Genome-sequence analysis of Acinetobacter johnsonii MB44 reveals potential nematode-virulent factors
© The Author(s) 2016
Received: 27 January 2016
Accepted: 25 June 2016
Published: 4 July 2016
Acinetobacter johnsonii is generally recognized as a nonpathogenic bacterium although it is often found in hospital environments. However, a newly identified isolate of this species from a frost-plant-tissue sample, namely, A. johnsonii MB44, showed significant nematicidal activity against the model organism Caenorhabditis elegans. To expand our understanding of this bacterial species, we generated a draft genome sequence of MB44 and analyzed its genomic features related to nematicidal attributes. The 3.36 Mb long genome contains 3636 predicted protein-coding genes and 95 RNA genes (including 14 rRNA genes), with a G + C content of 41.37 %. Genomic analysis of the prediction of nematicidal proteins using the software MP3 revealed a total of 108 potential virulence proteins. Some of these proteins were homologous to the known virulent proteins identified from Acinetobacter baumannii, a pathogenic species of the genus Acinetobacter. These virulent proteins included the outer membrane protein A, the phospholipase D, and penicillin-binding protein 7/8. Moreover, one siderophore biosynthesis gene cluster and one capsular polysaccharide gene cluster, which were predicted to be important virulence factors for C. elegans, were identified in the MB44 genome. The current study demonstrated that A. johnsonii MB44, with its nematicidal activity, could be an opportunistic pathogen to animals.
Bacterial species of the genus Acinetobacter are ubiquitous in nature and are usually found in the hospital environment; some of these species have been implicated in a variety of nosocomial infections (Bergogne-Berezin and Towner 1996). For instance, Acinetobacter baumannii is known as a global nosocomial pathogen for its ability to cause hospital outbreaks and develop antibiotic resistance (Dijkshoorn et al. 2007; Peleg et al. 2008); A. pittii and A. nosocomialis have been reported to be associated with human infections (Chuang et al. 2011; Wang et al. 2013). Certain Acinetobacter species are currently emphasized in discussions on pathogenicity and mechanisms of multidrug resistance. However, the species A. johnsonii, which was identified to encode an extended-spectrum β-lactamase that confers resistance against penicillins, cephalosporins, and monobactams (Zong 2014), has been scarcely reported to cause animal or human disease.
In this study, an A. johnsonii MB44 strain was isolated from a frost-plant-tissue sample in the process of screening for ice-nucleating bacteria (Li et al. 2012). Bioassay reveals the significant virulence of this strain against the model organism, Caenorhabditis elegans. To date, the human-pathogen A. baumannii and A. nosocomialis have been reported for their pathogenicity against C. elegans (Vila-Farres et al. 2015; Smith et al. 2004). Therefore, to identify the potential virulence factors and better understand the molecular mechanism of its ability to infect nematodes, we performed genome sequencing of A. johnsonii MB44. The genomic features and the potential nematode-virulent genes were reported herein.
Bacterial culture and genomic DNA preparation
A clonal population of A. johnsonii strain MB44 was derived from a single colony serially passaged three times. The bacterium was grown under incubation at 28 °C on Luria-Bertani (LB) agar plates (1.5 % agar) containing 0.5 % NaCl. Colonies were inoculated into 5 mL of LB medium with shaking at 28 °C for 24 h. Aliquots (250 μL) from the LB cultures were inoculated into 25 mL of LB broth in a 100 mL flask and incubated at 28 °C for 20 h. Cells were pelleted successively into one 1.5 mL centrifuge tube at 12,000 rpm. Genomic DNA was extracted using the Bacterial DNA Kit (GBCBIO), in accordance with the manufacturer’s protocol. DNA quality and quantity were determined with a Nanodrop spectrometer (Thermo Scientific, Wilmington, USA).
Nematode toxicity bioassay
For pathogenicity assay, C. elegans strain N2 was maintained at NGM agar with E. coli OP50 as food source. Assay was conducted with age synchronized L4 stage worms. A. johnsonii MB44 was grown in LB broth for 24 h. The cells were collected, re-suspended, and diluted in M9 buffer to make desired initial concentrations (based on OD600). Assay was conducted in 96 well plate such as each well contained 150 µL of cell suspension, 5 µL of 8 mM 5-fluorodeoxyuridine (FUdR), 40 µL M9 buffer and 40–50 L4 worms. Killing of worms was observed after 72 h.
The 16S rRNA gene sequences of the reference strains used for phylogenetic analysis were obtained from GenBank database of the National Center for Biotechnology Information (NCBI) (Benson et al. 2015). To construct the phylogenetic tree, these sequences were collected and nucleotide sequence alignment was carried out using ClustalW (Thompson et al. 1994). The software MEGA v.5.05 (Tamura et al. 2011) was used to generate phylogenetic trees based on 16S rRNA genes under the neighbor-joining approach (Saitou and Nei 1987).
