Characterization and antimicrobial activity of essential oils extracted from lemongrass (Cymbopogon flexuosus) using microwave-assisted hydro distillation.

Lemongrass (Cymbopogon flexuosus) essential oil (LGEO) contains α-citral, ß-citral and other phytochemicals extracted using various methods. This research extracted essential oils using steam distillation (SD) and microwave-assisted hydro distillation (MAHD) to maximize quantity and purity. LGEO was tested for antibacterial properties. LGEO was extracted using SD and compared to MAHD output based on oil production and chemical composition. We performed GCMS to characterize LGEO. Fourier transform infrared spectroscopy (FTIR) used for quantum chemical analysis. Spectroscopic analysis showed that SD extracted secondary metabolites (ethyl-linalool, isogeranial, ß-citral, α-citral, geranyl acetate, and caryophyllene) yielded 9.7 %, 11.5 %, 35.4 %, 13.4 %, 6.4 %, and 6.4 %, respectively, while MAHD yielded 10.2 %, 13.4 %, 43.2 %, 17.3 %, 6.9 %, and 7.3 %. MAHD extracted α and ß citral content was better than SD extraction technique. FTIR spectroscopy and quantum chemistry analysis showed extracted oil chemical composition, electronic structure of α and ß citral isomers. In the disc-diffusion experiment, both extracts were effective against Gram-positive and Gram-negative bacteria and harmful fungi. LGEO from SD and MAHD extraction (30 mg/mL) demonstrated disc diffusion assay antibacterial efficacy against microorganisms. The two extracts effectively inhibited microorganisms with MIC values of 3.75 and 7.5 µg/mL. It can be concluded that, LGEO have greater antimicrobial activity in MAHD extraction.

Pantoea tagorei sp. nov., a Rhizospheric Bacteria with Plant Growth-Promoting Activities.

A Gram-negative, short-rod, non-motile, facultatively anaerobic, potassium-solubilizing bacterium MR1 (Mine Rhizosphere) was isolated from rhizospheric soil of an open-cast coal mine of Jharia, Jharkhand, India. Isolate MR1 can grow in a broad range of temperature, pH, and NaCl concentrations. The 16S rRNA gene sequence of the strain showed 99.24% similarity with Pantoea septica LMG 5345T. However, maximum-likelihood tree constructed using 16S rRNA gene sequence, multilocus sequence analysis using concatenated sequences of ten housekeeping genes, whole-genome based phylogenetic reconstruction, digital DNA-DNA hybridization, and average nucleotide identity (ANIm and ANIb) values indicated segregation of MR1 from its closest relatives. Fatty acid profile of MR1 also suggested the same, with clear variation in major and minor fatty acid contents, having C13:0 anteiso (10-Methyldodecanoic acid) as the unique one. Thus, considering all polyphasic data, strain MR1T (= MTCC 13265T, where 'T' stands for Type strain) is presented as a novel species of the genus Pantoea, for which the name Pantoea tagorei sp. nov. is proposed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01147-9.

Genome shuffling improves pigment and other bioactive compound production in Monascus purpureus.

