A Promoter Region Binding Protein and DNA Gyrase Regulate Anaerobic Transcription of nifLA in Enterobacter cloacae

Abstract

Our work provides evidence that a sequence characteristic of FNR binding sites, when interacted with by a trans-acting factor, activates anaerobic transcription of the nifLA operon in Enterobacter cloacae. DNA gyrase activity has been found to be important for the anaerobic transcription of the nifLA promoter. Our results suggest that anaerobic regulation of the nifLA operon is mediated through the control of the promoter region-binding trans-acting factor at the transcriptional level, while DNA supercoiling functions in providing a topological requirement for the activation of transcription.In the nitrogen-fixing (nif) enteric bacteria Klebsiella pneumoniae and Enterobacter cloacae, the nif genes are regulated by the activity of the ntrC, ntrA, nifA, and nifL genes at the transcriptional level (2,3,4,24). nifL and nifA constitute an operon which is regulated by the product of ntrC or autoregulated by the product of nifA, NifA (5, 9). NifA acts as a positive regulator in conjunction with NtrA to activate nif genes (19), while the product of nifL, NifL, acts as a repressor of nif genes under oxygen or in an excess of fixed nitrogen (13).Our previous investigations showed that the nifLA promoter is highly sensitive to oxygen and that NifA is inactivated by NifL under oxygen (5,15,23). Accordingly, we hypothesized that nif regulation by oxygen is mediated at two different levels. First, at the transcriptional level, the oxygen sensitivity of the nifLA promoter ensures that the nifLA promoter and, hence, all other nif promoters are repressed by oxygen; second, at the posttranslational level, NifL interacts with NifA in the presence of oxygen. As the result of a shortage of active NifA, the expression of other nif genes is blocked.Direct interaction of NifL and NifA has been demonstrated by the two-hybrid system test (16, 23). As to the question of aerial regulation of the nifLA promoter, most investigators have claimed that neither FNR nor the oxrC gene product is involved in the expression of nifLA in response to oxygen (14). Since the activity of the K. pneumoniae nifLA promoter can be prevented by inhibition of DNA gyrase activity, they thus held that aerobic-anaerobic regulation of the nifLA promoter is mainly mediated through the level of DNA supercoiling (6,8). Our present work demonstrated that an upstream sequence of the nifLA promoter characteristic of the FNR binding sequence, when bound by the trans-acting factor from the bacterial cells, activates anaerobic expression of the nifLA operon. We also proved that DNA gyrase is truly important for anaerobic transcription of the nifLA promoter. A mechanism for aerobic regulation of the nifLA promoter is proposed. MATERIALS AND METHODSStrains and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. DNA manipulations.Preparation of plasmids DNA, endonuclease digestion, ligation, and transformation were carried out essentially as described by Sambrook et al. (21).Construction of nifLA promoter mutants. Initially, the E. cloacae nif...

Se(VI) Reduction and the Precipitation of Se(0) by the Facultative Bacterium Enterobacter cloacae SLD1a-1 Are Regulated by FNR

“…Complementation by the wild-type fnr sequence restored all abolished phenotypes. Based on GenBank entries, our nucleotide sequence analysis is the first to identify the fnr gene in Enterobacter; however, previous studies have reported an FNR-like protein in E. cloacae associated with the anaerobic transcription of 1-aminocyclopropane-1-carboxylate deaminase and the nifLA operon (9,12,23). Our data indicate that the fnr gene in E. cloacae SLD1a-1 is essential for Se(VI) reduction and is linked to anaerobic electron transfer processes.…”

Coordination of DNA replication and cell division in CyanobacteriaMicrocystis aeruginosa

“…Nalidixic acid is an inhibitor of DNA gyrase, which can unwind DNA supercoiling in a replication fork [3]. DNA replication blocking by nalidixic acid treatment caused Microcystis cells to inhibit the cell division and repress of ftsZ expression; however, it is known that DNA gyrase also selectively regulates the transcription of some genes [18]. We therefore used a different method of blocking chromosome replication, using hydroxyurea, which is an inhibitor of ribonucleotide reductase [2].…”

Diel cycling of DNA staining and nifH gene regulation in the unicellular cyanobacterium Crocosphaera watsonii strain WH 8501 (Cyanophyta)

“…The transcription of genes involved in nitrogen fixation has been shown to be sensitive to the supercoiling state of DNA in several different bacteria including Rhodopseudomonas capsulata, Klebsiella pneumoniae, Enterobacter cloacae, Sinorhizobium meliloti and the cyanobacterium Gloeothece (Nageli) sp. ATCC 27152 (Kranz and Haselkorn, 1986;Dimri and Das, 1988;Page and Gallon, 1992;Hu et al, 2000;Liu et al, 2005). Supercoiling of DNA is a topological change that can be assayed by changes in fluorescence using fluorescent DNA stains (Lebaron and Joux, 1994;Prosperi et al, 1994).…”