Employing a recombinant strain of Advenella mimigardefordensis for biotechnical production of Homopolythioesters from 3,3'-dithiodipropionic acid

Advenella mimigardefordensis strain DPN7(T) was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3'-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of the buk-ptb operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from Clostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaC(Am)). These measures yielded the potent PMP production strain A. mimigardefordensis strain SHX22. The deletion of mdo was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne buk-ptb operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of A. mimigardefordensis (phaC(Am)) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.     

Novel reaction of succinyl coenzyme A (Succinyl-CoA) synthetase: activation of 3-sulfinopropionate to 3-sulfinopropionyl-CoA in Advenella mimigardefordensis strain DPN7T during degradation of 3,3'-dithiodipropionic acid

The sucCD gene of Advenella mimigardefordensis strain DPN7^T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7^T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7^T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD_Am) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD_Am is Mg²⁺ or Mn²⁺ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (V_max = 9.85 ± 0.14 μmol min⁻¹ mg⁻¹, K_m = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (V_max = 0.12 ± 0.01 μmol min⁻¹ mg⁻¹) and the affinity was 6-fold lower (K_m = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCD_Am is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.     

Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7^T (Acd_DPN7) during degradation of 3,3′-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). Acd_DPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. Acd_S110, Acd_B4, Acd_H16, and Acd_KT2440 were able to dehydrogenate isobutyryl-CoA. Acd_KT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas Acd_DSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only Acd_TBEA6, Acd_N-1, and Acd_LB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for Acd_BL21 and Acd_WT001. Spectrophotometric assays provided apparent K_m and V_max values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of uncharacterized acyl-CoA dehydrogenases. Furthermore, C. necator N-1 was found to utilize 3SP as the sole source of carbon and energy.     

Dihydrolipoamide Dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha Catalyze Cleavage of 3,3′-Dithiodipropionic Acid into 3-Mercaptopropionic Acid

The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7^T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA_Am). LpdA_Am was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL_Re). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7^T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA_Am and pdhL_Re were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA_Am) or 1,667 mkat/kg of protein (PdhL_Re). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.In Advenella mimigardefordensis strain DPN7^T (15, 42), three independent mutants with an insertion of Tn5::mob in the lpdA gene coding for the E3 component of the pyruvate dehydrogenase multi-enzyme complex revealed an interesting phenotype: these mutants were fully impaired in utilizing 3,3′-dithiodipropionic acid (DTDP) as the sole carbon and energy source, whereas the growth on no other tested carbon sources was affected (41). Our main interest in the catabolism of DTDP is to unravel the pathway and to identify the involved enzymes. Furthermore, the application of this disulfide as precursor substrate for biotechnological production of polythioesters (PTE) (22) is of interest. Since poly(3-mercaptopropionate) (PMP) biosynthesis depends hitherto on supplying the harmful thiol 3-mercaptopropionic acid (3MP) (35), an improvement of the recombinant Escherichia coli system by heterologous expression of enzymes capable of cleaving the less toxic DTDP symmetrically into two molecules of 3MP, which are then polymerized, could be an important achievement toward large-scale biotechnological production of PMP.Two different enzyme systems catalyzing the conversion of disulfides into the corresponding thiols are already known and have been described in detail. (i) Enzymes belonging to the well-characterized family of pyridine-nucleotide disulfide oxidoreductases (25) contain a redox center formed by a disulfide bridge coupled to a flavin ring. They catalyze a simultaneous two-electron transfer via the enzymatic active disulfides associated with the pyridine nucleotides and flavin, toward the substrate (39, 40). (ii) An alternative disulfide reduction is catalyzed by enzymes using iron-sulfur clusters to cleave of disulfide substrates in two one-electron reduction steps (37). The disrupted gene in A. mimigardefordensis was designated lpdA_Am (EC 1.8.1.4), and it encodes a homodimeric flavoprotein, the dihydrolipoamide dehydrogenase LpdA_Am (i.e., the E3 component of the pyruvate dehydrogenase multi-enzyme complex of A. mimigardefordensis strain DPN7^T) belonging to the above-mentioned family of pyridine nucleotide-disulfide oxidoreductases. Enzymes of this class share high sequence and structural similarities and catalyze reduction of compounds which are linked by disulfide bonds (38). Alkylhydroperoxide reductases, coenzyme A disulfide reductases, glutathione reductases, mycothione reductases, thioredoxin reductases, and trypanothione reductases also, in addition to dihydrolipoamide dehydrogenases, belong to this family (3, 38). The physiological function of LpdA_Am is most probably the conversion of lipoamide to dihydrolipoamide, but the reduction of DTDP into two molecules of 3MP (Fig. ​(Fig.1)1) is also predicted, enabling the first step in DTDP catabolism in A. mimigardefordensis strain DPN7^T (41).Open in a separate windowFIG. 1.Reactions catalyzed by LpdA_Am and PdhL_Re. Presented are the enzymatic conversions of DTDP into two molecules of 3MP (A), lipoamide into dihydrolipoamide (B), and DTNB into two molecules of NTB (C). Abbreviations: DTDP, 3,3′-dithiodipropionic acid; 3MP, 3-mercaptopropionic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NTB, 2-nitro-5-thiobenzoic acid.Ralstonia eutropha H16 synthesizes copolymers of 3-hydroxybutyrate and 3MP, if 3MP (23) or DTDP (22) is supplied as a precursor in addition to a second utilizable carbon source. Although R. eutropha is not able to grow with DTDP as the sole carbon source, it must be capable of cleaving this organic disulfide symmetrically, because it synthesizes from it heteropolymers containing the resulting 3MP. Thus, R. eutropha must possess at least one gene encoding a DTDP-cleaving enzyme. Five genes coding for homologues of a dihydrolipoamide dehydrogenase (DHLDH), which in A. mimigardefordensis DPN7^T is obviously involved in DTDP degradation, are known to exist in the genome of R. eutropha H16 (27; M. Raberg, J. Bechmann, U. Brandt, J. Schlüter, B. Uischner, and A. Steinbüchel, unpublished data). Therefore, LpdA_Am and the five DHLDH paralogues of R. eutropha H16 were aligned and compared (Fig. ​(Fig.2).2). Subsequently, lpdA_Am and the gene encoding the DHLDH belonging to the pyruvate dehydrogenase complex of R. eutropha H16 (pdhL_Re) were cloned, heterologously expressed in Escherichia coli, purified, and assayed.Open in a separate windowFIG. 2.Phylogenetic relationships of the A. mimigardefordensis strain DPN7^T LpdA (boldface), R. eutropha H16 PdhL (boldface), and homologues. The neighbor-joining plot was derived from a CLUSTAL X alignment of amino acid sequences closely related to LpdA_Am. The amino acid sequence of the outer membrane protein P64K from Neisseria meningitidis was used as the outgroup. GenBank accession numbers are given in parentheses. Scale bar, 10% sequence divergence.     

