121. J Microbiol Methods. 2013 Nov;95(2):175-81. doi: 10.1016/j.mimet.2013.08.009.
Epub 2013 Aug 30.
Mosher JJ(1), Bernberg EL, Shevchenko O, Kan J, Kaplan LA.
(1)Stroud Water Research Center, Avondale, PA 19311, United States. Electronic
Longer sequences of the bacterial 16S rRNA gene could provide greater
phylogenetic and taxonomic resolutions and advance knowledge of population
dynamics within complex natural communities. We assessed the accuracy of a
Pacific Biosciences (PacBio) single molecule, real time (SMRT) sequencing based
on DNA polymerization, a promising 3rd generation high-throughput technique, and
compared this to the 2nd generation Roche 454 pyrosequencing platform. Amplicons
of the 16S rRNA gene from a known isolate, Shewanella oneidensis MR1, and
environmental samples from two streambed habitats, rocks and sediments, and a
riparian zone soil, were analyzed. On the PacBio we analyzed ~500 bp amplicons
that covered the V1-V3 regions and the full 1500 bp amplicons of the V1-V9
regions. On the Roche 454 we analyzed the ~500 bp amplicons. Error rates
associated with the isolate were lowest with the Roche 454 method (2%), increased
by more than 2-fold for the 500 bp amplicons with the PacBio SMRT chip (4-5%),
and by more than 8-fold for the full gene with the PacBio SMRT chip (17-18%).
Higher error rates with the PacBio SMRT chip artificially inflated estimates of
richness and lowered estimates of coverage for environmental samples. The 3rd
generation sequencing technology we evaluated does not provide greater
phylogenetic and taxonomic resolutions for studies of microbial ecology.
PMID: 23999276 [PubMed - indexed for MEDLINE]
122. J Clin Microbiol. 2014 May;52(5):1754-7. doi: 10.1128/JCM.00552-14. Epub 2014 Mar
Krohn S(1), Böhm S, Engelmann C, Hartmann J, Brodzinski A, Chatzinotas A, Zeller
K, Prywerek D, Fetzer I, Berg T.
(1)University Hospital Leipzig, Division of Gastroenterology and Hepatology,
Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct
sequencing were applied for rapid detection and identification of bacterial DNA
(bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a
mean of 3.24 log copies ml(-1). Direct sequencing of PCR products revealed 62%
mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal
restriction fragment length polymorphism (T-RFLP) results of a sample subset
confirmed sequence data showing polymicrobial DNA contents in 67% of
bactDNA-positive ascites samples.
PMID: 24622095 [PubMed - indexed for MEDLINE]
123. PLoS One. 2014 Sep 3;9(9):e106215. doi: 10.1371/journal.pone.0106215. eCollection
Rogers EE(1), Backus EA(1).
(1)United States Department of Agriculture, Agricultural Research Service, San
Joaquin Valley Agricultural Sciences Center, Parlier, California, United States
The glassy-winged sharpshooter (GWSS) is an invasive insect species that
transmits Xylella fastidiosa, the bacterium causing Pierce's disease of grapevine
and other leaf scorch diseases. X. fastidiosa has been shown to colonize the
anterior foregut (cibarium and precibarium) of sharpshooters, where it may
interact with other naturally-occurring bacterial species. To evaluate such
interactions, a comprehensive list of bacterial species associated with the
sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota
associated with the GWSS anterior foregut was conducted. Ninety-six individual
GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a
laboratory colony), were characterized for bacteria in dissected sharpshooter
cibaria and precibaria by amplification and sequencing of a portion of the 16S
rRNA gene using Illumina MiSeq technology. An average of approximately 150,000
sequence reads were obtained per insect. The most common genus detected was
Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup
B, one of two Wolbachia supergroups most commonly associated with arthropods. X.
fastidiosa was detected in all 96 individuals examined. By multilocus sequence
typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were
present in GWSS from California and the colony; only subspecies fastidiosa was
detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23
other bacterial genera were detected at or above an average incidence of 0.1%;
these included plant-associated microbes (Methylobacterium, Sphingomonas,
Agrobacterium, and Ralstonia) and soil- or water-associated microbes
(Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas). Sequences
belonging to species of the family Enterobacteriaceae also were detected but it
was not possible to assign these to individual genera. Many of these species
likely interact with X. fastidiosa in the cibarium and precibarium.
PMID: 25184624 [PubMed - indexed for MEDLINE]
124. Genet Mol Res. 2015 Aug 19;14(3):9703-21. doi: 10.4238/2015.August.19.3.
Bredow C(1), Azevedo JL(2), Pamphile JA(1), Mangolin CA(1), Rhoden SA(3).
(1)Departamento de Biotecnologia, Genética e Biologia Celular, Universidade
Estadual de Maringá, PR, Brasil. (2)Laboratório de Genética de Microrganismos
"Prof. Dr. João Lúcio de Azevedo", Departamento de Genética (LGN). (3)Intituto
Federal Catarinense, São Francisco do Sul, SC, Brasil.
Because of human population growth, increased food production and alternatives to
conventional methods of biocontrol and development of plants such as the use of
endophytic bacteria and fungi are required. One of the methods used to study
microorganism diversity is sequencing of the 16S rRNA gene, which has several
advantages, including universality, size, and availability of databases for
comparison. The objective of this study was to analyze endophytic bacterial
diversity in agricultural crops using published papers, sequence databases, and
phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S
rRNA gene was used to identify endophytic bacteria, in important agricultural
crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes,
located in different geographical regions (America, Europe, and Asia). The
corresponding 16S rRNA gene sequences were selected from the NCBI database,
aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The
most common orders present in the analyzed cultures were Bacillales,
Enterobacteriales, and Actinomycetales and the most frequently observed genera
were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that
only approximately 1.56% of the total sequences were not properly grouped,
demonstrating reliability in the identification of microorganisms. This study
identified the main genera found in endophytic bacterial cultures from plants,
providing data for future studies on improving plant agriculture, biotechnology,
endophytic bacterium prospecting, and to help understand relationships between
endophytic bacteria and their interactions with plants.
PMID: 26345903 [PubMed - indexed for MEDLINE]