GPL phenotypes of <i>mps1</i> complement.

<p>Alkaline-stable lipids were developed with the solvent system chloroform-methanol (90:10, vol/vol) on TLC. The GPLs were restored in the <i>mps1</i>-complemented J15cs mutant. The TLC plate was sprayed with 20% sulfuric acid in ethanol and charred at 180°C for 3 min.</p

Change of morphology due to expression of GPLs.

<p>The colony morphology was observed by an optical microscope after culturing. The J15cs strain formed rough, dry, and irregular colonies. The mc^<b>2</b>155 strain and the <i>mps1</i>-complemented J15cs mutant colonies were smoothly curved and wet (a). Scanning electron microscopy (b) and a comparison of bacterial cell length (c) are shown. The bacteria were grown on nutrient agar plates for 10 days at 37°C, collected, and filtered through a 5 μm membrane to remove aggregates. The average bacterial cell length was 2.11±0.46 μm, the mc^<b>2</b>155 strain; 5.70±1.65, the J15cs strain; 2.18±0.62 μm, the <i>mps1</i>-complemented J15cs mutant. The data are means±standard deviations (SD) for 20 bacteria. The separate experiments were done in duplicate. *, <i>p<</i>0.001.</p

Survival of <i>mps1</i>-complemented J15cs mutant in J774.1 cells.

<p>The J774.1 cells were grown on 24-well flat-bottom tissue culture plates. A bacterial single-cell suspension of approximately 1×10^<b>7</b> CFU/well was inoculated into the J774.1 cells at an approximate multiplicity of infection 10. After cocultivation for 3 h, the infected J774.1 cells were washed, and the infected J774.1 cells were further incubated for 2 h with the medium plus 200 μg/ml gentamicin. The medium was replaced with fresh medium containing 2 μg/ml of gentamicin (this time point was day 0), and the infected cells cultured for 8 days. At various time intervals after inoculation, the adherent cells were treated with 1% Triton-X100/PBS, and were sonicated. The bacterial burden was evaluated by counting CFU. The separate experiments were done in triplicate, and statistical analysis was performed using Dunnett’s test. * and ** indicate a <i>p</i> value of <0.05, compared to the J15cs strain inserted with a pNN2 vector only respectively.</p

Construction of the mc²155-<i>mps1</i> plasmid.

<p>The <i>mps1</i> of the mc^<b>2</b>155 strain was inserted into a pNN2 vector (kanamycin resistance). P<i>aphII</i>, the promoter of <i>aphII</i>; mc^<b>2</b>155-<i>mps1</i>, the open reading frame of <i>mps1</i> from <i>M</i>. <i>smegmatis</i> mc^<b>2</b>155; T<i>alpha</i>, the terminator region of the alpha antigen gene from <i>Mycobacterium kansasii</i>.</p

MALDI-TOF/MS spectra of each GPL.

<p>The molecular weights of GPLs derived from the <i>mps1</i>-complemented J15cs mutant were the same as those of the mc^<b>2</b>155 strain. The matrix was 2,5-dihydroxybenzoic acid in chloroform-methanol (1:1, vol/vol). It was analyzed in the Reflectron mode with an accelerating voltage operating in positive mode at 20 kV. The main molecule-related ions were detected as m/z, [M+Na]^<b>+</b>. Intens., intensity; a.u., arbitrary units. The mass numbers were fixed to the chemical structures and exact mass numbers.</p

An Unclassified Microorganism: Novel Pathogen Candidate Lurking in Human Airways

<div><p>During the assessments of the correlation of the diseases and the microbiota of various clinical specimens, unique 16S ribosomal RNA (rRNA) gene sequences (less than 80% similarity to known bacterial type strains) were predominantly detected in a bronchoalveolar lavage fluid (BALF) specimen from a patient with chronic lower respiratory tract infection. The origin of this unique sequence is suspected to be the causative agent of the infection. We temporarily named the owner organism of this sequence “IOLA” (Infectious Organism Lurking in Airways). In order to evaluate the significance of IOLA in human lung disorders, we performed several experiments. IOLA-16S rRNA genes were detected in 6 of 386 clone libraries constructed from clinical specimens of patients with respiratory diseases (in our study series). The gene sequences (1,427 bp) are identical, and no significantly similar sequence was found in public databases (using NCBI blastn) except for the 8 shorter sequences detected from patients with respiratory diseases in other studies from 2 other countries. Phylogenetic analyses revealed that the 16S rRNA gene of IOLA is more closely related to eukaryotic mitochondria than bacteria. However, the size and shape of IOLA seen by fluorescent <i>in-situ</i> hybridization are similar to small bacteria (approximately 1 µm with a spherical shape). Furthermore, features of both bacteria and mitochondria were observed in the genomic fragment (about 19 kb) of IOLA, and the GC ratio of the sequence was extremely low (20.5%). Two main conclusions were reached: (1) IOLA is a novel bacteria-like microorganism that, interestingly, possesses features of eukaryotic mitochondria. (2) IOLA is a novel pathogen candidate, and it may be the causative agent of human lung or airway disease. IOLA exists in BALF specimens from patients with remarkable symptoms; this information is an important piece for helping solve the elusive etiology of chronic respiratory disorders.</p></div

Blast search results of the CDSs on IOLA genomic fragment.

<p>Blast search results of the CDSs on IOLA genomic fragment.</p

Bacterial cell numbers and compositions in the specimens from patients, detected IOLA in clone library analysis.

<p><b>A,</b> Results of bacterial cell counts of the specimens from patient A using an epifluorescent staining method with ethidium bromid. Open circles indicate numbers of bacterial cells per ml of each the specimens. <b>B,</b> Percentage of the detected bacteria in the specimens with the clone library analysis of 16S rRNA gene. The percentages of IOLA-clones (orange box) in each of the clone libraries are shown in parentheses.</p

Patient characteristics.

a<p>WBC; white blood cell,</p>b<p>CRP; C-reactive protein,</p>c<p>CT; computed tomography,</p>d<p>GRNX; Garenoxacin,</p>e<p>MEPM; Meropenem,</p>f<p>N/A; not applicable.</p

GC content and annotation of IOLA genomic fragment (18,834 bp).

<p>High GC contents per 500-base pairs (kbp). The black box represents a 16S rRNA gene. The other boxes represent ORFs. The shaded boxes show similarities (amino-acid sequence) with known bacterial proteins. Annotation results of the ORFs are indicated via arrows. White boxes represent products of ORFs showing extremely low similarities with known proteins.</p