Survival in Soil of Different Toluene-Degrading Pseudomonas Strains after Solvent Shock

We assayed the tolerance to solvents of three toluene-degrading Pseudomonas putida strains and Pseudomonas mendocina KR1 in liquid and soil systems. P. putida DOT-T1 tolerated concentrations of heptane, propylbenzene, octanol, and toluene of at least 10% (vol/vol), while P. putida F1 and EEZ15 grew well in the presence of 1% (vol/vol) propylbenzene or 10% (vol/vol) heptane, but not in the presence of similar concentrations of octanol or toluene. P. mendocina KR1 grew only in the presence of heptane. All three P. putida strains were able to become established in a fluvisol soil from the Granada, Spain, area, whereas P. mendocina KR1 did not survive in this soil. The tolerance to organic solvents of all three P. putida strains was therefore assayed in soil. The addition to soil of 10% (vol/wt) heptane or 10% (vol/wt) propylbenzene did not affect the survival of the three P. putida strains. However, the addition of 10% (vol/wt) toluene led to an immediate decrease of several log units in the number of CFU per gram of soil for all of the strains, although P. putida F1 and DOT-T1 subsequently recovered. This recovery was influenced by the humidity of the soil and the incubation temperature. P. putida DOT-T1 recovered from the shock faster than P. putida F1; this allowed the former strain to become established at higher densities in polluted sites into which both strains had been introduced.     

Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N₂O) reductase (N₂OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N₂O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N₂O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N₂OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N₂OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.     

Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10⁻⁶). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

Selected Pseudomonas putida Strains Able To Grow in the Presence of High Butanol Concentrations

Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe. P. putida might be an alternative host for biobutanol production, overcoming the primary limitation of currently used strains—insufficient product titers due to low butanol tolerance.The focus of biofuel production research has recently shifted from ethanol to bioenergy carriers that are more compatible with existing infrastructure (e.g., refineries, transport, and car engines). At the forefront is n-butanol (hereafter referred to as butanol) for which large-scale production processes have been implemented (16, 35). Existing fermentations, however, are limited in energetically attractive butanol titers, because butanol inhibits microbial growth at concentrations above 16 g/liter (2, 10). As reported for other organic solvents with low logarithm of the partition coefficient in a two-phase octanol/water system (log P_ow), this toxicity is due primarily to accumulation of butanol (log P_ow, 0.8) in the cell membrane and subsequent impairment (4, 17, 30, 33). With the maximum aqueous solubility of 0.97 M (8.8% [vol/vol]), the maximum membrane concentration of butanol was calculated to be 1.59 M (17), spotlighting its potential toxicity. The low achievable butanol titers have necessitated large reactor volumes, resulting in high purification costs (8, 15). Recent metabolic engineering strategies for improving biobutanol fermentation have focused on maximization of butanol production rates (10, 19), reducing the levels of by-products (20), finding alternative substrates (20), or finding alternative hosts (2, 12, 21, 31). However, recently engineered microbial strains (1, 14) have not overcome butanol toxicity.High organic solvent concentrations are tolerated by strains of the bacterial species Pseudomonas putida reported to grow in a second phase of octanol (25), toluene (13), or styrene (32). This suggests that solvent-tolerant P. putida strains withstand high butanol titers and therefore warrant exploitation as host for butanol production. Indeed, viable solvent-tolerant P. putida S12 cells were observed at butanol concentrations of up to 10% (vol/vol) by live-dead staining and fluorescence microscopy (5) (see supplemental material). We used growth as the parameter of interest, because growth in the presence of butanol directly indicates the potential of selected P. putida strains as hosts for recombinant butanol production.Three solvent-tolerant P. putida strains, DOT-T1E (23), S12 (32), and Pseudomonas sp. strain VLB120 (18), and the solvent-sensitive P. putida reference strain KT2440 (24) were examined for their ability to grow in the presence of butanol. Toxicity assays were performed in 96-well microtiter plates (System Duetz [7]) at 30°C and 300 rpm using glucose-supplemented LB and M9 media (with 10 and 5 g/liter glucose, respectively) (26). Higher glucose concentrations in LB medium did not increase butanol tolerance (data not shown). Butanol was added in all experiments to cells in the mid-exponential phase. Cell growth was monitored by changes in optical density, and substrate and butanol concentrations were analyzed by high-pressure liquid chromatography (Trentec 308R-Gel.H; VWR Hitachi). Comparable low butanol concentrations were withstood by all P. putida strains, with butanol tolerance highly dependent on the medium composition (Table ​(Table1).1). Growth was observed at butanol concentrations up to 3% (vol/vol), occurring in a culture of Pseudomonas sp. strain VLB120 using glucose-supplemented LB medium.

