Mycobacterium Gilvum Research Articles

Energy metabolism in Mycobacterium gilvum PYR-GCK: insights from transcript expression analyses following two states of induction.

Mycobacterium gilvum PYR-GCK, a pyrene degrading bacterium, has been the subject of functional studies aimed at elucidating mechanisms related to its outstanding pollutant bioremediation/biodegradation activities. Several studies have investigated energy production and conservation in Mycobacterium, however, they all focused on the pathogenic strains using their various hosts as induction sources. To gain greater insight into Mycobacterium energy metabolism, mRNA expression studies focused on respiratory functions were performed under two different conditions using the toxic pollutant pyrene as a test substrate and glucose as a control substrate. This was done using two transcriptomic techniques: global transcriptomic RNA-sequencing and quantitative Real-Time PCR. Growth in the presence of pyrene resulted in upregulated expression of genes associated with limited oxygen or anaerobiosis in M.gilvum PYR-GCK. Upregulated genes included succinate dehydrogenases, nitrite reductase and various electron donors including formate dehydrogenases, fumarate reductases and NADH dehydrogenases. Oxidative phosphorylation genes (with respiratory chain complexes I, III –V) were expressed at low levels compared to the genes coding for the second molecular complex in the bacterial respiratory chain (fumarate reductase); which is highly functional during microaerophilic or anaerobic bacterial growth. This study reveals a molecular adaptation to a hypoxic mode of respiration during aerobic pyrene degradation. This is likely the result of a cellular oxygen shortage resulting from exhaustion of the oxygenase enzymes required for these degradation activities in M.gilvum PYR-GCK.

Toxic pyrene metabolism in Mycobacterium gilvum PYR-GCK results in the expression of mammalian cell entry genes as revealed by transcriptomics study.

Mycobacterium gilvum PYR-GCK is a bacterial strain under study for its bioremediation use on heavy hydrocarbon pollutants in the environment. During the course of our study, mammalian cell entry (mce) genes, known to facilitate pathogenicity in M. tuberculosis, were highly expressed during a comparative and substrate-related cultural global transcriptomic study. RNA sequencing of the global transcriptome of the test strain in two different substrates, pyrene and glucose, showed high expression of the mce genes based on the differential results. After validating the expression of these genes with quantitative real-time PCR, we arrived at the conclusion that the genes were expressed based on the pyrene substrate (a phytosterol compound), and sterol metabolism is said to activate the expression of the mce genes in some actinomycetes bacteria, M. gilvum PYR-GCK in this case. This study is believed to be important based on the fact that some mycobacterial strains are undergoing a continuous research as a result of their use in practical bioremediation of anthropogenic exposure of toxic organic wastes in the environment.

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondônia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

A Gene Expression Study of the Activities of Aromatic Ring-Cleavage Dioxygenases in Mycobacterium gilvum PYR-GCK to Changes in Salinity and pH during Pyrene Degradation

Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants found in the environment which can be removed through the use of physical and biological agents. The rate of PAH biodegradation is affected by environmental conditions of pH, salinity and temperature. Adaptation of the pyrene degrading bacteria, Mycobacterium gilvum PYR-GCK, to fluctuating environmental conditions during pyrene biodegrading activity was studied using the quantitative real time – Polymerase Chain Reaction (qRT-PCR) technique. Four aromatic ring-cleavage dioxygenase genes: phdF, phdI, pcaG and pcaH; critical to pyrene biodegradation, were studied in pH states of 5.5, 6.5, 7.5 and NaCl concentrations 0 M, 0.17 M, 0.5 M, 0.6 M, 1 M. First, we conducted a residual pyrene study using gas chromatography and flame ionization technologies. Central to a gene expression study is the use of a valid endogenous reference gene, making its determination our next approach, using the geNorm/NormFinder algorithms. Armed with a valid control gene, rpoB, we applied it to a gene expression study, using the comparative critical threshold (2ΔΔCT) quantification method. The pyrene degrading activity of the strain was strongly functional in all the NaCl concentration states, with the least activity found at 1M (∼70% degraded after 48 hours of cultivation). The transcripts quantification of three genes backed this observation with high expression levels. The gene expression levels also revealed pH 6.5 as optimal for pyrene degradation and weak degradation activity at pH of 5.5, corroborating the residual pyrene analysis. The expression of these genes as proteins has already been studied in our laboratory using proteomics techniques and this validates our current study.

