Extremely oligotrophic and complex-carbon-degrading microaerobic bacteria from Arabian Sea oxygen minimum zone sediments.

Sediments underlying marine hypoxic zones are huge sinks of unreacted complex organic matter, where despite acute O2 limitation, obligately aerobic bacteria thrive, and steady depletion of organic carbon takes place within a few meters below the seafloor. However, little knowledge exists about the sustenance and complex carbon degradation potentials of aerobic chemoorganotrophs in these sulfidic ecosystems. We isolated and characterized a number of aerobic bacterial chemoorganoheterotrophs from across a ~ 3 m sediment horizon underlying the perennial hypoxic zone of the eastern Arabian Sea. High levels of sequence correspondence between the isolates' genomes and the habitat's metagenomes and metatranscriptomes illustrated that the strains were widespread and active across the sediment cores explored. The isolates catabolized several complex organic compounds of marine and terrestrial origins in the presence of high or low, but not zero, O2. Some of them could also grow anaerobically on yeast extract or acetate by reducing nitrate and/or nitrite. Fermentation did not support growth, but enabled all the strains to maintain a fraction of their cell populations over prolonged anoxia. Under extreme oligotrophy, limited growth followed by protracted stationary phase was observed for all the isolates at low cell density, amid high or low, but not zero, O2 concentration. While population control and maintenance could be particularly useful for the strains' survival in the critically carbon-depleted layers below the explored sediment depths (core-bottom organic carbon: 0.5-1.0% w/w), metagenomic data suggested that in situ anoxia could be surmounted via potential supplies of cryptic O2 from previously reported sources such as Nitrosopumilus species.

Thermal Endurance by a Hot-Spring-Dwelling Phylogenetic Relative of the Mesophilic Paracoccus.

High temperature growth/survival was revealed in a phylogenetic relative (SMMA_5) of the mesophilic Paracoccus isolated from the 78 to 85°C water of a Trans-Himalayan sulfur-borax spring. After 12 h at 50°C, or 45 min at 70°C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained ~2% colony forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50°C and 70°C, respectively. After 12 h at 50°C, the thermally conditioned sibling SMMA_5_TC exhibited an ~1.5 time increase in CFU count; after 45 min at 70°C, SMMA_5_TC had 7% of the initial CFU count. 1,000-times diluted Reasoner's 2A medium, and MST supplemented with lithium, boron, or glycine-betaine, supported higher CFU-retention/CFU-growth than MST. Furthermore, with or without lithium/boron/glycine-betaine, a higher percentage of cells always remained metabolically active, compared with what percentage formed single colonies. SMMA_5, compared with other Paracoccus, contained 335 unique genes: of these, 186 encoded hypothetical proteins, and 83 belonged to orthology groups, which again corresponded mostly to DNA replication/recombination/repair, transcription, secondary metabolism, and inorganic ion transport/metabolism. The SMMA_5 genome was relatively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells' energy and resources toward system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped inherit those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCE For a holistic understanding of microbial life's high-temperature adaptation, it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat-specific inorganic/organic solutes, and potential genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multifaceted.

Paraburkholderia bengalensis sp. nov. isolated from roots of Oryza sativa, IR64.

Paraburkholderia bengalensis sp. nov. strain IR64_4_BI was isolated from rice roots cultivated in Madhyamgram field station of Bose Institute, West Bengal, India. IR64_4_BI is a Gram-negative, motile, nitrate-reducing, nitrogen-fixing bacterium. Whole-cell fatty acid analyses of IR64_4_BI show C16:0, summed feature 8 (comprising C18:1ω7c and/or C18:1 ω 6c) and summed feature 3(C16:1 w7c/C16:1 w6c or C16:1 ω 7c/C16:1 ω 6c) were the predominant fatty acids. 16S rRNA phylogeny showed that it was most similar to P. phymatum STM815T (98.5% identity), P. terrae KMY02T (98.44% identity) and P. hospita LMG 20598T (98.32% identity). The Average Nucleotide Identity-BLAST (ANIb) of P. bengalensis IR64_4_BI with P. hospita DSM 17164T, P. terrae DSM 17804T, P. phymatum STM815T and P. hospita LMG 20598T was 83.11, 83.52, 84.5 and 83.12% respectively. Comparison of genome sequence of IR64_4_BI with other species of Paraburkholderia using the Multi-locus species tree software show that P. bengalensis IR64_4_BI is a novel species. The ability of P. bengalensis IR64_4_BI to survive on nitrogen-free medium under microaerophilic conditions and the abundance of nitrogen metabolism-related genes makes this strain a potential candidate for developing a nitrogen-fixing system in rice. Based on genotypic, phenotypic and chemotaxonomic studies, we propose that IR64_4_BI (= MTCC 13051 = JCM 34777) is a new species of Paraburkholderia which has been assigned as Paraburkholderia bengalensis sp.nov.

