1 Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA
2 Microbial Food Safety Research Unit, Eastern Regional Research Center (ERRC), Agricultural Research Service, US Department of Agriculture, Wyndmoor, PA 19038, USA
Correspondence
Brian J. Wilkinson
bjwilkin{at}ilstu.edu
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AY138856.
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INTRODUCTION |
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Low temperature has profound effects on all aspects of microbial cell structure and function, involving the structural integrity of macromolecules, macromolecular assemblies, protein synthesis and nutrient uptake (Panoff et al., 1998; Weber & Marahiel, 2002
). Small, nucleic-acid-binding, cold-shock proteins play key roles in resuming growth under cold shock (Panoff et al., 1998
). Low temperature also reduces membrane fluidity, and causes membrane phase transitions from a liquid-crystalline state to a more rigid gel-like state. To restore membrane functionality at low temperatures, fatty acids with low melting points are incorporated into lipids. The two most common ways bacteria increase membrane fluidity are by incorporating unsaturated and branched-chain fatty acids (BCFAs), which have lower melting points than the corresponding saturated straight-chain fatty acids, into their lipids (Suutari & Laakso, 1994
). The L. monocytogenes membrane-fatty-acid composition is dominated to an unusual extent by BCFAs (>90 % of the total fatty acid content) (Annous et al., 1997
). Among the common BCFAs in L. monocytogenes, anteiso-C15 : 0 has the lowest melting point (Kaneda, 1991
). When the growth temperature declines, anteiso-C15 : 0 content in the membrane rises to maintain optimal membrane fluidity (Annous et al., 1997
; Edgcomb et al., 2000
; Nichols et al., 2002
).
Two Tn917 transposon-induced mutants (cld-1 and cld-2) have lost the ability to grow at 4 °C on solid media, but are not defective in the induction of cold-shock proteins (Bayles et al., 1996). These mutants are deficient in the production of odd-numbered BCFAs, and exhibit atypical amounts of even-numbered straight-chain and iso-BCFAs (Annous et al., 1997
; Edgcomb et al., 2000
). The membranes of strain cld-1 are significantly less fluid than those of the parent strain (Edgcomb et al., 2000
; Jones et al., 2002
). To further characterize this mechanism of psychrotolerance, it was important to identify the disrupted gene(s) that caused the cold-sensitive phenotype in the mutants. Based on the observations above, we speculated that the insertion of Tn917 into cld-1 and cld-2 may interrupt the gene(s) involved in BCFA biosynthesis (Annous et al., 1997
). In bacteria, the branched-chain portion of fatty acids derives primarily from the branched-chain amino acids (Kaneda, 1991
; de Mendoza et al., 2002
). Moreover, incorporation of a methyl branch can only be achieved by de novo biosynthesis of fatty acid (Suutari & Laakso, 1994
). Previous studies of BCFA biosynthesis have primarily focused on Bacillus subtilis (de Mendoza et al., 2002
). Two critical enzymes in the pathway from branched-chain amino acids to BCFA have been reported: branched-chain
-keto acid dehydrogenase (Bkd) and
-ketoacyl-acyl carrier protein synthase III (FabH) (Oku & Kaneda, 1988
; Choi et al., 2000
; Lu et al., 2004
). Isoleucine, valine and leucine are transaminated by branched-chain amino acid transaminase, and are subsequently oxidatively decarboxylated by the Bkd complex, resulting in production of the short branched-chain acyl-CoA derivatives 2-methylbutyryl-CoA, isobutyryl-CoA and isovaleryl-CoA (Fig. 1
). These acyl-CoA precursors are then utilized by FabH as starting units to initiate BCFA biosynthesis (Fig. 1
). Although the cold-regulated fatty acid adjustment is one universal adaptation to low temperature, the underlying mechanism of cold-regulated BCFA synthesis is not well characterized (de Mendoza et al., 2002
; Lu et al., 2004
) Here, we identified that the two L. monocytogenes cold-sensitive mutants (cld-1 and cld-2) had Tn917 inserted into the bkd cluster. We also attempted to determine the role of Bkd in biosynthesis and cold-regulation of BCFA.
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METHODS |
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Molecular biological methods.
Unless specified, all molecular biological methods were performed as described by Sambrook et al. (1989). Chromosomal DNA from L. monocytogenes was isolated using a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's manual, except that cells were lysed by lysozyme (20 µg ml1) at 37 °C for 60 min.
Cloning and sequencing the L. monocytogenes DNA adjacent to the transposon insertions.