Monascus purpureus, a pigment-producing ascomycetous fungus, has been traditionally used for red rice preparation using solid-state fermentation. The objective of this study was to develop an improved pigment-producing strain of M. purpureus MTCC 1090 through genome shuffling followed by detailed analytical estimations of pigments and other bioactive compounds produced by the fusant. Protoplast formation was optimum with 12 h-old mycelia incubated at 30 °C with cellulase, lyticase, and chitinase (40:1:1) for 5 h. Four UV-induced mutants that produced 13.1-39.5% higher amount of yellow, orange, and red pigments in fermented low-grade (cheap) broken rice were used as parents for genome shuffling. After the first round of fusion, four fusants with 35.9-60.52% higher pigment production capabilities were fused again, and finally the fusant F2-19 with distinct culture characteristic was selected under multi-selection pressure. It consistently produced 67%, 70%, and 76% higher content of yellow, orange, and red pigments respectively as compared to the wild-type. High-performance liquid chromatography (HPLC) analysis also reveals clear variation in pigment productions between wild-type and the fusant. Furthermore, HPLC analysis of F2-19 fermented rice extract confirms the production of 186 ± 8.71 and 3810 ± 29.81 mg/kg mevinolin and gamma-aminobutyric acid respectively. Citrinin was not detected. F2-19 fermented rice also has high antioxidant activity (7.92 ± 0.32 mg/g trolox equivalent), with good amount of phenolics (18.0 ± 0.95 mg/g gallic acid equivalent) and flavonoids (2.7 ± 0.26 mg/g quercetin equivalent). Thus, genome shuffling was successfully implemented on M. purpureus for the first time to develop a citrinin-free, better-performing fusant that holds future biotechnological potential. KEY POINTS: • Genome shuffling was performed by recursive protoplast fusion in Monascus purpureus. • The selected fusant, F2-19, was used in solid-state fermentation using low-grade rice. • It produced 67-76% higher content of yellow, orange, and red pigments than the wild-type. • HPLC detected 186 mg/kg mevinolin and 3810 mg/kg γ-aminobutyric acid, but no citrinin. • F2-19 shows high antioxidant activity with good amount of phenolics and flavonoids. Graphical abstract.

Poorly known microbial taxa dominate the microbiome of hypersaline Sambhar Lake salterns in India.

Inland athalassohaline solar salterns provide unique opportunity to study microbial successions along salinity gradients that resemble transition in natural hypersaline lakes. We analyzed for the first time 16S rRNA gene amplicon sequences of bacteria (V1-V2) and archaea (V4-V5) in saltern brines of India's largest inland hypersaline Sambhar Lake. Brines of the salterns (S1-S4) are alkaline (pH 9.5-10.5) with salinities of 130, 170, 280 and 350 gL-1 respectively. 16S rRNA gene copy-number of archaea outnumbered that of bacteria in all salterns. Their diversity also increased along S1 through S4, while that of bacteria decreased. Brines of S3 and S4 were dominated by specialized extreme halophilic bacterial (Halanaerobiales, Rhodothermaceae) and archaeal (Halobacteriales, Haloferacales) members with recognized salt-in strategy for osmoadaptation. Microbial assemblages positively correlated to saltern pH, total salinity, and ionic composition. Archaea in S1 and S2 were unprecedentedly represented by poorly known as-yet uncultivated groups, Woesearchaeota (90.35-93.51%) and Nanohaloarchaeota that belong to the newly proposed nano-sized superphylum DPANN. In fact, these taxa were identified in archaeal datasets of other athalassohaline salterns after re-analysis using latest RDP database. Thus, microbial compositions in hypersaline lakes are complex and need revisit particularly for their archaeal diversity to understand their hitherto unknown ecological function in extreme environments.

Moderate halophilic bacteria, but not extreme halophilic archaea can alleviate the toxicity of short-alkyl side chain imidazolium-based ionic liquids.

Imidazolium-based ionic liquids (IL) with short-alkyl side chain such as 1-ethyl-3-methyl-imidazolium chloride ([Emim]Cl) and 1-butyl-3-methyl-imidazolium chloride ([Bmim]Cl) has immense application potential including in lignocellulosic bioenergy production. But they are toxic to most microorganisms, and those isolated from different environments as IL-tolerant have salt tolerance capabilities. This study evaluates the relationship between salt and [Emim]Cl tolerance of microorganisms using different salinity sediments (2-19%) and brines (35%) of India's largest inland hypersaline lake, Sambhar in Rajasthan as the model system. While samples with 2% and 35% salinities do not yield any [Emim]Cl (100 mM) tolerant colonies, others have 6-50% colonies tolerant to the IL. Similar trend was observed with 50 mM [Bmim]Cl. Moderate halophilic isolates of genera Halomonas and Bacillus (growth in 0.7-3.0 M NaCl) isolated from the sediments could grow in as high as 375 mM [Emim]Cl, or 125 mM [Bmim]Cl facilitated by higher synthesis, and uptake of organic osmolytes; and up to 1.7-fold increased activity of active efflux pumps. [Bmim]Cl was more toxic than [Emim]Cl in all performed experiments. [Emim]Cl-adapted cells could trounce IL-induced stress. Interestingly, enrichment with 100 mM [Emim]Cl resulted in increase of IL-tolerant colonies in all sediments including the one with 2% salinity. However, the salt saturated brines (35%) do not yield any such colony even after repeated incubations. Extreme halophilic archaea, Natronomonas (growth in 3.0-4.0 M NaCl) isolated from such brines, were exceedingly sensitive to even 5 mM [Emim]Cl, or 1 mM [Bmim]Cl. Two additional extremophilic archaea, namely Haloferax and Haladaptatus were also sensitive to the tested ILs. Archaeal sensitivity is possibly due to the competitive interaction of [Emim]+ with their acidic proteome (15.4-17.5% aspartic and glutamic acids, against 10.7-12.9% in bacteria) that they maintain to stabilize the high amount of K+ ion accumulated by salt-in strategy. Thus, general salt adaptation strategies of moderate halophilic bacteria help them to restrain toxicity of these ILs, but extremophilic archaea are highly sensitive and demands meticulous use of these solvents to prevent environmental contamination.

