The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is important for pathogenesis and a target for vaccine development.

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and ß-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3'-propionic acid)-5α-hydroxy-7aß-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality.

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated DeltasupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the DeltasupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive.

Characterization of a second Rhodococcus erythropolis SQ1 3-ketosteroid 9alpha-hydroxylase activity comprising a terminal oxygenase homologue, KshA2, active with oxygenase-reductase component KshB.

Previously we have characterized 3-ketosteroid 9alpha-hydroxylase (KSH), a key enzyme in microbial steroid degradation in Rhodococcus erythropolis strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the kshA1 gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the kshA2 gene in mutant strain RG2 restored growth on AD and ADD, indicating that kshA2 also encodes KSH activity. The functional complementation was shown to be dependent on the presence of kshB. Transcriptional analysis showed that expression of kshA2 is induced in parent strain R. erythropolis SQ1 in the presence of AD. However, promoter activity studies, using beta-lactamase of Escherichia coli as a convenient transcription reporter protein for Rhodococcus, revealed that the kshA2 promoter in fact is highly induced in the presence of 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of kshA2 in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third kshA homologue was additionally identified in a kshA1 kshA2 double gene deletion mutant strain of R. erythropolis SQ1. The developed degenerate PCR primers for kshA may be useful for isolation of kshA homologues from other (actino) bacteria.

(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica.

Prephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-D-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-DL-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.

Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9alpha-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1.

9 alpha-Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9 alpha-hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9 alpha-hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two-component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9 alpha-hydroxy-4-androstene-3,17-dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30-50% conversion), confirming the involvement of KshA and KshB in AD(D) 9 alpha-hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.

Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Delta1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker.

This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus. Unmarked deletion of the kstD gene, encoding 3-ketosteroid Delta1-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sacB counter-selection system. Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R. erythropolis strain SQ1 was used to avoid its random genomic integration. The kstD gene deletion mutant, designated strain RG1, still possessed about 10% of the KSTD enzyme activity of wild-type and was not affected in its ability to grow on the steroid substrates 4-androstene-3,17-dione (AD) and 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD). Biochemical evidence subsequently was obtained for the presence of a second KSTD enzyme (KSTD2) in R. erythropolis SQ1. UV mutants of strain RG1 unable to grow on AD were isolated. One of these mutants, strain RG1-UV29, had lost all KSTD enzyme activity and was also unable to grow on 9OHAD. It stoichiometrically converted AD into 9OHAD in concentrations as high as 20 g x l(-1). The two KSTD enzymes apparently both function in AD and 9OHAD catabolism. These isoenzymes have been inactivated in strain RG1 (KSTD1 negative) and strain RG1-UV29 (KSTD1 and KSTD2 negative), respectively.

Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta(1)-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

Chorismate mutase and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of the methylotrophic actinomycete Amycolatopsis methanolica.

Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII and CMII synthesis to L-Phe repression was isolated. DSII was partially purified (a 42-kDa protein); its activity was strongly inhibited by L-Tyr. CMII was purified to homogeneity (93.6 fold) and characterized as a homodimeric protein with 16-kDa subunits, completely insensitive to feedback inhibition by L-Phe and L-Tyr. The activity of CMII was activated by CMIb; the activity of CMII plus CMIb was again inhibited by L-Phe and L-Tyr. A tightly blocked L-Phe- plus L-Tyr-requiring derivative of mutant strain GH141, GH141-19, that had lost both CMIa and CMII activities was isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning with a pMEA300-derived shuttle vector and characterization of the Amycolatopsis methanolica prephenate dehydratase gene.

An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized.

Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18,373 Da) and 416 amino acids (45,260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12,917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid-encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kilkor system in autonomous replication of pMEA300.

Biosynthesis of l-Phenylalanine and l-Tyrosine in the Actinomycete Amycolatopsis methanolica.

Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.

Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.

Purification and characterization of a dual function 3-dehydroquinate dehydratase from Amycolatopsis methanolica.

Studies on hydroaromatic metabolism in the actinomycete Amycolatopsis methanolica revealed that the organism grows rapidly on quinate (but not on shikimate) as sole carbon- and energy source. Quinate is initially converted into the shikimate pathway intermediate 3-dehydroquinate by an inducible NAD(+)-dependent quinate/shikimate dehydrogenase. 3-Dehydroquinate dehydratase subsequently converts 3-dehydroquinate into 3-dehydroshikimate, which is used partly for the biosynthesis of aromatic amino acids, and is partly catabolized via protocatechuate and the beta-ketoadipate pathway. Enzyme studies and analysis of mutants clearly showed that the single 3-dehydroquinate dehydratase present in A. methanolica has a dual function, the first example of a 3-dehydroquinate dehydratase enzyme involved in both the catabolism of quinate and the biosynthesis of aromatic amino acids. This enzyme was purified over 1700-fold to homogeneity. Its further characterization indicated that it is a Type II 3-dehydroquinate dehydratase, a thermostable enzyme with a large oligomeric structure (native M(r) 135 x 10(3)) and a subunit M(r) of 12 x 10(3). Characterization of aromatic amino acid auxotrophic mutants of A. methanolica suggested that genes encoding 3-dehydroquinate synthase and 3-dehydroquinate dehydratase are genetically linked but their transcription results in the synthesis of two separate proteins.