ligation products were electroporated into E. coli SURE and transformants were plated onto LBE supplemented with IPTG and X-Gal. White colonies were picked into LBE broth, plasmids were isolated using alkaline lysis and plasmids carrying the amplified product were identified by restriction digests and enzyme assays.
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ATase enzyme assays.
When screening E. coli derivatives for AspC activity, the Aspartate Aminotransferase UV-test kit of Sigma was used. For quantitative assays with each of the 20 common amino acids, the assay contained in 1·4 ml: CFE (12 µg protein), 83 mM Bistris (pH 6·8), 6·7 mM amino acid, 54 µM pyridoxal-5-phosphate and 6·7 mM 2-oxoglutarate. The reaction temperature was 30 °C. At 5, 10 and 15 min, 400 µl aliquots of the reaction mixture were withdrawn and added to 200 µl 0·25 M HCl. Zero minute time points were made by adding the components of the enzyme assay directly into 200 µl 0·25 M HCl. After all time points were taken, samples were centrifuged to remove precipitated protein and the glutamate concentration in each stopped reaction was measured using the colorimetric L-glutamic acid assay kit of Roche Biomolecular. Specific activities were calculated as µmol formazan formed min-1 (mg protein)-1, using the extinction coefficient of formazan at 492 nm (19·9 mmol-1 cm-1).
Protein concentrations were determined using the Micro Protein Determination kit of Sigma and BSA as the protein standard.
Construction of L. lactis JLS400.
An internal deletion was introduced into aspC as follows. The lactococcal aspC was amplified from pSUW421 using the primers AspC-up and AspC-down. After purification of the amplified product using a QIAquick PCR purification column (Qiagen), the DNA fragment was digested with BglII, ethanol precipitated and ligated at low concentrations [about 15 ng DNA (µl ligation reaction)-1] to favour intramolecular ligation. A 1 µl aliquot of the ligation reaction was used as template DNA in a PCR reaction containing the primers Del1 (5'-P-AAATCCGACCGCTGTTGCTC-3') and Del2 (5'-P-ATTCCTGTTGGATTTGAAGGC-3') (see Fig. 3). The same PCR cycling conditions as described above for the primers AspC-up and AspC-down were used. The resulting product was purified using a QIAquick column, and was intramolecularly ligated. A third round of PCR and column purification was performed as described above using the primers AspC-up and AspC-down. The resulting DNA fragment was digested with BglII and ligated into similarly digested pTRKL2. The ligation mixture was transformed into E. coli SURE. Restriction analysis was used to identify plasmids carrying the deleted gene (
aspC) and sequence analysis using primer 5'-TTTGCCCTCTACGCTTAC-3' was used to screen for plasmids containing the desired 270 bp internal deletion in aspC. A plasmid that contained the appropriate deletion was designated pSUW422. The
aspC fragment was liberated from pSUW422 with BglII and ligated into similarly digested pG+host5 (Biswas et al., 1993
), forming pSUW423.
The aspC fragment was introduced into the chromosome of L. lactis LM0230 using pSUW423 and the double-crossover homologous integration method of Biswas et al. (1993)
. Strains containing the 270 bp deletion in aspC were screened for by PCR using the primers AspC-up and AspC-down.
Growth studies in defined medium and skimmed milk.
L. lactis strains were grown from a 1% inoculum in M17-L containing Em until early stationary phase (911 h). A 1 ml aliquot of cells was centrifuged, washed twice with 0·85% NaCl and resuspended in 1 ml 0·85% NaCl. Cells were inoculated into 30 ml DM containing Em or 100 ml steamed milk containing Em to an initial OD600 of 0·01 or 0·001, respectively. Cells were incubated at 30 °C and OD600 readings were recorded from DM or clarified milk as described previously (Chen & Steele, 1998 ). If necessary, cell suspensions after milk clarification or from DM cultures were diluted in 100 mM Bistris, pH 6·5, or water respectively, to obtain an OD600 between 0·03 and 0·30. These values were determined to be within the linear range for cell density readings. Readings for pH were determined using an Orion Research model 410A pH meter equipped with a Mettler Toledo Ingold Electrode (Nelson-Jameson). Values for µmax are reported as the mean of 46 growth experiments±SD.
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RESULTS |
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Downstream of aspC, a partial ORF was identified (Fig. 3). A BLAST search of the deduced amino acid sequence indicated this ORF might encode the N-terminal 75 residues of an asparaginyl-tRNA synthetase.
