Oligotrophic Growth Research Articles

Adaptation mechanisms of Alcanivorax facilitating its predominance in marine environments

IntroductionAlcanivorax, a typical alkane-degrading bacterium, has demonstrated the ability to utilize inorganic electron donor in some reports. However, a comprehensive analysis of its potentiality to utilize inorganic electron donor is still lacking.MethodsIn this study, genomic and phylogenetic analyzes were used to explore the potential oxidative capacity of inorganic compounds in Alcanivorax. And its functions were verified through physiological experiments.ResultsThe sulfur oxidation-related genes sqr and tsdA are prevalent and have various evolutionary origins. Potential genes for CO oxidation were present in 39 strains, whereas genes associated with iron, hydrogen, and ammonia oxidation were either rare or absent. The physiological functions of Sqr and TsdA were confirmed in six representative strains under heterotrophic conditions. Adding thiosulfate enhanced Alcanivorax growth. However, Alcanivorax bacteria perform sulfide detoxification through Sqr rather than by gaining energy via sulfide oxidation Although no strain was confirmed to be chemoautotrophs, we discovered that the two clades, A. xenomutans and A. profundimaris, can grow under conditions with very low organic matter.DiscussionThe ability to utilize inorganic compounds as a supplementary energy source and adapt to carbon oligotrophic growth may contribute to the prevalence of Alcanivorax in marine ecosystems.

Mechanistic insights into sulfur source-driven physiological responses and metabolic reorganization in the fuel-biodesulfurizing Rhodococcus qingshengii IGTS8.

Comparative proteomics and untargeted metabolomics were combined to study the physiological and metabolic adaptations of Rhodococcus qingshengii IGTS8 under biodesulfurization conditions. After growth in a chemically defined medium with either dibenzothiophene (DBT) or MgSO4 as the sulfur source, many differentially produced proteins and metabolites associated with several metabolic and physiological processes were detected including the metabolism of carbohydrates, amino acids, lipids, nucleotides, vitamins, protein synthesis, transcriptional regulation, cell envelope biogenesis, and cell division. Increased production of the redox cofactor mycofactocin and associated proteins was one of the most striking adaptations under biodesulfurization conditions. While most central metabolic enzymes were less abundant in the presence of DBT, a key enzyme of the glyoxylate shunt, isocitrate lyase, was up to 26-fold more abundant. Several C1 metabolism and oligotrophy-related enzymes were significantly more abundant in the biodesulfurizing culture. R. qingshengii IGTS8 exhibited oligotrophic growth in liquid and solid media under carbon starvation. Moreover, the oligotrophic growth was faster on the solid medium in the presence of DBT compared to MgSO4 cultures. In the DBT culture, the cell envelope and phospholipids were remodeled, with lower levels of phosphatidylethanolamine and unsaturated and short-chain fatty acids being the most prominent changes. Biodesulfurization increased the biosynthesis of osmoprotectants (ectoine and mannosylglycerate) as well as glutamate and induced the stringent response. Our findings reveal highly diverse and overlapping stress responses that could protect the biodesulfurizing culture not only from the associated sulfate limitation but also from chemical, oxidative, and osmotic stress, allowing efficient resource management. IMPORTANCE Despite decades of research, a commercially viable bioprocess for fuel desulfurization has not been developed yet. This is mainly due to lack of knowledge of the physiology and metabolism of fuel-biodesulfurizing bacteria. Being a stressful condition, biodesulfurization could provoke several stress responses that are not understood. This is particularly important because a thorough understanding of the microbial stress response is essential for the development of environmentally friendly and industrially efficient microbial biocatalysts. Our comparative systems biology studies provide a mechanistic understanding of the biology of biodesulfurization, which is crucial for informed developments through the rational design of recombinant biodesulfurizers and optimization of the bioprocess conditions. Our findings enhance the understanding of the physiology, metabolism, and stress response not only in biodesulfurizing bacteria but also in rhodococci, a precious group of biotechnologically important bacteria.

Oligotrophic Gene Expression in Rhodococcus erythropolis N9T-4 under Various Nutrient Conditions.