The L. monocytogenes mutants in this study carry a Tn917 insertion (Camilli et al., 1990). Genomic DNA was digested with restriction enzyme EcoRI, self-ligated and transformed into E. coli Top10 (Invitrogen). Plasmid DNA was isolated from kanamycin-resistant transformants using a miniprep kit (Qiagen). Chromosomal DNA in the resultant plasmid was sequenced using the Dye Terminator Cycle Sequencing Kit and an ABI Prism 310 sequencer (Applied Biosystems). The sequencing reaction was performed using two oligonucleotides 5'-GGAGCATATCACTTTTCTTGGAGAG-3' and 5'-ACGGTTGAAAACTGTACC-3', respectively corresponding to the 5' and 3' ends of Tn917. Using oligonuleotides 5'-AGATGTTGGGAAAAAAGGTGGC-3' and 5'-CTTGAAGAATAGCGGCTTGTGG-3', the DNA region that spanned the portion of the genome between the two Tn917 insertion sites was cloned into the PCR cloning vector pCR2.1 (Invitrogen), and subsequently sequenced using primer walking (Fig. 2
). The inverse PCR procedure with oligonuleotides 5'-GAAACAATGCTAATGGCGAG and 5'-CTCCAATCGCATAGATGTG-3' was used to amplify the DNA segment beyond the EcoRI site flanking the Tn917 insertion site. Nucleic acid and deduced amino acid sequence data were analysed using MacVector 6.5 edition (Oxford Molecular) and the National Center for Biotechnology Information (NCBI) BLAST network service.
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RNA isolation and Northern blot analysis.
In the branched-chain -keto acid induction analysis, strain 10403S was grown in BHI medium supplemented with 500 µM
-ketomethylvalerate at 37 °C. In the cold-induction analysis, strain 10403S was grown in separate BHI cultures at 37 °C and 10 °C. When the OD600 reached 0·30·4, cells were chilled on ice, and harvested by centrifugation. RNA protection solution (Qiagen) was added to stabilize RNA immediately after harvesting. A 1 ml volume of RLT buffer (RNeasy Mini Kit, Qiagen) and 0·5 g glass beads were added to the cell pellet obtained from 2·5 ml of culture. A bead beater (Biospec Products) was used to break the cells (5000 r.p.m., 20 s, six times). Total RNA was isolated using an RNeasy Mini Kit according to the manufacturer's instructions. RNA was treated with DNase I (Invitrogen) prior to Northern blot analysis. Northern hybridization was performed using the NorthernMax kit according to the manufacturer's manual (Ambion).
Fatty acid analysis.
Cultures of L. monocytogenes were grown in 100 ml BHI medium at designated temperatures in the presence or absence of the following supplements: short BCFAs bypassing Bkd [2-methylbutyrate (MB), isobutyrate (IB) and isovalerate (IV)], substrates of Bkd [-ketomethylvalerate (KMV),
-ketoisovalerate (KIV) and
-ketoisocaproate (KIC)] and pyruvate. Cells were harvested in the mid-exponential phase (OD600, 0·50·7), and the pellet was washed three times with distilled water. The fatty acids in the cells (3040 mg wet weight) were saponified, methylated and extracted. The resulting methyl ester mixtures were separated by an Agilent 5890 dual-tower gas chromatograph. Fatty acids were identified by the MIDI microbial identification system (Sherlock 4.5 microbial identification system). This analysis was performed at Microbial ID (Newark, DE, USA).
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RESULTS |
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Genetic complementation restores the growth of cld-1 and its BCFA content
In order to confirm that the transposon insertion in the mutants was the cause of the cold-sensitive and BCFA-deficient phenotype, the mutation was complemented by a plasmid bearing the wild-type bkdB gene. Shuttle vector pME2K8tet contained the putative promoter region and the complete bkdB gene (Fig. 2). Transformant cld-1-bkdB grew normally on BHI agar at 37 °C, as did strain 10403S. Furthermore, cld-1-bkdB showed the same growth pattern as 10403S at 10 °C (Fig. 3
). As shown in Table 1
, functional bkdB also restored the normal BCFA content in cld-1 from 39 % to 98 % of total fatty acids. Clearly the BCFAs were closely associated with the growth ability of L. monocytogenes at low temperature. These data demonstrate that bkdB is important for the BCFA synthesis, and that the interrupted bkdB in strain cld-1causes the BCFA deficiency. As a negative control, the intact bkdB did not fully restore the BCFA and growth of cld-2, presumably because of a lack of a wild-type lpd gene in the pME2K8tet construct.
