scholarly journals High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide

2021 ◽  
Vol 22 (8) ◽  
pp. 4149
Author(s):  
Hye-sun Cho ◽  
Munjeong Choi ◽  
Yunjung Lee ◽  
Hyoim Jeon ◽  
Byeongyong Ahn ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Broad Spectrum ◽  
Bacterial Species ◽  
Rna Isolation ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
High Quality ◽  
Nucleic Acid Isolation

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

scholarly journals Characterization and Antimicrobial Efficacy of Bovine Dermcidin, a Novel Antimicrobial Peptide Gene

2019 ◽  
Author(s):  
Nevien Sabry ◽  
Tarek Moussa
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptide ◽  
Disulfide Bonds ◽  
Ph Value ◽  
Rna Isolation ◽  
Antimicrobial Effect ◽  
Blood Analysis ◽  
Bacterial Strains ◽  
Nacl Concentration ◽  
Peptide Gene

Description of a novel bovine antimicrobial peptide and its antimicrobial spectrum. RNA isolation and RT-PCR were done from various tissues. DCD peptide was synthesized, and antimicrobial activity was analyzed. Bovine dermcidin gene contains five exons intermittent by 4 introns. Bovine DCD-mRNA was 398 bp with ORF 336 bp. Bovine DCD was expressed in skin and blood. Analysis of the amino acid compositions revealed that cysteine was repeated 6 times indicating the presence of 3 disulfide bonds that play role in the peptide stability. Staphylococcus epidermidis, Streptococcus bovis, and Enterococcus faecalis were affected by Bovine DCD peptide. Highest antimicrobial effect was at 50 and 100 µg/ml. The effect on Escherichia coli and Candida albicans was slightly low. In all bacteria, Bovine DCD peptide activity did not affect by varying pH values, but in Staphylococcus aureus, the activity was affected greatly at pH 4.5 and 5.5. The optimum salt concentrations were 100 and 50 mM NaCl with all bacterial strains and E. coli, respectively. In case of C. albicans, the antimicrobial activity of Bovine DCD peptide decreased with increasing the pH value regardless the NaCl concentration. The pH 6.5 of the sweat buffer was the optimum for the Bovine DCD peptide activity.

Assessment of zinc solubilization potential of zinc-resistant Pseudomonas oleovorans strain ZSB13 isolated from contaminated soil

2023 ◽  
Vol 83 ◽  
Author(s):  
H. F. Rehman ◽  
A. Ashraf ◽  
S. Muzammil ◽  
M. H. Siddique ◽  
T. Ali
Keyword(s):  
Contaminated Soil ◽  
Bacterial Strain ◽  
Bacterial Species ◽  
Resistant Bacteria ◽  
Zn Deficiency ◽  
Rrna Gene ◽  
Growth Enhancement ◽  
Pseudomonas Oleovorans ◽  
Human Beings ◽  
Bacterial Strains

Abstract Zinc is an essential micronutrient that is required for optimum plant growth. It is present in soil in insoluble forms. Bacterial solubilization of soil unavailable form of Zn into available form, is an emerging approach to alleviate the Zn deficiency for plants and human beings. Zinc solubilizing bacteria (ZSB) could be a substitute for chemical Zn fertilizer. The present study aimed to isolate and characterize bacterial species from the contaminated soil and evaluate their Zn solubilizing potential. Zn resistant bacteria were isolated and evaluated for their MIC against Zn. Among the 13 isolated bacterial strains ZSB13 showed maximum MIC value upto 30mM/L. The bacterial strain with the highest resistance against Zn was selected for further analysis. Molecular characterization of ZSB13 was performed by 16S rRNA gene amplification which confirmed it as Pseudomonas oleovorans. Zn solubilization was determined through plate assay and broth medium. Four insoluble salts (zinc oxide (ZnO), zinc carbonate (ZnCO3), zinc sulphite (ZnS) and zinc phosphate (Zn3(PO4)2) were used for solubilization assay. Our results shows 11 mm clear halo zone on agar plates amended with ZnO. Likewise, ZSB13 showed significant release of Zn in broth amended with ZnCO3 (17 and 16.8 ppm) and ZnO (18.2 ppm). Furthermore, Zn resistance genes czcD was also enriched in ZSB13. In our study, bacterial strain comprising Zn solubilization potential has been isolated that could be further used for the growth enhancement of crops.