A global proteome study of Mycobacterium gilvum PYR-GCK grown on pyrene and glucose reveals the activation of glyoxylate, shikimate and gluconeogenetic pathways through the central carbon metabolism highway

Various hydrocarbons have been released into the environment as a result of industrialization. An effective way of removing these materials without further environmental contamination is microbial bioremediation. Mycobacterium gilvum PYR-GCK, a bacteria isolated from a PAH polluted estuary, was studied using comparative shotgun proteomics to gain insight on its molecular activity while using pyrene and glucose as sole carbon and energy sources. Based on annotated genomic information, a confirmation analysis was first performed to confirm its pyrene degradation activity, using gas chromatography-mass spectrometry technology. One dimensional gel electrophoresis and liquid chromatography-mass spectrometry technologies employed in the proteomics analysis revealed the expression of pyrene degrading gene products along with upregulated expression of proteins functioning in the glyoxylate and shikimate pathways, in the pyrene-induced cells. The study also revealed the pathway of pyrene degraded intermediates, via partial gluconeogenesis, into the pentose phosphate pathway to produce precursors for nucleotides and amino acids biosynthesis.

Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined.

Detection of antibacterial-like activity on a silica surface: fluoroquinolones and their environmental metabolites

Background, scope, and aims Antibacterial fluoroquinolones (FQs) are third-generation antibiotics that are commonly used as therapeutic treatments of respiratory and urinary tract infections. They are used far less in intensively farmed animal production systems, though their use may be permitted in the veterinary treatments of flocks or in medicated feeds. When used, only a fraction of ingested parent FQ actually reaches the in vivo target site of infection, while the remainder is excreted as the parent FQ and its metabolized products. In many species’ metabolism, enrofloxacin (EF) is converted into ciprofloxacin (CF) while both FQs are classified as parent FQs in human treatments. It is therefore likely that both FQs and their metabolic products will contribute to a common pool of metabolites in biological wastes. Wastes from intensive farming practices are either directly applied to agricultural land without treatment or may be temporarily stored prior to disposal. However, human waste is treated in sewage treatment plants (STPs) where it is converted into biosolids. In the storage or treatment process of STPs, FQs and their in vivo metabolites are further converted into other environmental metabolites (FQEMs) by ex vivo physicochemical processes that act and interact to produce complex mixtures of FQEMs, some of which have antibacterial-like activities. Biosolids are then often applied to agricultural land as a fertilizer amendment where FQs and FQEMs can be further converted into additional FQEMs by soil processes. It is therefore likely that FQ-contaminated biowaste-treated soils will contain complex mixtures of FQEMs, some of which may have antibacterial-like activities that may be expressed on bacteria endemic to the receiving agricultural soil environment. Concern has arisen in the scientific and in the general community that repeated use of FQ-contaminated biowaste as fertilizer amendments of nutrient-impoverished agricultural land may create a selective environment in which FQ-resistant bacteria might grow. The likelihood of this happening will depend, to some extent, on whether bioactive FQEMs are first synthesized from the parent FQs by the action and interaction of in vivo and ex vivo processes producing bioactive FQEMs in biowastes and biosolids. The postulated creation of a selective environment will also depend, in part, on whether such bioactive FQEMs are biologically available to bacteria, which may, in turn, be influenced by soil type, amendment regime, and the persistence of the bioactive FQEMs. Additionally, soil bacteria and soil processes may be affected in different ways or extents by bioactive FQEMs that could possibly act additively or synergistically at ecological targets in these non-target bacteria. This is an important consideration, since, while parent FQs have well-defined ecological targets (DNA gyrase and topoisomerase IV) and modes of bactericidal action, the FQEMs and their possible modes of action on the many different species of soil bacteria is less well studied. It is therefore understandable that there is a lack of conclusive evidence directly attributing biosolid usage to any increase in FQ-resistant bacteria detected in biowaste-amended agricultural soil. However, a lack of evidence may simply imply that a causal relationship between biosolid usage programs and any detection of low levels of FQ-resistant bacteria in soils has yet to be established, rather than an assumption of no relationship whatsoever. Based on results presented in this paper, the precautionary principle should be applied in the usage of FQ-contaminated biosolids as fertilizer amendments of agricultural land. The aim of this research was to test whether any bioactive FQEMs of EF could be synthesized by aerobic fermentation processes using Mycobacterium gilvum (American Tissue Culture Collection) and a mixed culture of microorganisms derived from an agricultural soil. High-performance thin-layer chromatography (HPTLC) and bioautography were tested as screening techniques in the detection and analysis of bioactive FQEMs.

