Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.     

Microbial desulfurization of gasoline in a Mycobacterium goodii X7B immobilized-cell system

Mycobacterium goodii X7B, which had been primarily isolated as a bacterial strain capable of desulfurizing dibenzothiophene to produce 2-hydroxybiphenyl via the 4S pathway, was also found to desulfurize benzothiophene. The desulfurization product was identified as o-hydroxystyrene by gas chromatography (GC)-mass spectrometry analysis. This strain appeared to have the ability to remove organic sulfur from a broad range of sulfur species in gasoline. When Dushanzi straight-run gasoline (DSRG227) containing various organic sulfur compounds was treated with immobilized cells of strain X7B for 24 h, the total sulfur content significantly decreased, from 227 to 71 ppm at 40 degrees C. GC flame ionization detection and GC atomic emission detection analysis were used to qualitatively evaluate the effects of M. goodii X7B treatment on the contents of gasoline. In addition, when immobilized cells were incubated at 40 degrees C with DSRG275, the sulfur content decreased from 275 to 54 ppm in two consecutive reactions. With this excellent efficiency, strain X7B is considered a good potential candidate for industrial applications for the biodesulfurization of gasoline.     

A Study of the Taxonomy of the Mycobacterium nonchromogenicum Complex and Report of Six Cases of Lung Infection Due to Mycobacterium nonchromogenicum

In numerical classification, four species of the Mycobacterium nonchromogenicum complex, Mycobacterium nonchromogenicum, M. terrae, M. novum, and M. triviale, formed one cluster. These four species appeared to be reduced to one species, Mycobacterium nonchromogenicum. Furthermore, relationships between the species were numerically analyzed by using the hypothetical median organism pattern. The results showed that the M. nonchromogenicum complex can be divided into two subgroups: M. nonchromogenicum and the other three. These two subgroups were differentiated from each other by scores based on two or more positive reactions in the following three characteristics: resistance to bleomycin (5 μg/ml); heat-stable acid phosphatase activity; nicotinamidase or pyrazinamidase activity or both activities. M. nonchromogenicum gave two or three positive reactions among these three, and M. terrae, M. novum, and M. triviale gave two or three negative reactions. Three cases of lung infection due to M. nonchromogenicum, as well as three other cases of probable lung infection due to M. nonchromogenicum, were observed in this study. Only one organism isolated from one doubtful case was M. terrae. Up to now, M. nonchromogenicum was considered a nonpathogen. It was shown, however, that this organism causes lung infection in humans.     

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.     

Role of Pasteurella pneumotropica and Mycoplasma pulmonis in Murine Pneumonia

Pneumonia caused by Mycoplasma pulmonis and Pasteurella pneumotropica was studied in conventional, specific pathogen-free (SPF), and germ-free mice. When P. pneumotropica was serially passed in conventional mice, M. pulmonis, as well as P. pneumotropica, was recovered from mice with gross lesions. When M. pulmonis was serially passed in conventional mice, both organisms were recovered. SPF mice given a nasal instillation of M. pulmonis alone, P. pneumotropica alone, or a combination of the two developed pneumonia when both organisms were present. These findings suggested that both organisms contribute to typical murine pneumonia. That M. pulmonis might be an L form of P. pneumotropica was suggested because some SPF mice inoculated with either organism yielded both on culture. This possibility was investigated with mole per cent guanine plus cytosine (GC) content and nucleic acid hybridization techniques. The GC content of P. pneumotropica is 42.2 mole per cent and that of M. pulmonis is 28.6 mole per cent. No specific hybrids between deoxyribonucleic acid (DNA) from M. pulmonis and DNA from P. pneumotropica were detected. This and the wide disparity in GC content showed that M. pulmonis is not an L form of P. pneumotropica. In germ-free mice, intranasal instillation with either organism alone produced pneumonia. The lesions produced when each organism was inoculated independently were characterized by areas of consolidation with perivascular and peribronchial lymphocytic infiltration. Qualitatively, the lesions produced when both organisms were inoculated simultaneously more closely resembled those seen in naturally occurring murine pneumonia. Statistical analysis indicated that the quantitative effect of the two organisms was additive. The indirect fluorescent antibody technique was used to locate organisms in lung tissue sections. M. pulmonis localized in the bronchial epithelium and P. pneumotropica localized in the alveolar lesions.     

