Freshwater Isolate Research Articles

Characterization of tailocins of Pragia fontium 24613 and the tailocin loci within the family Budviciaceae

Tailocins are nano-scale phage tail-like protein complexes that can mediate antagonistic interactions between closely related bacterial species. While the capacity to produce R-type tailocin was found widely across Gammaproteobacteria, the production of F-type tailocins seems comparatively rare. In this study, we examined the freshwater isolate, Pragia fontium 24613, which can produce both R- and F-type tailocins. We investigated their inhibition spectrum, focusing on clinically relevant enterobacteria, and identified the associated tailocin gene cluster. Transmission electron microscopy confirmed that inactivation of the tape measure protein within the tailocin cluster disrupted R-tailocin production. Comparative analysis of Budviciaceae gene clusters showed high conservation of R-type tailocin genes, whereas F-type tailocin genes were found in only a few species, with little conservation. Our findings indicate a high prevalence of bacteriocin production among underexplored Enterobacteriales species. Detected tailocins showed potential as antimicrobials targeting clinically significant pathogens.

Antibiotic Concentrations Affect the Virulence of Klebsiella quasipneumoniae subsp. similipneumoniae Isolates

Klebsiella species are becoming a major global public health concern. In particular, the increase in multidrug‐resistant strains is a cause for concern. This study was aimed at determining the antibiotic susceptibility of two different isolates of Klebsiella quasipneumoniae subsp. similipneumoniae and determining the virulence characteristics and bacterial morphology under subminimum inhibitory concentrations (sub‐MICs) of antibiotics. In this study, two multidrug‐resistant K. quasipneumoniae subsp. similipneumoniae isolates were identified, one of which was clinical, and the other was isolated from freshwater. The MICs of the antibiotics meropenem, chloramphenicol, ciprofloxacin, and kanamycin were determined for these isolates. The effects of the sub‐MICs on the virulence and morphological characteristics of the bacteria were investigated in comparison with K. pneumoniae (ATCC 13883). The MICs of meropenem, chloramphenicol, ciprofloxacin, and kanamycin were 0.04, 20, 2, and 8 μg/mL in the clinical isolate; 0.2, 15, 5, and 2 μg/mL in the freshwater isolate; and 0.03, 3, 0.1, and 0.3 μg/mL for K. pneumoniae. The biofilm‐forming ability of K. quasipneumoniae subsp. similipneumoniae isolates decreased with antibiotic sub‐MICs. Siderophore activity increased only with MIC/4 of kanamycin and MIC/2 of chloramphenicol in the clinical isolate (p > 0.05). Furthermore, bacterial morphology and expression of virulence genes were affected differently by the sub‐MICs. This study showed that biofilm formation decreased and that the changes in bacterial morphology and expression of virulence genes were very different in the presence of 1/2 and 1/4 sub‐MIC antibiotics.

Antimicrobial potential of Chlorella sorokiniana on MRSA – An in vitro study and an in silico analysis on ClpP protease

Antimicrobial potential of Chlorella sorokiniana on MRSA – An in vitro study and an in silico analysis on ClpP protease

Production of Single Cell Oils from Bacterial Sources

Single Cell Oils (SCO) is of profound interest for a variety of purposes ranging from biofuels to nutritional adjuvants, pharmaceutical applications and biotransformation for valuable products. A number of microorganisms have been shown to produce and accumulate SCO. In the present study a methodical attempt was made to isolate potential SCO producers from Indian water sources. Saltwater samples from the Arabian Sea and freshwater samples from an Indian cold-water river (Pindhari River, Uttarakhand) were collected and studied for the occurrence of lipid producing microbes. Of the several isolates shortlisted as lipid producers three isolates from this study were identified as potential SCO producers based on their lipid producing abilities. The types of fatty acids comprising the SCO from selected isolates were studied by Gas Chromatography (GC) and confirmed by Gas Chromatography/Mass Spectrometry (GC/MS). Lipid profiles from the GC analysis showed that the isolates in this study produced economically and nutritionally valuable Monounsaturated fatty acids (MUFA) like Palmitoleic acid and Oleic acid. Also, two isolates from the Arabian Sea were seen to produce a valuable omega-3 Polyunsaturated Fatty Acid (PUFA) like Eicosapentanoic acid. While a freshwater isolate produced Linoleic acid an omega-6 PUFA. Selected isolates were characterized for their biochemical characteristics and identified molecularly by 16S rRNA sequencing. Ornithinibacillus sp. Marseille-P3601 strain isolated in our study from the cold-water River Pindhari, Uttarakhand is found capable of producing PUFA.