A Bacillus strain TCL isolated from Jharia coalmine with remarkable stress responses, chromium reduction capability and bioremediation potential.

Microbial reduction of Cr(VI) to Cr(III) can mitigate environmental chromium toxicity. A chromium, cadmium and nickel tolerating strain TCL with 97% 16S rRNA gene sequence homology to Bacillus cereus was isolated from a derelict open-cast, Tasra Coalmine Lake of Jharia, India. It could tolerate up to Cr2000 [2,000 mg L-1 Cr(VI)] and completely reduce Cr200 within 16 h under heterotrophic condition. TCL grown in ≥ Cr500 exhibited multifarious stress responses particularly in its prolonged lag-phase, like cell aggregation, up to two-fold elongation, increased exopolysaccharide production, and stress enzyme activities. These were relieved by increasing inoculum size or nutrient content. Chromium reduction was constitutive, with maximum activities detected in loosely-bound exopolysaccharides and membrane fractions, followed by cytoplasm and spent media. Cr(VI) was efficiently reduced to Cr(III) and >90% was released in spent media. Cells also expressed Cr-induced active efflux pumps. Growing cells or its crude enzyme extracts could efficiently reduce Cr(VI) in diverse temperatures (15-45 °C), pH (5-9); and in presence of other metals (Cd, Cu, Mo, Ni, Pb), oxyanions (SO4-2, NO2-), and metabolic inhibitors (phenol, NaN3, EDTA). Growth and reduction were also detected in nutrient-limited minimal salt media, and contaminated leather industry effluent thereby making TCL a potential candidate for bioremediation.

Isolation of high molecular weight and humic acid-free metagenomic DNA from lignocellulose-rich samples compatible for direct fosmid cloning.

Activity-based screening of metagenomic DNA libraries is a promising approach to fish out genes encoding novel bioactive compounds/enzymes of industrial importance. The starting point of such functional screening in fosmid vectors is isolation of high molecular weight (HMW) DNA of sufficient purity from diverse environments. Metagenomic DNA isolation protocols mostly employ mechanical cell lysis that yields fragmented DNA. Those established for HMW DNA using enzymatic lysis have not considered samples with high lignocellulose or humic acid content. Enzymes from such environments are in great demand for bioenergy, paper, and related industries. Thus, an improved method was standardized that has three key features, i.e., use of harvested microbial biomass instead of raw samples, removal of humic substances prior to cell lysis by aluminum sulfate flocculation, and enzymatic/chemical lysis of cells with a lysozyme, mutanolysin, proteinase K, and SDS cocktail followed by phenol-chloroform extraction and precipitation of DNA by polyethylene glycol and NaCl. HMW DNA (~ 40 kb) was efficiently isolated from garden and forest soils, rice straw compost, and degrading wood from a hypersaline lake. The humic acid removal efficiency across samples was 96-98%. The isolated DNA was of high quality/purity and could be successfully used in downstream applications like PCR, ligation, and fosmid cloning. In fact, the DNA was directly used without any size selection, for fosmid library preparation with 70-90% efficiency as compared to the control insert. Thus, the method could suitably be used for HMW DNA isolation for the functional screening of enzymes from diverse humic acid-/lignocellulose-rich environments.

