Multiple transcriptional control of the Lactococcus lactis trp operon

Abstract

The Lactococcus lactis trpEGDCFBA operon is preceded by a noncoding leader region. Transcriptional studies of the trp operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire operon), 290 bases, and 160 bases (corresponding to parts of the leader region). These transcripts most likely result from initiation at the unique Ptrp promoter, transcription termination at either T1 (upstream of the trp operon) or T2 (downstream of the trp operon), and/or processing. Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly. The tryptophan-dependent transcription control is exerted through transcription antitermination.

FIG. 1

Identification of the trp transcripts. The upper part of the figure presents the genetic structure of the trpEGDCFBA operon (2, 3). The three open boxes represent DNA fragments used as probes in Northern blot experiments; they were synthesized by PCR and correspond to the sequence coordinates 592 to 3210, 3193 to 5866, and 5846 to 8632 in sequence M87483, respectively (2). Northern hybridization was carried out on RNA prepared from cells noninduced (+) or induced (−) with tryptophan (Trp) as described in Materials and Methods.

FIG. 2

Modulation of the amount of the trp transcripts in response to tryptophan availability. The amounts of the different transcripts in cells incubated for 30 min with (+) or without (−) tryptophan were compared in a Northern blot experiment. Oligonucleotide 6 was used as a probe.

FIG. 3

Structure and Northern blot analysis of the trp operon. (A) Structure of the leader and 3′ region of the trp operon. Numbers refer to nucleotides in sequence M87483 (2). Boxes correspond to genes. T1 and T2 indicate transcription terminators, and P_trp indicates the transcription promoter. (B) Northern analysis of the trp transcripts. Total RNA from induced cells was hybridized with either restriction fragments a to c or oligonucleotides 1 to 7 (see Materials and Methods). Hybridization (+) and absence of signal (−) are indicated. (C) Mapping of the trp transcripts. This transcription map combines results from Northern analysis and 5′ end mapping of the transcripts.

FIG. 4

Determination of the 5′ ends of the trp transcripts by primer extension. RNA isolated from noninduced cells (lanes 1) or induced cells (lanes 2) was used in a primer extension assay with oligonucleotide 5 as described in Materials and Methods. The extension products were run alongside sequencing reaction products obtained with the same primer. Only regions of the gel containing bands of extension product are shown. The localization of the 5′ ends within the sequence is indicated by arrows.

FIG. 5

Transcript production by different plasmids. (A and B) RNA extracted from L. lactis cells containing the indicated plasmids was hybridized in a Northern blot experiment with appropriate probes. (C) Schematic representation of the relevant regions of the plasmids used. Segments of trp leader are indicated by heavy lines. Since the plasmids used have high copy numbers, transcripts originating from plasmids outnumber transcripts originating from chromosome, which are therefore not visible in these experiments.

FIG. 6

Effect of tryptophan on the half-life of the 8-kb transcript. Production of the 8-kb transcript was induced by resuspending IL1403 cells for 30 min in CDM without tryptophan. Total RNA was isolated at time intervals after addition of 120 μg of rifampin per ml and either 100 μg of tryptophan per ml (+Trp) or no tryptophan (−Trp) and analyzed in Northern blot experiments with oligonucleotide 6 as the probe. Decay kinetics were measured in cells suspended in the presence (▪) or absence (□) of tryptophan.

FIG. 7

Fate of trp transcripts during growth cycle. (A) Growth curve of IL1403 in CDM with tryptophan (▪) or without tryptophan (□). (B) Fate of trp transcripts in cells grown in CDM with tryptophan. (C) Fate of trp transcripts in cells grown in CDM without tryptophan. Symbols: ▪ and □, 160-b transcript; ▴ and ▵, 290-b transcript; • and ○, 8-kb transcript. Downward arrows indicate that the amount of RNA is below the detection limit.