Mycobacterium fortuitum subspecies acetamidolyticum, a new subspecies of Mycobacterium fortuitum

Mycobacterium fortuitum subspecies acetamidolyticum is a new subspecies of M. fortuitum and has an intermediate growth rate. It is a nonphotochromogenic mycobacterium. It does not utilize glutamate but utilizes acetamide as a simultaneous nitrogen and carbon source. It is able to utilize acetate, malate, pyruvate, fumarate, glucose, fructose, and n-propanol as the sole sources of carbon in the presence of ammoniacal nitrogen, but does not utilize them in the presence of glutamate-nitrogen. It is easily differentiated from all rapidly growing mycobacteria by its inability to utilize glutamate as a simultaneous nitrogen and carbon source, and from all slowly growing mycobacteria by its capacity to utilize acetamide as a simultaneous nitrogen and carbon source. Its mycolic acid pattern is different from that of M. fortuitum. However, its deoxyribonucleic acid showed 94% relatedness with that of M. fortuitum. In view of the above findings, it has been designated as a new subspecies of M. fortuitum. The organism was isolated from sputum of a 56-year-old patient with lung disease and is considered to be a lung pathogen. The type strain is ATCC 35931 (NCH E11620).     

Human surfactant protein D (SP-D) binds Mycoplasma pneumoniae by high affinity interactions with lipids

Increasing evidence now identifies surfactant protein D (SP-D) as an important element of the innate immune system of the lung. In this study, we examined the interactions of rat and human SP-D with the human pathogen, Mycoplasma pneumoniae. Rat and human SP-D bound the organism with high affinity in a reaction that required Ca(2+) and was inhibited by EGTA. Membranes derived from the organism bound the proteins in a similar manner, except the rat SP-D also exhibited a significant level of Ca(2+)-independent binding. Pretreatment of membranes with proteases did not alter the Ca(2+)-dependent SP-D binding of membranes by either protein. Mannose, glucose, maltose, and inositol, at millimolar concentrations, competed for human SP-D binding to the bacterial membrane. Lipids extracted from membranes and separated by two-dimensional thin layer chromatography bound human SP-D with high affinity in a Ca(2+)-dependent reaction. A tandem mutant of SP-D with E321Q and N323D substitutions, failed to bind M. pneumoniae lipids, directly implicating the carbohydrate recognition domain in the interaction. The interaction of rat and human SP-D with M. pneumoniae was unaffected by the presence of surfactant lipids and the hydrophobic surfactant proteins. These findings demonstrate that M. pneumoniae is likely to be recognized by SP-D in the alveolar environment and that primary determinants recognized on the organism are lipid components of the cell membrane.     

Approaching the degradome in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating, lethal and currently untreatable lung disorder of unknown etiology. It is characterized by epithelial injury and activation, fibroblastic foci formation, and exaggerated accumulation of extracellular matrix (ECM) with the destruction of the lung parenchyma. Despite important progress in our understanding of the general mechanisms involved in lung fibrogenesis, the pathogenesis of the IPF remains unclear. Although the irreversible and progressive fibrosis in the lung suggests a decrease in lung degradative machinery, an increasing body of evidence, primarily obtained by global gene expression studies, demonstrates a significant upregulation of degrading enzymes in IPF. In this context, this review will focus on some families of the degradome, a term proposed for the complete set of proteases that are expressed at a specific time by a cell, tissue or an organism. In particular, we will approach recent progress in our understanding of the behavior of two families of metalloproteases M10 and M12 which are significantly changed in the IPF lungs. In general, evidence highlights the increasing diversity in both substrates and functions of these enzymes and the complexity of the processes in which they are involved, and indicate a critical role in the abnormal remodeling of IPF.     

Detection of Mycoplasma pulmonis by fluorogenic nuclease polymerase chain reaction analysis

Mycoplasma pulmonis induces persistent infections in laboratory mice and rats and can contaminate biological materials. We developed a fluorogenic nuclease polymerase chain reaction (fnPCR) assay to detect M. pulmonis specifically. Primer and probe sequences for the assay were targeted to 16S rRNA sequences specific to M. pulmonis. The assay consistently detected the equivalent of fewer than 10 copies of template DNA. When evaluated against a panel of 24 species of bacteria, the M. pulmonis assay detected only M. pulmonis isolates. Evaluation of 10-fold serial dilutions of cultured M. pulmonis showed that the M. pulmonis fnPCR assay and culture on Dutch agar had comparable sensitivity in detecting viable M. pulmonis organisms, whereas the mouse antibody production test displayed positive serologic results at dilutions higher than those in which viable organisms could be detected. Finally, the M. pulmonis fnPCR assay was able to detect M. pulmonis DNA in nasopharyngeal wash fluid and trachea, lung, and uterus tissue collected from mice naturally infected with M. pulmonis but did not detect the organism in similar samples collected from uninfected, negative control mice. The M. pulmonis fnPCR assay provides a high-throughput, PCR-based method to detect M. pulmonis in infected rodents and contaminated biological materials.     