Draft Genome Sequences of Four Novel Strains of Microbes Isolated from Lepidocephalichthys guntea.

We report the draft genome sequences of four bacterial strains (all of which are putatively novel species) belonging to four different genera. The Gram-positive Bacillus sp. strain GG161 and Rhodococcus sp. strain GG48 and the Gram-negative Achromobacter sp. strain GG226 and Shigella sp. strain GCP5 were all isolated from the gut of the optionally intestine-breathing freshwater fish Lepidocephalichthys guntea.

Structural-genetic insight and optimization of protease production from a novel strain of Aeromonas veronii CMF, a gut isolate of Chrysomya megacephala.

Structural-genetic characterization of protease producing genes and enzymes from microbial sources are seldom appreciated despite having its substantial utilization in protein engineering or genetic manipulation for biotechnological applications. Aeromonas veronii CMF, a mesophilic bacterium isolated from the gut of Chrysomya megacephala, was found to exhibited significant level of protease activity. For the revelation of genetic potential in relation to protease production, whole genome of this organism was sequenced and analysed while structure-function of different protease enzyme was predicated using various in silico analysis. The 4.5 mb CMF genome was found to encompass various types of protease and mostly they are neutral in nature. Enzyme production was highest in an optimum pH and temperature of 6.0 (32.09 ± 1.015 U/ml) and 35ºC (41.65 ± 1.152 U/ml), respectively. Other culture parameters for optimum production of protease were determined to be inoculum size (1%), incubation period (72 h), shaking condition (125 rpm), carbon and nitrogen source [2% lactose (92.21 ± 3.16 U/ml) and 0.5% urea (163.62 ± 4.31 U/ml), respectively] and effect of surfactants [0.02 mg/ml Tween 80 (174.72 ± 4.48 U/ml)]. Furthermore, A. veronii CMF exhibited significant enzyme production like serine protease (15.22 ± 0.563 U/ml), aspartate protease (33.16 ± 0.762 U/ml) and collagenase (17.26 ± 0.626 U/ml). Genomic information and results of physio-biochemical assays indicate its cost-effective potential use in different enzyme-industry.

Structural-Genetic Characterization Of Novel Butaryl co-A Dehydrogenase and Proposition of Butanol Biosynthesis Pathway in Pusillimonas ginsengisoli SBSA.

Despite extensive use in the biofuel industry, only butyryl co-A dehydrogenase enzymes from the Clostridia group have undergone extensive structural and genetic characterization. The present study, portrays the characterization of structural, functional and phylogenetic properties of butyryl co-A dehydrogenase identified within the genome of Pusillimonas ginsengisoli SBSA. In silico characterization, homology modelling and docking data indicates that this protein is a homo-tetramer and 388 amino acid residue long, rich in alanine and leucine residue; having molecular weight of 42347.69 dalton. Its isoelectric point value is 5.78; indicate its neutral nature while 38.38 instability index value indicate its stable nature. Its thermostable nature evidenced by its high aliphatic index (93.14); makes its suitable for industry-based use. The secondary structure prediction analysis of butyryl co-A dehydrogenase unveiled that the proteins has secondary arrangements of 54% α-helix, 13% ß-stand and 5% disordered conformation. However, phylogenetic analysis clearly indicates that probably horizontal gene transfer is the primary mechanism of spreading of this gene in this organism. Notably, multiple sequence alignment study of phylogenetically diverse butyryl co-A dehydrogenase sequence highlighted the presence of conserved amino acid residues i.e. YXV/LGXKXWXS/T. Physicochemical characterization of other relevant proteins involved in butanol metabolism of SBSA also has been carried out. However, metabolic construction of functional butanol biosynthesis pathway in SBSA, enlightened its cost-effective potential use in biofuel industry as an alternate to Clostridia system.

Aerobic microbial communities in the sediments of a marine oxygen minimum zone.