TABLE 1.

Tolerated butanol concentrations in different growth media

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Continuous Production of Long-Side-Chain Poly-β-Hydroxyalkanoates by Pseudomonas oleovorans

Shake flask experiments showed that Pseudomonas oleovorans began to be growth inhibited at 4.65 g of sodium octanoate liter^-1, with total inhibition at 6 g liter^-1. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter^-1 in the feed, the maximum specific growth rate was 0.51 h^-1, and the maximum specific rate of poly-β-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein^-1 h^-1 at a dilution rate (D) of 0.25 h^-1. When the specific growth rate (μ) was <0.3 h^-1, the PHA composition was relatively constant with a C₄/C₆/C₈/C₁₀ ratio of 0.1:1.7:20.7:1.0. At μ > 0.3 h^-1, a decrease in the percentage of C₈ with a concomitant increase in C₁₀ monomers as μ increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At D = 0.24 h^-1 and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate^-1 consumed, the yield of PHA was 0.085 g of PHA g of octanoate^-1 used, and 7.9 g of octanoate was consumed for each gram of NH₄⁺ supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass^-1 h^-1. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.     

Inactivation of Mycobacterium paratuberculosis by Pulsed Electric Fields

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50°C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log₁₀ CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50°C for 25 min or at 72°C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log₁₀ CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.     

Efflux Pumps Involved in Toluene Tolerance in Pseudomonas putida DOT-T1E

The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-′phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[¹⁴C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[¹⁴C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of ¹⁴C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.The sensitivity of microorganisms to toxic organic solvents is related to the logarithm of the partition coefficient of the solvent in a mixture of octanol and water (log P_ow). Aromatic hydrocarbons with a log P_ow of between 1.5 and 3.5 are extremely toxic to living organisms (47). These chemicals dissolve in the cytoplasmic membrane, disorganize it, and collapse the cell membrane potential; this, together with the induced loss of lipids and proteins, leads to irreversible damage resulting in the death of the cell (8, 47, 50).Independent laboratories have isolated Pseudomonas putida strains tolerant to different aromatic hydrocarbons such as toluene, styrene, and p-xylene (6, 15, 42, 48). All four isolated strains were able to grow in liquid culture medium to which a high concentration (1% [vol/vol]) of these aromatic hydrocarbons was added. Tolerance to organic solvents in these P. putida strains is achieved by a series of biochemical mechanisms that actively remove the organic solvent from cell membranes (16, 43) and by physical barriers that help the cell to become (to a certain degree) impermeable to the solvent (13, 37, 43, 48). The physical barriers involve the ordered organization of the cell surface lipopolysaccharides (37) together with modified phospholipids (4, 37, 43, 49). Modifications in phospholipids upon exposure to an organic solvent involve both a short-term response, in which the level of the trans isomers of unsaturated phospholipids increases, and a long-term response consisting of a modification of the polar head groups of phospholipids (4, 43, 49) and an increase in the total amount of phospholipids per dry weight (49). For P. putida DOT-T1, it was suggested that an energy-dependent exclusion system (such as an efflux pump) is critical for tolerance to solvents (43). This conclusion was based on the following findings: (i) P. putida DOT-T1 treated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone accumulated higher levels of 1,2,4-[¹⁴C]trichlorobenzene in cell membranes than did untreated cells, and (ii) P. putida DOT-T1 mutants which were sensitive to toluene, octanol, and other chemicals accumulated 5- to 20-fold-higher levels of 1,2,4-[¹⁴C]trichlorobenzene in cell membranes than did the wild-type strain. Similar observations have been reported for Pseudomonas sp. strain S12 (16).In this study, we report that P. putida DOT-T1 uses at least two efflux pumps for toluene exclusion, one that seems to be expressed constitutively and a second inducible one. A mini-Tn5′phoA-Km^r knocked out the constitutive efflux system of P. putida DOT-T1E. The mutant was shown to be hypersensitive to toluene but not to octanol. The Km^r marker of the mini-Tn5 and the 3′ adjacent chromosomal DNA were cloned, and the wild-type gene was rescued by colony screening hybridization and sequenced. Sequence analysis showed that the knocked-out gene in the mutant was a homolog of the mexB gene, which belongs to the efflux pump family of the resistant nodulator division type (34–36, 38–41).     