To confirm the identified ORF encodes the AspC activity detected in E. coli DL39(pSUW417), the ORF was amplified by PCR using primers Asp-up and Asp-down (Fig. 3). This fragment was cloned into pTRKL2 in both orientations with respect to the vector lacZ promoter, forming pSUW420 and pSUW421. AspC activity was detected in CFEs of both E. coli DL39(pSUW420) and DL39(pSUW421).
PCR reactions using the primers Asp-up and Asp-down successfully amplified a 1·5 kb fragment from the plasmid templates pSUW414, pSUW415 and pSUW416. Partial sequencing of these PCR products indicated all three of these plasmids encode aspC. No product was amplified when pSUW418 was used as template DNA.
The L. lactis LM0230 aspartate ATase has activity with Asp and no detectable activity with any other amino acids
The specific activity of AspC from CFEs of E. coli DL39(pSUW420) using Asp and 2-oxoglutarate as amino donor and amino acceptor, respectively, was calculated to be 1·07±0·06 µmol formazan formed min-1 (mg protein)-1. This value was determined from duplicate assays performed on two independent cultures of E. coli DL39(pSUW420). For all of the other common amino acids except for Cys, activities were below the quantifiable limit [0·06 µmol min-1 (mg protein)-1]. Assays using Cys as the substrate were inconclusive, as reducing agents interfere with the reaction catalysed by the L-glutamic acid assay kit.
A L. lactis aspC derivative requires Asp or Asn supplementation to grow in synthetic media or milk
A L. lactis LM0230 derivative containing a 270 bp internal deletion in aspC was constructed and designated JLS400. LM0230 and JLS400 were both transformed with pJK550, a plasmid carrying the genes for lactose utilization and PrtP of L. lactis C2O (Yu et al., 1996 ), which are essential for growth to high cell densities in milk. Additionally, the pJK550-containing strains were transformed with either pTRKL2 or pSUW421.
In DM, all four strains [LM0230(pJK550, pTRKL2), LM0230(pJK550, pSUW421), JLS400(pJK550, pTRKL2) and JLS400(pJK550, pSUW421)] entered exponential growth within 4·55·0 h after inoculation, produced acid at similar rates and reached a final OD600 of 2·22·4 (data not shown). Additionally, similar µmax values were calculated for all four strains (Table 2). Growth of LM0230(pJK550, pTRKL2), LM0230(pJK550, pSUW421) and JLS400(pJK550, pSUW421) occurred in MS15 minimal media (Table 2
). JLS400(pJK550, pTRKL2) grew in MS15 supplemented with Asn, but did not grow in MS15 or MS15 supplemented with Asp.
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DISCUSSION |
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AspCs are members of family I of the ATase superfamily (Jensen & Gu, 1996 ). Dendrograms aligning the sequences of ATases involved in the interconversion of aspartate and oxaloacetate reveal two distinct homology groups defined by Jensen & Gu (1996)
as subfamilies I
and I
(also defined as subfamilies Ia and Ib respectively by Okamoto et al., 1996
). These two families may differ slightly in their catalytic mechanism (Nakai et al., 1999
). Subfamily I
includes AspC from E. coli, and eukaryote cytosolic and mitochondrial AspCs. Subfamily I
includes the rest of the known eubacterial and archaeal AspCs. Based upon the results of BLAST searches, the lactococcal AspC is most similar to sequences within the I
group. The conservation of active site residues suggested to exist only within the I
subfamily (Nakai et al., 1999
), such as Lys109 (Lys102 in L. lactis) and Thr36-Ala37-Gly38 (Thr37-Leu38-Gly39 in L. lactis), supports this classification.
No rho-independent terminator was identified between the 3' end of aspC and the start of the ORF for a putative asparaginyl-tRNA synthetase (asnS). The genes for aspC and asnS in B. subtilis are also adjacent on the chromosome (Sorokin et al., 1996 ), although a transcriptional terminator with
G=-54·4 kJ mol-1 (Tinoco et al., 1973
) is found immediately 3' of the aspC ORF. However, it is unknown whether the lactococcal aspC is monocistronic or cotranscribed with asnS. The construct pSUW421 was capable of complementing the chromosomal aspC deletion in JLS400, suggesting cotranscription of aspC with the downstream asnS is not essential for expression of aspC in L. lactis. Further studies on the expression of aspC are needed to determine the mode of transcription of this gene.
