Abstract
The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.
Highlights
The 5’ and 3’ ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, ZuxG and ZuxH, were identified at the 3’ end of the lux operon
The transcriptional end points of the mRNA from the lux operon of V. harueyi have been defined, and two new genes encoded by the lux mRNA have been identified
Sl nuclease and primer extension mapping were the two techniques used to map the 5’ end of the mRNA and gave identical results providing that the amount of Sl nuclease was carefully controlled
Summary
Materials-Restriction enzymes were from Boehringer Mannheim or Pharmacia LKB Biotechnology. The probe, a loo-base &I-SspI single-stranded (ss) DNA fragment 5’ labeled at the SspI site with T4 polynucleotide kinase and [T-~‘P]ATP (0.1 pmol, 7.8 X 10’ cpm/pmol), was sealed in a glass microcapillary tube with RNA in a total volume of 10 ~1 containing 10 mM PIPES, pH 6.6, and 0.4 M NaCl. Hybridization was carried out at 55 “C for 12-16 h. 6 mM MgCl,, and 0.5 mM of the four dNTPs. The reaction products were phenol extracted, ethanol precipitated, resuspended in an 80% formamide, 10 mM NaOH dye solution, heat denatured, and resolved on an 8% acrylamide, 7 M urea sequencing gel. MRNA from cells grown in minimal medium containing arginine protected the DNA probe, and the same sized fragment was obtained as during induction in complex medium. The lOObase primer migrated as predicted with respect to the M13mp sequence ladder (data not shown), whereas the
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