Aerobic degradation of mercaptosuccinate by the gram-negative bacterium Variovorax paradoxus strain B4

The Gram-negative bacterium Variovorax paradoxus strain B4 was isolated from soil under mesophilic and aerobic conditions to elucidate the so far unknown catabolism of mercaptosuccinate (MS). During growth with MS this strain released significant amounts of sulfate into the medium. Tn5::mob-induced mutagenesis was successfully employed and yielded nine independent mutants incapable of using MS as a carbon source. In six of these mutants, Tn5::mob insertions were mapped in a putative gene encoding a molybdenum (Mo) cofactor biosynthesis protein (moeA). In two further mutants the Tn5::mob insertion was mapped in the gene coding for a putative molybdopterin (MPT) oxidoreductase. In contrast to the wild type, these eight mutants also showed no growth on taurine. In another mutant a gene putatively encoding a 3-hydroxyacyl-coenzyme A dehydrogenase (paaH2) was disrupted by transposon insertion. Upon subcellular fractionation of wild-type cells cultivated with MS as sole carbon and sulfur source, MPT oxidoreductase activity was detected in only the cytoplasmic fraction. Cells grown with succinate, taurine, or gluconate as a sole carbon source exhibited no activity or much lower activity. MPT oxidoreductase activity in the cytoplasmic fraction of the Tn5::mob-induced mutant Icr6 was 3-fold lower in comparison to the wild type. Therefore, a new pathway for MS catabolism in V. paradoxus strain B4 is proposed: (i) MPT oxidoreductase catalyzes the conversion of MS first into sulfinosuccinate (a putative organo-sulfur compound composed of succinate and a sulfino group) and then into sulfosuccinate by successive transfer of oxygen atoms, (ii) sulfosuccinate is cleaved into oxaloacetate and sulfite, and (iii) sulfite is oxidized to sulfate.     

The unstable melC operon of Streptomyces antibioticus is codeleted with a Tn4811-homologous locus.

The melC operon of Streptomyces antibioticus is unstable, undergoing frequent spontaneous deletions. All the delta melC mutants analyzed also lost 2-kb V1 DNA, which contained two open reading frames (ORFs) homologous to ORF4 (a putative oxidoreductase gene) and ORF5 (a putative AraC-type regulatory gene) of Tn4811. The two ORFs may constitute an accessory unit of a different transposon.     