Environmental metabolites of fluoroquinolones: synthesis, fractionation and toxicological assessment of some biologically active metabolites of ciprofloxacin

Biowastes produced by humans and animals are routinely disposed of on land, and concern is now growing that such practices provide a pathway for fluoroquinolone (FQs) antibacterial agents and their environmental metabolites (FQEMs) to contaminate the terrestrial environment. The focus of concern is that FQs and FQEMs may accumulate in amended soils to then adversely impact on the terrestrial environment. One postulated impact is the development of a selective environment in which FQ-resistant bacteria may grow. To find evidence in support of an accumulation of antibacterial-like activity, it was first necessary to establish whether any biologically active FQEMs could be synthesized by physicochemical factors that are normally present in the environment. However, many FQEMs are not commercially available to be used as standards in such studies. FQEMs were therefore synthesized using well-defined processes. They were subsequently analyzed using spectroscopy (UV-vis) and high performance liquid chromatography with mass spectral detection. The antibacterial-like activities of fractionated FQEMs were then assessed in novel bacterial growth inhibition bioassays, and results were compared to those obtained from instrumental analyses. Parent FQs were either exposed to sunlight or were synthesized using defined aerobic microbial (Mycobacterium gilvum or a mixed culture derived from an agricultural soil) fermentation processes. Mixtures of FQEMs derived from photo- and (intracellular) microbial processes were isolated by preparative chromatography and centrifugation techniques, respectively. Mixtures were subsequently fractionated using analytical high-performance thin layer chromatography (HPTLC), and excised analytes were tested in bioautography assays for their antibacterial-like activities. Two bacteria, Escherichia coli (E. coli) and Azospirillum brasilense (A. brasilense) were used as reporter organisms in testing FQ standards and any subtle differences between biologically active FQEMs of ciprofloxacin (CF). FQEMs produced in the photo-synthetic process had UV-vis profiles that were indistinguishable from the parent FQs, and yet mass spectral data revealed the presence of N-formylciprofloxacin (FCF). In contrast, the UV-vis profiles of FQEMs synthesized by M. gilvum and a mixed culture of microorganisms had UV-vis profiles that were similar to one another and markedly different to the parent fluoroquinolones. Mass spectral studies confirmed the presence of FCF and N-acetylciprofloxacin in both microbial ferments. In addition, a photo-FQEM (Cp 6), three M. gilvum FQEMs (Cm 5, Cm 8, and Cm 10) and a mixed culture FQEM (Cs 6) of CF and many other FQEMs of CF, norfloxacin (NF), and enrofloxacin (EF) were fractionated using HPTLC, although their identities have yet to be confirmed. Differences between bioautography results were obtained when E. coli or A. brasilense were used as reporter organisms. Parent FQs (CF and EF) and the FQEMs of CF (Cp 6, Cm 8, and Cs 6) displayed antibacterial-like activity when using E. coli as the reporter organism. In contrast, A. brasilense was insensitive to parent CF and sensitive to EF and all tested FQEMs of CF. Results are consistent with photo- and microbial processes modifying CF in different ways, with the latter changing the UV-vis chromophores. It can be inferred that a lack of detection of analytes (especially photo-FQEMs) when using UV-vis does not necessarily indicate an absence of analyte. Additionally, similarities between the UV-vis profiles of FQEMs extracted from the (monoculture) M. gilvum and the mixed culture microbial aerobic ferments are consistent with similar processes operating in both ferments. Results of HPTLC and bioautography studies revealed that mixtures of (photo- and microbial) FQEMs could be fractionated into individual components. Bioactive FQEMs of ciprofloxacin, as a representative FQ, can be synthesized by photo- and microbial processes, and their detection required the use of both instrumental and bioautography analytical techniques. It is likely that such FQEMs will also be present on agricultural land that has been repeatedly amended with FQ-contaminated biosolids. The use of instrumental analytical techniques alone and especially photometric detection techniques will underestimate antibacterial-like activities of FQEMs. Moreover, the extraction technique(s) and the selected toxicological endpoint(s) require careful consideration when assessing bioactivity. It is therefore recommended that instrumental analytical techniques and several bioautography assays be performed concurrently, and bioautography assays should use a variety of reporter organisms. Two types of bacterial growth bioassays are recommended in any assessment of antibacterial-like activity derived from CF (and possibly from other FQs). A standardized E. coli bioassay should be used as a general screening procedure to facilitate intra- and inter-laboratory exchange of data. Additionally, soil-specific (region-specific) growth inhibition bioassays should be undertaken using several species of endemic soil bacteria. It is likely that the two sets of data will be useful in future risk assessment processes.

