A human respiratory tract-associated bacterium with an extremely small genome

Abstract

Recent advances in culture-independent microbiological analyses have greatly expanded our understanding of the diversity of unculturable microbes. However, human pathogenic bacteria differing significantly from known taxa have rarely been discovered. Here, we present the complete genome sequence of an uncultured bacterium detected in human respiratory tract named IOLA, which was determined by developing a protocol to selectively amplify extremely AT-rich genomes. The IOLA genome is 303,838 bp in size with a 20.7% GC content, making it the smallest and most AT-rich genome among known human-associated bacterial genomes to our best knowledge and comparable to those of insect endosymbionts. While IOLA belongs to order Rickettsiales (mostly intracellular parasites), the gene content suggests an epicellular parasitic lifestyle. Surveillance of clinical samples provides evidence that IOLA can be predominantly detected in patients with respiratory bacterial infections and can persist for at least 15 months in the respiratory tract, suggesting that IOLA is a human respiratory tract-associated bacterium.

Fig. 1. Protocol for selective amplification of the IOLA genome.

The processes of DNA extraction and selective amplification of IOLA genomic DNA are shown. The proportions of IOLA DNA in the DNA preparations obtained with or without selective amplification were estimated by the 16S rRNA clone library-based method (61 and 75 clones were analyzed, respectively).

Fig. 2. CDSs in the IOLA genome and their homologies with known proteins.

a Circular representation of the IOLA genome and COG classification of the CDSs. Circles from outside to inside: 1, scale in kb; 2, forward-strand CDSs; 3, reverse-strand CDSs; 4, rRNA genes; 5, tRNA genes; 6, GC content (graphic scale; upper: 0.3, center: 0.2, lower: 0.1); and 7, GC skew (graphic scale; upper: 0.25, center: 0.05, lower: −0.15). The window size and the step size in circles 6 and 7 are 10,000 bp and 1000 bp, respectively. b Taxonomic affiliations of the top-hit proteins in the blastp search of each IOLA CDS. c Sequence identity (%) and overlap (%) of IOLA CDSs with the blastp top-hit proteins. Proteins from the top five bacterial taxa, other bacteria (bacterial taxa with proportions less than 5%), archaea, eukaryotes, and viruses are indicated by different colors. Eleven of the 14 eukaryote top-hit CDSs had the top hits to the proteins of mitochondria and chloroplasts.

Fig. 3. Maximum likelihood phylogenetic tree based on the concatenated sequences of 16 ribosomal proteins of Proteobacteria.

The tree includes 451 species belonging to phylum Proteobacteria, two species of Actinobacteria (used as outgroups) and IOLA (listed in Supplemental Data 2). A total of 454 sets of the 16 ribosomal proteins (RpL2, 3, 4, 5, 6, 14, 15, 16, 18, 22, and 24 and RpS3, 8, 10, 17, and 19) were used. Species belonging to the same class are presented in the same color. The tree was constructed using the 1895-position alignment by the maximum likelihood method with the LG + R10 model and 1000 ultrafast bootstrap replicates. The scale bar indicates substitutions per site. Black dots indicate divergence episodes with bootstrap values greater than 80%. Four species that have a small genome (<1.0 Mb) with a low GC content (<30%), such as IOLA, are indicated.

Fig. 4. Maximum likelihood phylogenetic trees based on the concatenated sequences of ribosomal and other housekeeping proteins of class Alphaproteobacteria.

a The tree includes 157 species belonging to class Alphaproteobacteria, 7 species of Zeta-proteobacteria (used as outgroups) and IOLA (listed in Supplemental Data 2). A total of 165 sets of the 16 ribosomal proteins were used (the same set as used in Fig. 3). b The 12 housekeeping proteins (SecA, SecY, TsaD, rpoA, RecA, DnaK, GroEL, GyrA, GyrB, PheS, SerS, and LeuS) of the 165 bacterial species analyzed in a were used. Both trees were constructed by the maximum likelihood method with the LG+F+R10 model and 1000 ultrafast bootstrap replicates and either the 1872-position (a) or 5048-position (b) alignment. Black dots indicate divergence episodes with bootstrap values greater than 80%. The scale bar indicates substitutions per site. In both trees, different genera belonging to order Rickettsiales are presented in different colors.

Fig. 5. Comparison of genomic features of bacteria with extremely small genomes and low GC contents.

a Scatter plot based on the genome sizes and GC contents of 9871 bacteria. Orange dots indicate bacteria with host names registered as “Homo sapiens”. IOLA is indicated by a red dot. b COG functional category proportions, hosts, and genome sizes of the host-associated bacteria with small genomes are shown. The 39 bacterial species with the smallest genome size in each genus were selected from 265 host-associated bacteria with genome sizes less than 1.2 Mb. The major clusters based on the heatmap of COG functional category proportions are colored. All information, such as the number of genes in each COG functional category, host names, genome sizes, and ecosystems, was collected from the JGI IMG database (http://img.jgi.doe.gov). The arrows in a show the human-associated bacteria selected in b. The generally known nonhuman vectors or hosts of the human-associated bacteria are shown as gray boxes in “host category”.

Fig. 6. Genomic diversity of IOLA.

a Twelve genomic segments (PCR1 to PCR275; approximately 1 kb in size, located in the IOLA genome at approximately 25-kb intervals) were amplified by nested PCR from 14 IOLA-positive clinical specimens and sequenced (see Supplementary Data 5 for the genomic positions of each segment). The numbers of SNPs in each segment (reference: KY-405) and the total number of SNPs and proportions of each SNP type are shown on the right-hand side. b An ML tree constructed based on the concatenated sequences of 12 genomic segments (12,410 bp in total). The scale bar indicates substitutions per site. The four specimens indicated in red were obtained from a single patient.

Fig. 7. Clinical course of a patient with long-term persistence of IOLA.

From the sampling date of KY-41, the clinical history of a patient who yielded four IOLA-positive BALF samples is shown. Black bars at the top represent the periods of hospitalization. Colored bars indicate the durations of antibiotic treatments. Asterisks indicate the dates of BALF specimen sampling. The results of IOLA-specific PCR detection, quantitative PCR analyses, and routine culture examination of BALF, sputum, and nasal swab specimens are presented under the clinical course. Total bacterial rDNA and IOLA 16 S rDNA were quantified by quantitative PCR. CT images of the patient’s lower bilateral lobes obtained on each sampling day are also shown. PSL prednisolone (corticosteroid), STFX: sitafloxacin, GRNX garenoxacin, DRPM doripenem, CZOP cefozopran, MEPM meropenem, TAZ/PIPC tazobactam/piperacillin, CAM clarithromycin. + PCR positive, − PCR negative. ND not done, GNR Gram-negative rod, Pa P. aeruginosa.