Increased productivity in poultry birds by sub-lethal dose of antibiotics is arbitrated by selective enrichment of gut microbiota, particularly short-chain fatty acid producers.

Antibiotics are widely used at sub-lethal concentrations as a feed supplement to enhance poultry productivity. To understand antibiotic-induced temporal changes in the structure and function of gut microbiota of chicken, two flocks were maintained for six weeks on a carbohydrate- and protein-rich diet. The feed in the conventional diet (CD) group was supplemented with sub-lethal doses of chlorotetracycline, virginiamycin and amoxicillin, while the organic diet (OD) had no such addition. Antibiotic-fed birds were more productive, with a lower feed conversion ratio (FCR). Their faecal samples also had higher total heterotrophic bacterial load and antibiotic resistance capability. Deep sequencing of 16S rDNA V1-V2 amplicons revealed Firmicutes as the most dominant phylum at all time points, with the predominant presence of Lactobacillales members in the OD group. The productivity indicator, i.e. higher Firmicutes:Bacteroidetes ratio, particularly in the late growth phase, was more marked in CD amplicon sequences, which was supported by culture-based enumerations on selective media. CD datasets also showed the prevalence of known butyrate-producing genera such as Faecalibacterium, Ruminococcus, Blautia, Coprococcus and Bacteroides, which correlates closely with their higher PICRUSt-based in silico predicted 'glycan biosynthesis and metabolism'-related Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologues. Semi-quantitative end-point PCR targeting of the butyryl-CoA: acetate CoA-transferase gene also confirmed butyrate producers as being late colonizers, particularly in antibiotic-fed birds in both the CD flocks and commercial rearing farms. Thus, antibiotics preferentially enrich bacterial populations, particularly short-chain fatty acid producers that can efficiently metabolize hitherto undigestable feed material such as glycans, thereby increasing the energy budget of the host and its productivity.

Ammonium induces differential expression of methane and nitrogen metabolism-related genes in Methylocystis sp. strain SC2.

Nitrogen source and concentration are major determinants of methanotrophic activity, but their effect on global gene expression is poorly studied. Methylocystis sp. strain SC2 produces two isozymes of particulate methane monooxygenase. These are encoded by pmoCAB1 (low-affinity pMMO1) and pmoCAB2 (high-affinity pMMO2). We used RNA-Seq to identify strain SC2 genes that respond to standard (10 mM) and high (30 mM) NH4(+) concentrations in the medium, compared with 10 mM NO3(-). While the expression of pmoCAB1 was unaffected, pmoCAB2 was significantly downregulated (log2 fold changes of -5.0 to -6.0). Among nitrogen metabolism-related processes, genes involved in hydroxylamine detoxification (haoAB) were highly upregulated, while those for assimilatory nitrate/nitrite reduction, high-affinity ammonium uptake and nitrogen regulatory protein PII were downregulated. Differential expression of pmoCAB2 and haoAB was independently validated by end-point reverse transcription polymerase chain reaction. Methane oxidation by SC2 cells exposed to 30 mM NH4(+) was inhibited at ≤ 400 ppmv CH4 , where pMMO2 but not pMMO1 is functional. When transferred back to standard nitrogen concentration, methane oxidation capability and pmoCAB2 expression were restored. Given that Methylocystis contributes to atmospheric methane oxidation in upland soils, differential expression of pmoCAB2 explains, at least to some extent, the strong inhibitory effect of ammonium fertilizers on this activity.

Genome analysis coupled with physiological studies reveals a diverse nitrogen metabolism in Methylocystis sp. strain SC2.

