Conjugative Type 4 Secretion System of a Novel Large Plasmid from the Chemoautotroph Tetrathiobacter kashmirensis and Construction of Shuttle Vectors for Alcaligenaceae

Tetrathiobacter spp. and other members of the Alcaligenaceae are metabolically versatile and environmentally significant. A novel, ∼60-kb conjugative plasmid, pBTK445, from the sulfur chemolithoautotroph Tetrathiobacter kashmirensis, was identified and characterized. This plasmid exists at a low copy number of 2 to 3 per host chromosome. The portion of pBTK445 sequenced so far (∼25 kb) harbors genes putatively involved in replication, transfer functions, partition, and UV damage repair. A 1,373-bp region was identified as the minimal replicon. This region contains a repA gene encoding a protein belonging to the RPA (replication protein A) superfamily and an upstream, iteron-based oriV. A contiguous 11-gene cluster homologous to various type 4 secretion systems (T4SSs) was identified. Insertional inactivation demonstrated that this cluster is involved in the conjugative transfer functions of pBTK445, and thus, it was named the tagB (transfer-associated gene homologous to virB) locus. The core and peripheral TagB components show different phylogenetic affinities, suggesting that this system has evolved by assembling components from evolutionarily divergent T4SSs. A virD4 homolog, putatively involved in nucleoprotein transfer, is also present downstream of the tagB locus. Although pBTK445 resembles IncP plasmids in terms of its genomic organization and the presence of an IncP-specific trbM homolog, it also shows several unique features. Unlike that of IncP, the oriT of pBTK445 is located in close proximity to the oriV, and a traL homolog, which is generally present in the TraI locus of IncP, is present in pBTK445 in isolation, upstream of the tagB locus. A significant outcome of this study is the construction of conjugative shuttle vectors for Tetrathiobacter and related members of the Alkaligenaceae.The genus Tetrathiobacter includes environmentally important betaproteobacteria belonging to the family Alcaligenaceae. Members of this family inhabit diverse habitats, ranging from animals and humans to soil, sewage, and sludge. They are also metabolically diverse and include facultative chemolithotrophs, versatile heterotrophs, xenobiotic degraders, fastidious parasites, and pathogens (15). While the type species, Tetrathiobacter kashmirensis, isolated from a temperate orchard soil, has been recognized as a thiosulfate- and tetrathionate-oxidizing facultative chemolithoautotroph (11, 15), Tetrathiobacter mimigardefordensis, isolated from compost, can utilize the organic disulfide 3,3′-dithiodipropionic acid for growth (42). More recently isolated soil-dwelling strains of T. kashmirensis can detoxify selenite by reducing it to insoluble elemental red selenium (18). Strains identified as T. kashmirensis on the basis of 16S rRNA gene sequence similarity (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EU523111","term_id":"170674436","term_text":"EU523111"}}EU523111) are allegedly involved in the biodegradation of thiodiglycol, the hydrolysis product of yperite, a highly hazardous derivative of mustard gas used in chemical weapons. In addition, bacteria isolated from a deep-sea environment and phylogenetically identified as T. kashmirensis (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EF619402","term_id":"148751435","term_text":"EF619402"}}EF619402) have been observed to degrade alkanes.Species of Alcaligenaceae possess a wide repertoire of plasmids (21, 32), a feature pertinent to their biodegradative and biogeochemical roles in the environment. Many of these plasmids are well known for harboring genes involved in biodegradation (14, 39, 44). However, not many of them have been studied at the molecular level. In the present study, we have identified, partially sequenced, and characterized a large (∼60-kb), low-copy-number, self-transmissible, novel plasmid, designated pBTK445, from T. kashmirensis strain WGT. We have characterized the minimal replication region of this plasmid and have subsequently constructed shuttle vectors that could be used for diverse members of the Alcaligenaceae, including Tetrathiobacter. A major part of the sequenced region was found to be occupied by genes homologous to constituents of various type 4 secretion systems (T4SSs) (5-7, 9). This locus was found to be involved in the conjugal transfer function of the new plasmid. Many features of pBTK445 resemble those of IncP plasmids, but the new plasmid also possesses several characteristics distinct from those of IncP plasmids. We discuss in detail those characteristics of pBTK445 that make it an interesting model for the study of the diversity and evolution of large plasmids.     

Molecular recognition of plant DNA: Does it differ from conventional animal DNA?

The recognition mechanism of DNA with small drugs/ligands is an important field of research from pharmacological point of view. Such studies are ample with DNAs extracted from animal cells, but are rare for those extracted from plant cells. However, such a study is strongly demanding for the formulation of pesticides and other agrochemicals. In this contribution, for the first time, we report the interaction of two well-known DNA binder ethidium bromide (EB) and Hoechst 33258 (H33258) with two genomic DNAs extracted from the leaves of Ricinus communis L. (castor bean) and Mangifera indica (mango) using steady-state and picosecond-resolved fluorescence spectroscopy. The purity of the extracted DNAs is confirmed from gel electrophoresis and optical absorption studies. As evidenced from the circular dichroism (CD) measurements the DNAs retain physiologically relevant B forms. The well-known DNA intercalator EB has been found to show an additional electrostatic mode of binding with the DNAs, which is not present in the conventional animal DNAs. The binding affinity of EB is found to be even weaker for the DNA extracted from M. indica compared to that in R. communis L. On the other hand, the binding affinity of H33258 with the plant DNAs is found to be comparable to that of animal DNAs. The difference in interaction could be rationalized from the possible differences in the base sequences.     

Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis

Fermentation of acid hydrolysate of water-hyacinth (Eichhornia crassipes), a free floating aquatic plant has been investigated for ethanol production. The dilute acid treatment has been applied to utilize the maximum hemicellulosic content of the water-hyacinth. The goal of this work was to investigate, both experimentally and theoretically using mathematical tools, a fermentative system utilizing water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate as a substrate for ethanol production using Pichia stipitis. It was found that 72.83% of xylose was converted to ethanol with a yield of 0.425 g_p/g_s and productivity of 0.176 g_p/L/h. An appropriate mathematical model was developed to explain theoretically the bioconversion of this hemicellulose acid hydrolysate to ethanol and the model was tested statistically to check the validity of the model.     

Syntheses and evaluation of glucosyl aryl thiosemicarbazide and glucosyl thiosemicarbazone derivatives as antioxidant and anti-dyslipidemic agents

A series of N-per-O-acetyl-glucosyl arylthiosemicarbazide and thiosemicarbazone derivatives have been synthesized and evaluated for their in vivo anti-dyslipidemic and in vitro antioxidant activities. Among 16 compounds tested, 3 compounds showed potent anti-dyslipidemic activity and 6 compounds showed potent antioxidant and scavenger of oxygen free radicals activity.     

Design,synthesis and bioactivity of catechin/epicatechin and 2-azetidinone derived chimeric molecules

A new class of chimeric molecules have been developed. These are based on polyphenols like catechin and epicatechin and monocyclic β-lactams. The two units are joined via a triazole linker using the ‘Click Chemistry’ conditions. The compounds showed good to weak antibacterial activity against Escherichia coli as well as moderate inhibition of RNase A.