scholarly journals Isolation and Characterization of “Dehalococcoides” sp. Strain MB, Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

2009 ◽  
Vol 75 (18) ◽  
pp. 5910-5918 ◽  
Author(s):  
Dan Cheng ◽  
Jianzhong He
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Anaerobic Respiration ◽  
Rrna Gene ◽  
Reductive Dehalogenase ◽  
Isolation And Characterization ◽  
Dehalococcoides Ethenogenes ◽  
Pure Cultures ◽  
Microbial Group ◽  
Rdase Genes

ABSTRACT In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H2 as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.

scholarly journals Identification of Anaerobic Selenate-Respiring Bacteria from Aquatic Sediments

2007 ◽  
Vol 73 (11) ◽  
pp. 3519-3527 ◽  
Author(s):  
Priya Narasingarao ◽  
Max M. Häggblom
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Anaerobic Respiration ◽  
Pseudomonas Stutzeri ◽  
Elemental Selenium ◽  
Rrna Gene ◽  
Terminal Electron Acceptor ◽  
Isolation And Characterization ◽  
Anaerobic Biotransformation ◽  
Gene Similarity

ABSTRACT The diversity population of microorganisms with the capability to use selenate as a terminal electron acceptor, reducing it to selenite and elemental selenium by the process known as dissimilatory selenate reduction, is largely unknown. The overall objective of this study was to gain an in-depth understanding of anaerobic biotransformation of selenium in the environment, particularly anaerobic respiration, and to characterize the microorganisms catalyzing this process. Here, we demonstrate the isolation and characterization of four novel anaerobic dissimilatory selenate-respiring bacteria enriched from a variety of sources, including sediments from three different water bodies in Chennai, India, and a tidal estuary in New Jersey. Strains S5 and S7 from India, strain KM from the Meadowlands, NJ, and strain pn1, categorized as a laboratory contaminant, were all phylogenetically distinct, belonging to various phyla in the bacterial domain. The 16S rRNA gene sequence shows that strain S5 constitutes a new genus belonging to Chrysiogenetes, while strain S7 belongs to the Deferribacteres, with greater than 98% 16S rRNA gene similarity to Geovibrio ferrireducens. Strain KM is related to Malonomonas rubra, Pelobacter acidigallici, and Desulfuromusa spp., with 96 to 97% 16S rRNA gene similarity. Strain pn1 is 99% similar to Pseudomonas stutzeri. Strains S5, S7, and KM are obligately anaerobic selenate-respiring microorganisms, while strain pn1 is facultatively anaerobic. Besides respiring selenate, all these strains also respire nitrate.

scholarly journals Monitoring Abundance and Expression of “Dehalococcoides” Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column

2008 ◽  
Vol 74 (18) ◽  
pp. 5695-5703 ◽  
Author(s):  
Sebastian Behrens ◽  
Mohammad F. Azizian ◽  
Paul J. McMurdie ◽  
Andrew Sabalowsky ◽  
Mark E. Dolan ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Reductive Dehalogenation ◽  
Gene Copy ◽  
Rrna Gene ◽  
Reductive Dehalogenase ◽  
Gene Copies ◽  
Donor And Acceptor ◽  
Rdase Genes

ABSTRACT We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “Dehalococcoides” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 106 (mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

scholarly journals Specific Detection, Isolation, and Characterization of Selected, Previously Uncultured Members of the Freshwater Bacterioplankton Community

2005 ◽  
Vol 71 (10) ◽  
pp. 5908-5919 ◽  
Author(s):  
Frederic Gich ◽  
Karin Schubert ◽  
Alke Bruns ◽  
Herbert Hoffelner ◽  
Jörg Overmann
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Light Harvesting Complexes ◽  
Probable Number ◽  
Bacterioplankton Community ◽  
Isolation And Characterization

ABSTRACT High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the α-Proteobacteria, β-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental α-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.

A PCR-based specific assay reveals a population of bacteria within the Chloroflexi associated with the reductive dehalogenation of polychlorinated biphenyls

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 2039-2046 ◽  
Author(s):  
Joy E. M. Watts ◽  
Sonja K. Fagervold ◽  
Harold D. May ◽  
Kevin R. Sowers
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Polychlorinated Biphenyls ◽  
Reductive Dehalogenation ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Specific Pcr ◽  
Pure Cultures ◽  
Primer Sets