Rhodococcus erythropolis N9T-4 is a super oligotroph that grows on an inorganic basal medium without any additional carbon and nitrogen sources and requires CO2 for its oligotrophic growth. Previously, we found that two genes, aldA and mnoA, encoding NAD-dependent aliphatic aldehyde dehydrogenase and N,N′-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase, respectively, were highly upregulated under oligotrophic conditions. In this study, we constructed reporter plasmids containing an enhanced green fluorescent protein gene under aldA or mnoA promoters (pAldA and pMnoA, respectively). Fluorescence analysis of N9T-4 cells with reporter plasmids revealed that tryptone and yeast extract strongly repressed the expression of oligotrophy-connected genes, whereas the effect of casamino acids was moderate. Furthermore, remarkably high expression of aldA and mnoA was observed when the reporter strains were grown in media containing primary alcohols, particularly ethanol. Malic acid repressed ethanol-induced gene expression, suggesting that C2 metabolism is involved in the oligotrophic growth of N9T-4. The regulation of oligotrophic gene expression elucidated in this study could provide appropriate conditions for the production of useful compounds in an oligotrophic microbial process.

Effects of Pb-, Cd-resistant bacterium Pantoea sp. on growth, heavy metal uptake and bacterial communities in oligotrophic growth substrates of Lolium multiflorum Lam.

Phosphate-solubilizing bacteria (PSB) can accelerate phytoremediation, especially in those fertilized soils. However, PSB function in oligotrophic growth substrates remains poorly studied. In this study, we isolated lead (Pb)- and cadmium (Cd)-resistant PSB from contaminated sandy soil at an abandoned lubricant plant. The isolated Pantoea sp. PP4 (PP4 hereafter) can produce organic acid and IAA (Indole-3-acetic acid) and dissolve up to 238mg/L of inorganic phosphate Ca2(PO4)3, exhibiting biosorption capability for Pb and bioprecipitation for Pb and Cd. In the sand pot experiment, inoculation of PP4 increased the accumulation of Pb and Cd in Lolium multiflorum Lam. by 28.9% and 95.5%, respectively, and increased the available phosphorous in oligotrophic river sand by 30.8% (P < 0.05). Meanwhile, the growth of Lolium multiflorum Lam. was also stimulated, resulting in 89.2%, 57.1%, 184.6%, and 28.5% increase in fresh weight, dry weight, root length, and shoot length, respectively. NMDS analysis showed that the bacterial communities in river sand were more clustered after inoculation with PP4. These results indicated that the application of Pantoea sp. PP4 can facilitate the phytoremediation of Pb and Cd in oligotrophic growth substrates, forming a convergent bacterial community. Our findings highlighted the importance of identifying ideal PSB to improve phytoremediation efficiency in oligotrophic environments.

Effect of csn2 gene deficiency on the starvation tolerance and the synthesis of extracellular polysaccharides in oligotrophic environment of Streptococcus mutans

Objective: To explore the effect of csn2 gene deficiency on starvation tolerance and extracellular polysaccharides (EPS) synthesis in an oligotrophic environment of Streptococcus mutans (Sm). Methods: The csn2 gene deletion strains and complementary strains of Sm were cultivated and then an oligotrophic growth environment for Sm growth by setting different concentration gradient media were created. Cell growth in oligotrophic environment was detected by growth curve. Biofilm volume was measured by crystalline violet staining. Scanning electron microscopy (SEM) and laser confocal microscope were performed to observe the biofilm structure of Sm. The synthesis of EPS was measured by the anthrone-sulfuric acid method. The expression of genes related to EPS synthesis was evaluated by quantitative real-time PCR (qRT-PCR). Results: The growth curve results showed that the deletion of csn2 gene inhibited the growth of Sm under starvation stress. Furthermore, the results of laser confocal microscope showed that the biofilm EPS/bacteria ratios produced by the wild-type strain, csn2 gene-deficient strain and complement strains under nutrient sufficient culture conditions were 0.44±0.07, 1.05±0.13 and 0.57±0.08 respectively, while the ratios of EPS/bacteria in an oligotrophic environment were 0.93±0.24, 3.05±0.21 and 1.32±0.46 respectively, indicating that the deletion of csn2 gene enhanced the ability of extracellular polysaccharide synthesis of Sm in the oligotrophic environment. The expression levels of EPS synthesis-related genes gtfB and gtfC were up-regulated by 2.5 fold and 1.8 fold respectively and the expression level of gtfD was down-regulated by two-thirds. Conclusions: The csn2 gene deficiency showed multiple effects on the physiological functions and virulence characteristics of Sm, including starvation tolerance and EPS synthesis. These changes might be related to the shift of the complex regulative network caused by csn2 gene deletion.