scholarly journals The Design and Construction of K11: A Novelα-Helical Antimicrobial Peptide

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Huang Jin-Jiang ◽  
Lu Jin-Chun ◽  
Lu Min ◽  
Huang Qing-Shan ◽  
Li Guo-Dong
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Broad Spectrum ◽  
Staphylococcus Epidermidis ◽  
Therapeutic Index ◽  
Klebsiella Pneumonia ◽  
Gram Negative Bacteria ◽  
Drug Resistant ◽  
Promising Candidate

Amphipathicα-helical antimicrobial peptides comprise a class of broad-spectrum agents that are used against pathogens. We designed a series of antimicrobial peptides, CP-P (KWKSFIKKLTSKFLHLAKKF) and its derivatives, and determined their minimum inhibitory concentrations (MICs) againstPseudomonas aeruginosa, their minimum hemolytic concentrations (MHCs) for human erythrocytes, and the Therapeutic Index (MHC/MIC ratio). We selected the derivative peptide K11, which had the highest therapeutic index (320) among the tested peptides, to determine the MICs against Gram-positive and Gram-negative bacteria and 22 clinical isolates includingAcinetobacter baumannii, methicillin-resistantStaphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis,andKlebsiella pneumonia. K11 exhibited low MICs (less than 10 μg/mL) and broad-spectrum antimicrobial activity, especially against clinically isolated drug-resistant pathogens. Therefore, these results indicate that K11 is a promising candidate antimicrobial peptide for further studies.

scholarly journals The Expanded Universe of Prokaryotic Argonaute Proteins

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Sergei Ryazansky ◽  
Andrey Kulbachinskiy ◽  
Alexei A. Aravin
Keyword(s):  
Rna Interference ◽  
Nucleic Acid ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Host Cells ◽  
Bacterial Strains ◽  
Domains Of Life ◽  
Genomic Studies ◽  
Basic Biology

ABSTRACT Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition—and sometimes cleavage—of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are involved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing. IMPORTANCE The eukaryotic Ago proteins and the RNA interference pathways they are involved in are widely used as a powerful tool in research and as potential therapeutics. In contrast, the properties and functions of prokaryotic Ago (pAgo) proteins have remained poorly understood. Understanding the diversity and functions of pAgos holds a huge potential for discovery of new cellular pathways and novel tools for genome manipulations. Only few pAgos have been characterized by structural or biochemical approaches, while previous genomic studies discovered about 300 proteins in archaeal and eubacterial genomes. Since that time the number of bacterial strains with sequenced genomes has greatly expanded, and many previously sequenced genomes have been revised. We undertook comprehensive analysis of pAgo proteins in sequenced genomes and almost tripled the number of known genes of this family. Our research thus forms a foundation for further experimental characterization of pAgo functions that will be important for understanding of the basic biology of these proteins and their adoption as a potential tool for genome engineering in the future.

scholarly journals Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

2017 ◽  
Vol 113 (5/6) ◽  
Author(s):  
Lehlohonolo B. Qhanya ◽  
Ntsane T. Mthakathi ◽  
Charlotte E. Boucher ◽  
Samson S. Mashele ◽  
Chrispian W. Theron ◽  
...  
Keyword(s):  
South Africa ◽  
Bacterial Species ◽  
Endocrine Disrupting Chemicals ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Mpumalanga Province ◽  
Synthetic Chemicals ◽  
Phylogenetic Identification ◽  
Rrna Gene Sequencing

Endocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa.

scholarly journals Characterization of Phascolarctobacterium succinatutens sp. nov., an Asaccharolytic, Succinate-Utilizing Bacterium Isolated from Human Feces

2011 ◽  
Vol 78 (2) ◽  
pp. 511-518 ◽  
Author(s):  
Yohei Watanabe ◽  
Fumiko Nagai ◽  
Masami Morotomi
Keyword(s):  
Bacterial Species ◽  
Short Chain Fatty Acids ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Gi Tract ◽  
Bacterial Strains ◽  
High Sequence Identity ◽  
Healthy Human ◽  
Content Type

