The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation - PubMed (original) (raw)

The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation

Lucia de la Riva et al. J Bacteriol. 2008 Dec.

Abstract

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO, and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on sigma(54), is mediated by the Ntr system. In contrast, neither NtrC nor the nitrogen assimilation control protein is involved in the nitrogen control of hpxDE, which is dependent on sigma(70) for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N(4)-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.

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Figures

FIG. 1.

FIG. 1.

Metabolic map for hypoxanthine assimilation to allantoin (A) and gene organization of the hpx system involved in this pathway in K. pneumoniae (B). The arrows show the extents and directions of transcription of the genes. Sequence similarity and function assignment of the _hpx_-encoded proteins are indicated in the table. XDH, xanthine dehydrogenase.

FIG. 2.

FIG. 2.

Promoter sequences of the divergently transcribed hpxD and hpxR genes and hpxO and hpxP genes. For each gene, the ATG initiation codon is boxed, the consensus for RNA polymerase (−10 and −35 sequences for σ70 recognition in hpxD, hpxR, and hpxO or the σ54 recognition sequence in hpxP) is boxed with dotted lines, and the 5′ end is shown by a black arrowhead labeled +1. Putative IHF or NtrC binding sites identified using Promscan and Virtual Footprint programs are indicated. Consensus sequences for LysR-type regulators (T-N11-A) are indicated. The sequence with partial dyad symmetry, TCTGC-N4-GCAAA, whose deletion abolished HpxR binding is indicated by arrows and labeled as the HpxR site, whereas the T-N11-A sequence, shown not to be involved in HpxR binding, is underlined with a dashed line.

FIG. 3.

FIG. 3.

Effect of an hpxR mutation on the expression of Φ(hpxD-lacZ), Φ(hpxR-lacZ), and Φ(hpxP-lacZ). Cells of the parental strain KC2653 (gray bars) and the hpxR mutant strain JA-K16 (black bars) bearing the indicated promoter fusion were grown in the indicated culture media. GNGln is a nitrogen-excess medium that contains 0.4% glucose and 0.2% each ammonium sulfate and

l

-Gln, and GGln is a nitrogen-limiting medium that contains 0.4% glucose and 0.04%

l

-Gln. Where indicated, 0.1% hypoxanthine (Hx) or 0.1% uric acid (Ur) was added to these media. β-Galactosidase activity is expressed as specific activity. One unit of β-galactosidase activity corresponds to the amount of enzyme that hydrolyzes 1 nmol of ONPG per min. Error bars indicate standard deviations.

FIG. 4.

FIG. 4.

Binding of HpxR to promoter fragments of the hpx system. (A) Diagrams of the hpxO-hpxP and hpxD-hpxR intergenic regions showing the transcription start site for each gene. The putative HpxR binding site (TCTGC-N4-GCAAA) in the divergent hpxD and hpxR promoters is indicated by a black box. The promoter fragments used as probes (Sop1, Sder1, Sder2, and Sder3) and their end positions with respect to the +1 position of the indicated gene are shown below each intergenic region. (B) Gel shift assays performed with recombinant HpxR. The 32P-labeled Sop1 probe encompassing the hpxO-hpxP intergenic region was added to binding mixtures containing increasing amounts of HpxR (0.1, 0.4, 0.8, or 2 μg) (left panel). Sder1, Sder2, or Sder3 32P-labeled probes were added to binding mixtures containing 0.5 μg of HpxR (middle panel). Hypoxanthine from 100 to 500 μM was added to binding reaction mixtures carried out with 0.5 μg of HpxR and probe Sder1 (right panel). All mixtures contained 20 ng of the indicated probe and a 500-fold molar excess of poly(dI-dC). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis. (C) Gel shift assays performed with probe Sder2 and crude extracts from cells of strain KC2653 grown with different nitrogen sources. Lane 1, no protein added; lane 2, recombinant HpxR as a control; lane 3, cell extract from GNGln cultures; lane 4, cell extract from GNGlnHx cultures; lane 5, cell extract from GGln cultures; lane 6, cell extract from GGlnHx cultures; lane 7, cell extract from GHx cultures. All mixtures contained 20 ng of the indicated probe, 5 μg of the cell extract, and a 500-fold molar excess of poly(dI-dC). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis. Retarded complexes attributed to HpxR or to nitrogen repression conditions (NR) are indicated.

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