BACKGROUND: Methylocystis sp. strain SC2 can adapt to a wide range of methane concentrations. This is due to the presence of two isozymes of particulate methane monooxygenase exhibiting different methane oxidation kinetics. To gain insight into the underlying genetic information, its genome was sequenced and found to comprise a 3.77 Mb chromosome and two large plasmids. PRINCIPAL FINDINGS: We report important features of the strain SC2 genome. Its sequence is compared with those of seven other methanotroph genomes, comprising members of the Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. While the pan-genome of all eight methanotroph genomes totals 19,358 CDS, only 154 CDS are shared. The number of core genes increased with phylogenetic relatedness: 328 CDS for proteobacterial methanotrophs and 1,853 CDS for the three alphaproteobacterial Methylocystaceae members, Methylocystis sp. strain SC2 and strain Rockwell, and Methylosinus trichosporium OB3b. The comparative study was coupled with physiological experiments to verify that strain SC2 has diverse nitrogen metabolism capabilities. In correspondence to a full complement of 34 genes involved in N2 fixation, strain SC2 was found to grow with atmospheric N2 as the sole nitrogen source, preferably at low oxygen concentrations. Denitrification-mediated accumulation of 0.7 nmol (30)N2/hr/mg dry weight of cells under anoxic conditions was detected by tracer analysis. N2 production is related to the activities of plasmid-borne nitric oxide and nitrous oxide reductases. CONCLUSIONS/PERSPECTIVES: Presence of a complete denitrification pathway in strain SC2, including the plasmid-encoded nosRZDFYX operon, is unique among known methanotrophs. However, the exact ecophysiological role of this pathway still needs to be elucidated. Detoxification of toxic nitrogen compounds and energy conservation under oxygen-limiting conditions are among the possible roles. Relevant features that may stimulate further research are, for example, absence of CRISPR/Cas systems in strain SC2, high number of iron acquisition systems in strain OB3b, and large number of transposases in strain Rockwell.

Complete genome sequence of Methylocystis sp. strain SC2, an aerobic methanotroph with high-affinity methane oxidation potential.

Methylocystis sp. strain SC2 is an aerobic type II methanotroph isolated from a highly polluted aquifer in Germany. A specific trait of the SC2 strain is the expression of two isozymes of particulate methane monooxygenase with different methane oxidation kinetics. Here we report the complete genome sequence of this methanotroph that contains not only a circular chromosome but also two large plasmids.

Complete sequence analysis of two methanotroph-specific repABC-containing plasmids from Methylocystis sp. strain SC2.

The complete nucleotide sequences of two large, low-copy-number plasmids of 229.6 kb (pBSC2-1) and 143.5 kb (pBSC2-2) were determined during assembly of the whole-genome shotgun sequences of the methane-oxidizing bacterium Methylocystis sp. strain SC2. The physical existence of the two plasmids in strain SC2 was confirmed by pulsed-field gel electrophoresis followed by Southern hybridization. Both plasmids have a conserved replication module of the repABC system and carry genes involved in their faithful maintenance and conjugation. In addition, they contain genes that might be involved in essential metabolic processes. These include several heavy metal resistance genes and copper transport genes in pBSC2-1 and a complete nitrous oxide reductase operon and a pmoC singleton in pBSC2-2, the latter encoding the PmoC subunit of particulate methane monooxygenase.

Linking activity, composition and seasonal dynamics of atmospheric methane oxidizers in a meadow soil.

Microbial oxidation is the only biological sink for atmospheric methane. We assessed seasonal changes in atmospheric methane oxidation and the underlying methanotrophic communities in grassland near Giessen (Germany), along a soil moisture gradient. Soil samples were taken from the surface layer (0-10 cm) of three sites in August 2007, November 2007, February 2008 and May 2008. The sites showed seasonal differences in hydrological parameters. Net uptake rates varied seasonally between 0 and 70 µg CH(4) m(-2) h(-1). Greatest uptake rates coincided with lowest soil moisture in spring and summer. Over all sites and seasons, the methanotrophic communities were dominated by uncultivated methanotrophs. These formed a monophyletic cluster defined by the RA14, MHP and JR1 clades, referred to as upland soil cluster alphaproteobacteria (USCα)-like group. The copy numbers of pmoA genes ranged between 3.8 × 10(5)-1.9 × 10(6) copies g(-1) of soil. Temperature was positively correlated with CH(4) uptake rates (P<0.001), but had no effect on methanotrophic population dynamics. The soil moisture was negatively correlated with CH(4) uptake rates (P<0.001), but showed a positive correlation with changes in USCα-like diversity (P<0.001) and pmoA gene abundance (P<0.05). These were greatest at low net CH(4) uptake rates during winter times and coincided with an overall increase in bacterial 16S rRNA gene abundances (P<0.05). Taken together, soil moisture had a significant but opposed effect on CH(4) uptake rates and methanotrophic population dynamics, the latter being increasingly stimulated by soil moisture contents >50 vol% and primarily related to members of the MHP clade.