The ecology of aerobic microorganisms is never explored in marine oxygen minimum zone (OMZ) sediments. Here we reveal aerobic bacterial communities along ∼3 m sediment-horizons of the eastern Arabian Sea OMZ. Sulfide-containing sediment-cores retrieved from 530 mbsl (meters beneath the sea-level) and 580 mbsl were explored at 15-30 cm intervals, using metagenomics, pure-culture-isolation, genomics and metatranscriptomics. Genes for aerobic respiration, and oxidation of methane/ammonia/alcohols/thiosulfate/sulfite/organosulfur-compounds, were detected in the metagenomes from all 25 sediment-samples explored. Most probable numbers for aerobic chemolithoautotrophs and chemoorganoheterotrophs at individual sample-sites were up to 1.1 × 107 (g sediment)-1. The sediment-sample collected from 275 cmbsf (centimeters beneath the seafloor) of the 530-mbsl-core yielded many such obligately aerobic isolates belonging to Cereibacter, Guyparkeria, Halomonas, Methylophaga, Pseudomonas and Sulfitobacter which died upon anaerobic incubation, despite being provided with all possible electron acceptors and fermentative substrates. High percentages of metatranscriptomic reads from the 275 cmbsf sediment-sample, and metagenomic reads from all 25 sediment-samples, matched the isolates' genomic sequences including those for aerobic metabolisms, genetic/environmental information processing and cell division, thereby illustrating the bacteria's in-situ activity, and ubiquity across the sediment-horizons, respectively. The findings hold critical implications for organic carbon sequestration/remineralization, and inorganic compounds oxidation, within the sediment realm of global marine OMZs.

Molecular mechanism of sulfur chemolithotrophy in the betaproteobacterium Pusillimonas ginsengisoli SBSA.

Chemolithotrophic sulfur oxidation represents a significant part of the biogeochemical cycling of this element. Due to its long evolutionary history, this ancient metabolism is well known for its extensive mechanistic and phylogenetic diversification across a diverse taxonomic spectrum. Here we carried out whole-genome sequencing and analysis of a new betaproteobacterial isolate, Pusillimonas ginsengisoli SBSA, which is found to oxidize thiosulfate via the formation of tetrathionate as an intermediate. The 4.7 Mb SBSA genome was found to encompass a soxCDYZAXOB operon, plus single thiosulfate dehydrogenase (tsdA) and sulfite : acceptor oxidoreductase (sorAB) genes. Recombination-based knockout of tsdA revealed that the entire thiosulfate is first converted to tetrathionate by the activity of thiosulfate dehydrogenase (TsdA) and the Sox pathway is not functional in this bacterium despite the presence of all necessary sox genes. The ∆soxYZ and ∆soxXA knockout mutants exhibited a wild-type-like phenotype for thiosulfate/tetrathionate oxidation, whereas ∆soxB, ∆soxCD and soxO::KanR mutants only oxidized thiosulfate up to tetrathionate intermediate and had complete impairment in tetrathionate oxidation. The substrate-dependent O2 consumption rate of whole cells and the sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, indicated that glutathione plays a key role in SBSA tetrathionate oxidation. The present findings collectively indicate that the potential glutathione : tetrathionate coupling in P. ginsengisoli involves a novel enzymatic component, which is different from the dual-functional thiol dehydrotransferase (ThdT), while subsequent oxidation of the sulfur intermediates produced (e.g. glutathione : sulfodisulfane molecules) may proceed via the iterative action of soxBCD .

Two pathways for thiosulfate oxidation in the alphaproteobacterial chemolithotroph Paracoccus thiocyanatus SST.

Chemolithotrophic bacteria oxidize various sulfur species for energy and electrons, thereby operationalizing biogeochemical sulfur cycles in nature. The best-studied pathway of bacterial sulfur-chemolithotrophy involves direct oxidation of thiosulfate (S2O32-) to sulfate (SO42-) without any free intermediate. This pathway mediated by SoxXAYZBCD is apparently the exclusive mechanism of thiosulfate oxidation in facultatively chemolithotrophic alphaproteobacteria. Here we explore the molecular mechanisms of sulfur oxidation in the thiosulfate- and tetrathionate(S4O62-)-oxidizing alphaproteobacterium Paracoccus thiocyanatus SST, and compare them with the prototypical Sox process of Paracoccus pantotrophus. Our results reveal a unique case where an alphaproteobacterium has Sox as its secondary pathway of thiosulfate oxidation converting ∼10% of the thiosulfate supplied, whilst ∼90% of the substrate is oxidized via a pathway that produces tetrathionate as an intermediate. Sulfur oxidation kinetics of a deletion mutant showed that thiosulfate-to-tetrathionate conversion, in SST, is catalyzed by a thiosulfate dehydrogenase (TsdA) homolog that has far-higher substrate-affinity than the Sox system of this bacterium, which in turn is also less efficient than the P. pantotrophus Sox. Deletion of soxB abolished sulfate-formation from thiosulfate/tetrathionate, while thiosulfate-to-tetrathionate conversion remained unperturbed. Physiological studies revealed the involvement of glutathione in SST tetrathionate oxidation. However, zero impact of the insertional mutation of a thiol dehydrotransferase (thdT) homolog, together with the absence of sulfite as an intermediate, indicated that SST tetrathionate oxidation is mechanistically novel, and distinct from its betaproteobacterial counterpart mediated by glutathione, ThdT, SoxBCD and sulfite:acceptor oxidoreductase. The present findings highlight extensive functional diversification of sulfur-oxidizing enzymes across phylogenetically close, as well as distant, bacteria.