Inactivation of Ascaris Eggs in Source-Separated Urine and Feces by Ammonia at Ambient Temperatures

Sustainable management of toilet waste must prevent disease transmission but allow reuse of plant nutrients. Inactivation of uterus-derived Ascaris suum eggs was studied in relation to ammonia in source-separated urine without additives and in human feces to which urea had been added, in order to evaluate ammonia-based sanitation for production of safe fertilizers from human excreta. Urine was used concentrated or diluted 1:1 and 1:3 with tap water at 4, 14, 24, and 34°C. Fecal material, with and without ash, was treated with 1% or 2% (wt/wt) urea at 24 and 34°C. At 34°C eggs were inactivated in less than 10 days in urine and in amended feces. At 24°C only feces with 2% (wt/wt) urea or 1% (wt/wt) urea at high pH (10) inactivated all eggs within 1 month, and no inactivation was observed after 75 days in urine diluted 1:3 (18 ± 11 mM NH₃). At temperatures of ≥24°C, NH₃ proved to be an efficient sanitizing agent in urine and feces at concentrations of ≥60 mM. Treating fecal material at 34°C can give a 6-log₁₀ egg inactivation within 1 month, whereas at 24°C 6 months of treatment is necessary for the same level of egg inactivation. At temperatures of 14°C and below, inactivation rates were low, with viable eggs after 6 months even in concentrated urine.     

Stenoxybacter acetivorans gen. nov., sp. nov., an Acetate-Oxidizing Obligate Microaerophile among Diverse O2-Consuming Bacteria from Termite Guts

In termite hindguts, fermentative production of acetate—a major carbon and energy source for the insect—depends on efficient removal of inwardly diffusing oxygen by microbes residing on and near the hindgut wall. However, little is known about the identity of these organisms or about the substrate(s) used to support their respiratory activity. A cultivation-based approach was used to isolate O₂-consuming organisms from hindguts of Reticulitermes flavipes. A consistently greater (albeit not statistically significant) number of colonies developed under hypoxia (2% [vol/vol] O₂) than under air, and the increase coincided with the appearance of morphologically distinct colonies of a novel, rod-shaped, obligately microaerophilic β-proteobacterium that was <95% similar (based on the 16S rRNA gene sequence) to its closest known relative (Eikenella corrodens). Nearly identical organisms (and/or their 16S rRNA genes) were obtained from geographically separated and genetically distinct populations of Reticulitermes. PCR-based procedures implied that the novel isolates were autochthonous to the hindgut of R. flavipes and comprised ca. 2 to 7% of the hindgut prokaryote community. Representative strain TAM-DN1 utilized acetate and a limited range of other organic and amino acids as energy sources and possessed catalase and superoxide dismutase. On solid medium, the optimal O₂ concentration for growth was about 2%, and no growth occurred with O₂ concentrations above 4% or under anoxia. However, cells in liquid medium could grow with higher O₂ concentrations (up to 16%), but only after proportionately extended lag phases. The genetic and physiological distinctiveness of TAM-DN1 and related strains supports their recognition as a new genus and species, for which the name Stenoxybacter acetivorans gen. nov., sp. nov. is proposed.     