ABSTRACTIsolation, cultivation, and characterization of the intestinal microorganisms are important for understanding the comprehensive physiology of the human gastrointestinal (GI) tract microbiota. Here, we isolated two novel bacterial strains, YIT 12067Tand YIT 12068, from the feces of healthy human adults. Phylogenetic analysis indicated that they belonged to the same species and were most closely related toPhascolarctobacterium faeciumACM 3679T, with 91.4% to 91.5% 16S rRNA gene sequence similarities, respectively. Substrate availability tests revealed that the isolates used only succinate; they did not ferment any other short-chain fatty acids or carbohydrates tested. When these strains were cocultured with the xylan-utilizing and succinate-producing bacteriumParaprevotella xylaniphilaYIT 11841T, in medium supplemented with xylan but not succinate, their cell numbers became 2 to 3 orders of magnitude higher than those of the monoculture; succinate became undetectable, and propionate was formed. Database analysis revealed that over 200 uncultured bacterial clones from the feces of humans and other mammals showed high sequence identity (>98.7%) to YIT 12067T. Real-time PCR analysis also revealed that YIT 12067T-like bacteria were present in 21% of human fecal samples, at an average level of 3.34 × 108cells/g feces. These results indicate that YIT 12067T-like bacteria are distributed broadly in the GI tract as subdominant members that may adapt to the intestinal environment by specializing to utilize the succinate generated by other bacterial species. The phylogenetic and physiological properties of YIT 12067Tand YIT 12068 suggest that these strains represent a novel species, which we have designatedPhascolarctobacterium succinatutenssp. nov.

scholarly journals Biodiversity of Actinomycetes from Heavy Metal Contaminated Technosols

2021 ◽  
Vol 9 (8) ◽  
pp. 1635
Author(s):  
Michaela Cimermanova ◽  
Peter Pristas ◽  
Maria Piknova
Keyword(s):  
Heavy Metal ◽  
Bacterial Colonization ◽  
Bacterial Species ◽  
Chemical Properties ◽  
Metal Resistance ◽  
Rrna Gene ◽  
Toxic Substances ◽  
Bacterial Strains ◽  
16S Rrna Gene Analysis ◽  
Genus Streptomyces

Technosols are artificial soils generated by diverse human activities and frequently contain toxic substances resulting from industrial processes. Due to lack of nutrients and extreme physico-chemical properties, they represent environments with limited bacterial colonization. Bacterial populations of technosols are dominated usually by Actinobacteria, including streptomycetes, known as a tremendous source of biotechnologically important molecules. In this study, the biodiversity of streptomycete-like isolates from several technosols, mainly mine soils and wastes (landfills and sludge) in Slovakia, was investigated. The combination of basic morphological and biochemical characterisations, including heavy metal resistance determination, and molecular approaches based on 16S rRNA gene analysis were used for the identification of the bacterial strains. From nine isolates of Actinobacteria collected from different habitats, one was found to represent a new species within the Crossiella genus. Eight other isolates were assigned to the genus Streptomyces, of which at least one could represent a new bacterial species. Some isolates showed high resistance to Pb, Zn, Cu or Ni. The most tolerated metal was Pb. The results obtained in this study indicate that technosols are a prospective source of new actinomycete species resistant to heavy metals what underlines their bioremediation potential.

scholarly journals An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260002
Author(s):  
María José Cárdenas Espinosa ◽  
Tabea Schmidgall ◽  
Georg Wagner ◽  
Uwe Kappelmeyer ◽  
Stephan Schreiber ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Bacterial Degradation ◽  
Rna Seq ◽  
High Quality ◽  
Total Rna ◽  
Aromatic Substrates ◽  
Next Generation Sequencing Ngs ◽  
Total Rna Extraction

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

scholarly journals Prevalence of Diversified Antibiotic Resistant Bacteria within Sanitation Related Facilities of Human Populated Workplaces in Abbottabad

2020 ◽  
Author(s):  
Jawad Ali ◽  
Malik Owais Ullah Awan ◽  
Gulcin Akca ◽  
Iftikhar Zeb ◽  
Bilal AZ Amin ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Resistance ◽  
Bacterial Species ◽  
Resistant Bacteria ◽  
Rrna Gene ◽  
Beta Lactam ◽  
Bacterial Strains ◽  
Antibiotic Resistant Bacteria ◽  
Sensitivity Testing ◽  
Antibiotic Resistant

