Genetic organization of the citCDEF locus and identification of mae and clyR genes from Leuconostoc mesenteroides

Abstract

In this paper, we describe two open reading frames coding for a NAD-dependent malic enzyme (mae) and a putative regulatory protein (clyR) found in the upstream region of citCDEFG of Leuconostoc mesenteroides subsp. cremoris 195. The transcriptional analysis of the citrate lyase locus revealed one polycistronic mRNA covering the mae and citCDEF genes. This transcript was detected only on RNA prepared from cells grown in the presence of citrate. Primer extension experiments suggest that clyR and the citrate lyase operon are expressed from a bidirectional A-T-rich promoter region located between mae and clyR.

FIG. 1

(A) Organization of the CL genes in L. mesenteroides. Every gene is drawn to scale, and orientations of transcription are indicated by arrows. The Sau3A DNA fragments cloned in plasmids pCL9 and pCg5 are shown as thin lines at the bottom. Transcription initiation points are indicated by small open circles, and the transcripts are indicated by a double broken line. Probes used for Northern blotting are shown as solid bars. (B) Northern blot autoradiogram. Total RNAs prepared from L. mesenteroides cells grown in the presence of citrate (lanes 1, 3, and 5) or in its absence (lanes 2, 4, and 6) were analyzed by electrophoresis on agarose-formamide gels, blotted onto a nylon membrane, and hybridized with mae (lane 1 and 2), citF (lane 3 and 4), or citG (lane 5 and 6). Arrows indicate the migration positions of 23S and 16S rRNAs and the 5.2- and 4.0-kb transcripts. Sizes were determined by using an RNA ladder (9.5 to 0.25 kb; Gibco-BRL).

FIG. 2

Comparison of part of the deduced amino acid sequence of mae from L. mesenteroides (LEUCM) with parts of the sequences of the malic enzymes from B. stearothermophilus (BACST; protein sequence accession no. P16468), Streptococcus bovis (SBOV; accession no. U35659), B. subtilis (YTSJ; accession no. Z99118), A. fulgidus (ARFUL; accession no. AE000984), and P. horikoshii (PYHOR; accession no. 3257695). Identical amino acids are indicated by asterisks. The conserved region corresponding to an NAD-binding domain and the malic enzyme signature are enclosed in boxes I and II, respectively. The numbers on the right are amino acid positions in the protein sequences. The percentages on the right are the levels of identity between the entire protein sequence deduced from mae and the sequences of the previously described proteins.

FIG. 3

Multiple-sequence alignment of ClyR with proteins belonging to the SorC family of transcriptional regulators: SorC, from K. pneumoniae (protein sequence accession no. P37078); DeoR, a deoxyribonucleoside regulator from B. subtilis (accession no. P39140); YdeW, a hypothetical transcriptional regulator in the HipB-UxaB intergenic region of E. coli (accession no. P76141); YgaP, a hypothetical transcriptional regulator in the gap 5′ region of Bacillus megaterium (accession no. P35168); YjhU, a hypothetical transcriptional regulator in the fecI-fimB intergenic region of E. coli (accession no. P39356). Sequence identities (percentages) between ClyR and the individual proteins are as follows: SorC, 23.3%; YjhU, 26.2% in a 221-amino-acid overlap; DeoR, 22.6%; YdeW, 20.7%; YgaP, 18.7%. The deduced primary sequences were aligned by the multiple-sequence alignment program Multalign (5). Amino acids conserved in at least three of the six sequences shown are shaded. The helix-turn-helix domains are white letters in black boxes. The derived consensus (Cons) sequence corresponding to amino acids conserved in at least four sequences is in the bottom row. All sequences are numbered from Met-1.

FIG. 4

Determination of the transcription start site of mae and clyR. (A) Primer extension performed with clyR primer 1. (B) Primer extension performed with mae primer 4. Primer extension reactions were performed as described in the text. Primer extension products were obtained by using RNA isolated from L. mesenteroides cells grown in the presence of citrate (lane 1) or in its absence (lane 2). The same oligonucleotide primer used for the primer extension analysis was used for sequencing by the dideoxy-chain termination method. G, guanine; A, adenosine; T, thymine; C, cytosine. A sequence complementary to that read from the ladder is shown, and the start site of the transcript is indicated by an arrow. (C) Nucleotide sequence of the mae-clyR intergenic region containing the bidirectional promoter region of the mae-citCDEF and clyR genes. The 239-bp intergenic region between translation codons of the mae and clyR genes is in boldface. The −10 and −35 regions and the transcription initiation sites (+1) are white letters in black boxes. The putative RBSs are shaded. The ATGs of the mae and clyR genes are underlined.