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Temperature affects the growth-stimulatory activities of the BCFA precursors
As described above, addition of short BCFAs extensively changed the fatty acid composition in bkd mutant cld-2 (Table 2). To clarify the influence of different fatty acid compositions on the growth of L. monocytogenes at low temperature, cld-2 was grown at different temperatures in medium supplemented with these BCFA precursors. The stimulatory activities of the short BCFAs showed significant temperature dependence. At 26 °C, MB and IB restored the growth of cld-2 to the same level as the wild-type, but there was a prolonged lag phase not observed with strain 10403S (Fig. 4b
). IV exhibited no apparent growth-stimulatory activity at 26 °C. Only MB restored the growth of cld-2 at 10 °C (Fig. 4c
). Furthermore, the temperature-dependent activity prompted us to examine whether IB and IV could depress the stimulatory activity of MB at low temperature. When IB, IV and MB were added at the same concentrations, IB and IV exhibited little or no effect (data not shown). However, an excess of IB and IV significantly depressed the growth-stimulatory activity of MB at 10 °C (Fig. 5
). IB produced a stronger depression than IV, which was consistent with the fatty acid data showing that IB caused a greater decrease in anteiso-C15 : 0 (Table 2
). In contrast, at 37 °C, the same excess of IB and IV did not depress the MB-stimulated growth, but stimulated growth to a level above that with MB alone (data not shown). These data demonstrate that although L. monocytogenes is able to achieve optimal growth at 37 °C when it has a high content of either anteiso-BCFAs or iso-BCFAs, there must be a substantial amount of anteiso-C15 : 0 for growth at low temperatures.
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bkd transcription is induced by substrates, but not low temperatures
The role of the Bkd substrates in the regulation of bkd expression was investigated by Northern analysis. As shown in Fig. 6, a 7 kb transcript was detected, which might cover the whole bkd cluster, suggesting that ptb, buk, lpd, bkdA1, bkdA2 and bkdB were co-transcribed as an operon. The 7 kb bkd transcript was induced by exogenous branched-chain
-keto acids (Fig. 6a
), which was similar to bkd induction in B. subtils and E. faecalis (Debarbouille et al., 1999
; Ward et al., 2000
). Since cold-regulated BCFA synthesis is one of the major adaptive responses to low temperature, it was of interest to determine whether bkd expression was regulated by temperature. RNA isolated from strain 10403S grown at 37 °C and 10 °C was used to determine the role of low temperature in bkd transcription. As shown in Fig. 6(b)
, there was no significant difference between the bkd transcripts at 37 °C and 10 °C, indicating that the transcription of bkd remained at the same level when L. monocytogenes was grown at 37 °C and 10 °C.
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DISCUSSION |
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Since we sequenced the 5·6 kb bkd region of L. monocytogenes 10403S, the genome sequences of several L. monocytogenes strains have been published (Glaser et al., 2001; Nelson et al., 2004
). The bkd regions in L. monocytogenes strains EGD-e and F2365 are composed of the genes lmo1369lmo1374, and lmof2365_1386lmof2365_1391, respectively. Genetic comparison of the bkd regions from the three L. monocytogenes strains showed synteny between the strains, with nearly complete identity at the DNA level. Northern blot analysis indicated that the six genes in this cluster (ptb, buk, lpd, bkdA1, bkdA2 and bkdB) were co-transcribed, which was similar to what has been described for Pseudomonas putida, B. subtilis and E. faecalis (Burns et al., 1989
; Debarbouille et al., 1999
; Ward et al., 2000
).
Homology searches also revealed that the Bkd in L. monocytogenes exhibited considerable homology to pyruvate dehydrogenase (PDH) from various bacteria (data not shown). PDH and Bkd were the only two members of the 2-keto acid dehydrogenase complex family in L. monocytogenes (Glaser et al., 2001) that catalysed the decarboxylation of 2-keto acids, and generated acyl-CoA derivatives. The Bkd mutations in L. monocytogenes were not lethal, whereas a B. subtilis strain with mutation in both Bkd and PDH required addition of BCFA precursors for growth (Willecke & Pardee, 1971
). This suggests that PDH may compensate for the loss of Bkd activity to some extent. Indeed, in B. subtilis, PDH showed enzymic activity with branched-chain
-keto acids (Oku & Kaneda, 1988
). The PDH enzymic preference (pyruvate>KIV>KMV>KIC) is perfectly consistent with the fatty acid profile in bkd mutants, where straight-chain and KIV-derived, even-numbered iso fatty acids rose to compensate for the decreased production of KMV- and KIC-derived odd-numbered fatty acids (Table 1
). In addition, KIV was more efficient than KMV and KIC in stimulating growth, and restoring BCFA content (Table 2
, Fig. 4
). These observations did not match Bkd enzymic preference (KMV>KIV>KIC>pyruvate) (Oku & Kaneda, 1988
). The further decrease in BCFA content to 27 % in mutant cld-2 via pyruvate was presumably because pyruvate competed with branched-chain
-keto acids for PDH. A level of 27 % BCFA might be close to the minimum BCFA necessary for growth of L. monocytogenes, in that further increases of pyruvate did not further decrease the BCFA in cld-2. Similarly, B. subtilis must have a minimum of 28 % BCFAs in the membrane in order to survive (Kaneda, 1991
), and E. coli needs at least 20 % unsaturated fatty acids (Nunn et al., 1983
).