Biodesulfurization of DBT in tetradecane and crude oil by a facultative thermophilic bacterium Mycobacterium goodii X7B

Mycobacterium goodii X7B, a facultative thermophilic bacterium, cleaving the C-S bond of dibenzothiophene via a sulfur-specific pathway, was investigated for DBT in tetradecane and crude oil desulfurization. The extent of growth was improved by fed-batch culture controlled at a constant pH. The total sulfur level of dibenzothiophene in tetradecane, was reduced by 99%, from 200 to 2 ppm within 24h at 40 degrees C. After 72 h treatment, 59% of the total sulfur content in Liaoning crude oil was removed, from 3600 to 1478 ppm.     

Investigation of spa pools associated with lung disorders caused by Mycobacterium avium complex in immunocompetent adults

Three cases of Mycobacterium avium complex-related lung disorders were associated with two poorly maintained spa pools by genotypic investigations. Inadequate disinfection of the two spas had reduced the load of environmental bacteria to less than 1 CFU/ml but allowed levels of M. avium complex of 4.3 x 10(4) and 4.5 x 10(3) CFU/ml. Persistence of the disease-associated genotype was demonstrated in one spa pool for over 5 months until repeated treatments with greater than 10 mg of chlorine per liter for 1-h intervals eliminated M. avium complex from the spa pool. A fourth case of Mycobacterium avium complex-related lung disease was associated epidemiologically but not genotypically with another spa pool that had had no maintenance undertaken. This spa pool contained low numbers of mycobacteria by smear and was culture positive for M. avium complex, and the nonmycobacterial organism count was 5.2 x 10(6) CFU/ml. Public awareness about the proper maintenance of private (residential) spa pools must be promoted by health departments in partnership with spa pool retailers.     

Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response

Moraxella catarrhalis is an important pathogen in patients with chronic obstructive lung disease (COPD). While M. catarrhalis has been categorized as an extracellular bacterium so far, the potential to invade human respiratory epithelium has not yet been explored. Our results obtained by electron and confocal microscopy demonstrated a considerable potential of M. catarrhalis to invade bronchial epithelial (BEAS-2B) cells, type II pneumocytes (A549) and primary small airway epithelial cells (SAEC). Moraxella invasion was dependent on cellular microfilament as well as on bacterial viability, and characterized by macropinocytosis leading to the formation of lamellipodia and engulfment of the invading organism into macropinosomes, thus indicating a trigger-like uptake mechanism. In addition, the cells examined expressed TLR2 as well as NOD1, a recently found cytosolic protein implicated in the intracellular recognition of bacterial cell wall components. Importantly, inhibition of TLR2 or NOD1 expression by RNAi significantly reduced the M. catarrhalis-induced IL-8 secretion. The role of TLR2 and NOD1 was further confirmed by overexpression assays in HEK293 cells. Overall, M. catarrhalis may employ lung epithelial cell invasion to colonize and to infect the respiratory tract, nonetheless, the bacteria are recognized by cell surface TLR2 and the intracellular surveillance molecule NOD1.     

Surfactant protein A binds Mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols

Surfactant Protein A (SP-A) is an abundant, multifunctional lectin that resides within the bronchoalveolar compartment of the lung and plays an important role in the innate immunity of the organ. Mycoplasma pneumoniae is a human pathogen that resides in the same compartment as SP-A, and we examined the interaction between the two. Preparations of human and rat SP-A recognized the mycoplasma with high affinity in the presence of Ca(2+), exhibiting apparent K(')(d) values in the nanomolar range. Membranes prepared from the microbe also bound human and rat SP-A with similar characteristics and affinity to the intact cells. The ligand for SP-A was insensitive to proteolysis. Lipid extracts prepared from the mycoplasma, bound SP-A with high affinity when examined by ligand blot analysis. These lipid extracts were also potent competitive inhibitors (IC(50) = 0.2 nM) of human SP-A binding to mycoplasma membranes. The major lipid ligands for the protein identified by mass spectrometry are a group of disaturated phosphatidylglycerols. The addition of SP-A to cultures of M. pneumoniae markedly attenuated the growth of the organism assessed by colony formation, metabolic activity, and DNA replication. The bacteriostatic effects of SP-A were reversed by dipalmitoylphosphatidylglycerol. These findings demonstrate that human SP-A can play a direct role in antibody-independent immunity to M. pneumoniae by interacting with lipid ligands expressed on the surface of the organism and implicate SP-A in the immediate host response to the bacteria.     

