Interplay between three global regulatory proteins mediates oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon.

The Escherichia coli cydAB operon, encoding the subunits of the high-affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen-responsive regulators Fnr and ArcA. Here, we report that the histone-like protein H-NS is an aerobic repressor of cydAB expression. ArcA is shown to antagonize H-NS action to render cydAB expression insensitive to H-NS repression in anaerobiosis. The targets for H-NS-mediated aerobic repression are the four oxygen-regulated promoters, designated P1, P2, P3 and P4. H-NS control is the result of H-NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters. We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein-DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions. According to this model, ArcA-P plays a central role in cydAB regulation by antagonizing H-NS repression of cydAB transcription when oxygen becomes limiting. This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.

Oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon: roles of multiple promoters and the Fnr-1 and Fnr-2 binding sites.

The Escherichia coli cydAB operon encodes the high-affinity terminal oxidase of the oxygen respiratory chain, cytochrome d oxidase. The sensor-regulator pair, ArcB-ArcA, is responsible for the microaerobic activation of the cydAB operon, whereas the anaerobic regulator Fnr represses its expression in the absence of oxygen. Fnr binds in vitro at two sites within the cydAB promoter element. To discern whether these two regions have an in vivo function in the anaerobic regulation of cydAB, the Fnr-binding motifs were mutagenized individually and in combination. The effects of these mutations on in vivo gene expression were determined by lac fusion and primer extension analysis. Our results show that the Fnr-2 site is critical for Fnr-mediated anaerobic repression of the two main cydAB promoters, P1 and P2. In contrast, the Fnr-1 site has an auxiliary role in the anaerobic repression of P1, but not of P2. Transcription from P1 did not affect ArcA-mediated activation or Fnr-mediated repression of P2, indicating that oxygen regulation is exerted on both promoters in an independent fashion. In addition, three new promoters were identified in the cydAB control region, and the 5' ends of the corresponding transcripts were mapped. Two of these promoters, designated P3 and P4, are co-ordinately regulated with P1 and P2 in response to oxygen, ArcA and Fnr. The P5 promoter is not Fnr regulated and is only weakly activated by ArcA. The contribution of these three additional promoters to the overall cydAB expression is most relevant under aerobic conditions. Our results suggest a unique repression model, in which one Fnr dimer bound to one single site (Fnr-2) is sufficient to downregulate transcription from four cydAB promoters. In conclusion, transcription of the cydAB operon is driven by a complex regulatory element containing at least five promoters that act in unison to provide adequate oxygen control of gene expression.

Coordinate intracellular expression of Salmonella genes induced during infection.

Salmonella typhimurium in vivo-induced (ivi) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg2+, Fe2+, and PhoPQ. All of the seven ivi fusions that are induced by both low pH and low Mg2+ (e.g., iviVI-A) are activated by the PhoPQ regulatory system. Iron-responsive ivi fusions include those induced under iron limitation (e.g., entF) as well as one induced by iron excess but only in the absence of PhoP (pdu). Intracellular expression studies showed that each of the pH- and Mg2+-responsive fusions is induced upon entry into and growth within three distinct mammalian cell lines: RAW 264.7 murine macrophages and two cultured human epithelial cell lines: HEp-2 and Henle-407. Each ivi fusion has a characteristic level of induction consistent within all three cell types, suggesting that this class of coordinately expressed ivi genes responds to general intracellular signals that are present both in initial and in progressive stages of infection and may reflect their responses to similar vacuolar microenvironments in these cell types. Investigation of ivi expression patterns reveals not only the inherent versatility of pathogens to express a given gene(s) at various host sites but also the ability to modify their expression within the context of different animal hosts, tissues, cell types, or subcellular compartments.

Mechanism of translational coupling in the nifLA operon of Klebsiella pneumoniae.

The nifLA operon of Klebsiella pneumoniae encodes the sensor-activator pair involved in the regulation of other nif genes. Balanced synthesis of both proteins, which is required for correct regulation, is achieved by coupling translation of nifA to that of nifL. The mechanism of translational coupling at the nifLA operon was analysed using a specialized ribosome system, and the effect of substituting the natural Shine-Dalgarno of nifL or nifA for specialized Shine-Dalgarno sequences was determined. Our results indicate that translational coupling occurs in this operon by a reinitiation mechanism. Additionally, reinitiation at the nifA can happen even in the absence of good Shine-Dalgarno recognition by the reinitiating ribosome, although its efficiency is lower. The effect of a putative translational enhancer sequence (downstream box) on translational coupling efficiency was also determined. Mutations that reduce the homology of the putative downstream box to the consensus had only a minor effect on nifA translation by wild-type ribosomes. However, they had a significant effect on nifA translation by specialized ribosomes, suggesting that recognition of the downstream box may compensate inefficient ribosomal interactions with the Shine-Dalgarno sequence.

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae.

The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA. Results showed that a balanced synthesis of both gene products is essential for correct regulation. Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the phi10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA. In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low. This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL. Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA. Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes.

Transcription termination within the regulatory nifLA operon of Klebsiella pneumoniae.

The effect of premature stop codons in the nifL gene on the expression of nifA-lacZ operon and protein fusions in Klebsiella pneumoniae was analysed in detail. Our results revealed transcriptional polarity in this operon. By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified. As shown for other Rho-dependent terminators, their cytosine content is much higher than the incidence of guanines. However, other regions of the operon that have this feature did not show termination activity, suggesting that, contrary to previous reports, a correlation between these parameters cannot readily be established. Some of our results alos suggested that, in addition to polarity, other mechanisms may prevent expression of nifA when translation of nifL is altered. Their importance for efficient regulation of nitrogen fixation genes is discussed.

Geometry of the process of transcription activation at the sigma 54-dependent nifH promoter of Klebsiella pneumoniae.

The Klebsiella pneumoniae nifH promoter is very strictly controlled by nitrogen availability and highly dependent on sigma 54 and integration host factor (IHF) for expression. This promoter region has been used to examine the role of IHF in the activation of transcription from sigma 54-dependent promoters and to analyze the positional restrictions which may exist for an activation mechanism from distant sites such as this one. By functionally replacing the binding site of IHF by sequence-directed curved DNA fragments, it has been shown that the role of IHF in stimulating transcription is structural; it brings the molecules directly involved in the process into close proximity. Unlike other promoter regions with an activation mechanism at a distance, this IHF-dependent promoter requires a precise geometry for efficient transcription. In this sense, it resembles an activation mechanism from near sites. However, alternative functional structures which are very different from the native one can be isolated.