Growth of Bacteria Degrading Naphthalene and Salicylate at Low Temperatures

A total of 58 bacterial strains degrading naphthalene and salicylate were isolated from soil samples polluted with oil products, collected in different regions of Russia during winter and summer. The isolates were assessed for their ability to grow at low temperatures (4, 8, and 15°C); bacteria growing at 4°C in the presence of naphthalene or salicylate accounted for 65 and 53%, respectively, of the strains isolated. The strains differed in the temperature dependence of their growth rates. It was demonstrated that the type of expression of the Nah⁺ phenotype at low temperatures depended on the combination of host bacterium and plasmid.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

Preliminary study on relationships among strains forming a bacterial community selected on naphthalene from a marine sediment

Two bacterial strains were isolated from a bacterial community formed of nine strains, selected from a marine sediment on a seawater medium with naphthalene as sole carbon source. The two strains studied in the present work were the only strains of this community able to grow in pure culture on naphthalene; therefore, they were called "primary" strains. The seven other strains were maintained in the community by using metabolic intermediates of the two primary strains; they were called "auxiliary" strains. Regulation of naphthalene metabolism was studied for the two primary strains. They oxidized naphthalene into catechol, which was degraded only by the meta pathway. For Pseudomonas Lav. 4, naphthalene oxygenase and salicylate hydroxylase were inducible; catechol 2,3-dioxygenase was constitutive. For Moraxella Lav. 7, naphthalene oxygenase was constitutive; salicylate hydroxylase and catechol 2,3-oxygenase were inducible. The Moraxella strain carries two cryptic plasmids, about 63- and 85-kb in molecular size. In the bacterial community culture medium, Moraxella Lav. 7 prevented accumulation of 2-hydroxymuconate semialdehyde formed by Pseudomonas Lav. 4. The auxiliary strains take up formic, acetic, pyruvic, propionic, and succinic acids released by the two primary strains.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40 degrees C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Virulence and resistance markers in clinical and environmental Aeromonas strains isolated in Romania

The virulence and resistance (R) features of 37 Aeromonas strains from diarrheal cases and 150 from the aquatic environment (isolated during cold and warm season) were tested at different incubation temperatures (4 degrees C, 28 degrees C and 37 degrees C). When incubated at 4 degrees C temperature, the Aeromonasstrains isolated during the cold season expressed the highest number of virulence factors by comparison with the strains isolated during warm season and from diarrhoeal cases, the virulence spectrum increasing simultaneously with the incubation temperature (i.e. 28 degrees C and 37 degrees C) for all strains. Mucinase was the unique virulence factor constantly present in all three categories of strains at all three incubation temperatures. The aquatic as well as clinical strains exhibited similar R levels to ampicillin and colistin, while for the other tested antibiotics, the aquatic strains generally proved higher R levels than clinical strains, excepting cephtazidime. Plasmids of molecular weights ranging between 1904-21226 bp, were isolated in 36.5% of Aeromonas strains, some of them being correlated with specific R patterns. The large virulence spectrum correlated with high R in Aeromonas strains isolated from the aquatic medium is pleading for the significant role of these bacteria in the pathogenic potential of the natural reservoir possibly implicated in human pathology.     

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil.

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.     

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil.

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.     

IncP-7 naphthalene-degradative plasmids from Pseudomonas putida

Abstract Seven naphthalene-degrading and two naphthalene and camphor-degrading Pseudomonas putida strains were isolated from marine sediments. Most of them carried two plasmids, of molecular size 60 and 200 kb. The naphthalene and salicylate metabolism determinants were transferred to a P. putida strain by conjugation, and the transconjugants acquired either both plasmids or only the 200-kb one. These plasmids appear to belong to the IncP-7 group and encode for catabolism of naphthalene and salicylate, but not camphor.     

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

Nitrogen-fixing pseudomonads isolated from roots of plants grown in the canadian high arctic

Root-associated bacteria capable of reducing acetylene to ethylene (biological nitrogen fixation) were isolated from various native plants grown in the Canadian High Arctic. All the strains belonged to the genus Pseudomonas but varied in several physiological characteristics. The rates of acetylene reduction at 14 or 20 degrees C were higher than at 25 or 9 degrees C. Six strains reduced acetylene at 4 degrees C. All the strains exhibited chemotaxis to l-asparagine in semisolid agar at 4 to 25 degrees C. Eleven strains colonized roots of canola (Brassica campestris cv. Tobin) in field soil at population densities of log 4.3 to log 5.1 CFU/g of fresh root at 14 degrees C and log 4.0 to log 5.2 CFU/g of fresh root at 25 degrees C. Some of these nitrogen-fixing pseudomonad strains demonstrated a competitive advantage for root colonization over other rhizosphere bacteria at low temperatures. The combined capabilities of nitrogen fixation and root colonization by diazotrophic pseudomonads may be useful for the development of a biofertilizer inoculant for temperate and cold regions.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Structural and functional variability of genetic systems for catabolizing polycyclic aromatic hydrocarbons in Pseudomonas putida strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1 operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Effect of temperature,pH, and initial cell number on luxCDABE and nah gene expression during naphthalene and salicylate catabolism in the bioreporter organism Pseudomonas putida RB1353

One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V(max)) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V(max) during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V(max) during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1 degrees C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at approximately 35 degrees C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.     

Characterization of psychrotolerant heterotrophic bacteria from Finnish Lapland

A total of 331 aerobic heterotrophic bacterial strains were isolated from various ecosystems of Finnish Lapland (68-69 degrees N) including forest soil, arctic alpine-tundra soil, stream water, lake and mire sediments, lichen and snow algae. Whole cell fatty acid and 16S rRNA gene sequence analysis and microscopy indicated that the isolates were dominated by Gram-negative bacteria, while only 20 Gram-positive strains were isolated. Based on 16S rRNA gene sequences the isolates were members of alpha-, beta-, gamma-Proteobacteria, Gram-positives with low G+C content, Actinobacteria and the Cytophaga/Flexibacter/Bacteroides group. More than one-third of the isolates could be tentatively identified as Pseudomonas spp. which were particularly abundant in the alpine-tundra soils where they represented 60% of all isolates. Other frequently isolated Gram-negative taxa were Burkholderia sp., Collimonas sp., Pedobacter sp., Janthinobacter sp., Duganella sp., Dyella sp. and Sphingomonas sp. Growth temperature ranges and hydrolytic enzyme activities of selected ca.100 strains were screened. The strains were psychrotolerant growing generally at temperatures ranging from 0 to 30 degrees C, as 82% of the isolates grew at 0 degrees C while only 7% grew at 35 degrees C. Protease and lipase activities at 5 degrees C were detected in more than half of the strains while approximately 20% of the strains possessed amylase and/or cellulase activities.     

Protein Arp and protein H from group A streptococci. Ig binding and dimerization are regulated by temperature.

Cell surface proteins that bind to the Fc part of Ig are expressed by many strains of group A streptococci, an important human pathogen. Two such bacterial strains, AP4 and AP1, were shown to bind IgA and IgG, respectively, in a temperature-dependent manner. The binding of radiolabeled Ig to the bacterial cells was lower at 37 degrees C than at 22 and 4 degrees C. Similarly, protein Arp, the IgA-binding protein isolated from strain AP4, and protein H, the IgG-binding protein isolated from strain AP1, displayed a strong Ig-binding at 22 degrees C and lower temperatures, and virtually no binding at all at 37 degrees C. The effect was reversible: lowering of the temperature restored the binding and vice versa. A gradual shift between binding and nonbinding took place between 27 and 37 degrees C. Gel chromatography and velocity sedimentation centrifugation showed that protein Arp and protein H appeared as noncovalently associated dimers at 10 and 22 degrees C, and as monomers at 37 degrees C. These results strongly suggest that the dimerization of protein Arp and protein H, rather than the low temperature itself, yielded the strong Ig-binding of the proteins at 10 and 22 degrees C. Indeed, after covalent cross-linking of the dimers at 10 degrees C by incubation with low concentrations of glutaraldehyde, full Ig-binding was achieved even at 37 degrees C. A carboxyl-terminal proteolytic fragment of protein Arp, which completely lacked the IgA-binding capacity at any temperature, showed the same temperature-dependent dimerization as intact protein Arp, suggesting that the Ig-binding part of the protein is not required for dimerization. The implications of these results for the function of Ig-binding group A streptococcal proteins, and their role in the host-parasite relationship are discussed.     

Diversity of genetic systems responsible for biodegradation of naphthalene in Pseudomonas fluorescens strains

The genetic systems that are responsible for naphthalene catabolism were analyzed in 18 naphthalene-degrading Pseudomonas fluorescens strains isolated from oil-contaminated soils in different regions of Russia. It was found that thirteen strains contain plasmids, from 20 to 120 kb in size, at least five of which are conjugative and bear the catabolic genes responsible for the complete utilization of naphthalene and salicylate. Five plasmids belong to the P-7 incompatibility group, and two plasmids belong to the P-9 incompatibility group. The naphthalene biodegradation genes of P. fluorescens are highly homologous to each other. The study revealed a new group of the nahAc genes and two new variants of the nahG gene. The suggestion is made that the key genes of naphthalene biodegradation, nahAc and nahG, evolve independently and occur in P. fluorescens strains in different combinations.     

Cold stress induces switchover of respiratory pathway to lactate glycolysis in psychrotrophicRhizobium strains

Two psychrotrophic strains of Rhizobium, DDSS69, a non-cold acclimated strain, and ATR1, a cold acclimated strain, were subjected to cold stress. A 4-fold increase in the specific activity of lactate dehydrogenase (LDH) was characteristic for cold stressed cells of DDSS69, whereas ATR1 showed a higher LDH activity in general, which increased 1.5-fold under cold stress. Cold sensitive mutants of DDSS69 which could not grow below 15 degrees C, in contrast to the wild type which could grow at 5 degrees C, were isolated using Tn5-tagged mutagenesis. These mutants showed a 40% lower LDH activity than the wild type grown at 5 degrees C that was comparable to the wild type grown at 15 degrees C. High specific activity of succinic dehydrogenase (SDH) at 28 degrees C in both strains and mutants indicated that aerobic respiration via the citrate cycle is the normal mode of saccharide utilization. Shifts to lower temperatures decreased the specific activity of SDH. However, alcohol dehydrogenase (ADH) activity remained very low in both the strains and the mutants at low temperatures indicating that a shift from aerobic saccharide metabolism to anaerobic one under cold stress involves lactate glycolysis rather than alcohol fermentation. There was an increase in membrane-bound ATPase activity under cold stress which is correlated to higher LDH activity. These data show that, in psychrotrophic Rhizobium strains, cold stress induces a switchover of respiratory metabolism from aerobic to anaerobic pathway, especially lactate glycolysis.     

Isolation and characterization of two hydrocarbon-degrading Bacillus subtilis strains from oil contaminated soil of Kuwait

Two strains of hydrocarbon-utilizing bacteria were isolated from soil samples of the Kuwait Burqan oil field at a temperature of 37 degrees C. The bacteria were motile endospore-forming rods with slight differences in their metabolic patterns and 16S rRNA sequence. Vegetative cells of the strains designated as AHI and AHII had an ultrastructure typical of gram-positive bacteria and showed gram-positive staining. The bacteria did not show pigmentation. Best growth was observed at 37 degrees C at neutral pH and NaCl concentrations in the range of 5-10 g per l. Both strains were obligatory aerobic and developed on synthetic media with either Diesel fuel, n-decan or naphthalene as the sole carbon and energy source. No specific growth factors were required. On the basis of their morphological, physiological and biochemical features, as well as their 16S rRNA analysis and electron microscope study, both strains were assigned to the species of Bacillus subtilis.     

Bacteria--degraders of polycyclic aromatic hydrocarbons, isolated from soil and bottom sediments in salt-mining areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical and salt producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1-1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.