Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli

Abstract

Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the narG operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [Pichia] and plant [Nicotiana] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of narG, narK, and dnrE but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide. The promoters of narG, narK, and dnrE carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for Escherichia coli. The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions. Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of P. stutzeri for selective regulation of nitrate respiration.

FIG. 1

Organization of the narXL region of P. stutzeri and physical map of mutants. The map covers approximately 9.2 kb. narX overlaps narL by 71 bp. The maps for the narX and narL mutants are shown with the extent of deletions and orientation (arrowheads) of the kanamycin cassette. P_L, NarL-regulated promoters. ORFs 235 and 134 are labeled according to the number of amino acids of their hypothetical gene products.

FIG. 2

Structural features of NarL and NarX. Sequences were aligned with the CLUSTAL W program (37). Identical amino acids are marked by asterisks; similar amino acids are marked by colons. (A) Alignment of NarL and NarP proteins, identified in the bottom row. The structural predictions for NarL_Ps as deduced from the E. coli protein (4) are shown for the 10 α-helices and 5 β-strands. Helices 8 and 9 form the DNA-binding region. Boldfaced letters, E. coli residues important for phosphoryl transfer and the equivalent positions of NarL_Ps (aspartic acid residues 13, 14, and 59 and lysine 109) and homologous proteins. (B) Alignment of NarX and NarQ proteins, identified in the bottom row. The regions forming distinct structural elements are boxed and are discussed in the text. The predicted transmembrane helices for P. stutzeri and E. coli are boldfaced and are labeled TM1 and TM2.

FIG. 3

NarX functions as a sensory component with a preference for nitrate. Immunolabeling was done with polyclonal antisera raised against the purified nitrate reductase (NarGH; upper bands represent the NarG subunit, and lower bands represent the NarH subunit), cytochrome cd₁ nitrite reductase (NirS), the cytochrome b subunit of NO reductase (NorB), and N₂O reductase (NosZ). Lanes 1 and 2, cells cultured for 8 h with nitrate and nitrite, respectively (see Materials and Methods). Each panel shows the results obtained with strain MK21 (WT) and strain MRX119 (NarX⁻). Detection was carried out with a protein A-peroxidase conjugate and chloronaphthol (28). The NarG levels obtained by nitrate or nitrite induction differ in this experiment by a factor of 2.3. Amounts of cell extracts used were 48 μg each for NarGH and NorB and 6 μg each for NirS and NosZ. Mass references (in kilodaltons) were derived from the SeeBlue standard (Novex).

FIG. 4

NarL acts as a transcriptional activator for the narG operon. The DNA probe used for Northern blot analysis is given at the top of each panel. Total RNA was extracted from MK21 (lanes 1), MRL118 (ΔnarL) (lanes 2), and MRX119 (ΔnarX) (lanes 3). Cells were grown for 3 h with oxygen in the absence of nitrate (+O₂) and then shifted to nitrate-denitrifying conditions and extracted 1 h after the shift (denitrifying). Transcripts from the nir operon are found as monocistronic nirS and polycistronic nirSTB messages (22). Size standards are the RNA molecular weight marker no. I (Boehringer GmbH, Mannheim, Germany) and the 16S and 23S rRNA species.

FIG. 5

Determination of the 5′ ends of the narG and narK transcripts by primer extension analysis. Total RNA was obtained from wild-type cells (MK21) grown aerobically (lane 1), under O₂ limitation (lane 2), or under nitrate-denitrifying conditions (O₂ limitation in the presence of nitrate) (lanes 3). The right panel for narK shows the lack of extension products of RNA from MRL118 (ΔnarL) (lane 4), which had been induced for denitrification identical to that of the wild type. Primer extension was performed with oligonucleotides complementary to the 5′ ends of the coding regions of narG and narK shown in Fig. 6. Lanes A, C, G, and T show the results of dideoxy sequencing reactions carried out with the same primers. For MRL118 only the dideoxyadenine reaction is shown.

FIG. 6

NarL-dependent promoters of narG (A) and narK (B). The transcription start sites obtained from primer extension analysis are marked +1. Potential NarL sites are marked by half-arrows; nucleotides that correspond to the E. coli consensus are boldfaced. Putative FNR sites are boxed, and nucleotides within those sites that correspond to the E. coli consensus are highlighted. The oligonucleotides used for primer extension are underlined. RBS, ribosome binding site. The amino acid sequence obtained from the purified NarG subunit is shown in boldface in panel A.