The hpx Genetic System for Hypoxanthine Assimilation as a Nitrogen Source in Klebsiella pneumoniae : Gene Organization and Transcriptional Regulation

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 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 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.

Purine Utilization byKlebsiella oxytocaM5al: Genes for Ring-Oxidizing and -Opening Enzymes

“…We conclude that K. oxytoca quantitatively assimilates all N atoms from purines. An identical conclusion was drawn by de la Riva et al, who used adenine instead of guanine (24).…”

“…The genetic nomenclature extends that used by de la Riva et al (24). The mnemonic hpx (hypoxanthine) is applied to all genes whose products are involved in utilization of hypoxanthine or its catabolites.…”

“…While this paper was in review, the paper by de la Riva et al (24), describing the hpxDE, hpxR, hpxO, and hpxPQT genes from Klebsiella pneumoniae W70, was posted in the "JB Accepts" section of the Journal of Bacteriology online edition. Results and conclusions concerning these seven genes are congruent between the two studies.…”

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase

“…Purine oxidation in nature is catalyzed by three distinct classes of enzymes: the molybdenum-containing hydroxylases such as xanthine oxidoreductase (1,2), the Fe II -and ␣-ketoglutarate-dependent xanthine hydroxylases (3,4), and a newly described two-component system, HpxDE, consisting of a [2Fe-2S]/flavin-containing reductase and a Rieske/non-heme iron-containing oxygenase (5,6). The first class is by far the most broadly distributed, with members found throughout the eubacteria, archaea, and eukaryota.…”

Substrate Selectivity of YgfU, a Uric Acid Transporter from Escherichia coli

“…On the other hand, a complete gene cluster for purine utilization (hpx cluster) has been recently identified in Klebsiella pneumoniae (39) and Klebsiella oxytoca (40) containing separate genes for all three enzymatic steps of uric acid oxidation, namely hpxO (urate hydroxylase), hpxT (HIU hydrolase), and hpxQ (OHCU decarboxylase). Apart from HpxT (HIU hydrolase), which is homologous to E. coli HIU hydrolase and B. subtilis PucM, HpxO was found to function as a novel FAD-dependent urate oxidase (41), distinct from the classical urate oxidase (uricase) known for B. subtilis (36) and other species with complete purine utilization pathways (42).…”