Incidence, management, and reporting of severe and fatal Plasmodium falciparum malaria in secondary and tertiary health facilities of Alipurduar, India in 2009.

BACKGROUND & OBJECTIVES: The proportion of malaria cases that are complicated and fatal are not well described in India. Alipurduar sub-division of Jalpaiguri district in West Bengal is highly endemic for malaria. We constructed a retrospective cohort of severe malaria patients admitted in the secondary and tertiary care facilities in Alipurduar to determine the incidence, assess the management, and evaluate the reporting of severe and fatal malaria. METHODS: We reviewed routine surveillance data and the case records of all the malaria patients admitted in all secondary and tertiary care facilities, both public and private. We defined severe malaria cases as Plasmodium falciparum infection with clinical signs and symptoms of organ involvement in a resident of Alipurduar admitted during January to December 2009. We compared clinical and demographic characteristics of severe malaria cases that died with those who survived. We also reviewed human resources and laboratory facilities available for the treatment of severe malaria in these health facilities. RESULTS: During 2009, 6191 cases of P. falciparum in Alipurduar were reported to the malaria surveillance system. We identified 336 (5.4%) cases of severe malaria among which 33 (9.8%) patients died. Four malaria deaths were also recorded from primary health centres. Only 17 of the 37 (46%) total deaths recorded were reported to the routine surveillance system. Most severe cases were males (65%), aged >15 years (72%), and nearly half were admitted to secondary care hospitals (48%). In multivariate analysis, the risk factors associated with death included increased delay fever onset and hospitalization, treatment in a secondary level hospital, younger age, and multi-organ involvement. The secondary level public hospital had too few physicians and nurses for supporting severe malaria patients as well as inadequate laboratory facilities for monitoring such patients. CONCLUSIONS: Severe and fatal malaria continue to burden Alipurduar and record keeping in health facilities was poor. Many malaria deaths were not routinely reported even in the public sector. Improved surveillance and increased human and laboratory resources are needed to reduce malaria mortality.

Factors associated with low utilization of x-ray facilities among the sputum negative chest symptomatics in Jalpaiguri district (West Bengal) 2009.

New sputum negative (NSN) tuberculosis case detection in Jalpaiguri district has been consistently low. Availability and accessibility of health facilities with chet x-rays is key for the diagnosis of NSN cases. To identify factors associated with utilisation of x-ray facilities in the district, we interviewed 4,875 chest symptomatics who were sputum negative on two occasions with an antibiotics course in between. Chest radiography was available in only three public health facilities in the district. Low income, long distance from the public health facilities with chest radiography and high cost of x-rays at private hospitals were key factors associated with symptomatics not undergoing X-ray. It is necessary to increase facilities for radiological diagnosis and provide mobility support for the symptomatics in Jalpaiguri.

Risk factors for malaria deaths in Jalpaiguri district, West Bengal, India: evidence for further action.

BACKGROUND: In 2006, a cluster of malaria deaths in the highly endemic Jalpaiguri district, West Bengal, India, led to assignment of additional resources. Malaria deaths decreased, but continued to occur. A study was conducted to identify the risk factors for residual malaria deaths. METHODS: Malaria death was defined as a death from fever with microscopically confirmed Plasmodium falciparum among residents of Jalpaiguri during 2007-2008. For each case, three age-, sex- and locality-matched controls were recruited among microscopically confirmed falciparum malaria patients cured during the same period. Clinical and treatment information was abstracted from records. Information about knowledge about malaria, presence of bed nets and DDT spraying was collected through interviews of the close relatives of study subjects. Odds ratio (OR) were calculated using multivariate methods. RESULTS: 51 malaria deaths were matched with 153 controls, which did not differ by age (median: 35 versus 36 years) and proportion of males (63% versus 63%). On multiple logistic regression analysis, compared with survivors, malaria deaths were more likely to have been admitted with already existing complications [OR = 4.1, 95% confidence interval (CI) = 1.6-10)], treated at a private facility (OR = 3.7, 95% CI = 1.2-12), received treatment after 48 hours of fever onset (OR = 14, 95% CI = 2.9-64), received chloroquine (OR = 13.3, 95% CI = 3.7-47). Households of the deceased were also more likely to miss bed nets (OR = 6.3, 95% CI = 1.9-24) and DDT spraying (OR = 9.2, 95% CI = 2.8-31). CONCLUSION: Elimination of malaria deaths will require education of providers for prompt referral before complications, engagement of the private sector, community awareness for early treatment as well as scaled-up use of bed nets use and DDT. Use of newer generation anti-malarials must to be generalized.