Molecular Cloning and Characterization of a Surface-Localized Adhesion Protein in Mycoplasma bovis Hubei-1 Strain

Mycoplasma bovis (M. bovis) is an important pathogen that causes various bovine diseases, such as mastitis in cows and pneumonia in calves. The surface proteins are generally thought to play a central role in the pathogenesis of this organism. We screened the entire genome of M. bovis Hubei-1 and discovered a gene named vpmaX that encodes the 25 kDa variable surface lipoprotein A (VpmaX). Sequence analysis revealed that VpmaX contains several repetitive units and a typical bacterial lipoprotein signal sequence. The vpmaX gene was cloned and expressed in E. coli to obtain recombinant VpmaX (rVpmaX). Western blot analysis using a rabbit antibody against rVpmaX demonstrated that VpmaX is a membrane protein. Immunostaining visualized via confocal laser scanning microscopy showed that rVpmaX was able to adhere to embryonic bovine lung cells (EBL), and this was also confirmed by a sandwich ELISA. In summary, a surface-localized adhesion protein was identified in M. bovis Hubei-1.     

Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.     

Effect of thermal selection of the male gametes on the resistance to temperature and chlorophos of the body and muscles in tadpoles of the common frog

A study was made of the effect of thermal selection of parental spermatozoa on the heat resistance of the whole organism and of muscles in progeny of Rana temporaria, as well as of the organismal resistance to pesticides (0.0025 M chlorophos). The thermal selection of spermatozoa was performed by exposing their suspension to 36 degrees C for 30-40 minutes. With tadpoles, the resistance of muscles to 38 degrees C, was determined, in addition to the resistance of the organism to 34 degrees and to 0.0025 M chlorophos. The results of selection were stage--specific: at stages of growth (stages 39, 42, 50) the heat resistance of the organism was lower and that of muscles was higher than in controls. At the stage of metamorphosis proper (stage 52) the heat resistance of both the organism and muscles increased. The thermal selection of parental spermatozoa exerted no effect on the resistance of the progeny to 0.0025 M chlorophos.     

Characteristics of S Organism Isolated from Methanobacillus omelianskii

Previous work showed that Methanobacillus omelianskii was a mixed culture of an ethanol-oxidizing organism called S organism and a hydrogen-utilizing methane bacterium, strain MOH. S organism grows poorly on ethanol unless a hydrogen-utilizing methanogenic bacterium is included to utilize the H(2) produced during growth. Further studies have shown that, among many substrates tested, only ethanol, n-propanol, n-butanol, isobutanol, n-pentanol, acetaldehyde, oxalacetate, and pyruvate are fermented by S organism, either alone or in combination with Methanobacterium ruminantium. It grew better in pure culture with pyruvate than with alcohols. H(2) gas phase inhibited growth on pyruvate as well as on alcohol. When grown alone on pyruvate, S organism produced mainly acetate, ethanol, and CO(2), in addition to a small amount of H(2). When combined with M. ruminantium, no H(2) and very little ethanol were produced and acetate production was increased. When M. ruminantium was present, electrons from pyruvate oxidation by S organism were channeled almost entirely to H(2) and hence to methane formation rather than ethanol. Also, S organism utilized more pyruvate when grown with M. ruminantium. Attempts to obtain better growth of S organism on ethanol by addition of many possible electron acceptors were unsuccessful. It grew best between 32 and 45 C, had a per cent guanine plus cytosine content of deoxyribonucleic acid bases of 47.27 +/- 0.1, contained no cytochrome, and could be grown on a defined medium with pyruvate as the energy and carbon source and with (NH(4))(2)SO(4) as the main nitrogen source. These and other results suggest that S organism belongs in a new genus, but assignment of a definite taxonomic status should await isolation and characterization of more strains.     

The reducing antioxidant capacity of Mycoplasma fermentans

Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.     

Re-mapping Robert Rosen's (M,R)-systems

The central concern of this paper is to re-evaluate Rosen's replicating (M,R)-systems, presented in his book 'Life Itself ', where M and R signify metabolism and repair, respectively. We look anew at Rosen's model of an organism in the light of extensive research into natural hierarchical systems, and the paper presents conclusions drawn from a comparison between Rosen's relational model and that of a birational complementary natural hierarchy. We accept that Rosen's relational model provides a useful stepping stone to understanding the nature of life, but also suggest that it induces potentially digressive conclusions. We conclude that a binary segregation of relational assemblies into mechanisms and organisms is insufficient, and indicate how a threefold segregation throws new light on Rosen's model. An organism is not 'the complement of a mechanism': the complement of a mechanism is its ecosystem. An organism is the 'complex interface' between mechanism and ecosystem.