Bacterial cells typically contain a number of ATases with overlapping substrate specificity (Jensen & Calhoun, 1981 ). For example, in E. coli both AspC and TyrB, an ATase that is preferentially active on aromatic amino acids, catalyse the transamination of oxaloacetate to Asp. However, growth studies in MS15 and milk suggest no other L. lactis enzyme can transaminate oxaloacetate in vivo efficiently enough to support growth under Asp-limiting conditions. This is consistent with the substrate specificity of the lactococcal aromatic (Gao & Steele, 1998
; Yvon et al., 1997
) and branched-chain (Atiles et al., 2000
; Yvon et al., 2000
) ATases, which lack detectable activity with Asp. Asp also appears to be the only amino acid substrate for AspC. Many other subfamily I
AspC homologues also have relative activities with Asp that are at least 100-fold higher than the activities with other amino acids (Marino et al., 1988
; Sung et al., 1990
; Xing & Whitman, 1992
; Okamoto et al., 1996
). However, the inability to detect activity of the lactococcal AspC with amino acids other than Asp may also have been due to the lack of sensitivity of the assay used, which was approximately 6% relative activity to that observed with Asp.
In a previous study a lactococcal strain was isolated after acriflavine mutagenesis which was unable to acidify 11% reconstituted skimmed milk to a pH less than approximately 5·0 in 6 h unless supplemented with Asp (Wang et al., 1998 ). This strain, designated KB4, produces decreased levels of the protein pyruvate carboxylase (Wang et al., 2000
), has approximately 5% the pyruvate carboxylase activity of the wild-type strain (Wang et al., 1998
) and lacks a 12 MDa plasmid present in the parent strain. The exact molecular basis of the mutation or mutations in KB4 has not been reported. As pyruvate carboxylase catalyses the formation of oxaloacetate, the biosynthetic precursor to Asp, this data suggested the growth defect was due to a decreased level of Asp production by KB4. This current study used homologous recombination to construct isogenic strains and demonstrate that a mutation in the L. lactis Asp biosynthetic pathway is sufficient to eliminate this strains ability to grow in milk. During six growth curve replicates, the strain which lacked both the wild-type chromosomal and plasmid copy of aspC never acidified milk to more than 0·20 pH units below that of the blank and did not grow at a detectable rate. As oligopeptides liberated from caseins by the lactococcal PrtP serve as the major nitrogen source for L. lactis in milk (Juillard et al., 1995a
), and L. lactis can apparently transport and hydrolyse at least one Asp-containing peptide from ß-casein (Kunji et al., 1998
), the inability of JLS400(pJK550, pTRKL2) to rapidly grow in or acidify milk suggests L. lactis is unable to liberate an adequate level of Asp or Asn from milk caseins to support optimal growth. As Asp is the biosynthetic precursor for Asn, threonine, lysine and methionine, as well as pyrimidine and purine nucleotides, Asp biosynthesis may also be essential for supplying the cell with sufficient levels of these biological compounds. It has previously been shown that L. lactis purine auxotrophs are unable to grow in milk (Dickely et al., 1995
); however, the necessity of threonine, lysine or pyrimidine biosynthesis is unknown. Asp is probably not diverted towards methionine biosynthesis in dairy lactococci, as these organisms are typically auxotrophic for methionine (Chopin, 1993
). Studies using milk supplemented with amino acids and nucleotides will be necessary to determine whether the growth defect of JLS400(pJK550, pTRKL2) is the direct result of the Asp auxotrophy, and/or is an indirect effect on other biosynthetic pathways.
One surprising result from the growth studies was that JLS400(pJK550, pTRKL2) failed to grow in MS15 media supplemented with Asp, but did grow when supplemented with Asn. The most likely explanation is that L. lactis LM0230 can convert Asn to Asp via an asparagine synthetase or related enzyme, and transport of free Asn is energetically more favourable than Asp transport. This is supported by previous studies which have shown that lactococci do not readily transport free Asp (Hillier et al., 1978 ) and that the affinity constant of the Asp transporter is approximately 80-fold higher than the affinity constant of the Asn transporter, for their respective substrates (250 µM vs 3 µM, respectively) (Konings et al., 1989
). This hypothesis is additionally supported by the fact that JLS400(pJK550, pTRKL2) acidifies Asp-supplemented milk slower than JLS400(pJK550, pSUW421) (Fig. 5
), while these two strains acidify Asn-supplemented milk at the same rate (data not shown).
Experiments to clone other ATase genes from L. lactis are ongoing. Construction of single and multiple ATase mutants will be used to further study the physiological role of these enzymes during growth in milk as well as their potential role in the generation of flavour precursors in fermented products.
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ACKNOWLEDGEMENTS |
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We thank Myrta Atiles and Cedric Mendiola for assistance in construction of the L. lactis LM0230 genomic library.
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Received 21 June 2000;
revised 20 September 2000;
accepted 27 September 2000.