Polychlorinated biphenyls (PCBs) accumulate and persist in sediments posing a risk to human health and the environment. Highly chlorinated PCBs are reductively dechlorinated in anaerobic sediments and two bacteria, designated o-17 and DF-1, from a novel phylogenetic group that reductively dechlorinate PCBs have recently been identified. However, there is a paucity of knowledge about the distribution, diversity and ecology of PCB-dechlorinating bacteria due to difficulty in obtaining pure cultures and the lack of detection by universal PCR 16S rRNA gene primer sets in sediments. A specific PCR primer was developed and optimized for detection of o-17/DF-1 and other closely related bacteria in the environment. Using this primer set it was determined that bacteria of this group were enriched in sediment microcosms from Baltimore Harbour concurrent with active dechlorination of 2,2′,3,4,4′,5′-hexachlorobiphenyl. Additional 16S rRNA gene sequences that had high levels of similarity to described PCB dechlorinators were detected in sediments from the Elizabeth River tributary of Chesapeake Bay, which had confirmed PCB-dechlorinating activities. Phylogenetic comparison of these detected 16S rRNA gene sequences revealed a relatively diverse group of organisms within the dehalogenating Chloroflexi that are distinct from the Dehalococcoides spp. Results from this study indicate that reductive PCB dechlorination activity may be catalysed by a previously undescribed group of micro-organisms that appear to be prevalent in PCB-impacted sites.

scholarly journals Isolation and Characterization of Endophytic Bacteria from Tembelekan (Lantana camara L.) as Antibacterial Compounds Producer

2015 ◽  
Vol 2 (2) ◽  
pp. 86-98
Author(s):  
Dina Dyah Saputri ◽  
Maria Bintang ◽  
Fachriyan H Pasaribu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Endophytic Bacteria ◽  
Pathogenic Bacteria ◽  
Lantana Camara ◽  
Rrna Gene ◽  
Antibacterial Compounds ◽  
Isolation And Characterization ◽  
Ms Analysis

Endophytic bacteria are microorganisms that live in the internal tissues of plants and have symbiotic mutualism with their host plants. Endophytic bacteria may produce secondary metabolites that can be developed for medical, agricultural, and industrial purposes. Lantana camara is a medicinal plant that has therapeutic potential to treat a variety of diseases such as fever, tuberculosis, rheumatism, asthma, and skin disease. The purpose of this study was to isolate and characterize endophytic bacteria from Lantana camara which has potential to produce antibacterial compounds. The method of this research include isolation of endophytic bacteria of Lantana camara. Antibacterial activity assay was done against four types of pathogenic bacteria i.e. Bacillus cereus, Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis. Characterization of endophytic bacteria was by 16S rRNA gene analysis and identification of antibacterial compounds by GC-MS analysis. Isolation of endophytic bacteria from Lantana camara resulted in BT22 as a potential isolate. Analysis of 16S rRNA gene showed that the BT22 isolate was similar to Bacillus amyloliquefaciens YB-1402 with 99% identity. The results of GC-MS analysis showed some antibacterial compounds such as: Cyclohexanone, 2-[2-(1,3-dithiolan-2-yl)propyl]-6-methyl-3-(1-methylethyl), Octadecane (CAS) n-Octadecane and Tetracosane (CAS) n-Tetracosane.

scholarly journals Molecular characterization of Nocardiopsis species from Didwana dry salt lake of Rajasthan, India

2021 ◽  
Vol 13 (1) ◽  
pp. 396-401
Author(s):  
Khushbu Parihar ◽  
Alkesh Tak ◽  
Praveen Gehlot ◽  
Rakesh Pathak ◽  
Sunil Kumar Singh
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bioactive Compounds ◽  
Salt Lake ◽  
Bioactive Compound ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Multiple Sequence ◽  
Isolation And Characterization

The genus Nocardiopsis is well known to produce secondary metabolites especially antibacterial bioactive compound. Isolation and characterization of bioactive compounds producing novel isolates from unusual habitats are crucial. The present study was aimed to explore Didwana dry salt lake of Rajasthan state in India for the isolation and characterization of actinomycetes. The isolated actinomycetes isolates were characterized based on culture characteristics, biochemical tests and 16S rRNA gene sequencing. The 16S rRNA gene sequence analysis revealed that all the five isolates inhabiting soil of the said dry salt lake of Didwana, Rajasthan belonged to four species of Nocardiopsis viz., N. synnemataformans, N. potens, N. prasina and N. dassonvillei subsp. albirubida. The molecular identification based on 16S rRNA gene sequences was found accurate and robust. The phylogram generated through multiple sequence alignment of all the test isolates of Nocardiopsis revealed that the isolates aroused from a single branch and validated monophyletic association. The present study is the first report of exploring Nocardiopsis isolates from the dry salt lake. These characterized Nocardiopsis isolates isolated from Didwana dry salt lake habitat are novel stains and can be of significance in the detection and utilization of novel bioactive compounds.