Biochar reduced extractable dieldrin concentrations and promoted oligotrophic growth including microbial degraders of chlorinated pollutants

The role of organic amendments for natural degradation of aged persistent organic pollutants (POPs) in agricultural soils remains controversial. We hypothesised that organic amendments enhance bacterial activity and function at the community level, facilitating the degradation of aged POPs. An incubation study was conducted in a closed chamber over 12 months to assess the effects of selected organic amendments on extractable residues of aged dieldrin. The role of bacterial diversity and changes in community function was explored through sequenced marker genes. Linear mixed effect models indicated that, independent of amendment type, cumulative CO2 release was negatively associated with decreases in dieldrin concentration, by up to 7% per µmol CO2–C respired by microorganisms. The addition of poultry litter led to the highest daily carbon mineralisation, which was associated with low dieldrin dissipation after 9 months. In comparison, biochar resulted in significant decreases in extractable dieldrin residues over time, which coincided with shifts towards aerobic, oligotrophic, gram-negative bacteria, some with dehalogenation metabolism, and with increased potentials for biosynthesis of membrane components such as fatty acids and high redox quinones. The results supported an alternative theory that labile carbon promoted blooms of copiotrophic growth, which suppressed the required community-level traits and oligotrophic diversity to degrade chlorinated pollutants.

Cprp-An Unusual, Repetitive Protein Which Impacts Pleuromutilin Biosynthesis in the Basidiomycete Clitopilus passeckerianus.

Interrogation of an EST database for Clitopilus passeckerianus identified a putative homolog to the unusual stress response gene from yeast; ddr48, as being upregulated under pleuromutilin production conditions. Silencing of this gene, named cprp, produced a population of transformants which demonstrated significantly reduced pleuromutilin production. Attempts to complement a Saccharomyces cerevisiae ddr48 mutant strain (strain Y16748) with cprp were hampered by the lack of a clearly identifiable mutant phenotype, but interestingly, overexpression of either ddr48 or cprp in S. cerevisiae Y16748 led to a conspicuous and comparable reduction in growth rate. This observation, combined with the known role of DDR48 proteins from a range of fungal species in nutrient starvation and stress responses, raises the possibility that this family of proteins plays a role in triggering oligotrophic growth. Localization studies via the production of a Cprp:GFP fusion protein in C. passeckerianus showed clear localization adjacent to the hyphal septa and, to a lesser extent, cell walls, which is consistent with the identification of DDR48 as a cell wall-associated protein in various yeast species. To our knowledge this is the first study demonstrating that a DDR48-like protein plays a role in the regulation of a secondary metabolite, and represents the first DDR48-like protein from a basidiomycete. Potential homologs can be identified across much of the Dikarya, suggesting that this unusual protein may play a central role in regulating both primary and secondary metabolism in fungi.

Light stimulates anoxic and oligotrophic growth of glacial Flavobacterium strains that produce zeaxanthin.

Bacteria that inhabit glaciers usually produce carotenoids. Here, we report that a group of zeaxanthin-producing glacial Flavobacterium exhibited light-promoted growth. Of the tested 47 strains, 45 showed increased growths but two died under illumination at 50 μmol photon m-2 s-1. Light stimulation occurred mainly in either anoxic or nutrient-poor cultures, while the same levels of light promotion were found for that grown at 14 and 7 °C. Pigment assays identified overrepresentative zeaxanthin but trace retinal in the light promoted 45 strains, while flexirubin was exclusively in the light-lethal two. Genomic analysis revealed the gene cluster for zeaxanthin synthesis in the 45 strains, in which 37 strains also harbored the proteorhodopsin gene prd. Transcriptomic analysis found that light-induced expressions of both the zeaxanthin synthesis and proteorhodopsin genes. Whereas, deletion of the prd gene in one strain did not diminish light promotion, inhibition of zeaxanthin synthesis did. In comparison, no light promotion was determined in a glacier Cryobacterium luteum that produced a non-zeaxanthin-type carotenoid. Therefore, light stimulation on the glacial Flavobacterium is mostly likely related to zeaxanthin, which could provide better photoprotection and sustain membrane integrity for the organisms living in cold environments.

Identification of a transcriptional regulator for oligotrophy-responsive promoter in Rhodococcus erythropolis N9T-4.

Two genes, aldA, and mnoA, encoding an NAD-dependent aliphatic dehydrogenase and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase, respectively, are strongly expressed when Rhodococcus erythropolis N9T-4 is grown under oligotrophic conditions. In this study, we found a transcriptional regulator required for the transcription of both aldA and mnoA. The transcriptional regulator was also found to be essential for the oligotrophic growth of N9T-4.

Functional analysis of putative transporters involved in oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, which is an extremely oligotrophic bacterium, can survive in a completely inorganic medium with no additional carbon source. This bacterium utilizes atmospheric CO2, but does not require any additional energy source such as light and hydrogen gas, required by autotrophic microorganisms. However, its CO2 fixation and energy-acquisition systems in the oligotrophic growth remain unrevealed. We expected N9T-4 to have the transporter(s) that imports essential compound(s) for its oligotrophic growth. Three putative ATP-binding cassette (ABC) transporters were found to be highly upregulated under oligotrophic conditions. We constructed the gene-deletion mutants of a gene encoding the substrate-binding protein for each ABC transporter (∆sbp1, ∆sbp2, and ∆sbp3). Among these mutants, ∆sbp1 showed growth defects on oligotrophic medium without carbon source. We examined the growth of the mutants on the oligotrophic medium containing 1% trehalose as a sole carbon source. The results exhibited worse growth of ∆sbp3 than that of the control strain (∆ligD), whereas intracellular trehalose content of all mutants decreased compared with that of ∆ligD. It was reported that trehalose functions as the mycolate carrier to the arabinogalactan layer in the cell wall of Mycobacterium tuberculosis. Transmission electron microscopic analysis of ∆sbp1 cells showed that an outermost envelope of the ∆sbp1 cell diminished, which was expected to be mycolate layer. From these results, we suggest that the same trehalose-recycling system as that in a Mycobacterium cell functions in the oligotrophic growth of N9T-4, and the ABC transporter comprising Sbp1 as the substrate-binding protein is strongly involved in the oligotrophic growth of N9T-4.

Extreme slow growth as alternative strategy to survive deep starvation in bacteria

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call ‘oligotrophic growth state’, provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions.

Establishment of an effective oligotrophic cultivation system for Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4 grows on an inorganic solid-state medium with no additional carbon and energy sources; however, it is unable to grow well in a liquid culture medium under the oligotrophic conditions. We examined submerged cultivations of N9T-4 using a polyurethane foam sponge to achieve approximately 10 times of the oligotrophic growth of the bacterium in the liquid culture medium.

Aura-biomes are present in the water layer above coral reef benthic macro-organisms.

As coral reef habitats decline worldwide, some reefs are transitioning from coral- to algal-dominated benthos with the exact cause for this shift remaining elusive. Increases in the abundance of microbes in the water column has been correlated with an increase in coral disease and reduction in coral cover. Here we investigated how multiple reef organisms influence microbial communities in the surrounding water column. Our study consisted of a field assessment of microbial communities above replicate patches dominated by a single macro-organism. Metagenomes were constructed from 20 L of water above distinct macro-organisms, including (1) the coral Mussismilia braziliensis, (2) fleshy macroalgae (Stypopodium, Dictota and Canistrocarpus), (3) turf algae, and (4) the zoanthid Palythoa caribaeorum and were compared to the water microbes collected 3 m above the reef. Microbial genera and functional potential were annotated using MG-RAST and showed that the dominant benthic macro-organisms influence the taxa and functions of microbes in the water column surrounding them, developing a specific “aura-biome”. The coral aura-biome reflected the open water column, and was associated with Synechococcus and functions suggesting oligotrophic growth, while the fleshy macroalgae aura-biome was associated with Ruegeria, Pseudomonas, and microbial functions suggesting low oxygen conditions. The turf algae aura-biome was associated with Vibrio, Flavobacterium, and functions suggesting pathogenic activity, while zoanthids were associated with Alteromonas and functions suggesting a stressful environment. Because each benthic organism has a distinct aura-biome, a change in benthic cover will change the microbial community of the water, which may lead to either the stimulation or suppression of the recruitment of benthic organisms.

A unique intracellular compartment formed during the oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, isolated from stored crude oil, shows extremely oligotrophic features and can grow on a basal medium without any additional carbon, nitrogen, sulfur, and energy sources, but requires CO2 for its oligotrophic growth. Transmission electron microscopic observation showed that a relatively large and spherical compartment was observed in a N9T-4 cell grown under oligotrophic conditions. In most cases, only one compartment was observed per cell, but in some cases, it was localized at each pole of the cell, suggesting that it divides at cell division. We termed this unique bacterial compartment an oligobody. The oligobody was not observed or very rarely observed in small sizes under nutrient rich conditions, whereas additional carbon sources did not affect oligobody formation. Energy dispersive X-ray spectroscopy analysis revealed remarkable peaks corresponding to phosphorus and potassium in the oligobody. The oligobodies in N9T-4 cells could be stained by Toluidine blue, suggesting that the oligobody is composed of inorganic polyphosphate and is a type of acidocalcisome. Two genes-encoding polyphosphate kinases, ppk1 and ppk2, were found in the N9T-4 genome: ppk1 disruption caused a negative effect on the formation of the oligobody. Although it was suggested that the oligobody plays an important role for the oligotrophic growth, both ppk-deleted mutants showed the same level of oligotrophic growth as the wild-type strain.

Bioprospecting of facultatively oligotrophic bacteria from non-rhizospheric soils

Conventional cultivation method relies on the use of nutrient-rich medium, which might limit the exploration of oligotrophic bacteria from soil ecosystems. In the present study attempts were made to isolate oligotrophic bacteria from non-rhizospheric samples collected from mountain roadside, beach, bay and wetland using 1000-fold diluted nutrient agar. Phylogenetic analysis and biogeographic studies of these isolates were performed based on their 16S rDNA sequences. Besides, tests for the utilization of methanol or agar as their carbon source (methylotrophic or agarolytic), light as their energy source (photoheterotrophic), fixation of nitrogen or solubilization of tricalcium phosphate were conducted. A total of 74 isolates with facultatively oligotrophic behavior were obtained, since they can proliferate on both 1000-fold diluted and conventional nutrient agar. 16S rDNA sequence analyses assigned them mainly to class Actinobacteria and α-Proteobacteria. All these isolates belonged to 24 genera which encompassed 35 species, demonstrating that these facultatively oligotrophic bacteria occupied a wide range of bacterial lineages. Fifty-seven out of 74 isolates formed colonies on methanol-containing agar, while only 21 showed better growth in methanol-containing medium. Nine isolates were verified to utilize agar instead of methanol for growth. A total of 4 isolates showed photoheterotrophic trait and the pufLM gene was successfully PCR amplified and sequenced. The methylotrophic or photoheterotrophic behaviors might provide advantages for them to inhabit oligotrophic environments. Members affiliated with many genera were first demonstrated to fix nitrogen or solubilize phosphate, and isolates with profound activities have potentials to be developed as bioinoculants used to promote plant growth. In addition, the use of diluted nutrient agar helped to explore several unrecognized species from non-rhizospheric soils. All these isolates may provide opportunities for studying their oligotrophic growth, metabolism and interaction with plants in a near future.

Rufibacter quisquiliarum sp. nov., a new member of the phylum Bacteroidetes isolated from a bioreactor treating landfill leachate.

A novel bacterium, CAI-18bT, was isolated from a bioreactor that treated landfill leachate using an oligotrophic growth medium. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CAI-18bT is a member of the genus Rufibacter, showing 97.1 % pairwise similarity to Rufibacter roseus H359T, 96.4 % to Rufibacter tibetensis 1351T, 96.4 % to Rufibacter glacialis MDT1-10-3T and 96.0 % to Rufibacter immobilis MCC P1T. Strain CAI-18bT was rod-shaped, motile, oxidase- and catalase-positive. The predominant fatty acids were iso-C15 : 0 (24.1 %) and iso-C17 : 1 I (22.3 %), the major respiratory quinone was MK-7, and the predominant polar lipids were phosphatidylethanolamine and an unknown aminophospholipid. The G+C content of the genomic DNA of strain CAI-18bT was 50.7 mol%. The novel bacterium can be distinguished from related type strains based on its ability to assimilate N-acetylglucosamine and gentiobiose. On the basis of the phenotypic, chemotaxonomic and molecular data, strain CAI-18bT represents a novel species of the genus Rufibacter, for which the name Rufibacter quisquiliarum sp. nov. is proposed. The type strain is CAI-18bT (=DSM 29854T=NCAIM B.02614T).

The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO2 and forms its colonies on an inorganic basal medium (BM) without any additional carbon source. Screening of a random mutation library constructed by a unique genome deletion method that we established indicated that the aceA, aceB, and pckG genes encoding isocitrate lyase, malate synthase, and phosphoenolpyruvate carboxykinase, respectively, were requisite for survival on BM plates. The aceA- and aceB deletion mutants and the pckG deletion mutant grew well on BM plates containing L-malate and D-glucose, respectively, suggesting that the glyoxylate (GO) shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4. Interestingly, most of the enzyme activities in the TCA cycle were observed in the cell-free extract of N9T-4, with perhaps the most important exception being α-ketoglutarate dehydrogenase (KGDH) activity. Instead of the KGDH activity, we detected a remarkable level of α-ketoglutarate decarboxylase (KGD) activity, which is the activity exhibited by the E1 component of the KGDH complex in Mycobacterium tuberculosis. The recombinant KGD of N9T-4 catalyzed the decarboxylation of α-ketoglutarate to form succinic semialdehyde (SSA) in a time-dependent manner. Since N9T-4 also showed a detectable SSA dehydrogenase activity, we concluded that N9T-4 possesses a variant TCA cycle, which uses SSA rather than succinyl-CoA. These results suggest that oligotrophic N9T-4 cells utilize the GO shunt to avoid the loss of carbons as CO2 and to conserve CoA units in the TCA cycle.

Utilization of atmospheric ammonia by an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth and requires CO2 for its growth. In this report, nitrogen sources for the oligotrophic growth of N9T-4 were examined. As is true for most other bacteria, N9T-4 preferred ammonium salt to nitrate as the nitrogen source on an inorganic minimum medium without carbon sources. Interestingly, N9T-4 could also grow on the minimal medium solidified by agarose or silica gel without carbon and nitrogen sources, suggesting that this bacterium is also oligotrophic for nitrogen. We can rule out the possibility of diazotrophic growth of this bacterium, because nitrogenase activity was not detected in the cells and the putative gene encoding nitrogenase was not found in N9T-4 genome. DNA microarray analysis revealed that one of the ammonium transporter genes (amtB) was strongly upregulated 40-50 fold higher under oligotrophic conditions than under heterotrophic conditions. Disruption of amtB led to a growth defect under nitrogen-limiting conditions. Furthermore, additional ammonia vapor enhanced the growth of N9T-4 on the minimum medium without nitrogen sources in a closed culture system. These results suggest that N9T-4 utilizes the trace amount of atmospheric ammonia as the nitrogen source.

Reversible and Irreversible Pollutant-Induced Bacterial Cellular Stress Effects Measured by Ethidium Bromide Uptake and Efflux

Chemical pollution is known to affect microbial community composition but it is poorly understood how toxic compounds influence physiology of single cells that may lay at the basis of loss of reproductive fitness. Here we analyze physiological disturbances of a variety of chemical pollutants at single cell level using the bacterium Pseudomonas fluorescens in an oligotrophic growth assay. As a proxy for physiological disturbance we measured changes in geometric mean ethidium bromide (EB) fluorescence intensities in subpopulations of live and dividing cells exposed or not exposed to different dosages of tetradecane, 4-chlorophenol, 2-chlorobiphenyl, naphthalene, benzene, mercury chloride, or water-dissolved oil fractions. Because ethidium bromide efflux is an energy-dependent process any disturbance in cellular energy generation is visible as an increased cytoplasmic fluorescence. Interestingly, all pollutants even at the lowest dosage of 1 nmol/mL culture produced significantly increased ethidium bromide fluorescence compared to nonexposed controls. Ethidium bromide fluorescence intensities increased upon pollutant exposure dosage up to a saturation level, and were weakly (r(2) = 0.3905) inversely correlated to the proportion of live cells at that time point in culture. Temporal increase in EB fluorescence of growing cells is indicative for toxic but reversible effects. Cells displaying high continued EB fluorescence levels experience constant and permanent damage, and no longer contribute to population growth. The procedure developed here using bacterial ethidium bromide efflux pump activity may be a useful complement to screen sublethal toxicity effects of chemicals.

Gene Expression Analysis of Methylotrophic Oxidoreductases Involved in the Oligotrophic Growth ofRhodococcus erythropolisN9T-4

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO₂ without any additional carbon or energy source. We performed a gene expression analysis of the oxidoreductases, which are involved in methanol metabolism, under various growth and induction conditions in N9T-4. A real-time PCR analysis revealed that the genes encoding NAD-dependent formaldehyde dehydrogenase (nFADH) and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase (MDH) were strongly expressed under the oligotrophic conditions at levels of 20-100 fold those under heterotrophic conditions, in which n-tetradecane was used as the sole carbon source, while glucose did not affect the gene expression pattern in a minimum medium. The genes encoding mycothiol-dependent formaldehyde dehydrogenase (mFADH) and formate dehydrogenase were not induced under oligotrophic conditions, although mFADH expression was observed when formaldehyde was added to the induction medium. These results suggest that N9T-4 had three distinct formaldehyde oxidation systems, and that MDH and nFADH were the key enzymes in its oligotrophic growth.