Complete genome, catabolic sub-proteomes and key-metabolites of Desulfobacula toluolica Tol2, a marine, aromatic compound-degrading, sulfate-reducing bacterium.

Among the dominant deltaproteobacterial sulfate-reducing bacteria (SRB), members of the genus Desulfobacula are not only present in (hydrocarbon-rich) marine sediments, but occur also frequently in the anoxic water bodies encountered in marine upwelling areas. Here, we present the 5.2 Mbp genome of Desulfobacula toluolica Tol2, which is the first of an aromatic compound-degrading, marine SRB. The genome has apparently been shaped by viral attacks (e.g. CRISPRs) and its high plasticity is reflected by 163 detected genes related to transposases and integrases, a total of 494 paralogous genes and 24 group II introns. Prediction of the catabolic network of strain Tol2 was refined by differential proteome and metabolite analysis of substrate-adapted cells. Toluene and p-cresol are degraded by separate suites of specific enzymes for initial arylsuccinate formation via addition to fumarate (p-cresol-specific enzyme HbsA represents a new phylogenetic branch) as well as for subsequent modified ß-oxidation of arylsuccinates to the central intermediate benzoyl-CoA. Proteogenomic evidence suggests specific electron transfer (EtfAB) and membrane proteins to channel electrons from dehydrogenation of both arylsuccinates directly to the membrane redox pool. In contrast to the known anaerobic degradation pathways in other bacteria, strain Tol2 deaminates phenylalanine non-oxidatively to cinnamate by phenylalanine ammonia-lyase and subsequently forms phenylacetate (both metabolites identified in (13) C-labelling experiments). Benzoate degradation involves CoA activation, reductive dearomatization by a class II benzoyl-CoA reductase and hydrolytic ring cleavage as found in the obligate anaerobe Geobacter metallireducens GS-15. The catabolic sub-proteomes displayed high substrate specificity, reflecting the genomically predicted complex and fine-tuned regulatory network of strain Tol2. Despite the genetic equipment for a TCA cycle, proteomic evidence supports complete oxidation of acetyl-CoA to CO2 via the Wood-Ljungdahl pathway. Strain Tol2 possesses transmembrane redox complexes similar to that of other Desulfobacteraceae members. The multiple heterodisulfide reductase-like proteins (more than described for Desulfobacterium autotrophicum HRM2) may constitute a multifaceted cytoplasmic electron transfer network.

Detection of antibiotic-producing Actinobacteria in the sediment and water of Ma'in thermal springs (Jordan).

INTRODUCTION: Detection of new Actinobacteria is significant to discover new antibiotics because development of new antibiotics is connected to the characterization of novel bacterial taxa. This study has focused on the identification and isolation of antibiotic-producing Actinobacteria from the sediment and the water of Ma'in thermal springs (48-59°C) situated in the center area of Jordan. METHODS: Samples of sediment and water were transferred to glucose yeast malt agar medium and Actinobacteria were cultivated, isolated and identified according to scanning electron microscopy and 16S rRNA gene analysis. Antibacterial activities of the isolates were then tested against different test bacteria by agar well diffusion method. RESULTS: Three different species of Actinobacteria were isolated (M1-1, M2-2, M3-2) from sediment samples. Based on 16S rRNA gene analysis, isolate M1-1 was found to have only 90% identity percentage with Nocardiopsis sp., however, isolates M2-2 and M3-2 were found to be closely related Streptomyces sp. (97%) and Nocardioides luteus (99%), respectively. The antibacterial activity showed that strain M1-1 is active against P. aeruginosa ATCC 2785 (inhibition zone, 9 mm). Strain M2-2 was found to be active against S. aureus ATCC 29213 (12 mm), B. cereus ATCC 11778 (11 mm), and E. coli ATCC 25922 (9 mm). In respect to strain M3-2, it was found to be active against S. aureus ATCC 29213 (14 mm) and B. cereus ATCC 11778 (9 mm). There were no actinobacterial isolates obtained from water samples despite their significant diversity revealed by our previous metagenomic analysis, which showed the presence of 13 different species dominated by Arthrobacter (an Actinobacterium belonging to family Actinomycetales). CONCLUSION: There were 17 different Actinobacteria that could be detected in Ma'in thermal springs (13 unculturable species and 3 culturable species). The culturable Actinobacteria were found to have some antimicrobial activity. Further chemical analysis of the bioactive compounds is recommended.