scholarly journals Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes

2006 ◽  
Vol 72 (9) ◽  
pp. 5877-5883 ◽  
Author(s):  
Victor F. Holmes ◽  
Jianzhong He ◽  
Patrick K. H. Lee ◽  
Lisa Alvarez-Cohen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sequence Matching ◽  
Relative Copy Number ◽  
Reductive Dehalogenase ◽  
Copy Numbers ◽  
Reductase Gene ◽  
Reductive Dehalogenase Genes

ABSTRACT While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.

scholarly journals Implementation of a Non-Invasive Bioprospecting Protocol for Isolation of Lactobacillus from Feces of Hens Under Foraging Conditions

2018 ◽  
Vol 14 (28) ◽  
pp. 93-111
Author(s):  
Simón Robledo-Cardona ◽  
Sabina Ramírez-Hincapié ◽  
Javier Correa-Álvarez
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Lactobacillus Species ◽  
Rrna Gene ◽  
Isolation And Identification ◽  
Maldi Tof Ms ◽  
Non Invasive ◽  
Maldi Tof ◽  
Isolation And Characterization ◽  
Tof Ms

In animal production, probiotics seek to replace the use of antibiotics, while diminishing mortality and morbidity rates to raise productivity. Probiotics constitute a natural alternative that, in contrast with antibiotics, neither produces pathogen resistance, nor leaves chemical residues in the final product. Several bacteria, including some belonging to the genus Lactobacillus have been described as probiotics with high potential. A non-invasive bioprospecting protocol aimed for the isolation and characterization of lactobacilli from chicken feces was established. Fecal samples were collected from the ground. These were diluted and cultured in LAB selective medium. Colonies were identified by three methods: Gram stain, MALDI-TOF MS and sequencing of 16S rRNA gene. An initial probiotic potential of lactobacilli isolates was determined via antagonism tests using five enteropathogen reference strains: Staphylococcus aureus, Enterococcus faecium, Candida albicans, Pseudomonas spp. and Salmonella spp. 24 isolates belonging to four Lactobacillus species were identified by MALDITOF MS. BLAST of 16S rRNA gene of eight randomly selected isolates, confirmed MALDI-TOF MS identification. Five of these eight isolates inhibited the growth of at least one of the pathogenic strains used, three isolates of Lactobacillus plantarum and two of Lactobacillus salivarius. Our protocol achieved 21 lactobacilli per 100 isolates performance, greatly surpassing the normal percentage of lactobacilli in chicken gut microbiome, that so, its implementation would facilitate the isolation and identification of new probiotic strains from feces.

scholarly journals The first report and biological characteristics of Helcococcus ovis isolated from clinical bovine mastitis in a Chinese dairy herd

2021 ◽  
Author(s):  
Kai Liu ◽  
Zhaoju Deng ◽  
Limei Zhang ◽  
Xiaolong Gu ◽  
Gang Liu ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bovine Mastitis ◽  
Infection Model ◽  
Growth Speed ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Diagnostic Laboratory ◽  
Milk Samples ◽  
Isolation And Characterization

Helcococcus ovis (H. ovis) was first reported in ovine subclinical mastitis milk and post-mortem examinated organs in Spain and the United Kingdom in 1999, subsequently, it appeared in cattle, horse, goat, and human. However, isolation and characterization of the strain in clinical bovine mastitis is unknown. Therefore, our objective was to identify the pathogen in clinical bovine mastitis. A total of 4 strains from 34 bovine mastitis milk samples were identified, there are tiny and transparent colonies from clinical bovine mastitis milk samples in a Chinese dairy farm, however, these colonies could not be identified using on-farm biochemical tests. The isolates were transported to Mastitis Diagnostic Laboratory of China Agricultural University in Beijing. The colonies were identified as a mixture of H. ovis and Arcanobacterium pyogenes according to microscopic examination and 16S rRNA gene sequencing and  the phylogenetic tree was constructed using 16S rRNA gene sequence of H. ovis isolates. In addition, the growth curve and biochemistry test were performed, we also examined the antimicrobial resistance profiles and constructed murine mammary infection model. Our results showed that the H. ovis were closely related to the strains isolated from China and Japan, growth speed of H. ovis was relatively slower than Strep.agalactiae, the phenotypic characteristics were similar to CCUG37441 and CCUG39041 except to lactose, isolates were sensitive to most of antimicrobials except daptomycin, H. ovis could lead to murine mastitis. In this report, we firstly described the characteristics of H. ovis that are associated with clinical bovine mastitis in China.

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