AbstractAntibiotics discovery was a significant breakthrough in the field of therapeutic medicines, but the over (mis)use of such antibiotics (n parallel) caused the increasing number of resistant bacterial species at an ever-higher rate. This study was thus devised to assess the multi-drug resistant bacteria present in sanitation-related facilities in human workplaces. In this regard, samples were collected from different gender, location, and source-based facilities, and subsequent antibiotic sensitivity testing was performed on isolated bacterial strains. Four classes of the most commonly used antibiotics i.e., β-lactam, Aminoglycosides, Macrolides, and Sulphonamides, were evaluated against the isolated bacteria.The antibiotic resistance profile of different (70) bacterial strains showed that the antibiotic resistance-based clusters also followed the grouping based on their isolation sources, mainly the gender. Twenty-three bacterial strains were further selected for their 16s rRNA gene based molecular identification and for phylogenetic analysis to evaluate the taxonomic evolution of antibiotic resistant bacteria. Moreover, the bacterial resistance to Sulphonamides and beta lactam was observed to be the most and to Aminoglycosides and macrolides as the least. Plasmid curing was also performed for MDR bacterial strains, which significantly abolished the resistance potential of bacterial strains for different antibiotics. These curing results suggested that the antibiotic resistance determinants in these purified bacterial strains are present on respective plasmids. Altogether, the data suggested that the human workplaces are the hotspot for the prevalence of MDR bacteria and thus may serve the source of horizontal gene transfer and further transmission to other environments.

Reclassification of Herbaspirillum putei as a later heterotypic synonym of Herbaspirillum huttiense, with the description of H. huttiense subsp. huttiense subsp. nov. and H. huttiense subsp. putei subsp. nov., comb. nov., and description of Herbaspirillum aquaticum sp. nov.

2010 ◽  
Vol 60 (6) ◽  
pp. 1418-1426 ◽  
Author(s):  
Anatoly P. Dobritsa ◽  
M. C. S. Reddy ◽  
Mansour Samadpour
Keyword(s):  
Dna Hybridization ◽  
Type Strain ◽  
Novel Species ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Phenotypic Characteristics ◽  
Phylogenetic Relatedness ◽  
The 16S Rrna Gene

Resequencing of the 16S rRNA gene of the type strain of Herbaspirillum putei Ding and Yokota 2004 revealed 99.9 % sequence similarity to that of the type strain of Herbaspirillum huttiense (Leifson 1962) Ding and Yokota 2004. This high phylogenetic relatedness of H. putei and H. huttiense was confirmed by the results of DNA–DNA hybridization between H. huttiense DSM 10281T and H. putei ATCC BAA-806T (reassociation value 96 %). Therefore, it is proposed to reclassify the type strain of H. putei as a strain of H. huttiense. However, the genome of the type strain of H. putei is about 0.9 Mb larger than that of the H. huttiense type strain. This results in a decrease in the reassociation value in the reciprocal DNA–DNA hybridization to 72 %, a level slightly above the threshold for delineating bacterial species. These data and distinctive phenotypic characteristics indicate that the name Herbaspirillum putei is a later heterotypic synonym of Herbaspirillum huttiense and permit the description of two novel subspecies, Herbaspirillum huttiense subsp. huttiense subsp. nov. (type strain ATCC 14670T =JCM 21423T =DSM 10281T) and Herbaspirillum huttiense subsp. putei subsp. nov., comb. nov. (type strain 7-2T =JCM 21495T =ATCC BAA-806T). Three bacterial strains, IEH 4430T, IEH 4515 and IEH 8757, isolated from water were found to be the closest relatives of these strains. Strain IEH 8757 was classified as a strain of H. huttiense subsp. putei. Studies of genotypic and phenotypic features of strains IEH 4430T and IEH 4515 showed that the strains represent a novel species, which is most closely related to H. huttiense and for which the name Herbaspirillum aquaticum sp. nov. is proposed (type strain IEH 4430T =DSM 21191T =ATCC BAA-1628T).

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