Oxidation of Methyl tert-Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n-Octane-Grown Pseudomonas putida GPo1

The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K_s value (20 to 40 mM) for MTBE.     

Biodesulfurization in Biphasic Systems Containing Organic Solvents

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium, Pseudomonas putida A4, was constructed by introducing the biodesulfurizing gene cluster dszABCD, which was from Rhodococcus erythropolis XP, into the solvent-tolerant strain P. putida Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents. P. putida A4 had the same substrate range as R. erythropolis XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells⁻¹ h⁻¹ for the first 2 h in the presence of 10% (vol/vol) p-xylene. P. putida A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.     

Method To Immobilize the Aphid-Pathogenic Fungus Erynia neoaphidis in an Alginate Matrix for Biocontrol

Erynia neoaphidis is an important fungal pathogen of aphid pests worldwide. There have been few reported attempts to formulate this natural agent for use in biocontrol. In the current study, factors involved in the immobilization of E. neoaphidis hyphae in an alginate matrix were investigated. Hyphae of two isolates cultured in liquid medium were 220 to 620 μm in length and 7 to 19 μm in diameter with a 74 to 83% cytoplasmic content. The optimal concentration of low-viscosity sodium alginate for production of conidia from entrapped hyphae was 1.5% (wt/vol), and 0.1 and 0.25 M calcium chloride were equally suitable for use as the gelling solution. Alginate beads were rinsed with 10% sucrose after gelling. However, beads should not be left for longer than 40 min in 0.1 M calcium chloride or 10% sucrose to prevent a 10% loss in conidial production. A 40% (vol/vol) concentration of fungal biomass produced significantly more conidia than either 20% or the standard concentration of 10%. This effect persisted even after beads were dried overnight in a laminar flow hood and stored at 4°C for 4 days. Conidia from freshly produced alginate beads caused 27 to 32% infection in Pea aphids as determined by standardized laboratory bioassays. This finding was not significantly different from infections in aphids inoculated with fresh mycelial mats or plugs from Petri dish cultures. In conclusion, algination appears to be a promising technique for utilizing E. neoaphidis in the biocontrol of aphid pests.     

Escherichia coli and Salmonella enterica Are Protected against Acetic Acid,but Not Hydrochloric Acid,by Hypertonicity