Bioconversion of Fish Hatchery Waste as Feed in the Production of Live Feed

Purple Non-Sulfur Bacteria (PNSB), also known as phototrophic bacteria are widely distributed in both freshwater and marine environment and capable to grow in wide range of substrates. In this study, Bacterium Rhodobacter sphaeroides strain UMS2, a freshwater isolate was used in this study in utilization of fish hatchery waste. This study was conducted to determine the nutritional values of bioprocess product that was grown in fish hatchery waste. Finally, the waste bio-converted product was used as feed supplement to monitor the growth performance of live feed Tubifex spp. Inoculum of Rhodobacter sphaeroides strain UMS2 was developed in 112 synthetic media and 48-h culture of 30% (v/v) inoculum was used in fish hatchery waste during the bioprocess. The nutritional values of bio-converted product, except total ash (%), were not significantly improved with 30% (v/v) inoculum of Rhodobacter sphaeroides, strain UMS2. Feeding trial in bloodworm (Tubifex spp.) with bioconversion product conducted for 15 days to monitor growth (w/v) of live feed. Initial growth 1.42 ± 0.001 g/L of Tubifex spp. was stocked in 15 × 75 × 15 cm plastic tray connected with recirculated system. Tubifex spp. was observed to be comparatively higher (1.55 ± 0.12 g/L) while fed in the product that contained bacterium than the growth (1.44 ± 0.15 g/L) of Tubifex spp. fed in the bioconversion product of without bacterium. The inoculums size (30%) of bacterium not enough to support the growth of Rhodobacter sphaeroides, strain UMS2 in the bioconversion process to improve the nutritional values. However, while used as feed supplement it improved the growth performance of the Tubifex spp. So, bacterium Rhodobacter sphaeroides, strain UMS2 has potentiality to be used as feed supplement in the production of live feed.

Transmission of a Freshwater Isolate of Ichthyophonus (Clade C) to Two Marine Fish Species.

The ingestion of infected prey is the most recognizable mode of transmission for Ichthyophonus, but because this mode of transmission is unidirectional from small prey fish to larger predators, it cannot sustain the parasite within or among populations nor does it explain transmission to planktivores. Recently, waterborne transmission was demonstrated in cultured Rainbow Trout Oncorhynchus mykiss, which could explain how the parasite is transmitted without piscivory. However, it is possible that this is an adaptation to aquaculture conditions, and may not occur among wild fish. To address this question, experiments were conducted to determine if a freshwater isolate of Ichthyophonus is infectious and pathogenic to marine species, as well as if transmission is possible between different marine species. Pacific Staghorn Sculpins Leptocottus armatus were fed a freshwater isolate of Ichthyophonus (clade C) and then housed with susceptible sentinel Rock Soles Lepidopsetta bilineata. Ninety two percent of the orally exposed sculpins and 30% of the sentinel soles were Ichthyophonus-positive at the end of the study, with 0% infected controls. These results demonstrate that a freshwater isolate of Ichthyophonus is infectious and pathogenic to marine species and can be transmitted in seawater in the absence of piscivory. It also provides a plausible mechanism for transmission to small prey fish and planktivores, as well as within a population of piscivores when infected prey is not available.