Northern blot analysis showed that transcription of the bkd operon was not elevated by low temperature. This was consistent with the observation that total BCFA content was largely unchanged regardless of growth temperatures (Table 3). The major change in fatty acid composition was the increase of anteiso C15 : 0 when L. monocytogenes was grown at low temperatures compared to 37 °C. The mechanism of cold-regulated synthesis of BCFAs remains elusive even in B. subtilis (de Mendoza et al., 2002
; Lu et al., 2004
). Strain cld-2 was able to accomplish a similar adjustment of fatty acid composition in response to low temperatures when BCFA precursors were provided. It seems that cold-regulated shortening of BCFA, in the presence of precursors, is not significantly impaired by the Bkd defect, indicating that Bkd is probably not the major cold-regulation point of fatty acid biosynthesis. Since incorporation of methyl-branches in BCFA could only be achieved by de novo biosynthesis of fatty acids (Suutari & Laakso, 1994
), the major cold-regulation point is likely to be located downstream of Bkd in the fatty acid biosynthesis pathway.
The substrate preferences of Bkd are involved in determining the ultimate cellular BCFAs, in that the precursor pool produced by Bkd is one major determinant in this pathway (Kaneda, 1991; Willecke & Pardee, 1971
). The predominance of anteiso fatty acids in L. monocytogenes implies that, in this organism, Bkd has a higher affinity for isoleucine-derived substrates. Impaired Bkd could no longer preferentially produce anteiso precursors, and the fatty acid composition in cld-2 was largely dependent on the exogenous precursor pool (Table 3
). Compared to 10403S (81 %), the decreased total anteiso fatty acid content in cld-2 (55 %) in the presence of a mixture of MB, IB and IV suggests that preference for anteiso precursors is partially lost due to the Bkd defect (Table 3
). Therefore the role of Bkd in preferential synthesis of anteiso precursor may make Bkd essential for growth of L. monocytogenes at low temperatures.
The activities of MB, IB and IV in stimulating growth of Bkd mutants at 37 °C were also reported in B. subtilis and Myxococcus xanthus (Willecke & Pardee, 1971; Toal et al., 1995
). Short BCFAs were activated to their acyl-CoA esters by acyl-CoA synthetase, and therefore compensated for the deficiency of BCFA precursors (Rock & Jackowski, 1985
) (Fig. 1
). Nevertheless, only the anteiso-BCFA precursor MB stimulated growth of Bkd mutants at 10 °C. The varying ability of BCFA precursors to support growth at different temperatures probably reflected the membrane fluidity requirements at that particular temperature. Although both iso-BCFAs and anteiso-BCFAs could provide sufficient membrane fluidity at 37 °C, only anteiso-C15 : 0 could provide the proper fluidity at low temperatures (Edgcomb et al., 2000
; Jones et al., 2002
). As a result, a substantial amount of anteiso-C15 : 0 was necessary for growth of L. monocytogenes at low temperatures. Supplementation with IB and IV led to increased iso-BCFAs at the expense of anteiso-BCFAs. The inhibitory activity of IB and IV on the growth of cld-2 in the presence of MB at low temperatures was probably due to competitive inhibition of enzymes in the fatty acid biosynthesis pathway, including the initiation enzyme FabH. Preference of these enzymes for isoleucine-derived substrates might explain why only large excesses of IB and IV showed activity. In conclusion, introduction of iso-BCFA-related metabolites should decrease anteiso-BCFA synthesis, and therefore depress or delay the growth at low temperature. This finding has the potential to be used to control the growth of L. monocytogenes, specifically at refrigeration temperatures.
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ACKNOWLEDGEMENTS |
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Received 19 September 2004;
revised 8 November 2004;
accepted 8 November 2004.
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