Comparison of Growth Performance, Pigment Synthesis, and Esterase Activity of Synechococcus sp. HS01 and Limnothrix sp. KO01 in Response to Cadmium Toxicity.

Various studies have been conducted to understand the impact of environmental pollutants on cyanobacteria due to their abundant presence in aquatic and terrestrial environments, specific morphological and physiological characteristics, and high ecological flexibility in response to environmental changes. Here, the effect of different concentrations of cadmium on two native strains of cyanobacteria, namely Synechococcus sp. HS01 and Limnothrix sp. KO01 was studied and compared with each other. In this regard, the cyanobacterial growth, pigment contents, and esterase enzyme activity were evaluated after exposure of the cells to different concentrations of cadmium (II). The toxic effects of Cd(II) on the growth rate of Limnothrix sp. KO01, even at low concentrations, tended to be higher than those for Synechococcus sp. HS01. The content of pigments decreased by an increase in Cd(II) concentration. In compliance with the cell growth, the changes occurred in pigment contents of Limnothrix sp. KO01 was more sensitive than Synechococcus sp. HS01 in the presence of different concentrations of cadmium. Flow cytometry analysis of Cd(II) effects on esterase activity of both strains after 6, 24, 48, and 72 h of exposure to Cd(II) concentrations of 9, 27, 63, and 90 µM showed that tolerance to Cd(II) toxicity in Limnothrix sp. KO01 is less than Synechococcus sp. HS01. The results obtained in this study suggest high potentials of Synechococcus sp. HS01 for heavy metal bioaccumulation due to its considerable tolerance to cadmium.

High phenol degradation capacity of a newly characterized Acinetobacter sp. SA01: Bacterial cell viability and membrane impairment in respect to the phenol toxicity.

An efficient phenol-degrading bacterial strain, belonging to Acinetobacter genus, was isolated and selected to study the impact of different environmentally relevant phenol concentrations on the degradation process. The bacterial isolate, labeled as Acinetobacter sp. SA01 was able to degrade the maximum phenol concentration of 1 g/l during 60 h at optimum condition of pH 7, 30 °C and 180 rpm. Aeration and initial cell density, the two important factors, were carefully examined in the optimal growth conditions. The results showed that these two variables related proportionally with phenol degradation rate. Further investigations showed no effect of inoculum size on the enhancement of degradation of phenol at over 1 g/l. Flow cytometry (FCM) study was performed to find out the relationship between phenol-induced damages and phenol degradation process. Single staining using propidium iodide (PI) showed increased cell membrane permeability with an increase of phenol concentration, while single staining with carboxyfluorescein diacetate (cFDA) demonstrated a considerable reduction in esterase activity of the cells treated with phenol at more than 1 g/l. A detailed investigation of cellular viability using concurrent double staining of cFDA/PI revealed that the cell death increases in cells exposed to phenol at more than 1 g/l. The rate of cell death was low but noticeable in the presence of phenol concentration of 2 g/l, over time. Phenol at concentrations of 3 and 4 g/l caused strong toxicity in living cells of Acinetobacter sp. SA01. The plate count method and microscopy analysis of the cells treated with phenol at 1.5 and 2 g/l confirmed an apparent reduction in cell number over time. It was assumed that the phenol concentrations higher than 1 g/l have destructive effects on membrane integrity of Acinetobacter sp. SA01. Our results also revealed that the toxicity did not reduce by increasing initial cell density. Scanning electron microscopy (SEM) examination of bacterial cells revealed the surface morphological changes following exposure to phenol. The bacterial cells, with wizened appearance and wrinkled surface, were observed by exposing to phenol (1 g/l) at lag phase. A morphological change occurred in the mid-logarithmic phase as the bacterial cells demonstrated coccobacilli form as well as elongated filamentous shape. The wrinkled cell surface were totally disappeared in mid-stationary phase, suggesting that the complete degradation of phenol relieve the stress and direct bacterial cells toward possessing smoother cell membrane.

Effect of MA01 rhamnolipid on cell viability and expression of quorum-sensing (QS) genes involved in biofilm formation by methicillin-resistant Staphylococcus aureus.

A group of biosurfactants, called rhamnolipids, have been shown to have antibacterial and antibiofilm activity against multidrug-resistant bacteria. Here, we examined the effect of rhamnolipid biosurfactants extracted from Pseudomonas aeruginosa MA01 on cell growth/viability, biofilm formation, and membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) ATCC6538 bacterial cells. The results obtained from flow cytometry analysis showed that by increasing the concentration of rhamnolipid from 30 to 120 mg/mL, the cell viability decreased by about 70%, and the cell membrane permeability increased by approximately 20%. In fact, increasing rhamnolipid concentration was directly related to cell membrane permeability and inversely related to cell survival. Microtiter plate biofilm assay and laser scanning confocal microscopy analysis revealed that rhamnolipid, at a concentration of 60 mg/mL, exerts a reducing effect on the biofilm formation of Staphylococcus aureus. Real-time PCR analysis for monitoring the relative changes in the expression of agrA, agrC, icaA, and icaD genes involved in biofilm formation and related to the quorum-sensing pathway after treatment with rhamnolipid indicated a reduced expression level of these genes, as well as sortase A gene. The results of the present study deepen our knowledge regarding the use of microbial natural products as promising candidates for therapeutic applications.

Screening of anti-Acinetobacter baumannii phytochemicals, based on the potential inhibitory effect on OmpA and OmpW functions.

Therapeutic options including last-line or combined antibiotic therapies for multi-drug-resistant strains of Acinetobacter baumannii are ineffective. The outer membrane protein A (OmpA) and outer membrane protein W (OmpW) are two porins known for their different cellular functions. Identification of natural compounds with the potentials to block these putative porins can attenuate the growth of the bacteria and control the relating diseases. The current work aimed to screen a library of 384 phytochemicals according to their potentials to be used as a drug, and potentials to inhibit the function of OmpA and OmpW in A. baumannii. The phytocompounds were initially screened based on their physico-chemical, absorption, distribution, metabolism, excretion and toxicity (ADMET) drug-like properties. Afterwards, the selected ligands were subjected to standard docking calculations against the predicted three-dimensional structure of OmpA and OmpW in A. baumannii. We identified three phytochemicals (isosakuranetin, aloe-emodin and pinocembrin) possessing appreciable binding affinity towards the selected binding pocket of OmpA and OmpW. Molecular dynamics simulation analysis confirmed the stability of the complexes. Among them, isosakuranetin was suggested as the best phytocompound for further in vitro and in vivo study.

New Provisional Function of OmpA from Acinetobacter sp. Strain SA01 Based on Environmental Challenges.

An outer membrane protein A (OmpA) from Acinetobacter sp. strain SA01 was identified and characterized in-depth based on the structural and functional characteristics already known of its homologues. In silico structural studies showed that this protein can be a slow porin, binds to peptidoglycan, and exhibits emulsifying properties. Characterization of the recombinant SA01-OmpA, based on its emulsifying properties, represented its promising potentials in biotechnology. Also, the presence of SA01-OmpA in outer membrane vesicles (OMV) and biofilm showed that this protein, like its homologues in Acinetobacter baumannii, can be secreted into the extracellular environment through OMVs and play a role in the formation of biofilm. After ensuring the correct selection of the protein of interest, the role of oxidative stress induced by cell nutritional parameters (utilization of specific carbon sources) on the expression level of OmpA was carefully studied. For this purpose, the oxidative stress level of SA01 cell cultures in the presence of three nonrelevant carbon sources (sodium acetate, ethanol, and phenol) was examined under each condition. High expression of SA01-OmpA in ethanol- and phenol-fed cells with higher levels of oxidative stress than acetate suggested that oxidative stress could be a substantial factor in the regulation of SA01-OmpA expression. The significant association of SA01-OmpA expression with the levels of oxidative stress induced by cadmium and H2O2, with oxidative stress-inducing properties and lack of nutritional value, confirmed that the cells tend to harness their capacities with a possible increase in OmpA production. Collectively, this study suggests a homeostasis role for OmpA in Acinetobacter sp. SA01 under oxidative stress besides assuming many other roles hitherto attributed to this protein.IMPORTANCE Acinetobacter OmpA is known as a multifaceted protein with multiple functions, including emulsifying properties. Bioemulsifiers are surface-active compounds that can disperse hydrophobic compounds in water and help increase the bioavailability of hydrophobic hydrocarbons to be used by degrading microorganisms. In this study, an OmpA from Acinetobacter sp. SA01 was identified and introduced as an emulsifier with a higher emulsifying capacity than Pseudomonas aeruginosa rhamnolipid. We also showed that the expression of this protein is not dependent on the nutritional requirements but is more influenced by the oxidative stress caused by stressors. This finding, along with the structural role of this protein as a slow porin or its role in OMV biogenesis and biofilm formation, suggests that this protein can play an important role in maintaining cellular homeostasis under oxidative stress conditions. Altogether, the present study provides a new perspective on the functional performance of Acinetobacter OmpA, which can be used both to optimize its production as an emulsifier and a target in the treatment of multidrug-resistant strains.

Identification and characterization of a bacterial core methionine synthase.

Methionine synthases are essential enzymes for amino acid and methyl group metabolism in all domains of life. Here, we describe a putatively anciently derived type of methionine synthase yet unknown in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In Dehalococcoides mccartyi strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class Dehalococcoidia lack metF encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-metE gene. We heterologously expressed core-MetECBDB in E. coli and purified the 38 kDa protein. Core-MetECBDB exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (KM ≈ 240 µM) and L-homocysteine (KM ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a kcat ≈ 60 s-1. Core-MetECBDB did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetECBDB and the canonical MetE enzyme from E. coli have different enzymatic specificities also in vivo. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain.

Limnothrix sp. KO05: A newly characterized cyanobacterial biosorbent for cadmium removal: the enzymatic and non-enzymatic antioxidant reactions to cadmium toxicity.

In this study, we isolated five indigenous cyanobacterial strains from different aqueous environments, with heavy metals contamination, in East Azerbaijan Province (northwest portion of Iran). A strain was identified by morphological and 16S rRNA sequence analysis as Limnothrix sp. KO05 and selected for further studies as having the greatest potential for cadmium uptake. Scanning electron microscopy (SEM) demonstrated cyanobacterium Limnothrix sp. KO05 forms filamentous structures and is straight or curved to some extent. The utmost biosorption capacity was found to be 82.18±1.22mgg-1 at a Cd (II) concentration level of 150mgL-1. Langmuir adsorption isotherm indicated a better fit to the experimental data. Response surface methodology (RSM) on the basis of four independent variables and the predicted maximum biosorption efficiency was 98.7% under the optimum condition. FT-IR spectroscopy profile of the Cd treated sample as demonstrated in confirmation of the benefits of various functional groups of proteins and polysaccharides of cyanobacterial biomass, involved in surface binding of Cd. The determination of catalase (CAT) activity in strain KO05 exposed to Cd (II) concentrations of 2, 5 and 10mgL-1 showed an increase in enzyme activity after 24h exposure compared to unexposed cells. Correspondingly, CAT activity showed a significant decrease after 48h of treatment with Cd (II) concentrations of 5 and 10mgL-1. CAT activity was decreased significantly at all concentrations within 72h after exposure to Cd. On the contrary, while ascorbate peroxidase (APX) gave the expected lower activity compared to the CAT within 24h after Cd treatment, its activity lasted up to 72h. Limnothrix sp. KO05 cells treated with 5 and 10mgL-1 Cd (II) over 72h exposure showed a reduction in chlorophyll a contents compared to the controls. However, following exposure to Cd, chlorophyll a and carotenoid contents is reduced and after overcoming stress and deployment of an adaptation mechanism, the amounts of these pigments is gradually increased in the cells. The reduction was slower for chlorophyll a pigment compared to carotenoids that may be an indication of the physiological importance of chlorophyll pigment for the phtosynthetic cells. Results related to lipid peroxidation in Limnothrix sp. KO05 represent a significant increase of MDA in the first 24h after exposure to the different concentrations of Cd (2, 5 and 10mgL-1). However, the MDA levels were decreased over time and no significant difference attained after 72h exposure to Cd concentrations of 2 and 10mgL-1 compared to control.