Comparative genome analysis reveals the molecular basis of nicotine degradation and survival capacities of Arthrobacter

Abstract

Arthrobacter is one of the most prevalent genera of nicotine-degrading bacteria; however, studies of nicotine degradation in Arthrobacter species remain at the plasmid level (plasmid pAO1). Here, we report the bioinformatic analysis of a nicotine-degrading Arthrobacter aurescens M2012083, and show that the moeB and mogA genes that are essential for nicotine degradation in Arthrobacter are absent from plasmid pAO1. Homologues of all the nicotine degradation-related genes of plasmid pAO1 were found to be located on a 68,622-bp DNA segment (nic segment-1) in the M2012083 genome, showing 98.1% nucleotide acid sequence identity to the 69,252-bp nic segment of plasmid pAO1. However, the rest sequence of plasmid pAO1 other than the nic segment shows no significant similarity to the genome sequence of strain M2012083. Taken together, our data suggest that the nicotine degradation-related genes of strain M2012083 are located on the chromosome or a plasmid other than pAO1. Based on the genomic sequence comparison of strain M2012083 and six other Arthrobacter strains, we have identified 17 σ(70) transcription factors reported to be involved in stress responses and 109 genes involved in environmental adaptability of strain M2012083. These results reveal the molecular basis of nicotine degradation and survival capacities of Arthrobacter species.

Figure 1. 16S rDNA phylogenetic analysis of Arthrobacter strains.

The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.82 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Neighbour-Joining (NJ) method and are in the units of the number of base substitutions per site. The analysis involved 55 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1,327 positions in the final DataSet. Evolutionary analyses were conducted in MEGA5. Only bootstrap values greater than 50% are shown. The strains used in genome comparison are shown in red, the type strains are indicated by superscript T and the strain M2012083 is highlighted in blue. The accession numbers of the sequences used are listed in the parentheses.

Figure 2. Genome similarity analysis of strain M2012083.

The genomes of the seven Arthrobacter strains were submitted to the web service RAST for comparison. The genome of M2012083 was compared with the other six complete genomes and the comparison scores were shown. Higher score means higher similarity.

Figure 3. Comparison of the genome of Arthrobacter aurescens M2012083 with other Arthrobacter genomes.

The outermost circle (circle 1) represents the scale. Circle 2, the chromosomal (blue) and plasmids (orange and green) open reading frames (ORFs) of A. aurescens TC1 as references; circle 3, ORFs of A. chlorophenolicus A6 complete genome; circle 4, ORFs of A. nitroguajacolicus Rue61a complete genome; circle 5, ORFs of A. arilaitensis Re117 complete genome; circle 6, ORFs of A. phenanthrenivorans Sphe3 complete genome; circle 7, ORFs of A. sp. FB24 complete genome; circle 8, ORFs of A. aurescens M2012083 complete genome. ORFs are represented by colorful sticks (red-to-blue were assigned according to the similarity of the ORF to the homolog in TC1 genome) in circles 3 to 8.

Figure 4. Comparison of COG categories among seven subject Arthrobacter strains.

Functional comparisons among the genomes of Arthrobacter sp. FB24, A. aurescens TC1, A. chlorophenolicus A6, A. arilaitensis Re117, A. phenanthrenivorans Sphe3, A. nitroguajacolicus Rue61a and A. aurescens M2012083 were performed based on the functional classifications of COG database. The ordinate axis represents the number of genes in each COG functional category. The 22 COGs categories are as following: RNA processing and modification (A); chromatin structure and dynamics (B); energy production and conversion (C); cell cycle control, cell division, chromosome partitioning (D); amino acid transport and metabolism (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (H); lipid transport and metabolism (I); translation, ribosomal structure and biogenesis (J); transcription (K); replication, recombination and repair (L); cell wall, membrane, envelope biogenesis (M); cell motility (N); posttranslational modification, protein turnover, chaperones (O); inorganic transport and metabolism (P); secondary metabolites biosynthesis, transport and catabolism (Q); general function prediction only (R); function unknown (S); signal transduction mechanisms (T); intracellular trafficking, secretion and vesicular transport (U); defense mechanisms (V).

Figure 5. Numbers of conversed and specific groups in M2012083 compared with six other Arthrobacter genomes.

This analysis is based on the results of orthologous relationships analysis which is shown in DataSet1. Green rectangle, orthologous groups specific in M2012083 compared with the strain(s) of the horizontal axis; brown rectangle, orthologous groups conserved in M2012083 compared with the strain(s) of the horizontal axis but were not found in other studied strain(s); blue rectangle, orthologous groups conserved in M2012083 compared with the strain(s) of the horizontal axis and also were found in some other studied strain(s); studied strains, strains M2012083, FB24, TC1, A6, Re117, Sphe3 and Rue61a; Ndb, N-heterocyclic compounds degradation bacteria TC1 and Rue61a; Evb, environmental bacteria FB24, TC1, A6, Sphe3 and Rue61a.

Figure 6. Comparison among the genome of M2012083 and plasmid pAO1.

The synteny plot of plasmid pAO1 versus M2012083 genome was generated by using MUMmer program. Red ringlets indicate the forward conserved genes between M2012083 genome and plasmid pAO1; blue ringlets indicate the reverse conserved genes between M2012083 genome and plasmid pAO1. The names, arrangements and functions of genes, and the nicotine-degrading pathway in Arthrobacter are shown according to Ganas P, Igloi GL and Brandsch R³⁸. The rectangles in different colors represent gene clusters involved in carbohydrate catabolism (ch), plasmid function (plf), γ-N-methylaminobutyrate catabolism (mgaba), assembly and quality control of the (αβγ)₂ holoenzyme complexes of NDH and KDH (aqc), nicotine degradation (nic1 and nic2), molybdenum cofactor biosynthesis (moco) and transposons (Tn554), and some insertion sequences (IS and IS1473). ORFs were indicated by arrows, and the hollow arrows represent hypothetical protein genes (hyp). See text and DataSet3 for details.