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish.

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.

A type Ib plasmid segregation machinery of the Advenella kashmirensis plasmid pBTK445.

pBTK445 is a newly described large (∼60Kb), low-copy number, conjugative plasmid indigenous to the sulfur-chemolithoautotroph Advenella kashmirensis. Based on its minimal replication region, a shuttle vector, pBTKS was constructed which can be used for diverse Alcaligenaceae members. The construct was found to be stably maintained both in the native host as well as in Escherichia coli in the absence of selective pressure which indicated that pBTKS harbors the stabilizing system of pBTK445, that are commonly coded by low-copy-number plasmids. Deletion analyzes of pBTKS confirmed the essentiality of parA (encoding a Walker-type ATPase of 214 amino acids) and the downstream located small parB (encoding an 85 amino acid protein having no sequence homolog in the database) in the faithful partitioning of pBTK445. A 1075bp PCR product, containing parA, parB and an upstream sequence having nine 11bp direct repeats (parS site) was found to comprise the partition functions of pBTK445, stabilizing both low-copy or high-copy number homologous and heterologous replicons in diverse hosts. The incompatibility determinant and the par promoter, P(par) were both found to be present within a 191bp iterated sequence present upstream of parA. ParB was found to regulate the expression of the Par proteins from P(par). The presence of a typical Walker-type ATPase motif in ParA, a short phylogenetically unrelated ParB, that acts as a repressor of P(par), and location of the iterated parS site upstream of parA, confirm that the active partition system of pBTK445 belongs to the type Ib.

Biochemistry and molecular biology of lithotrophic sulfur oxidation by taxonomically and ecologically diverse bacteria and archaea.

Lithotrophic sulfur oxidation is an ancient metabolic process. Ecologically and taxonomically diverged prokaryotes have differential abilities to utilize different reduced sulfur compounds as lithotrophic substrates. Different phototrophic or chemotrophic species use different enzymes, pathways and mechanisms of electron transport and energy conservation for the oxidation of any given substrate. While the mechanisms of sulfur oxidation in obligately chemolithotrophic bacteria, predominantly belonging to Beta- (e.g. Thiobacillus) and Gammaproteobacteria (e.g. Thiomicrospira), are not well established, the Sox system is the central pathway in the facultative bacteria from Alphaproteobacteria (e.g. Paracoccus). Interestingly, photolithotrophs such as Rhodovulum belonging to Alphaproteobacteria also use the Sox system, whereas those from Chromatiaceae and Chlorobi use a truncated Sox complex alongside reverse-acting sulfate-reducing systems. Certain chemotrophic magnetotactic Alphaproteobacteria allegedly utilize such a combined mechanism. Sulfur-chemolithotrophic metabolism in Archaea, largely restricted to Sulfolobales, is distinct from those in Bacteria. Phylogenetic and biomolecular fossil data suggest that the ubiquity of sox genes could be due to horizontal transfer, and coupled sulfate reduction/sulfide oxidation pathways, originating in planktonic ancestors of Chromatiaceae or Chlorobi, could be ancestral to all sulfur-lithotrophic processes. However, the possibility that chemolithotrophy, originating in deep sea, is the actual ancestral form of sulfur oxidation cannot be ruled out.

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, approximately 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 ( approximately 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 S4-intermediate pathway for the oxidation of thiosulfate by the chemolithoautotroph Tetrathiobacter kashmirensis and inhibition of tetrathionate oxidation by sulfite.