Chapman et al. (B. Chapman, N. Jensen, T Ross, and M. B. Cole, Appl. Environ. Microbiol. 72:5165-5172, 2006) demonstrated that an increased NaCl concentration prolongs survival of Escherichia coli O157 SERL 2 in a broth model simulating the aqueous phase of a food dressing or sauce containing acetic acid. We examined the responses of five other E. coli strains and four Salmonella enterica strains to increasing concentrations of NaCl under conditions of lethal acidity and observed that the average “lag” time prior to inactivation decreases in the presence of hydrochloric acid but not in the presence of acetic acid. For E. coli in the presence of acetic acid, the lag time increased with increasing NaCl concentrations up to 2 to 4% at pH 4.0, up to 4 to 6% at pH 3.8, and up to 4 to 7% (wt/wt of water) NaCl at pH 3.6. Salmonella was inactivated more rapidly by combined acetic acid and NaCl stresses than E. coli, but increasing NaCl concentrations still decreased the lag time prior to inactivation in the presence of acetic acid; at pH 4.0 up to 1 to 4% NaCl was protective, and at pH 3.8 up to 1 to 2% NaCl delayed the onset of inactivation. Sublethal injury kinetics suggest that this complex response is a balance between the lethal effects of acetic acid, against which NaCl is apparently protective, and the lethal effects of the NaCl itself. Compared against 3% NaCl, 10% (wt/wt of water) sucrose with 0.5% NaCl (which has similar osmotic potential) was found to be equally protective against adverse acetic acid conditions. We propose that hypertonicity may directly affect the rate of diffusion of acetic acid into cells and hence cell survival.We previously observed that inactivation of Escherichia coli O157 SERL 2 by acetic acid at adverse pH in a broth model simulating the aqueous phase of acidic sauces and dressings was reduced by the presence of NaCl (4). Specifically, the time to a 3-log₁₀-unit reduction (t_3D) of E. coli SERL 2 as function of NaCl concentration was significantly nonmonotonic; that is, the t_3D initially increased when NaCl was increased (from 1 to 3% [wt/wt] of solution), but the t_3D decreased upon a further increase in NaCl concentration (to 8% [wt/wt] of solution) (4). The statistical significance of this “nonmonotonic” response increased with increasing exposure time from 24 to 72 h (at 23°C), primarily due to a proportionally greater increase in inactivation at 1% (wt/wt) NaCl with increasing treatment time than that which was observed at higher NaCl concentrations (4).The combination of acid and NaCl is a common example of the food industry''s “hurdle” approach, which is used to preserve a large and diverse range of foods, including acidic dressings and sauces, fermented meats, cheeses, and preserved vegetables. Given the widespread use of this hurdle combination in food manufacturing, the first aim of this study was to determine whether the observed protection of E. coli SERL 2 from acid inactivation by NaCl is common among E. coli and Salmonella enterica and at what NaCl concentration maximum protection is achieved. A second aim was to determine whether NaCl protection is specific against acid pH in general or against acetic acid in particular. Third, possible protection against acid inactivation by another osmolyte, sucrose, was assessed to resolve whether the effect is solute specific.When cells are placed in hypertonic environments, plasmolysis occurs as the cytoplasmic volume reduces due to water loss by osmosis. The thin peptidoglycan layer of gram-negative microorganisms is anchored to the cytoplasmic membrane and can be distended by plasmolysis or even ruptured when plasmolysis is more extreme. Decad and Nikaido (5) observed that the cytoplasmic volume in gram-negative microorganisms was reduced to ∼50% at ∼0.3 M NaCl but that the plasmolysis-induced cell wall damage was minimal. At 0.5 M (2.9%, wt/wt) NaCl, however, they observed cell wall damage in a large fraction of cells. Thus, in the experiments described here we explored the mechanism of the protective effect of NaCl, and specifically cell wall damage in E. coli populations simultaneously exposed to NaCl and either acetic or hydrochloric acid (HCl), by enumeration of both injured and noninjured survivors by culture on media with and without bile salts.     

Sensitization of Listeria monocytogenes to Low pH, Organic Acids, and Osmotic Stress by Ethanol

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.     

Composition of Toluene-Degrading Microbial Communities from Soil at Different Concentrations of Toluene

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus Pseudomonas. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that Pseudomonas strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by Pseudomonas strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.     

A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter⁻¹. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C₈ fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.     

Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C₅ to C₁₀) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O₂ uptake activities, while acetate- and 3-methylcatechol-dependent O₂ uptake activities were unaffected. Toluene-dependent O₂ uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min⁻¹ to 2.45 min⁻¹. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H₂O₂, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent K_s and V_max values of 39.7 μM and 112.3 nmol min⁻¹ mg of protein⁻¹ for ethylene and 32.3 μM and 89.2 nmol min⁻¹ mg of protein⁻¹ for propylene.     

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T₅₀ in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k_cat/K_m ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter⁻¹. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.     

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “Protaminobacter rubrum”

“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a ³²⁵RLDRD³²⁹ motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg³²⁵ and Arg³²⁸ in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R³²⁵Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter⁻¹ and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter⁻¹) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g⁻¹ with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.