Microbial community analysis of the hypersaline water of the Dead Sea using high-throughput amplicon sequencing.

Amplicon sequencing using next-generation technology (bTEFAP® ) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well-known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high-throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity.

Exploring the microbial diversity in Jordanian hot springs by comparative metagenomic analysis.

A culture-independent approach was utilized in this study to reveal the microbial diversity in Jordanian hot springs represented by Ma'in and Afra hot springs. Water samples from Ma'in and Afra hot springs were collected in June 2015. The in situ temperature of water samples range was 38-59°C and the pH range was 7.4-8.4. The metagenome was extracted and analyzed using the next generation technology (bTEFAP® ). A total of 314,310 sequences were parsed and 288,452 were then clustered. The sequences were predominated by bacteria (>84%) and the relative abundance of archaea in each sample was <1%. Eukaryotic microorganisms were detected but with varying abundances (0.6%-15%). Because most of the detected sequences were found to belong to the domain of bacteria (196,936 sequences out 288,452), the bacterial sequences were utilized for further microbial analyses. With respect to alpha and beta diversity, samples were rarefied to 30,000 sequences and bootstrapped at 10,000 sequences. The Shannon-Wiener Index curve plot reaches a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. By examining the relative abundance of phyla detected in each sample, it appears that the biota of both Jordanian hot springs sampled are compositionally similar, with over 50% of the microbial community of each sample being comprised of the phylum Proteobacteria. The second most abundant phylum was the phylum Bacteroidetes which represents more than 13% in each sample. The phylum Firmicutes was also detected with a significant abundance. However, lower abundance of Deinococcus, Verrucomicrobia, Planctomycetes, and Chloroflexi was detected. A principal coordinate analysis plot was generated based upon the weighted UniFrac distance matrix. By utilizing Monte Carlo simulations, we were able to determine that there were no significant differences in the microbial diversity between each sample.

Microbiological analysis, antimicrobial activity, and heavy-metals content of Jordanian Ma'in hot-springs water.

Ma'in hot springs are known as sites of balneotherapy. However, little is known about their microbiology and chemistry. In this study, we aim at evaluating the antimicrobial activity of Ma'in hot-springs water (MHSW), studying its microbiology, and determining its physicochemical properties including the heavy metals content. Therefore, water samples were collected from Ma'in hot springs and tested for antimicrobial activity using agar diffusion method. Water was then cultivated on nutrient agar to isolate and identify the dominant bacteria by chemical and molecular methods. The identified strains were tested by cross streak method to evaluate their antimicrobial activity against different clinical and standard strains. Finally, water samples were chemically analyzed and the heavy-metals content was assessed. Results revealed that MHSW was not active against any of the clinical isolates. Nevertheless, MHSW was found to be active against five standard bacterial strains, namely, Staphylococcus epidermidis ATCC 12228 (inhibition zone: 20mm), Staphylococcus aureus ATCC 29213 (inhibition zone: 19mm), Micrococcus luteus ATCC 9341 (inhibition zone: 15.3mm), and Bacillus cereus ATCC 11778 (inhibition zone: 12.3mm). After cultivation of MHSW, five bacterial isolates were obtained and identified based on 16S rRNA gene analysis as new strains of Anoxybacillus flavithermus (identity percentage ranges between 96-99%). Physicochemical analysis revealed that the in situ temperature was 59°C, pH was 7.8, salinity was 1.6ppt, and dissolved oxygen was 3.8mgl-1. In respect to heavy-metals content in MHSW, the following metals were present in the order: Cr (0.571ppm)>Mn(0.169ppm)>Fe (0.124ppm)>Zn (0.095)>Cu(0.070ppm)>Ni(0.058ppm)>Cd (0.023ppm)>Pb (0ppm). Cd, Cr, Ni and Mn were found to be higher than permissible levels set by international organizations and countries. This study highlights new chemical and microbiological data about Ma'in hot springs.