Chemolithotrophic oxidation of reduced sulfur compounds was studied in the betaproteobacterium Tetrathiobacter kashmirensis in correlation with its transposon (Tn5-mob)-inserted mutants impaired in sulfur oxidation (Sox(-)) and found to be carried out via the tetrathionate intermediate (S(4)I) pathway. The group of physiologically identical Sox(-) mutant strains presently examined could fully oxidize thiosulfate supplied in the media to equivalent amounts of tetrathionate but could only convert 5-10% of the latter to equivalent amounts of sulfite (equivalences in terms of mug atoms of S ml(-1)). These mutants were found to possess intact thiosulfate dehydrogenase, but defunct sulfite dehydrogenase, activities. Single copies of Tn5-mob in the genomes of the Sox(-) mutants were found inserted within the moeA gene, responsible for molybdopterin cofactor biosynthesis. This explained the inactivity of sulfite dehydrogenase. Chemolithotrophic oxidation of tetrathionate and sulfite by T. kashmirensis was found to be inhibited by 12 mM tungstate, whose effect could however be reversed by further addition of 15 mM molybdate. In mixotrophic medium, the mutants showed uninterrupted utilization of maltose but inhibition of tetrathionate utilization due to accumulation of sulfite. When sulfite was added to wild type cultures growing on tetrathionate-containing chemolithoautotrophic medium, it was found to render concentration-dependent inhibition of oxidation of tetrathionate. Our findings indicate that sulfite molecules negatively regulate their own synthesis by plausible inhibitory interaction(s) with enzyme(s) responsible for the oxidation of tetrathionate to sulfite; thereby clearly suggesting that one of the control mechanisms of chemolithotrophic sulfur oxidation could be at the level of sulfite.

The dimeric repressor SoxR binds cooperatively to the promoter(s) regulating expression of the sulfur oxidation (sox) operon of Pseudaminobacter salicylatoxidans KCT001.

Sulfur oxidation in Pseudaminobacter salicylatoxidans KCT001 is rendered by the combined action of several enzymes encoded by a thiosulfate-inducible sox operon. In this study it has been conclusively demonstrated by insertional mutagenesis that the regulatory gene of this operon is soxR, which encodes a DNA-binding protein belonging to the ArsR-SmtB family. SoxR was found to bind to two promoter-operator segments within the sox cluster, of which the one (wx) located between soxW and soxX controls the expression of sulfur-oxidation genes soxX through soxD while the other, a bi-directional element (sv) located between soxS and soxV, controls the expression of soxVW in one direction and the putative regulatory cluster soxSRT in the other. In the case of the wx promoter the repressor was found to bind in a cooperative manner to two distinct binding sites having different affinities, while in the case of the sv promoter binding occurred at a symmetric dimeric site and involved a higher degree of cooperativity. The high degree of cooperativity observed in the binding of SoxR to its target sites seemed to be due to the propensity of SoxR monomers to form dimers. The apparent dissociation constants of the SoxR-operator complexes were in the nanomolar range, indicating relatively strong interactions. It was demonstrated using a reporter system in Escherichia coli that this high-affinity binding of SoxR led to efficient repression in trans. Thus the role of SoxR as a repressor of the sox operon has not only been conclusively established but it has also been shown that this repression is brought about through cooperative interactions of SoxR with dimeric binding sites that occlude the operon promoters.

A novel gene cluster soxSRT is essential for the chemolithotrophic oxidation of thiosulfate and tetrathionate by Pseudaminobacter salicylatoxidans KCT001.

Chemolithotrophic sulfur oxidation (Sox) in the alpha-proteobacterium Pseudaminobacter salicylatoxidans KCT001 was found to be governed by the gene cluster soxSRT-soxVWXYZABCD. Independent transposon-insertion mutations in the genes soxB, soxC, soxD, and also in a novel open reading frame (ORF), designated as soxT, afforded revelation of the entire sox locus of this bacterium. The deduced amino acid sequence of the novel ORF soxT comprised 362 residues and exhibited significant homology with hypothetical proteins of diverse origin, including a permease-like transport protein of Escherichia coli. Two contiguous ORFs, soxR and soxS, immediately preceded the soxT gene. The gene cluster soxSRT was located upstream of soxVWXYZABCD and was transcribed divergently with respect to the latter. Chemolithotrophic utilization of both thiosulfate and tetrathionate was observed to have been impaired in all of these Sox- mutants, implicating the involvement of the gene cluster soxSRT-soxVWXYZABCD in the oxidation of both thiosulfate and tetrathionate.