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and ¹H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.Abbreviations DCP 2,4-dichlorophenol - IAA indole-3-acetic acid - IM indole-3-methanol     

Aconitase B is required for optimal growth of Xanthomonas campestris pv. vesicatoria in pepper plants

The aerobic plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) colonizes the intercellular spaces of pepper and tomato. One enzyme that might contribute to the successful proliferation of Xcv in the host is the iron-sulfur protein aconitase, which catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle and might also sense reactive oxygen species (ROS) and changes in cellular iron levels. Xcv contains three putative aconitases, two of which, acnA and acnB, are encoded by a single chromosomal locus. The focus of this study is aconitase B (AcnB). acnB is co-transcribed with two genes, XCV1925 and XCV1926, encoding putative nucleic acid-binding proteins. In vitro growth of acnB mutants was like wild type, whereas in planta growth and symptom formation in pepper plants were impaired. While acnA, XCV1925 or XCV1926 mutants showed a wild-type phenotype with respect to bacterial growth and in planta symptom formation, proliferation of the acnB mutant in susceptible pepper plants was significantly impaired. Furthermore, the deletion of acnB led to reduced HR induction in resistant pepper plants and an increased susceptibility to the superoxide-generating compound menadione. As AcnB complemented the growth deficiency of an Escherichia coli aconitase mutant, it is likely to be an active aconitase. We therefore propose that optimal growth and survival of Xcv in pepper plants depends on AcnB, which might be required for the utilization of citrate as carbon source and could also help protect the bacterium against oxidative stress.     

Sialic acid metabolism's dual function in Haemophilus influenzae

Many bacterial commensals and pathogens use the sialic acids as carbon and nitrogen sources. In Escherichia coli, the breakdown of these sugars is catalysed by gene products of the nan (Nacylneuraminate) operon; other microorganisms may use a similar catabolic strategy. Despite the known ligand and antirecognition functions of the sialic acids, the contribution of their catabolism to infection or host colonization has never been directly investigated. We addressed these questions with Haemophilus influenzae type b, which metabolizes relatively few carbohydrates, using the infant-rat infection model. The predicted H. influenzae homologue (HI0142) of the E. coli sialic acid aldolase structural gene, nanA, was subcloned and mutagenized by insertion of a kanamycin resistance cassette. Phenotypic investigation of the resulting H. influenzae aldolase mutants showed that: (i) HI0142 is essential for sialic acid degradation; (ii) the products of the open reading frames (ORFs) flanking HI0142 (HI0140, 41, 44 and 45) are likely to have the same functions as those of their counterparts in E. coli; (iii) sialylation of the lipooligosaccharide (LOS) epitope recognized by monoclonal antibody 3F11 is dependent on an environmental source of sialic acid; (iv) a nanA mutant hypersialylates its LOS sialyl acceptor, corresponding to an apparent increased fitness of the mutant in the infant-rat model; and (v) expression of the LOS sialyl acceptor is altered in cells grown without exogenous sialic acid, indicating the direct or indirect effect of sialic acid metabolism on LOS antigenicity. Taken together the data show the dual role of sialic acid catabolism in nutrition and cell surface modulation.     

Transposition of Tn916 in the four replicons of the Butyrivibrio proteoclasticus B316(T) genome

The rumen bacterium Butyrivibrio proteoclasticus B316(T) has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316(T) was performed with the broad host-range conjugative transposon Tn916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316(T) . A search of the B316(T) genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn916 insertions identified in B316(T) mutants during our conjugation experiments.     

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.     

Plasmids for biphenyl, chlorobiphenyl and metatoluylate degradation from Pseudomonas putida

Pseudomonas putida strain SU83, harbors the pBS311 plasmid coding for the degradation of biphenyl, 2- and 4-chlorbiphenyl, meta- and paratoluylate. The insertional mutants of the plasmid obtained by the transposon Tn5 insertion were isolated. One of the mutants was used for cloning of the biphenyl degradation genes. The plasmid pBS311:: Tn5 DNA was inserted into the BamHI site of the plasmid pBR322 and cloned. 11 recombinants of 354 tested were treated with 0.1% solution of 2,3-dioxybiphenyl. One of them has acquired the yellow colour testifying to conversion of 2,3-dioxyphenyl to "2-hydroxy-6-keto-6-phenylhexa-2,4-diene acid. The recombinant plasmid pBS312 from this clone is 10.5 kb in size, the size of the insert being 6.2 kb. Escherichia coli SU185 cells harbouring pBS312 are able to support metacleavage of 2,3-dioxybiphenyl, 3-methylcatechol and catechol, but not of 4-methylcatechol. The results suggest the cloned fragment to contain a gene for 2,3-dioxybiphenyl-1,2-dioxygenase, the third enzyme for biphenyl catabolism.     