Complete genome sequence of Mycobacterium sp. strain (Spyr1) and reclassification to Mycobacterium gilvum Spyr1

Mycobacterium sp.Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated.

Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) Protein Domains Target LipY Lipases of Pathogenic Mycobacteria to the Cell Surface via the ESX-5 Pathway

The type VII secretion system ESX-5 is a major pathway for export of PE and PPE proteins in pathogenic mycobacteria. These mycobacteria-specific protein families are characterized by conserved N-terminal domains of 100 and 180 amino acids, which contain the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs after which they are named. Here we investigated secretion of the triacylglycerol lipase LipY, which in fast-growing mycobacteria contains a signal sequence, but in slow-growing species appears to have replaced the signal peptide with a PE or PPE domain. Selected LipY homologues were expressed in wild-type Mycobacterium marinum and its corresponding ESX-5 mutant, and localization of the proteins was investigated by immunoblotting and electron microscopy. Our study shows that Mycobacterium tuberculosis PE-LipY (LipY(tub)) and M. marinum PPE-LipY (LipY(mar)) are both secreted to the bacterial surface in an ESX-5-dependent fashion. After transport, the PE/PPE domains are removed by proteolytic cleavage. In contrast, Mycobacterium gilvum LipY, which has a signal sequence, is not transported to the cell surface. Furthermore, we show that LipY(tub) and LipY(mar) require their respective PE and PPE domains for ESX-5-dependent secretion. The role of the PE domain in ESX-5 secretion was confirmed in a whole cell lipase assay, in which wild-type bacteria expressing full-length LipY(tub), but not LipY(tub) lacking its PE domain, were shown to hydrolyze extracellular lipids. In conclusion, both PE and PPE domains contain a signal required for secretion of LipY by the ESX-5 system, and these domains are proteolytically removed upon translocation.

Accelerated biodegradation of pyrene and benzo[ a]pyrene in the Phragmites australis rhizosphere by bacteria–root exudate interactions

We investigated the biodegradation of pyrene and benzo[ a]pyrene in Phragmites australis rhizosphere sediment. We collected P. australis plants, rhizosphere sediments, and unvegetated sediments from natural aquatic sites and conducted degradation experiments using sediments spiked with pyrene or benzo[ a]pyrene. Accelerated removal of pyrene and benzo[ a]pyrene was observed in P. australis rhizosphere sediments with plants, whereas both compounds persisted in unvegetated sediments without plants and in autoclaved rhizosphere sediments with sterilized plants, suggesting that the accelerated removal resulted largely from biodegradation by rhizosphere bacteria. Initial densities of pyrene-utilizing bacteria were substantially higher in the rhizosphere than in unvegetated sediments, but benzo[ a]pyrene-utilizing bacteria were not detected in rhizosphere sediments. Mycobacterium gilvum strains isolated from rhizosphere sediments utilized pyrene aerobically as a sole carbon source and were able to degrade benzo[ a]pyrene when induced with pyrene. Phragmites australis root exudates containing phenolic compounds supported growth as a carbon source for the one Mycobacterium strain tested, and induced benzo[ a]pyrene-degrading activity of the strain. The stimulatory effect on benzo[ a]pyrene biodegradation and the amounts of phenolic compounds in root exudates increased when P. australis was exposed to pyrene. Our results show that Mycobacterium–root exudate interactions can accelerate biodegradation of pyrene and benzo[ a]pyrene in P. australis rhizosphere sediments.

Mycobacterium insubricum sp. nov.

