Theoretical Prediction and Experimental Verification of Protein-Coding Genes in Plant Pathogen Genome Agrobacterium tumefaciens Strain C58

Abstract

Agrobacterium tumefaciens strain C58 is a Gram-negative soil bacterium capable of inducing tumors (crown galls) on many dicotyledonous plants. The genome of A. tumefaciens strain C58 was re-annotated based on the Z-curve method. First, all the ‘hypothetical genes’ were re-identified, and 29 originally annotated ‘hypothetical genes’ were recognized to be non-coding open reading frames (ORFs). Theoretical evidence obtained from principal component analysis, clusters of orthologous groups of proteins occupation, and average length distribution showed that these non-coding ORFs were highly unlikely to encode proteins. Results from the reverse transcription-polymerase chain reaction (RT-PCR) experiments on three different growth stages of A. tumefaciens C58 confirmed that 23 (79%) of the identified non-coding ORFs have no transcripts in these growth stages. In addition, using theoretical prediction, 19 potential protein-coding genes were predicted to be new protein-coding genes. Fifteen (79%) of these genes were verified with RT-PCR experiments. The RT-PCR experimental results confirmed the reliability of our theoretical prediction, indicating that false-positive prediction and missing genes always exist in the annotation of A. tumefaciens C58 genome. The improved annotation will serve as a valuable resource for the research of the lifestyle, metabolism, and pathogenicity of A. tumefaciens C58. The re-annotation of A. tumefaciens C58 can be obtained from http://211.69.128.148/Atum/.

Effects upon metabolic pathways and energy production by Sb(III) and As(III)/Sb(III)-oxidase gene aioA in Agrobacterium tumefaciens GW4

“…The quality and quantity of the RNA were determined with a spectrophotometer (NanoDrop 2000, Thermo). Reverse transcription was performed with a RevertAid First Strand cDNA Synthesis Kit (Thermo) with 300 ng of total RNA for each sample [ 31 ]. Then the obtained cDNA was diluted 10-fold for quantitative RT-PCR analysis by ABI VIIA7 in a 0.1 mL Fast Optical 96-well Reaction Plate (ABI) using SYBR ® Green Realtime PCR Master Mix (Toyobo).…”

An Oxidoreductase AioE is Responsible for Bacterial Arsenite Oxidation and Resistance

“…Total RNA was extracted used Trizol Kit (Invitrogen) and incubated with RNase-free DNase I (Takara) at 37 °C to remove the genomic DNA. Then, the reaction was terminated by addition of 50 mM EDTA at 65 °C for 10 min 45 . After confirming the negative DNA contamination and determining the concentration of RNA by spectrophotometer (NanoDrop 2000, Thermo), RT-PCR for testing the co-transcribe of the ars gene cluster was performed using the primers listed in Table S2 , while 300 ng total RNA was reverse transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (Thermo).…”

Disrupting ROS-protection mechanism allows hydrogen peroxide to accumulate and oxidize Sb(III) to Sb(V) in Pseudomonas stutzeri TS44

“…Total RNA was extracted using the TRIzol® Reagent (Invitrogen) and treated with DNaseI following the manufacturer’s instructions. The synthesis of cDNA from 300 ng of total RNA was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo) [41]. The resulting cDNA was used as a template for qRT-PCR with the SYBR-Green® qPCR Master Mix (Takara).…”