Genome-based insights into a putative novel Methylomonas species (strain Kb3), isolated from an Indian rice field

Abstract Methylomonas is one of the most important and frequently detected genera in aquatic habitats and rice fields. A strain of Methylomonas, Kb3, an aerobic methanotroph, was isolated from a rice field from the Western parts of India. Kb3 cells were composed of short, motile rods and formed pink colored colonies and pink pellicles in liquid. On the basis of 16S rRNA gene, the closest valid species was Methylomonas methanica strain S1 (97.9% sequence similarity). Kb3 also showed close affiliation to “Methylomonas denitrificans” FJG1, a recently described, but not yet valid species (99.1% sequence similarity) and to strain LW13, a freshwater lake Washington isolate (99.5% sequence similarity). To understand the correct taxonomical position and metabolic properties of Kb3, the whole genome was sequenced. Kb3 genome showed a complete methane oxidation pathway, the presence of pMMO and sMMO genes, genes for nitrogen fixation and potential to convert nitrite to nitric oxide and nitric oxide to nitrous oxide. Numerous genes for chemotaxis and motility were detected. Genome comparison of Kb3 with the genomes of Methylomonas methanica S1 and “Methylomonas denitrificans” FJG1 showed ortho-ANI values of 86–87%, much less than 95%, the current cut-off for species. Digital DNA-DNA hybridization also showed values of 41–42%, much lesser than the 70% cut-off, both indicating that Kb3 did not belong to any of these species. Methylomonas LW13 and Methylomonas Kb3 showed close genome level similarities, i.e. OrthoANI value of 97.9% and a DDH of 87.3% and close branching in the phylogenomic tree confirming that Kb3 and LW13 can together form a new species.

Letting go: bacterial genome reduction solves the dilemma of adapting to predation mortality in a substrate-restricted environment.

Resource limitation and predation mortality are major determinants of microbial population dynamics, and optimization for either aspect is considered to imply a trade-off with respect to the other. Adaptation to these selective factors may, moreover, lead to disadvantages at rich growth conditions. We present an example of a concomitant evolutionary optimization to both, substrate limitation and predation in an aggregate-forming freshwater bacterial isolate, and we elucidate an underlying genomic mechanism. Bacteria were propagated in serial batch culture in a nutrient-restricted environment either with or without a bacterivorous flagellate. Strains isolated after 26 growth cycles of the predator-prey co-cultures formed as much total biomass as the ancestor at ancestral growth conditions, albeit largely reallocated to cell aggregates. A ~273 kbp genome fragment was lost in three strains that had independently evolved with predators. These strains had significantly higher growth yield on substrate-restricted media than others that were isolated from the same treatment before the excision event. Under predation pressure, the isolates with the deletion outcompeted both, the ancestor and the strains evolved without predators even at rich growth conditions. At the same time, genome reduction led to a growth disadvantage in the presence of benzoate due to the loss of the respective degradation pathway, suggesting that niche constriction might be the price for the bidirectional optimization.

Protistan predation interferes with bacterial long-term adaptation to substrate restriction by selecting for defence morphotypes.

Bacteria that are introduced into aquatic habitats face a low substrate environment interspersed with rare productive 'hotspots', as well as high protistan grazing. Whereas the former condition should select for growth performance, the latter should favour traits that reduce predation mortality, such as the formation of large cell aggregates. However, protected morphotypes often convey a growth disadvantage, and bacteria thus face a trade-off between investing in growth or defence traits. We set up an evolutionary experiment with the freshwater isolate Sphingobium sp. strain Z007 that conditionally increases aggregate formation in supernatants from a predator-prey coculture. We hypothesized that low substrate levels would favour growth performance and reduce the aggregated subpopulation, but that the concomitant presence of a flagellate predator might conserve the defence trait. After 26 (1-week) growth cycles either with (P+) or without (P-) predators, bacteria had evolved into strikingly different phenotypes. Strains from P- had low numbers of aggregates and increased growth yield, both at the original rich growth conditions and on various single carbon sources. By contrast, isolates from the P+ treatment formed elevated proportions of defence morphotypes, but exhibited lower growth yield and metabolic versatility. Moreover, the evolved strains from both treatments had lost phenotypic plasticity of aggregate formation. In summary, the (transient) residence of bacteria at oligotrophic conditions may promote a facultative oligotrophic life style, which is advantageous for survival in aquatic habitats. However, the investment in defence against predation mortality may constrain microbial adaptation to the abiotic environment.

Isolation and Genomic Characterization of ‘Desulfuromonas soudanensis WTL’, a Metal- and Electrode-Respiring Bacterium from Anoxic Deep Subsurface Brine

Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, ‘Desulfuromonas soudanensis’ strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that ‘D. soudanensis’ releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. ‘D. soudanensis’ contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of ‘D. soudanensis’ underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats.