Five independent strains, isolated from clinical samples but probably not responsible for disease, revealed phenotypic and genotypic features that appeared to exclude their belonging to any of the recognized Mycobacterium species. The strains, which are non-pigmented rapid growers, presented a cell-wall lipid pattern resembling those of Mycobacterium brumae and Mycobacterium fallax. Sequencing of the 16S rRNA, hsp65 and rpoB genes and the 16S-23S rRNA gene internal transcribed spacer (ITS) revealed that the strains are clearly distinct from every other Mycobacterium species. While the 16S rRNA and rpoB genes were characterized by a single sequevar, two sequevars were detected in hsp65 and three in the ITS. The divergence shown in the latter region was striking, in which only two short regions (less than 150 bp in all) were comparable with other mycobacteria, apart from Mycobacterium monacense and Mycobacterium gilvum. The PCR restriction analysis pattern of the novel strains also differs from any reported to date. The name Mycobacterium insubricum sp. nov. is proposed for the novel species; the type strain is FI-06250(T) (=DSM 45132(T) =CIP 109609(T)).

Surface Spreading Motility Shown by a Group of Phylogenetically Related, Rapidly Growing Pigmented Mycobacteria Suggests that Motility Is a Common Property of Mycobacterial Species but Is Restricted to Smooth Colonies

Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.

Surface motility of polycyclic aromatic hydrocarbon (PAH)-degrading mycobacteria

Surface motility of the polycyclic aromatic hydrocarbon (PAH)-degrading Mycobacterium gilvum VM552 was tested on agar and agarose plates prepared with varying amounts of gelling agents in the presence and absence of phenanthrene. Extensive spreading, originating from the point of inoculation, was observed on the surfaces of plates prepared with up to 0.3% agar and up to 0.6% agarose. The spreading velocities were 15.8 mm d −1 on 0.3% agar and 19.5 mm d −1 on 0.3% agarose plates. No evidence was found of accelerated or directed surface motility towards PAH crystals. The morphology of spreading M. gilvum VM552 colonies depended on both the carbon source and the type and concentration of the gelling agent. In 0.3% agar plates, M. gilvum VM552 cells were organized in 1–2-mm-wide branches of 1–5 cm length, while on agarose they slid as a homogenous monolayer across the surface. Microscopic inspection of the colonies on agar surfaces suggested that formation of branches was the combined effect of: (i) cell division and growth at the tip of a branch; (ii) propulsion of cells from the mature basal parts of a branch towards the tip; and (iii) physiologically induced reduced friction between cells and agar. Similar surface migration patterns were observed for the anthracene-degrading M. frederiksbergense LB501T.

Acetylation and nitrosation of ciprofloxacin by environmental strains of mycobacteria

To determine the ability of environmental bacteria to metabolize the frequently prescribed fluoroquinolone drug ciprofloxacin, eight Mycobacterium spp. cultures were grown for 4 days in a medium containing sorbitol and yeast extract with 100 mg x L(-1) ciprofloxacin. After the cultures had been centrifuged and the supernatants extracted with ethyl acetate, two metabolites were purified by using high-performance liquid chromatography. They were identified with liquid chromatography/electrospray ionization mass spectrometry and proton nuclear magnetic resonance spectroscopy. Ciprofloxacin was transformed to both N-acetylciprofloxacin (2.5%-5.5% of the total peak area at 280 nm) and N-nitrosociprofloxacin (6.0%-8.0% of the peak area) by Mycobacterium gilvum PYR-GCK and Mycobacterium sp. PYR100 but it was transformed only to N-acetylciprofloxacin by Mycobacterium frederiksbergense FAn9, M. gilvum ATCC 43909, M. gilvum BB1, Mycobacterium smegmatis mc2155, Mycobacterium sp. 7E1B1W, and Mycobacterium sp. RJGII-135. The results suggest that biotransformation may serve as a ciprofloxacin resistance mechanism for these bacteria.

ENHANCED KINETICS OF SOLID-PHASE MICROEXTRACTION AND BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN THE PRESENCE OF DISSOLVED ORGANIC MATTER

