Mycobacterium tuberculosis Is a Natural Ornithine Aminotransferase (rocD) Mutant and Depends on Rv2323c for Growth on Arginine.

Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using 13C- or 15N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb

Scheme of the arginase pathway and the arginine decarboxylase pathway.

<p>In <i>Bacillus subtilis</i>, the first reaction of the arginase pathway, the formation of ornithine (Orn), is catalyzed by arginase (<i>rocF</i>). RocD, an ornithine aminotransferase, utilizes ornithine to form glutamate semialdehyde (GSA) or pyrroline-5-carboxylate (P5C). P5C is the substrate of RocA, a pyrroline-5-carboxylate dehydrogenase, which produces glutamate (Glu). In <i>Yersinia pestis</i>, an arginine decarboxylase (<i>speA</i>) forms agmatine (Agma). Agma is further degraded to N-carbamoylputrescine (CP) and putrescine (Put) by <i>aguA</i>, <i>aguB</i>, and <i>speB</i>. The genome of Mtb includes homologues for the arginase pathway (<i>rocD</i>, <i>rocA</i>) and the arginine decarboxylase pathway (<i>speA / adi</i>).</p

Alignment of partial <i>rocD</i> sequence from members of Mtb complex (MTC) and non-tuberculous mycobacteria (NTM).

<p>Besides published genome data for <i>M</i>. <i>tuberculosis</i> H37Rv, <i>M</i>. <i>ulcerans</i>, <i>M</i>. <i>avium</i>, <i>M</i>. <i>marinum</i>, and <i>M</i>. <i>smegmatis</i>, sequence data obtained from clinical isolates (<i>M</i>. <i>tuberculosis</i>, <i>M</i>. <i>bovis bovis</i>, <i>M</i>. <i>africanum</i>, and <i>M</i>. <i>canettii</i>) and from <i>M</i>. <i>bovis</i> BCG, <i>M</i>. <i>simiae</i>, <i>M</i>. <i>scrofulaceum</i>, <i>M</i>. <i>abscessus</i>, <i>M</i>. <i>phlei</i>, and <i>M</i>. <i>chelonae</i> were included. The genome sequence of Mtb H37Rv was used as a reference. The partial <i>rocD</i> sequence encompasses base pairs 615 to 681. Members of the Mtb complex (MTC), in contrast to non-tuberculous mycobacteria (NTM), carry a 13 bps deletion within <i>rocD</i> resulting in a premature stop (underlined).</p

Growth of Mtb knockout strains lacking Rv2323c, <i>rocD</i>, or <i>adi</i> on arginine as nitrogen source.

<p>Mtb wild type (Mtb), Mtb knockout mutants of Rv2323c (Rv2323c-KO) <b>(A)</b>, <i>rocD</i> (<i>rocD</i>-KO) <b>(B)</b>, <i>adi</i> (<i>adi</i>-KO) <b>(C)</b>, and the complemented strain for the Rv2323c knockout mutant (Compl.) <b>(A)</b> are shown. All strains were cultured for 11 days in minimal medium with glycerol and glucose as carbon sources, and with 5 mM of arginine as a sole nitrogen source (solid line). Mtb wild type was also cultured without arginine as a control (broken line). Growth was analyzed measuring absorbance (OD₆₀₀) at day 3, 5, 7, 9, and 11. Mean values and standard deviations are shown for three independent biological experiments.</p

Relative isotopologue composition of amino acids derived from Mtb grown in presence of [U-¹³C₆]arginine (A) and cultivation of Mtb on arginine as carbon source (B).

<p><b>A)</b> Mtb wild type was cultured for 7 days in minimal medium with glycerol and glucose as carbon sources and with 5 mM of a 1:1 mixture of [U-¹³C₆]arginine) and unlabeled arginine as sole nitrogen source. ¹³C-Enrichments of ornithine, proline, glutamate, and aspartate were determined by GC-MS. Labeled ornithine and proline contained mainly M+5 isotopomers. Glutamate and aspartate showed more complex mixtures of multiple isotopomers. M+1, M+2, M+3, M+4, and M+5 indicate molecules with one to five ¹³C- labeled atoms, respectively (for ¹³C-excess of labeled amino acids see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136914#pone.0136914.s003" target="_blank">S2 Table</a>). Mean values and standard deviations are shown for two independent biological experiments each measured in triplicate. <b>B)</b> Mtb wild type (Mtb) was cultured for 11 days in Sauton’s modified medium with 5 mM, 10 mM, or 25 mM of arginine (open symbols), or with 10 mM or 50 mM of glycerol (closed symbols) as single carbon source. Mean values and standard deviations are shown for three independent biological experiments.</p

Growth (A) and glutamate formation (B) of a <i>rocD</i> mutant in Msmeg cultured on arginine as nitrogen source.

<p>Msmeg wild type (Msmeg), Msmeg knockout mutant for <i>rocD</i> (<i>rocD</i>-KO), the Msmeg <i>rocD</i>-KO mutant complemented with <i>rocD</i> wild type gene from Msmeg (<i>rocD</i>-KO::<i>rocD</i>_Msmeg), and the Msmeg <i>rocD</i>-KO mutant complemented with <i>rocD</i> wild type gene from Mtb (<i>rocD</i>-KO::<i>rocD</i>_Mtb) are shown. All strains were cultured for 40 hours in minimal medium with glycerol and glucose as carbon sources and with 5 mM of arginine as a sole nitrogen source (solid line). Msmeg wild type was also cultured without arginine as a control (broken line). <b>A)</b> Growth was analyzed measuring absorbance (OD₆₀₀) at 3h, 6h, 17h, 20h, 23h, and 40h. Mean values and standard deviations are shown for three independent biological experiments. <b>B)</b> Intracellular glutamate was determined in bacterial cell extracts after 0, 6, and 20 hours of growth. Mean values and standard deviations are shown for three independent biological experiments, p- values were calculated using student’s <i>t</i> test (* p < 0.05, ** p < 0.01).</p

Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.Autosomal dominant polycystic kidney disease (ADPKD) is typically a late-onset disease caused by mutations in PKD1 or PKD2, but about 2% of patients with ADPKD show an early and severe phenotype that can be clinically indistinguishable from autosomal recessive polycystic kidney disease (ARPKD). The high recurrence risk in pedigrees with early and severe PKD strongly suggests a common familial modifying background, but the mechanisms underlying the extensive phenotypic variability observed among affected family members remain unknown. Here, we describe severely affected patients with PKD who carry, in addition to their expected familial germ-line defect, additional mutations in PKD genes, including HNF-1β, which likely aggravate the phenotype. Our findings are consistent with a common pathogenesis and dosage theory for PKD and may propose a general concept for the modification of disease expression in other so-called monogenic disorders

Mutations in Multiple PKD Genes May Explain Early and Severe Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is typically a late-onset disease caused by mutations in PKD1 or PKD2, but about 2% of patients with ADPKD show an early and severe phenotype that can be clinically indistinguishable from autosomal recessive polycystic kidney disease (ARPKD). The high recurrence risk in pedigrees with early and severe PKD strongly suggests a common familial modifying background, but the mechanisms underlying the extensive phenotypic variability observed among affected family members remain unknown. Here, we describe severely affected patients with PKD who carry, in addition to their expected familial germ-line defect, additional mutations in PKD genes, including HNF-1 beta, which likely aggravate the phenotype. Our findings are consistent with a common pathogenesis and dosage theory for PKD and may propose a general concept for the modification of disease expression in other so-called monogenic disorders