Complete nucleotide sequence of the 113-kilobase linear catabolic plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a and transcriptional analysis of genes involved in quinaldine degradation

The nucleotide sequence of the linear catabolic plasmid pAL1 from the 2-methylquinoline (quinaldine)-degrading strain Arthrobacter nitroguajacolicus Rü61a comprises 112,992 bp. A total of 103 open reading frames (ORFs) were identified on pAL1, 49 of which had no annotatable function. The ORFs were assigned to the following functional groups: (i) catabolism of quinaldine and anthranilate, (ii) conjugation, and (iii) plasmid maintenance and DNA replication and repair. The genes for conversion of quinaldine to anthranilate are organized in two operons that include ORFs presumed to code for proteins involved in assembly of the quinaldine-4-oxidase holoenzyme, namely, a MobA-like putative molybdopterin cytosine dinucleotide synthase and an XdhC-like protein that could be required for insertion of the molybdenum cofactor. Genes possibly coding for enzymes involved in anthranilate degradation via 2-aminobenzoyl coenzyme A form another operon. These operons were expressed when cells were grown on quinaldine or on aromatic compounds downstream in the catabolic pathway. Single-stranded 3' overhangs of putative replication intermediates of pAL1 were predicted to form elaborate secondary structures due to palindromic and superpalindromic terminal sequences; however, the two telomeres appear to form different structures. Sequence analysis of ORFs 101 to 103 suggested that pAL1 codes for one or two putative terminal proteins, presumed to be covalently bound to the 5' termini, and a multidomain telomere-associated protein (Tap) comprising 1,707 amino acids. Even if the putative proteins encoded by ORFs 101 to 103 share motifs with the Tap and terminal proteins involved in telomere patching of Streptomyces linear replicons, their overall sequences and domain structures differ significantly.     

Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation.

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.     

Organization and regulation of the mannopine cyclase-associated opine catabolism genes in Agrobacterium tumefaciens 15955.

We have isolated and characterized Tn3HoHo1- and Tn5-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR). Characterization of the transposon-induced lacZ fusion mutants by beta-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines. Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus. The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements. Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid. Analysis of beta-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source. The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen. This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability.     

Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires addition of the substrates for induction and is oxygen dependent. The highest activity is obtained when the concentration of inducer is 0.2 mM. Spectrophotometric data are consistent with the suggestion that the indole ring is broken during degradation of IAA. We hypothesize that the enzyme catalyzes an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation product in the proposed pathway.     

Transposon Tn5403, a mobilization-helper element: Complete nucleotide sequence and distribution in aquatic strains

Abstract Tn 5403 , a new transposable element, was found in a Klebsiella pneumoniae strain (KIIIA) isolated from an aquatic environment following a pollution accident. It was identified by its ability to mobilize ori T-deleted recombinant plasmids and thus can be proposed as a ‘helper’ element participating in the transfer mechanisms of non-conjugative plasmids. Nucleotides sequence analysis shows that the element consists of 3663 bp. Two open reading frames (ORFs) are found: one ( tnp A) encoding a putative transposase and the other ( tnp R) a putative resolvase. The two ORFs are transcribed in opposite directions and are separated by a putative recombination site (res). Results obtained with a specific probe suggest that Tn 5403 or a similar element is present in different Enterobacteriaceae strains isolated from the same river three years after the isolation of the Tn 5403 -harbouring KIIIA strain.     

Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha

A novel class of biopolymers referred to as polythioesters (PTE) was recently detected when the polyhydroxyalkanoate (PHA) accumulating bacterium Ralstonia eutropha was cultivated in the presence of 3-mercaptopropionic acid (3MP) or 3,3'-thiodipropionic acid (TDP). In this study, 3,3'-dithiodipropionic acid (DTDP) and 3-mercaptovaleric acid (3MV) were identified as two additional precursor carbon sources for in vivo biosynthesis of PTE in R. eutropha. Biosynthesis of copolymers of 3-hydroxybutyrate (3HB) and 3MP, which contributed 19-25% of cell dry matter, was compared referring to the different precursor substrates. Using DTDP as carbon source, which is probably cleaved into two molecules 3MP, yielded an about 2.3-fold higher molar 3MP content of the copolyester than TDP, which is probably cleaved into only one molecule 3MP. Furthermore, cultivation of R. eutropha in the presence of 3MV resulted in biosynthesis of copolymers consisting predominantly of 3HB with low amounts of 3MV and 3-hydroxyvalerate, each contributing less than 5 mol% of the constituents. In contrast, 4-mercaptobutyric acid could be not incorporated into PHAs, although - as documented in this study - five different strategies, various precursor substrates, R. eutropha and also a recombinant strain of Escherichia coli were employed. Therefore, this study not only extended the range of substrates suitable for PTE biosynthesis and also the range of PTE constituents in R. eutropha, it also demonstrates limits for PTE biosynthesis in this bacterium.     

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874–2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via β-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.