Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.     

Use of chromosomal gene fusions to investigate the role of repetitive DNA in regulation of genes involved in lipopolysaccharide biosynthesis in Haemophilus influenzae.

The lic3 locus of Haemophilus influenzae consists of four open reading frames. The derived amino acid sequences of orf2 and orf4 exhibit homology to Escherichia coli GalE and AdK, respectively. The functions of orf1 and orf3 remain unknown. orf1 contains multiple tandem repeats of the tetrameric DNA sequence CAAT near the 5' end. Two possible translational starts (ATG1 and ATG2) lie upstream. We have used lacZ fusions to investigate whether changes in the number of CAAT repeats in conjunction with differential usage of the upstream frames control the expression of lic3-orf1. Phase-variable expression of lacZ was observed for individual colonies and could be related to variable numbers of CAAT repeats. Of the three possible upstream frames, only one, containing the more downstream of the two possible ATG start codons (ATG2), is used for strong expression of lacZ. Utilization of the more upstream ATG (ATG1) or ATG2 was observed with medium-level expression, while utilization of any of the three possible frames was observed when lacZ was expressed at low to undetectable levels, indicating that other mechanisms may affect expression. To investigate this, lacZ was fused in frame with ATG2 of lic3-orf1, with concomitant deletion of the repeats. Phase-variable expression was still observed, supporting the view that an alternative level of control operates in conjunction with the repeat mechanism.