Comparison of methods for isolating Campylobacter jejuni from raw milk.

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Comparison of enrichment broths for isolation of Campylobacter jejuni

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

Comparison of enrichment broths for isolation of Campylobacter jejuni.

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

Comparison of enrichment methods and atmosphere modification procedures for isolating Campylobacter jejuni from foods.

A comparison was made of enrichment broths for recovery of Campylobacter jejuni from food by the methods of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353) and of Park et al. (Can. J. Microbiol. 27:841-842). No significant differences were found between the results obtained with the two broths. Recovery was greater, however, with a constant gas flow into the broths than with an evacuation-replacement method.     

Comparison of enrichment methods and atmosphere modification procedures for isolating Campylobacter jejuni from foods

A comparison was made of enrichment broths for recovery of Campylobacter jejuni from food by the methods of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353) and of Park et al. (Can. J. Microbiol. 27:841-842). No significant differences were found between the results obtained with the two broths. Recovery was greater, however, with a constant gas flow into the broths than with an evacuation-replacement method.     

Use of the valine biosynthetic pathway to convert glucose into isobutanol

Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259–2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also—as has been demonstrated previously (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009)—used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.     

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).     

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).     

Substrate induction and metabolite accumulation during anaerobic toluene utilization by the denitrifying strain T1.

The denitrifying strain T1 utilizes toluene anaerobically. We now report that anaerobic toluene degradation is inducible in strain T1. Fluoracetate treatment of cell suspensions inhibited both the rate of toluene metabolism and the formation of the toluene dead-end products benzylsuccinate and benzylfumarate, which is consistent with the pathway proposed by Evans et al. (Appl. Environ. Microbiol. 58:496-501, 1992). In addition, when either nitrate was limiting or fluoroacetate was added, benzoate was detected during toluene metabolism.     

Evaluation of a fluorescent lectin-based staining technique for some acidophilic mining bacteria

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245-2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.     

Viability of heterotrophic bacteria in the Bay of Marseilles

Marine microorganism activities are commonly assessed by bulk methods and assigned to the total cell count. The presence in significant amounts of ghost, dead, and damaged cells makes such as assignment a non-correct one. A Nucleic Acid Double Staining protocol (NADS) of fresh water bacteria (Barbesti et al., Cytometry 40 (2000) 214-218) has been adapted to resolve viable, damaged and dead cells in marine environments (Grégori et al., Appl. Environ. Microbiol. 67 (2001) 4662-4670). The present reports the first in situ application of this approach, conducted in the Bay of Marseilles in winter and spring periods at two sites with contrasted features.     

Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD?? of 1.62 to 5.05 μg, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab α-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of α-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.     

Heterologous gene expression in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: β-galactosidase,dibenzothiophene monooxygenase,PNB carboxy esterase, 2-aminobiphenyl-2,3-diol dioxygenase,and chloramphenicol acetyl transferase

Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus (Fridjonsson et al. in J Bacteriol 184:3385–3391, 2002, Koyama et al. in Appl Environ Microbiol 56:2251–225, 1990, Lasa et al. in J Bacteriol 174:6424–6431, 1992, Mathew et al. in Appl Environ Microbiol 58:421–425, 1992, Takagi et al. in J Ind Microbiol Biotechnol 23:214–217, 1999, Tamakoshi et al. in Extremophiles 5:17–22 2001), the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile (bglA from Pyrococcus woesei), a cofactor-requiring enzyme (dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme (carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.     

Optimized generation of vectors for the construction of Haloferax volcanii deletion mutants

A method for the construction of in frame deletions in chromosomal genes of the archaeon Haloferax volcanii has been described recently (Allers et al. (2004), Appl. Environ. Microbiol. 70:943-953). Two steps of the procedure for deletion vector construction were optimized. First, the deletion version of the gene of interest was generated by fusion of two PCR products, which were comprised of the upstream and downstream regions of the gene, respectively, using a third PCR reaction. Second, the fusion PCR product was ligated with the optimized vector pMH101 in the presence of one of four possible restriction enzymes ("restriction selection cloning"). Taken together, the optimized procedure omits one cloning step and enhances both speed and efficiency of deletion vector construction considerably.     

Transduction of porcine enteropathogenic Escherichia coli with a derivative of a shiga toxin 2-encoding bacteriophage in a porcine ligated ileal loop system

In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Phi3538 (Deltastx(2)::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Phi3538 (Deltastx(2)::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.     

Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity.     

Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity.