Publications

Publications

Publications After 2021

84. "Interplay between Sulfur Assimilation and Biodesulfurization Activity in Rhodococcus qingshengii IGTS8: Insights into a Regulatory Role of the Reverse Transsulfuration Pathway" O. Martzoukou, P.D. Glekas, M. Avgeris, D. Mamma, A. Scorilas, D. Kekos, S. Amillis, D.G. Hatzinikolaou
doi: (2022). Request copy
Abstract: Biodesulfurization is a process that selectively removes sulfur from dibenzothiophene and its derivatives. Several natural biocatalysts harboring the highly conserved desulfurization operon dszABC, which is significantly repressed by methionine, cysteine, and inorganic sulfate, have been isolated. However, the available information on the metabolic regulation of gene expression is still limited. In this study, scarless knockouts of the reverse transsulfuration pathway enzyme genes cbs and metB were constructed in the desulfurizing strain Rhodococcus sp. strain IGTS8. We provide sequence analyses and report the enzymes' involvement in the sulfate- and methionine-dependent repression of biodesulfurization activity. Sulfate addition in the bacterial culture did not repress the desulfurization activity of the Delta cbs strain, whereas deletion of metB promoted a significant biodesulfurization activity for sulfate-based growth and an even higher desulfurization activity for methionine-grown cells. In contrast, growth on cysteine completely repressed the desulfurization activity of all strains. Transcript level comparison uncovered a positive effect of cbs and metB gene deletions on dsz gene expression in the presence of sulfate and methionine, but not cysteine, offering insights into a critical role of cystathionine beta-synthase (C beta S) and MetB in desulfurization activity regulation. IMPORTANCE Precise genome editing of the model biocatalyst Rhodococcus qingshengii IGTS8 was performed for the first time, more than 3 decades after its initial discovery. We thus gained insight into the regulation of dsz gene expression and biocatalyst activity, depending on the presence of two reverse transsulfuration enzymes, C beta S and MetB. Moreover, we observed an enhancement of biodesulfurization capability in the presence of otherwise repressive sulfur sources, such as sulfate and L-methionine. The interconnection of cellular sulfur assimilation strategies was revealed and validated.

83. “Bacterial Isolates from Greek Sites and Their Efficacy in Degrading Petroleum”
G.C. Ntroumpogianni, E. Giannoutsou, A.D. Karagouni, A.L. Savvides
Sustainability doi: 10.3390/su14159562 (2022). Request copy
Abstract: Polycyclic aromatic hydrocarbons (PAHs) are a major organic pollutant, not only because they do not self-degenerate but also because they accumulate in the food chain and give rise to serious repercussions in terms of biodiversity sustainability. Petroleum-degrading bacteria have long been used as a promising solution in the effort to biodegrade crude oil. In this study, new isolates from specific Greek environments displaying various levels of crude oil contamination, as well as isolates belonging to the ATHUBA collection, were thoroughly investigated for their capacity to degrade crude oil. Furthermore, the presence of nahH and alkJ genes in the above bacterial isolates, as well as their ability to form agglomerates or release surfactants, was investigated. Two consortia were formed, and their ability to degrade crude oil was tested, achieving similar degrading capacities as those observed with the individual strains. A Pseudomonas plecoglossicida isolate demonstrated the highest percentage (76.7%) ability to degrade crude oil. The biodegradation rate of this isolate was further evaluated by measuring the alkanes/hopanes ratio over a period of ten days, exhibiting a higher degradation rate in short-chain (C11-C21) alkanes, whereas a decrease in the ratio was observed when the number of carbons in petroleum increased. This is the first detailed report on bacterial communities in oil-polluted areas of Greece that contain a variety of bacteria with the ability to degrade PAHs in contaminated sites and may provide a novel alternative to various bioremediation processes or be used as inocula in autochthonous bioaugmentation procedures for crude oil biodegradation.

82. "Biochemical and Thermodynamic Studies on a Novel Thermotolerant GH10 Xylanase from Bacillus safensis"
P.D. Glekas, S. Kalantzi, A. Dalios, D.G. Hatzinikolaou, D. Mamma
Biomolecules, doi:10.3390/biom12060790 (2022). Request copy
Abstract: Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 degrees C, stable over a broad range of pH (5.0-8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. K-M, v(max), and k(cat )for oat spelt xylan hydrolysis were found to be 1.96 g.L-1, 58.6 mu mole.min(-1) (mg protein)(-1), and 49 s(-1), respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 degrees C were Delta S* = -61.9 J.mol(-1).K-1, Delta H* = 37.0 kJ.mol(-1) and Delta G* = 57.6 kJ.mol(-1). BsXyn10 retained high levels of activity at temperatures up to 60 degrees C. The thermodynamic parameters (Delta H-D*, Delta G(D)*, AS(D)*) for the thermal deactivation of BsXyn10 at a temperature range of 40-80 degrees C were: 192.5 <= Delta H-D* <= 192.8 kJ.mol(-1), 262.1 <= Delta S-D* <= 265.8 J.mol(-1).K-1, and 99.9 <= Delta G(D)* <= 109.6 kJ.mol(-1). The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2-6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications.

81. "Antibacterial Activity of Copper Nanoparticles against Xanthomonas campestris pv. vesicatoria in Tomato Plants"
A. Varympopi, A. Dimopoulou, D. Papafotis, P. Avramidis, I. Sarris, T. Karamanidou, A.K. Kerou, A. Vlachou, E. Vellis, A. Giannopoulos
International Journal of Molecular Sciences, doi10.3390/ijms23084080 (2022). Request copy
Abstract: Copper-based bactericides have appeared as a new tool in crop protection and offer an effective solution to combat bacterial resistance. In this work, two copper nanoparticle products that were previously synthesized and evaluated against major bacterial and fungal pathogens were tested on their ability to control the bacterial spot disease of tomato. Growth of Xanthomonas campestris pv. vesicatoria, the causal agent of the disease, was significantly suppressed by both nanoparticles, which had superior function compared to conventional commercial formulations of copper. X-ray fluorescence spectrometry measurements in tomato leaves revealed that bioavailability of copper is superior in the case of nanoparticles compared to conventional formulations and is dependent on synthesis rather than size. This is the first report correlating bioavailability of copper to nanoparticle efficacy.

80. "Deciphering the biodesulfurization potential of two novel Rhodococcus isolates from a unique Greek environment"
P.D. Glekas, O. Martzoukou, M.E. Mastrodima, E. Zarkadoulas, D.S. Kanakoglou, D. Kekos, M. Pachnos, G. Mavridis, D. Mamma, D.G. Hatzinikolaou
AIMS Microbiology, doi:10.3934/microbiol.2022032 (2022). Request copy
Abstract: Sustainable biodesulfurization (BDS) processes require the use of microbial biocatalysts that display high activity against the recalcitrant heterocyclic sulfur compounds and can simultaneously withstand the harsh conditions of contact with petroleum products, inherent to any industrial biphasic BDS system. In this framework, the functional microbial BDS-related diversity in a naturally oil-exposed ecosystem, was examined through a 4,6-dimethyl-dibenzothiophene based enrichment process. Two new Rhodococcus sp. strains were isolated, which during a medium optimization process revealed a significantly enhanced BDS activity profile when compared to the model strain R. qingshengii IGTS8. In biocatalyst stability studies conducted in biphasic mode using partially hydrodesulfurized diesel under various process conditions, the new strains also presented an enhanced stability phenotype. In these studies, it was also demonstrated for all strains, that the BDS activity losses were decoupled from the overall cells' viability, in addition to the fact that the use of whole-broth biocatalyst positively affected BDS performance.

79. "Biodesulfurization of Dibenzothiophene and Its Alkylated Derivatives in a Two-Phase Bubble Column Bioreactor by Resting Cells of Rhodococcus erythropolis IGTS8"
G. Prasoulas, K. Dimos, P. Glekas, S. Kalantzi, S. Sarris, C. Templis, K. Vavitsas, D.G. Hatzinikolaou, N. Papayannakos, D. Kekos
Processes, doi:10.3390/pr9112064 (2021). Request copy
Abstract:Biodesulfurization (BDS) is considered a complementary technology to the traditional hydrodesulfurization treatment for the removal of recalcitrant sulfur compounds from petroleum products. BDS was investigated in a bubble column bioreactor using two-phase media. The effects of various process parameters, such as biocatalyst age and concentration, organic fraction percentage (OFP), and type of sulfur compound-namely, dibenzothiophene (DBT), 4-methyldibenzothiophene (4-MDBT), 4,6-dimethyldibenzothiophene (4,6-DMDBT), and 4,6-diethyldibenzothiophene (4,6-DEDBT)-were evaluated, using resting cells of Rhodococcus erythropolis IGTS8. Cells derived from the beginning of the exponential growth phase of the bacterium exhibited the highest biodesulfurization efficiency and rate. The biocatalyst performed better in an OFP of 50% v/v. The extent of DBT desulfurization was dependent on cell concentration, with the desulfurization rate reaching its maximum at intermediate cell concentrations. A new semi-empirical model for the biphasic BDS was developed, based on the overall Michaelis-Menten kinetics and taking into consideration the deactivation of the biocatalyst over time, as well as the underlying mass transfer phenomena. The model fitted experimental data on DBT consumption and 2-hydroxibyphenyl (2-HBP) accumulation in the organic phase for various initial DBT concentrations and different organosulfur compounds. For constant OFP and biocatalyst concentration, the most important parameter that affects BDS efficiency seems to be biocatalyst deactivation, while the phenomenon is controlled by the affinities of biodesulfurizing enzymes for the different organosulfur compounds. Thus, desulfurization efficiency decreased with increasing initial DBT concentration, and in inverse proportion to increases in the carbon number of alkyl substituent groups.

78. "Streptomyces, Greek Habitats and Novel Pharmaceuticals: A Promising Challenge"
P. Laskaris, A.D. Karagouni
Microbiology Research, doi:10.3390/microbiolres12040061 (2021). Request copy
Abstract: Bacteria of the genus Streptomyces produce a very large number of secondary metabolites, many of which are of vital importance to modern medicine. There is great interest in the discovery of novel pharmaceutical compounds derived from strepomycetes, since novel antibiotics, anticancer and compounds for treating other conditions are urgently needed. Greece, as proven by recent research, possesses microbial reservoirs with a high diversity of Streptomyces populations, which provide a rich pool of strains with potential pharmaceutical value. This review examines the compounds of pharmaceutical interest that have been derived from Greek Streptomyces isolates. The compounds reported in the literature include antibiotics, antitumor compounds, biofilm inhibitors, antiparasitics, bacterial toxin production inhibitors and antioxidants. The streptomycete biodiversity of Greek environments remains relatively unexamined and is therefore a very promising resource for potential novel pharmaceuticals.

77. "Doing synthetic biology with photosynthetic microorganisms"
K. Vavitsas, A. Kugler, S. Satta, D.G. Hatzinikolaou, P. Lindblad, D.P. Fewer, P. Lindberg, M. Toivari, K. Stensjo
Physiologia Plantarum, doi:10.1111/ppl.13455 (2021). Request copy
Abstract: The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.