In vitro cytogenetic testing of an organoselenium compound and its sulfur analogue in cultured rat bone marrow cells.

BACKGROUND: Selenium (Se) is a non-metal element, occurring in varying degrees in the environment and it has been found to be a component of several enzymes. Different selenium compounds have been associated with carcinogenicity, toxicity, modification of metal toxicity and prevention of cancer. Organoselenium compounds had substantially greater bioavailability and less toxicity than that of inorganic selenium. From a chemical point of view, Se resembles sulfur (S) in many of its properties, thus, Se and S may be considered to be isosteric. The ability of a synthetic organoselenium compound; cyclopenta-dienyldicarbonyl ironselenoterephthalic acid (CSe) and its sulfur analogue (CS) in the range of 10-8 to 10-5 M, to induce sister-chormatid exchanges (SCE) and alter cell division expressed as mitotic index (MI) as well as cell survival has been investigated. METHODS: Rat bone marrow cells were cultured in the presence of CSe and CS in the range of 10-8 to 10-5 M with a total exposure time of 4, 16 or 28 h at 37 degrees C. Fluorescence-plus-Giemsa (FPG) technique was used to visualize chromosomes for SCE analysis and MI determination. Trypan blue exclusion technique was used to determine cell viability. RESULTS: At the three exposure times, cell survival progressively decreased with increasing concentration, but the effect of either chemical was not significant (ANOVA; P < 0.05) as compared to the negative control. Significant reductions in MI were calculated at the highest concentration (10-5 M) when either chemical was applied for 16 or 28 h. Furthermore, the mean SCE increased with longer exposure times and, in general, CSe had slightly greater effect on cell survival and caused higher frequencies of SCE than CS. The exception was the 10-8 M treatment. However, both CSe and CS failed to induce 2-fold SCE as that of the negative control and therefore they are not considered as mutagens. CONCLUSION: Both CSe and CS in the range of 10-8 to 10-5 M could not double the SCE rate of the negative control and therefore not considered as mutagens at these experimental conditions.

Phenol content, antioxidant capacity and antibacterial activity of methanolic extracts derived from four Jordanian medicinal plants.

This study was performed to assess the antioxidant and antibacterial properties of methanolic extracts derived from aerial parts of four Jordanian medicinal plants (Artemisia sieberi, Peganum harmala, Rosmarinus officinalis (Green-Flowered) and Sarcopterium spinosium). The possible relationship between these biological properties and the total phenolic concentrations of these extracts were also be determined. The antioxidant capacity and total phenolic concentrations were assessed by the ABTS method and Folin-Ciocalteu method, respectively. The amount of the extract required to scavenge 50% of ABTS (IC50) was also measured. Broth dilution and disc diffusion assays were performed to measure the antibacterial activity of these extracts against available bacterial strains. Variations were observed among the examined plants in antioxidant and antibacterial activities as well as in their phenol contents. According to ABTS assay and IC50 value, the highest free radical scavenging potential was found in Sarcopterium spinosium, followed by Rosmarinus officinalis, Peganum harmala and Artemisia sieberi, respectively. Similarly, the results of antibacterial assays showed that Sarcopterium spinosium exhibited the highest antibacterial activity against all tested bacterial strains as compared to Rosmarinus officinalis, Peganum harmala and Artemisia sieberi. Moreover, Sarcopterium spinosium contained the highest amount of phenolic compounds followed by, Rosmarinus officinalis, Artemisia sieberi and Peganum harmala, respectively. In conclusion, these plants are not only interesting sources for antimicrobial agents but also have a considerable amount of antioxidants. In addition, these findings revealed that the antioxidant capacity and antibacterial activity of these plant extracts do not necessary be attributed to their total phenolic concentrations.