The cytochromes c-550 of Paracoccus denitrificans and Thiosphaera pantotropha: a need for re-evaluation of the history of Paracoccus cultures

Abstract The c -type cytochrome and protein profiles were compared for a number of cultures of Paracoccus denitrificans obtained from a range of culture collections. The cultures fell into two groups corresponding to the two original isolates of this bacterial species. One group, which included NCIMB 8944, ATCC 13543, ATCC 17741, ATCC 19367, Pd 1222 and DSM 413, were similar or identical to LMD 22.21. The second group, including DSM 65 and LMG 4218, were similar or identical to LMD 52.44. These groupings were not compatible with the recorded history of culture deposition. Mass spectrometry and amino acid sequence comparisons showed that the cytochrome c -550 of the LMD 52.44 culture group differed by 16% from that of the LMD 22.21 group, and yet was only 1% different from the cytochrome c -550 of Thiosphaera pantotropha . These results suggest that consideration should be given to creation of a new species of Paracoccus pantotropha , which would include Thiosphaera pantotropha and Paracoccus denitrificans LMD 52.44.     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.     

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Linkage of X-linked retinitis pigmentosa to the hypervariable DNA marker M27β (DXS255)

Summary A hypervariable DNA marker is closely linked to one of the most severe forms of night blindness, X-linked retinitis pigmentosa (RP). Affected individuals with X-linked RP, obligate carriers, and ophthalmologically identifiable carriers of the disease were included in a linkage study. The diagnosis was established in five sibships by funduscopic and electrophysiological investigations. When the X-linked probe M27 was used, 2 recombinants out of 29 informative meioses were detected (=0.07 at a maximum lod of 4.75). The hypervariable probe detected two different alleles in 38 of 39 females tested. M27 is therefore a potentially very useful probe for carrier detection and prenatal diagnosis, as well as for addressing the question of heterogeneity of X-linked RP.     

Crystal structure of MshB from Mycobacterium tuberculosis, a deacetylase involved in mycothiol biosynthesis

All living species require protection against the damaging effects of the reactive oxygen species that are a natural by-product of aerobic life. In most organisms, glutathione is a critical component of these defences, maintaining a reducing environment inside cells. Some bacteria, however, including pathogenic mycobacteria, use an alternative low molecular mass thiol compound called mycothiol (MSH) for this purpose. Enzymes that synthesize MSH are attractive candidates for the design of novel anti-TB drugs because of the importance of MSH for mycobacterial life and the absence of such enzymes in humans. We have determined the three-dimensional structure of MshB (Rv1170), a metal-dependent deacetylase from Mycobacterium tuberculosis that catalyses the second step in MSH biosynthesis. The structure, determined at 1.9A resolution by X-ray crystallography (R=19.0%, R(free)=21.4%), reveals an alpha/beta fold in which helices pack against a seven-stranded mostly parallel beta-sheet. Large loops emanating from the C termini of the beta-strands enclose a deep cavity, which is the location of the putative active site. At the bottom of this cavity is a metal-binding site associated with a sequence motif AHPDDE that is invariant in all homologues. An adventitiously bound beta-octylglucoside molecule, used in crystallization, enables us to model the binding of the true substrate and propose a metal-dependent mechanistic model for deacetylation. Sequence comparisons indicate that MshB is representative of a wider family of enzymes that act on substituted N-acetylglucosamine residues, including a deacetylase involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors in eukaryotes.     

Whole genome sequencing of a natural recombinant Toxoplasma gondii strain reveals chromosome sorting and local allelic variants

Background  

Toxoplasma gondii is a zoonotic parasite of global importance. In common with many protozoan parasites it has the capacity for sexual recombination, but current evidence suggests this is rarely employed. The global population structure is dominated by a small number of clonal genotypes, which exhibit biallelic variation and limited intralineage divergence. Little is known of the genotypes present in Africa despite the importance of AIDS-associated toxoplasmosis.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection