Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp.

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.

Effects of low temperature, cold shock, and various carbon sources on esterase and lipase activities and exopolysaccharide production by a psychrotrophic Acinetobacter sp.

The activities of isocitrate lyase, esterase, and lipase by the psychrotrophic Acinetobacter sp. strain HH1-1 were monitored during incubation at 25 degreesC, 5 degreesC, and after a 25 degreesC to 5 degreesC down shift in growth temperature. During growth at 25 degreesC, isocitrate lyase activity was detected in cell-free extracts, but at 5 degreesC and after cold shock, activity was measured primarily in the cell culture supernatant. Strain HH1-1 produced two cell-associated esterases and an extracellular esterase and lipase. Activities of the extracellular esterase and lipase were reduced when cells were grown at 5 degreesC and after cold shock. In contrast, an increased synthesis of a 53-kDa cell-associated esterase was observed 50 h after cold shock. An extracellular polysaccharide was also produced, indicated by a decrease in surface tension in cell culture supernatant when cells were incubated at 25 degreesC; but like extracellular enzyme activity, production of the exopolymer was reduced when cells were subjected to low temperatures. These results indicated that the intracellular enzyme, isocitrate lyase, leaked out of the cell after cold shock and during growth at 5 degreesC. The increased activity of a cell-associated esterase suggested this enzyme is required for growth at low temperatures. In contrast, activities of extracellular lipolytic enzymes and production of an extracellular polysaccharide were negatively affected at the lower temperatures.

Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15.

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.

Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.

The psychorotrophic Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 degrees C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 degrees C. Mineralization of hexadecane at 5 degrees C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis the A gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 degrees C.

Cold shock proteins and cold acclimation proteins in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant.

The production of cold shock proteins (csps) and cold acclimation proteins (caps) was characterized in the psychrotrophic bacterium Pseudomonas putida Q5 and its transconjugant P. putida Q5T which contains the toluene-degradative TOL (pWWO) plasmid, using two-dimensional gel electrophoresis and computing scanning laser densitometry. Similar growth rates for the psychrotrophic bacterium P. putida Q5 and the transconjugant were found at temperatures ranging from 30 to 0 degree C. Sixteen proteins were quantified and compared in P. putida Q5 and P. putida Q5T following a 25 to 5 degrees C cold shock or constant growth at 5 degrees C. During constant growth at 25 degrees C, a decrease in the synthesis of various proteins occurred in the transconjugant. Following cold shock to 5 degrees C or constant growth at 5 degrees C, csps and caps were produced with a greater number occurring in the transconjugant. This may suggest an additional stress response in the transconjugant owing to metabolic load exerted by the TOL plasmid. Growth of P. putida Q5T with toluate produced seven proteins that appeared to be TOL-plasmid mediated and of which some were also designated as caps.

Assessment of the biodegradation potential of psychrotrophic microorganisms.

Bioremediation of polluted temperate and cold temperature environments may require the activity of psychrotrophic bacteria, because their low temperature growth range parallels the ambient temperatures encountered in these environments. In the present study, 135 psychrotrophic microorganisms isolated from a variety of ecosystems in Canada were examined for their ability to mineralize 14C-labelled toluene, naphthalene, dodecane, hexadecane, 2-chlorobiphenyl, and pentachlorophenol. A number of the psychrotrophic strains mineralized toluene, naphthalene, dodecane, and hexadecane. None of the psychrotrophs were capable of mineralizing 2-chlorobiphenyl or pentachlorophenol. Those strains demonstrating mineralization activity were subsequently screened by the polymerase chain reaction (PCR) and Southern hybridization of PCR products for the presence of catabolic genes (alkB, ndoB, todCl, and xylE) involved in known bacterial biodegradative pathways for these compounds. Some of the psychrotrophs able to mineralize toluene and naphthalene possessed catabolic genes that hybridized to xylE or todCl, and ndoB, respectively. The alkB PCR fragments obtained from the strains that mineralized dodecane and hexadecane did not hybridize to an alkB gene probe derived from Pseudomonas oleovorans. Psychrotrophic strain Q15, identified as a Rhodococcus sp., also mineralized the C28 n-paraffin octacosane. A gene probe constructed from the "alkB" PCR fragment from strain Q15 did hybridize with the alkB PCR fragments from most of the psychrotrophic alkane biodegraders, indicating that the alkB primers may be amplifying another gene(s), perhaps with low homology to P. oleovorans alkB, which may be involved in the biodegradation of both short chain (dodecane) and longer chain alkanes (hexadecane, octacosane). All of the psychrotrophic biodegradative isolates examined were capable of mineralization activity at both 23 and 5 degrees C, indicating their potential for low temperature bioremediation of petroleum hydrocarbon contaminated sites.

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium.

A psychrotrophic bacterium, originally isolated from a natural aquatic environment, was characterized and identified as Pseudomonas putida Q5 for use as a representative recipient for biodegradative genes from a mesophilic microorganism. The TOL plasmid pWWO of the mesophile P. putida PaW1 was successfully transferred by conjugation to the naturally isolated psychrotroph P. putida Q5, as shown by plasmid analysis by agarose gel electrophoresis. Expression of the genes encoded by the mesophilic TOL plasmid in the psychrotroph was shown by the fact that the transconjugant (designated P. putida Q5T) had the capacity to degrade and utilize toluate (1,000 mg/liter) as a sole source of carbon at temperatures as low as 0 degrees C. Comparison of growth rates over a wide temperature range (0 to 30 degrees C) indicated that the physiological activity of the transconjugant was not reduced and that the plasmid DNA from the mesophile and its encoded enzymes functioned effectively in the psychrotroph at temperatures well below those at which the mesophile could grow. The production and demonstrated functioning of P. putida Q5T illustrates the possibility of developing specific degradative capacities in bacteria which can readily function at low temperatures in chemically contaminated environments or in industrial wastewater treatment systems.

Microbial decomposition of leaf material at 0°C.

The microbial decomposition of leaves (both fresh and autumnshed) at 0°C using stream sediment-water was investigated. The maximum rates of loss of leaf carbohydrate and protein at 0°C were considerable, being about 40% of those at 20°C. These rates were only slightly affected by the type of leaf material present being 1.3-fold higher with fresh leaves as compared with autumn-shed leaves. In addition, an epifluorescence microscopic counting technique was developed and utilized to enumerate the microbial populations colonizing the decomposing leaves. The average microbial densities on fresh and autumn-shed leaves after 35 days of incubation were 1.3 × 10(6) and 9.0 × 10(5) microorganisms cm(-2) at 0°C as compared with 5.5 × 10(6) and 3.3 × 10(6) microorganisms cm(-2) at 20°C, respectively. Antibacterial and antifungal antibiotics were used to estimate the comparative involvement of sediment bacteria and fungi in leaf degradation.

Effect of sulfide on nitrogen fixation in a stream sediment-water system.

Nitrogen fixation (C(2)H(2) reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C(2)H(4) production occurring in the presence of 8 mumol of S per ml of stream water. Sodium sulfide at concentrations of 16 mumol of S per ml or greater inhibited nitrogen fixation, with 64 mumol of S per ml being completely inhibitory. Sulfide at levels of 16 mumol/ml or above inhibited CO(2) production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO(2) production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 mumol of S per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H(2), H(2)S, and other sulfur species at various pH values, it appeared that H(2)S was the toxic entity and that HS was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS from H(2)S.

Effect of sequence of exposure to chlorophenols in short-term bacterial bioassays.

A bioassay using Pseudomonas fluorescens was affected by the sequence of exposure to pentachlorophenol (PCP) and 2,3,4,5-tetrachlorophenol (TCP). Surviving cells from standardized cell suspensions initially treated with PCP at concentrations ranging from 10 to 75 micrograms/ml, followed by removal of the toxicant, were not affected by a second dose of PCP at the same concentration. However, if the second dose was TCP, the test organism was sensitive to the second exposure. The most toxic sequence was an initial exposure to TCP followed by a second exposure to PCP. The response of the test organism to PCP and TCP was clearly dependent upon both the toxicant concentrations used and the sequence of toxicant addition.

Measurement of Electron Transport System (ETS) activity in soil.

Electron transport system (ETS) activity was measured in amended and nonamended soil by measuring the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan), which can be easily extracted with methanol without interference from other compounds found in soil. A high correlation between ETS activity and oxygen consumption was observed. This technique allows rapid quantitative measurements of microbial ETS activity in soil.

Nitrogen fixation and methane metabolism in a stream sediment-water system amended with leaf material.

The reduction of acetylene and the production and oxidation of methane in a stream sediment--water system amended with either fresh leaves or autumn-shed leaves in the presence and absence of air were studied. Net methane production by the sediment--water system occurred only when leaf material was added, with fresh leaves giving 2.2 times the methane accumulation as autumn-shed leaves. Static incubation in the presence of air had little effect on net methane production, with such production being about the same as (with fresh leaves) or 79% of (with autumn-shed leaves) the anaerobic rates. Acetylene reduction was more affected by the presence of air, with anaerobic rates being at very low levels. The presence of fresh leaves was again stimulatory, with the acetylene reduction rate being 5.5-fold greater than that occurring with autumn-shed leaves.

Perturbation of lipid membranes by organic pollutants.

The ability of a range of organic pollutants--hexachlorobenzene, mirex(1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachlorooctahydro-1,3,4-metheno-1H cyclobuta(cd) pentalene), 1,3,5-trichlorobenzene, 2,4,6-trichlorophenol, p-nitrophenol, p-chlorophenol, DDT, and pentachlorophenol--to perturb liposomes of dipalmitoyl phosphatidylcholine (DPPC) has been measured by differential scanning calorimetry. The degree of perturbation was measured by the increase in breadth of the main DPPC phase transition in both heating and cooling scans. DDT and the phenol derivatives were effective perturbers of phospholipid, broadening the transition by as much as 12-fold. Hexachlorobenzene and mirex did not perturb at all when mixed with DPPC at concentrations as high as 20 mol%, although 1,3,5-trichlorobenzene caused slight broadening of the main transition at this concentration. Perturbation is facilitated by the presence of a hydroxyl group on the benzene ring and hindered by increasing degrees of chloride substitution. An apparent correlation exists between the extent of phospholipid perturbation measured by differential scanning calorimetry and LD50 values for these compounds taken from the literature. This suggests the possibility of formulating an "index of perturbation" which could be used to screen certain classes of organic compounds for potential biological toxicity on a routine basis.

Cold-sensitive mutant with defective growth at 5 degrees C from a psychrotrophic bacterium.

A cold-sensitive (CS) mutant of the psychrotroph, Bacillus psychrophilus, was obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenization and penicillin counterselection. In the presence of citrate, the wild-type grew well at both 5 and 20 degrees C whereas the CS mutant grew well at 20 degrees C (the permissive temperature) but, at 5 degrees C (the restrictive temperature), grew at a reduced rate for two to three generations followed by a complete plateau in growth. Upon return of the CS mutant to 20 degrees C, after a delay of about 40 h, growth resumed at the appropriate rate. The CS mutant exhibited growth rates similar to parental rates on a wide variety of carbon sources at 5 degrees C, but when Krebs cycle intermediates were used as substrates and in the presence of an equimolar amount of citrate, the typical cold-sensitive growth pattern occurred. Comparison of oxidative phosphorylation in the parent and CS mutant indicated that no phosphorylation occurred at 5 degrees C in the CS mutant during the plateau in growth. Examination of the effect of temperature on ATPase activity showed that at 5 degrees C the specific activity of ATPase isolated from the CS mutant grows at 5 degrees C was 15-fold less than the ATPases isolated from wild-type cells grown at either 5 or 20 degrees C and 10.5-fold lower than ATPase from CS mutant cells grown at 20 degrees C. The large reduction in CS mutant ATPase activity at 5 degrees C appears to be at least partly due to an effect on synthesis since citrate did not inhibit preformed ATPase.