Acetophenone derivatives from a freshwater fungal isolate of recently described Lindgomyces madisonensis (G416)

The exploration of freshwater ascomycetes, which have undergone only limited investigation, may provide opportunities both to characterize new genera/species of fungi and to uncover new chemical diversity. In this study, seven acetophenone derivatives, madisone, 4'-methoxymadisone, dehydromadisone, 2″-methoxymadisone, dihydroallovisnaginone, dimadisone, and 4'-methoxydimadisone were characterized from an organic extract of a recently described Lindgomyces madisonensis (G416) culture, which was isolated from submerged wood collected in a stream in North Carolina. Madisone, dehydromadisone, 2″-methoxymadisone, dimadisone and 4'-demethoxydimadisone have not been reported previously, while 4'-methoxymadisone and dihydroallovisnaginone were previously unknown as natural products. Their structures were assigned on the basis of NMR and HRESIMS data, with the structure of madisone supported by X-ray crystallography. The antimicrobial activities of madisone, 4'-methoxymadisone and dihydroallovisnaginone were evaluated against a panel of bacteria and fungi. A heat map analysis of the surface of a G416 culture showed that most of the isolated compounds concentrated in the guttate compared with the vegetative mycelium of the fungus.

Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.

Genome Sequence Analysis Reveals Evidence of Quorum-Sensing Genes Present in Aeromonas hydrophila Strain M062, Isolated from Freshwater.

Aeromonas hydrophila has emerged worldwide as a human pathogen. Here, we report the draft whole-genome sequence of a freshwater isolate from Malaysia, A. hydrophila strain M062, and its N-acylhomoserine lactone genes are also reported here.

Whole-Genome Sequencing Analysis of Quorum-Sensing Aeromonas hydrophila Strain M023 from Freshwater.

Aeromonas hydrophila is a well-known waterborne pathogen that recently was found to infect humans. Here, we report the draft genome of a freshwater isolate from a Malaysian waterfall, A. hydrophila strain M023, which portrays N-acylhomoserine lactone-dependent quorum sensing.

Genomic Insights of Pectobacterium carotovorum Strain M022 Quorum-Sensing Activity through Whole-Genome Sequencing.

Pectobacterium carotovorum is known to cause serious damage to various major crops worldwide. Here, we report the draft genome of Pectobacterium carotovorum strain M022, a freshwater isolate from a Malaysian waterfall, which has been reported as a plant pathogen and is able to communicate with N-acylhomoserine lactone-mediated quorum sensing.

Studies on interfacial interactions of TiO2 nanoparticles with bacterial cells under light and dark conditions

The probable underlying mechanism(s) of bacterial cell–TiO2 nanoparticles (TiO2 NPs) interaction in the absence of photo-irradiation has been less studied since most of the prior cytotoxicity studies focused on irradiated TiO2. The present study draws attention to the possible role of cell surface–TiO2 NP interactions under dark conditions, through an array of spectroscopic and microscopic investigations. A dominant freshwater bacterial isolate, Bacillus licheniformis, which interacted with environmentally relevant concentrations of TiO2 NPs (1 μg/mL), was analysed and compared under both light and dark conditions. Aggregation of cells upon NP interaction and adsorption of NPs onto the cell membrane was evident from the scanning electron micrographs under both light and dark conditions. The FT–IR and FT–Raman spectra suggested stress response of bacterial cells by elevated protein and polysaccharide content in the cell–NP interaction. The Xray photoelectron spectroscopic data substantiated the reduction of titanium from Ti(IV) to Ti(III) species which might have contributed to the redox interactions on the cell surface under light as well as dark conditions. The internalization of NPs in the cytoplasm were obvious from the transmission electron micrographs. The consequent cell death/damage was confirmed through fluorescence spectroscopy and microscopy. To conclude, the current study established the substantial role of interfacial interactions in cytotoxicity of the TiO2 NPs irrespective of the irradiation conditions.

