During denitrification, the production and consumption of nitric oxide (NO), an obligatory and freely diffusible intermediate, must be tightly regulated in order to prevent accumulation of this highly reactive nitrogen oxide. Sequencing upstream of norCB, the structural genes for NO reductase, in the denitrifying bacterium Rhodobacter sphaeroides 2.4.3, we have identified a gene, designated nnrR, which encodes a protein that is a member of the cyclic AMP receptor family of transcriptional regulators. Insertional inactivation of nnrR prevents growth on nitrite, as well as the reduction of nitrite and NO, but has no effect on reduction of nitrate or photosynthetic growth. By using nirK-lacZ and norB-lacZ fusions, we have shown that NnrR is a positive transcriptional regulator of these genes. nnrR is expressed at a low constitutive level throughout the growth of R. sphaeroides 2.4.3. These results show that NnrR is not a global regulator but is instead a regulator of genes whose products are directly responsible for production and reduction of NO. Evidence is also presented suggesting that an NnrR homolog may be present in the nondenitrifying bacterium R. sphaeroides 2.4.1. The likely effector of NnrR activity, as determined on the basis of work detailed in this paper and other studies, is discussed.

Nitrite reductase catalyzes the reduction of nitrite to nitric oxide, the first step in denitrification to produce a gaseous product. We have cloned the gene nirK, which encodes the copper-type nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides, strain 2.4.3. The deduced open reading frame has significant identity with other copper-type nitrite reductases. Analysis of the promoter region shows that transcription initiates 31 bases upstream of the translation start codon. The transcription initiation site is 43.5 bases downstream of a putative binding site for a transcriptional activator. Maximal expression of a nirK-lacZ construct in 2.4.3 requires both a low level of oxygen and the presence of a nitrogen oxide. nirK-lacZ expression was severely impaired in a nitrite reductase-deficient strain of 2.4.3. This suggests that nirK expression is dependent on nitrite reduction. The inability of microaerobically grown nitrite reductase-deficient cells to induce nirK-lacZ expression above basal levels in medium unamended with nitrate demonstrates that changes in oxygen concentrations are not sufficient to modulate nirK expression.The availability of fixed nitrogen is often a major factor controlling the biological productivity of ecosystems. During the cycling of nitrogen in the biosphere, a significant sink for fixed nitrogen is denitrification, the reduction of nitrate to gaseous forms of nitrogen, primarily nitrogen gas (22). Gaseous forms of nitrogen are unavailable for use by the majority of organisms. Because denitrification is a process that results in the conversion of fixed forms of nitrogen to gaseous forms, it can have a significant impact on the productivity of an ecosystem. For example, nitrate concentrations in the ocean have been found to be a major factor limiting biological productivity (19). Denitrification is the major sink for ocean nitrate, establishing a direct link between denitrification and biological productivity in the marine environment (6). This has been dramatically demonstrated in recent studies that have shown that decreases in the rate of denitrification during the last glacial maximum may have increased the productivity of the ocean enough to lower the partial pressure of carbon dioxide in the atmosphere (1, 9).Denitrification is a respiratory process in which bacteria utilize nitrate and other inorganic oxides of nitrogen as alternate electron acceptors when oxygen concentrations are limiting. In the first step of denitrification, nitrate is reduced to nitrite (12). This reaction is not unique to denitrification, however, since it occurs during ammonification and assimilatory nitrate reduction. The next step in denitrification is the reduction of nitrite to nitric oxide (NO). This reaction is catalyzed by nitrite reductase and is the defining reaction of denitrification, since it produces the first gaseous intermediate (38). Moreover, nitric oxide-producing nitrite reductases are associated only with denitrification (12). There are two classes of nitrite reductase...

SummaryThe Escherichia coli Tat system mediates Secindependent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding b-galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat-dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be

During catabolic diseases such as sepsis, inflammation, and infection, a state of growth hormone (GH) resistance develops in liver. This has been attributed in part to increased production of the proinflammatory cytokine interleukin-1␤ (IL-1␤). To determine how IL-1␤ induces GH resistance, we studied the acid-labile subunit (ALS) gene whose hepatic transcription is increased by GH via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. IL-1␤ reduced the ability of GH to stimulate ALS mRNA in rat primary hepatocytes and ALS promoter activity in H4-II-E rat hepatoma cells. This inhibition was dependent on ALSGAS1, an element resembling a ␥-interferon activated sequence that mediates the transcriptional effects of GH. Inhibition by IL-1␤ was also associated with a reduction of GH-dependent binding of STAT5 to this element after chronic (8 and 24 h), but not after acute treatment (15 min). Because these results indicated that the inhibition by IL-1␤ was indirect, expression of the recently discovered suppressors of cytokine action (SOCS) was examined in liver cells. IL-1␤ did not alter the expression of SOCS1, SOCS2, and CIS, indicating that they are not involved. In contrast, IL-1␤ increased SOCS3 mRNA by 8-fold after 24 h of treatment, whereas GH had no effect. Forced expression of SOCS3 was just as effective as IL-1␤ in reducing the GH induction of ALS promoter activity in H4-II-E rat hepatoma cells. Similar results were observed in primary rat hepatocytes. We conclude that the induction of SOCS3 by IL-1␤ contributes to the development of GH resistance in liver, and represents a mechanism by which cytokines such as IL-1␤ cross-talk with cytokines using the JAK-STAT pathway.

A gene cluster which includes genes required for the expression of nitric oxide reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.Denitrification is the reduction of nitrate (NO 3 Ϫ ) to gaseous intermediates, principally nitrogen gas. Nitric oxide (NO), an obligatory intermediate during denitrification, is generated from the one-electron reduction of nitrite (NO 2 Ϫ ) (41). NO reduction is coupled to energy generation (18,29). NO is also a well-known cytotoxic compound, so its production during denitrification has the potential of causing significant cell damage. To mitigate the toxicity of NO, its steady-state concentration during denitrification is maintained at low-nanomolar levels (11). The protein responsible for NO reduction, NO reductase (Nor), catalyzes the reaction 2NO ϩ 2H ϩ 3N 2 O ϩ H 2 O. Nitrous oxide (N 2 O) is an inert, nontoxic intermediate that is frequently the terminal product of denitrification (42). Nor has been purified and shown to be a heterodimeric membrane protein (9,14,16). Metal analysis has shown that it contains only iron in stoichiometric amounts. Recently, the Nor structural genes have been characterized, and sequence analysis revealed that Nor was related to the cytochrome c oxidase superfamily (36). In particular, Nor is most closely related to the heme b-containing oxidases, which are expressed under conditions of low oxygen concentration. It has been suggested that Nor was the original member of this family and that the other members arose by modifying the Nor structure (26).The genetic organization of the region of the chromosome encoding the nor structural genes varies among denitrifiers. In Pseudomonas stutzeri, the two structural genes form a distinct transcriptional unit (43). In Pseudomonas aeruginosa, the two structural genes ...