Diliana D. Simeonova

Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

Life based on phosphite: a genome-guided analysis of Desulfotignum phosphitoxidans

BackgroundThe Delta-Proteobacterium Desulfotignum phosphitoxidans is a type strain of the genus Desulfotignum, which comprises to date only three species together with D. balticum and D. toluenicum. D. phosphitoxidans oxidizes phosphite to phosphate as its only source of electrons, with either sulfate or CO2 as electron acceptor to gain its metabolic energy, which is of exclusive interest. Sequencing of the genome of this bacterium was undertaken to elucidate the genomic basis of this so far unique type of energy metabolism.ResultsThe genome contains 4,998,761 base pairs and 4646 genes of which 3609 were assigned to a function, and 1037 are without function prediction. Metabolic reconstruction revealed that most biosynthetic pathways of Gram negative, autotrophic sulfate reducers were present. Autotrophic CO2 assimilation proceeds through the Wood-Ljungdahl pathway. Additionally, we have found and confirmed the ability of the strain to couple phosphite oxidation to dissimilatory nitrate reduction to ammonia, which in itself is a new type of energy metabolism. Surprisingly, only two pathways for uptake, assimilation and utilization of inorganic and organic phosphonates were found in the genome. The unique for D. phosphitoxidans Ptx-Ptd cluster is involved in inorganic phosphite oxidation and an atypical C-P lyase-coding cluster (Phn) is involved in utilization of organophosphonates.ConclusionsWe present the whole genome sequence of the first bacterium able to gain metabolic energy via phosphite oxidation. The data obtained provide initial information on the composition and architecture of the phosphite–utilizing and energy-transducing systems needed to live with phosphite as an unusual electron donor.

Draft Genome Sequence of the Methanotrophic Gammaproteobacterium Methyloglobulus morosus DSM 22980 Strain KoM1

ABSTRACT Here, we report the draft genome sequence of the methanotrophic gammaproteobacterium Methyloglobulus morosus DSM 22980 strain KoM1, which is proposed to be the type species for the novel genus Methyloglobulus. The genome (4.143 Mb) consists of a single circular chromosome and harbors genes for 2-aminoethylphosphonate (ciliatine) biosynthesis.

“Unknown Genome” Proteomics A New NAD(P)-dependent Epimerase/Dehydratase Revealed by N-terminal Sequencing, Inverted PCR, and High Resolution Mass Spectrometry

We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of ∼35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23–47 and 91–118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

Draft Genome Sequence of Serratia sp. Strain DD3, Isolated from the Guts of Daphnia magna

ABSTRACT We report the draft genome sequence of Serratia sp. strain DD3, a gammaproteobacterium from the family Enterobacteriaceae. It was isolated from homogenized guts of Daphnia magna. The genome size is 5,274 Mb.

Draft genome sequence of Desulfotignum phosphitoxidans DSM 13687 strain FiPS-3

ABSTRACT We report the 5.008-Mbp assembled draft genome sequence of Desulfotignum phosphitoxidans strain FiPS-3 (DSM 13687), which gains metabolic energy from the oxidation of phosphite to phosphate. Its genome provides insights into the composition and architecture of the phosphite-utilizing and energy-transducing systems required to live with phosphite as electron donor.

Draft Genome Sequence of Flavobacterium succinicans Strain DD5b.

ABSTRACT We present the first 3.315-Mbp assembled draft genome sequence of Flavobacterium succinicans strain DD5b. This bacterium is a phosphite-assimilating representative of the genus Flavobacterium isolated from guts of the zooplankton Daphnia magna.

Draft Genome Sequence of Klebsiella pneumoniae subsp. pneumoniae ATCC 9621

ABSTRACT We present here the 5.561-Mbp assembled draft genome sequence of Klebsiella pneumoniae subsp. pneumoniae ATCC 9621, a phosphite- and organophosphonate-assimilating Gammaproteobacterium. The genome harbors 5,179 predicted protein-coding genes.

“Unknown-genome” proteomics- based identification of a new NADP-epimerase/dehydratase from Desulf. phosphitoxidans by inverted-PCR, Edman-sequencing and high resolution mass spectrometry

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.