Facile and Novel Strategy for Methods of Extraction of Biofuel Grade Lipids from Microalgae- an Experimental Report

The structural features of microalgal cell make it too difficult to extract the total lipid content of the cell as such. Thus, the cell disruption before lipid extraction becomes mandatory and has to be cost-effective. In the present study various methods and combination of few methods were adopted for effective extraction in order to choose the most effective cell disruption method for the complete extraction of lipids from a selected indigenous freshwater isolate, Scenedesmus sp. NTEB03. Interestingly, we found that grinding and bead-beating method showed two fold increased lipid productivity (23.2%) than the other methods tested. Biomass and lipid productivity of Scenedesmus sp., was found to be 0.0418 g L -1 d -1 and 4.3 mg L -1 d -1 respectively. Fatty acid profiles revealed that oleic (C18:1) and linoleic acid (C18:2) content being higher in the lipids, which are most appropriate for the biodiesel production. A novel strategy for most effective, simple method for cell disruption in Scenedesmus sp., was grinding/bead-beating, which is the most suitable method for complete extraction of biofuel grade lipids.

Serological Cross-Reaction Between O-Antigens of Shigella dysenteriae Type 4 and an Environmental Escherichia albertii Isolate

An environmental freshwater bacterial isolate, DM104, appearing as Shigella-like colonies on selective agar plates was found to show strong and specific serological cross-reactivity with Shigella dysenteriae type 4. Biochemical identification according to the analytical profile index, molecular serotyping by restriction of the amplified O-antigen gene cluster (rfb-RFLP), together with phylogenetic analysis of the 16S rRNA gene and multi-locus sequence analysis, identified the isolate as Escherichia albertii. rfb-RFLP of DM104, revealed a profile different from that of S. dysenteriae type 4. However, western blot analysis of extracted lipopolysaccharides demonstrated strong cross-reactivity with S. dysenteriae type 4 using specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. dysenteriae type 4. The observed O-antigen cross-reaction between an E. albertii isolate and S. dysenteriae extends our knowledge of the extent of O-antigen cross-reaction within the Escherichia/Shigella group of organisms, and offers the possibility of using DM104 and similar cross-reacting strains as shigellosis vaccine candidates.

Cytotoxicity of TiO2 nanoparticles and their detoxification in a freshwater system

In the current study, two aspects concerning (i) the cytotoxicity potential of TiO₂ nanoparticles (NPs) toward freshwater algal isolate Scenedesmus obliquus and (ii) the potential detoxification of NPs by the microalgae were assessed under light (UV-illumination) and dark conditions at low exposure levels (≤1 μg/mL), using sterile freshwater as the test medium. The statistically significant reduction in cell viability, increase in reactive oxygen species production and membrane permeability (light vs. dark) suggested photo-induced toxicity of TiO₂ NPs. The electron micrographs demonstrated adsorption of the NPs onto the cell surface and substantiated their internalization/uptake. The fluorescence micrographs and the confocal laser scanning (CLSM) images suggested the absence of a definite/intact nucleus in the light treated cells pointing toward the probable genotoxic effects of NPs. In a separate three cycle experiment, a continuous decrease in the cytotoxicity was observed, whereas, at the end of each cycle only fresh algae were added to the supernatant containing NPs from the previous cycle. The decreasing concentrations of the NPs in the subsequent cycles owing to agglomeration-sedimentation processes exacerbated by the algal interactions played a crucial role in the detoxification. In addition, the exo-polymeric substances produced by the cells could have rendered the available NPs less reactive, thereby, enhancing the detoxification effects.

Geometry of Zn(II) complexes with dissolved organic matter: X-ray studies at variable pH

The structure of Zn(II) complexes with dissolved organic matter (DOM) is an important consideration in developing molecular-level models of Zn(II) speciation, but recent reports favoring the tetrahedral geometry differ from earlier findings that geometry was largely octahedral. In general, the presence of thiolate ligands favors the tetrahedral geometry, while O and N ligands favor the octahedral geometry. This work presents extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) spectroscopic results, indicating an octahedral geometry over the pH range of 5–9 for a freshwater DOM isolate. Changes in XANES derivatives as a function of pH can be explained in terms of ligand protonation and/or changing ligand groups. Tetrahedral Zn(II)–DOM geometry may be restricted to binding environments containing thiol groups.