Genome sequencing and assembly
The genome of MB44 was sequenced by a commercial service at Beijing BerryGenomics Co., Ltd. using the Illumina HiSeq 2000 platform. Genomic DNA was sequenced with the Illumina sequencing platform by the paired-end strategy (2 × 125 bp) and the details of library construction and sequencing can be found at the Illumina website, yielding 8,593,104 total reads and providing 137-fold coverage of the genome. ABySS v.1.3.7 (Simpson et al. 2009) was employed for sequence assembly and the optimal value of k-mer is 90. The final draft assembly contained 75 contigs and the total size of the genome is 3.36 Mb. Contigs were ordered based upon Acinetobacter lwoffii WJ10621 (Hu et al. 2011) as reference genome using Mauve (Darling et al. 2004). The circular genome of A. johnsonii MB44 was generated using Artemis (Rutherford et al. 2000).
Automated genome annotation was completed by the NCBI Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The coding sequences (CDSs) were predicted using software Glimmer v.3.02 (Delcher et al. 2007). The predicted CDSs were translated and used to search the NCBI non-redundant database, UniProt, and Clusters of Orthologous Groups (COG) databases. The whole genomic tRNAs were identified using tRNAscan-SE v.1.21 (Lowe and Eddy 1997), and rRNAs were found by RNAmmer v.1.2 Server (Lagesen et al. 2007). Genes with signal peptides were predicted by SignalP (Petersen et al. 2011). In addition, genes carrying trans-membrane helices were predicted by TMHMM (Moller et al. 2001); and CRISPR repeats were searched using CRISPRFinder (Grissa et al. 2007).
The draft genome sequence of A. johnsonii MB44 (GenBank accession no. LBMO00000000.1) was compared with the available complete genome of A. baumannii AB307-0294 (GenBank: NC_011595.1) and A. pittii ANC4052 (GenBank: APQO00000000.1). A web server, named OrthoVenn (Wang et al. 2015), was adopted to identify orthologous clusters among the genomes of these species. The function of each orthologous cluster was deduced by BLASTP (Altschul et al. 1997) analysis against UniProt databases. A Venn diagram was created using the web application Venny (http://bioinfogp.cnb.csic.es/tools/venny/) with the orthologous cluster ID list. The average nucleotide identity among these species was calculated by the ANI (average nucleotide identity) calculator (Goris et al. 2007).
This whole-genome shotgun project was deposited at DDBJ/EMBL/GenBank under the accession LBMO00000000. The version described in this paper is version LBMO01000000.
Results and discussion
Microbial features, classification, and nematode toxicity bioassay of MB44
General features of the A. johnsonii MB44 genome sequence
Number of genes associated with general COG functional categories
Information storage and processing
Translation, ribosomal structure and biogenesis
Replication, recombination and repair
Cellular processes and signaling
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Comparison among the genome characteristics of A. baumannii AB307-0294, A. pittii ANC 4052, and A. johnsonii MB44
A. johnsonii MB44
A. baumannii AB307-0294
A. pittii ANC 4052
Frost plant tissue
Genome size (bp)
G + C Content (mol%)
Genes predicted virulent to C. elegans
Prediction of pathogenic proteins in A. johnsonii MB44 using MP3
Pathogenic proteins (HS)a
Type I secretion system
Type II secretion system
Potential virulent proteins in A. johnsonii MB44 and known homologous A. baumannii virulent proteins
Gene accession number
Amino acid similarity (%)
Penicillin-binding protein 7/8
A. baumannii strain 307-0294
A. baumannii strain 98-37-09
A. baumannii strain 98-37-09
Outer membrane protein A
A. baumannii ATCC 19606
Putative genes involved in siderophore biosynthesis and transport
Capsular polysaccharide gene cluster
Summary of homology searches for the open reading frames found in the putative capsule cluster of A. johnsonii MB44
ORF (aa) (A. johnsonii MB44)
Homologous protein (aa) (A. baumannii AB307-0294)
Identity/similarity (%) (aa overlap)
Peptidyl-prolyl cis-trans isomerase
Protein tyrosine kinase
Polysaccharide export outer membrane protein
In this study, we presented a whole-genome analysis of A. johnsonii MB44 to identify its potential virulence factors against C. elegans. The MB44 genome contained 108 virulent proteins predicted by MP3, and four proteins showed high identity to the known virulent proteins in the pathogenic A. baumannii. Furthermore, one siderophore biosynthesis gene cluster and one capsular polysaccharide gene cluster were identified, which were relevant to nematicidal activity of pathogenic bacteria. The current study demonstrated that A. johnsonii, which was generally recognized as a nonpathogenic bacterium, could be an opportunistic pathogen to animals.
ST performed most of the experiments, made most of the data evaluation and drafted parts of the manuscript. MA and LX participated in the analysis and interpretation of the data. LL conceived and directed the study and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by a grant from the National Basic Research Program of China (973 Program, Grant 2013CB127504) and grants from the National Natural Science Foundation of China (Grant Nos. 31570123 and 31270158).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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