The uptake kinetics of fluorene, phenanthrene, fluoranthene, pyrene, and benzo[e]pyrene by solid-phase microextraction fibers was studied in the presence of dissolved organic matter (DOM) obtained from sediment pore water and resulted in increased fiber absorption and desorption rate coefficients. Compared to the control without DOM, these rate coefficients were increased at a DOM concentration of 36.62 mg/L by a factor of 1.27 to 2.21 and 1.31 to 2.10 for fluorene and benzo[e]pyrene, respectively. The calculated values for the fiber absorption and desorption rate coefficients show that diffusion through an unstirred boundary layer (UBL) surrounding the fiber probably forms the rate-limiting step of the process. The mineralization of aqueous-phase phenanthrene and pyrene by a representative polycyclic aromatic hydrocarbon (PAH)-degrading bacterium (Mycobacterium gilvum VM552) also was found to be enhanced by DOM. The initial degradation rates of phenanthrene (9.03 (microg/L) and pyrene (1.96 microg/L) were significantly higher compared to the control values and were enhanced by a factor of 1.32 and 1.26 at a DOM concentration of 43.14 and 42.15 mg/L, respectively. We suggest that such an enhancement results from the combination of faster uptake kinetics of the water-dissolved compounds in the UBL surrounding microbial cells and direct access of the bacteria to DOM-associated PAHs. These enhanced kinetic effects of DOM may have strong implications in sediment processes like desorption, nonequilibrium exposure, and biodegradation.

The Quantitative Structure-Mutagenicity Relationship of Polycylic Aromatic Hydrocarbon Metabolites

Quantitative structure-activity relationships (QSARs) for benz[a]anthracene (BA)mutagens using 73 descriptors were searched. The mutagenicity data was obtained fromAmes assays for Mycobacterium vanbaalenii, Mycobacterium gilvum and Mycobacteriumflavescens strains. These data were fitted using a mutagenicity-cytotoxicity competitionmodel which defines the mutagenic potencies of BA metabolites, and include oxides,phenols, quinones, and dihydrodiols. The QSAR equations were derived using the moleculardescriptor set (charged partial surface area, spatial, thermodynamic and electronicdescriptors) and semi-empirical energetic and charge descriptors. Genetic functionapproximation was used to reduce and fit independent variables, including linear- andquadratic-based functions. Multiple QSAR equations were generated and a separate QSARequation was chosen and evaluated for each strain using conventional r2, F-test, and cross-validated r2. Each strain exhibited its own characteristic descriptors.

Diversity of soil mycobacterium isolates from three sites that degrade polycyclic aromatic hydrocarbons

This paper investigates the diversity of polycyclic aromatic hydrocarbon (PAH)-degrading mycobacterium isolates from three different sites within United States: Montana, Texas and Indiana. All five mycobacterium isolates differed in chromosomal restriction enzyme-fragmentation patterns; three isolates possessed linear plasmids. The DNA sequence between the murA and rRNA genes were divergent but the sequence upstream of nidBA genes, encoding a dioxygenase involved in pyrene oxidation, was more highly conserved. Long-chain fatty acid analysis showed most similarity between three isolates from the same Montana site. All isolates were sensitive to rifampicin and isoniazid, used in tuberculosis treatment, and to syringopeptins, produced by plant-associated pseudomonads. Biofilm growth was least for isolate MCS that grew on plate medium as rough-edged colonies. The patterns of substrate utilization in Biolog plates showed clustering of the Montana isolates compared with Mycobacterium vanbaalenii and Mycobacterium gilvum. The five PAH-degrading mycobacterium isolates studied differ in genetic and biochemical properties. Different properties with respect to antibiotic susceptibility, substrate utilization and biofilm formation could influence the survival in soil of the microbe and their suitability for use in bioaugmentation.

Characterization of IS 1110-like sequences found in Mycobacterium species other than Mycobacterium avium

During study of a polycyclic aromatic hydrocarbon-degrading gene cluster in Mycobacterium sp. SNP11, which is a fast-growing strain related to Mycobacterium gilvum, a DNA segment with very high similarities to the IS 1110 element of Mycobacterium avium was identified. Insertion sequence IS 1110 was discovered for the first time in 1994 during a study of plasmid incidence in AIDS-derived M. avium strains. This element had thus far been detected in most human, veterinary, and environmental M. avium isolates, but not in other Mycobacterium species such as M. bovis, M. tuberculosis, M. xenopi, M. kansasii or M. gordonae. In the present paper, we describe the isolation and characterization of IS Mysp1, an IS 1110-like element present in several copies in the genome of Mycobacterium sp. strain SNP11. PCR and hybridization experiments revealed that this element is commonly found in fast-growing Mycobacterium strains. Moreover, Blast searches against the recently sequenced genome of M. smegmatis mc 2155 revealed that this strain contains four IS 1110-like elements. Analysis and sequence comparison of the whole of the IS 1110-like elements revealed several common features not found in the most closely related mycobacterial members, IS 900, IS 901, IS 902, IS 1626 and IS 1547.