Janet L. Schottel

Painting and printing living bacteria: Engineering nanoporous biocatalytic coatings to preserve microbial viability and intensify reactivity

Latex biocatalytic coatings containing ∼50% by volume of microorganisms stabilize, concentrate and preserve cell viability on surfaces at ambient temperature. Coatings can be formed on a variety of surfaces, delaminated to generate stand‐alone membranes or formulated as reactive inks for piezoelectric deposition of viable microbes. As the latex emulsion dries, cell preservation by partial desiccation occurs simultaneously with the formation of pores and adhesion to the substrate. The result is living cells permanently entrapped, surrounded by nanopores generated by partially coalesced polymer particles. Nanoporosity is essential for preserving microbial viability and coating reactivity. Cryo‐SEM methods have been developed to visualize hydrated coating microstructure, confocal microscopy and dispersible coating methods have been developed to quantify the activity of the entrapped cells, and FTIR methods are being developed to determine the structure of vitrified biomolecules within and surrounding the cells in dry coatings. Coating microstructure, stability and reactivity are investigated using small patch or strip coatings where bacteria are concentrated 102‐ to 103‐fold in 5–75 μm thick layers with pores formed by carbohydrate porogens. The carbohydrate porogens also function as osmoprotectants and are postulated to preserve microbial viability by formation of glasses inside the microbes during coat drying; however, the molecular mechanism of cell preservation by latex coatings is not known. Emerging applications include coatings for multistep oxidations, photoreactive coatings, stabilization of hyperthermophiles, environmental biosensors, microbial fuel cells, as reaction zones in microfluidic devices, or as very high intensity (>100 g·L‐1 coating volume·h‐1) industrial or environmental biocatalysts. We anticipate expanded use of nanoporous adhesive coatings for prokaryotic and eukaryotic cell preservation at ambient temperature and the design of highly reactive “living” paints and inks.

Production and regulation of potato-scab-inducing phytotoxins by Streptomyces scabies

Summary: Phytotoxins with potato-scab-inducing activity were produced by the pathogenic Streptomyces scabies strain RB2 when grown on oatmeal agar medium or in oatmeal broth medium. Five compounds isolated from cell filtrates were identified as thaxtomin compounds 1-5, previously reported as produced by S. scabies grown on potato slices. The optimum temperature for phytotoxin production in oatmeal broth medium was 28 °C. Production of thaxtomin A, the major product, was repressed at least 130-fold when S. scabies RB2 was grown in oatmeal broth medium supplemented with 0.5% glucose. Thaxtomin A production was also repressed by tryptophan and tyrosine, precursors of which may be involved in feedback inhibition of early steps in biosynthesis. Phytotoxins were secreted by the organism when the cells reached late exponential to early stationary phases of culture growth. The time during growth at which each thaxtomin compound was produced suggests a pathway for the latter steps in thaxtomin biosynthesis.

A single-use luciferase-based mercury biosensor using Escherichia coli HB101 immobilized in a latex copolymer film.

A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 μm thick whereas the bottom layer of copolymer plus E. coli cells was 30 μm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl2 in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl2. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at −20°C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl2.

Effects of alterations in the translation control region on bacterial gene expression use of cat gene constructs transcribed from the lac promoter as a model system

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-to-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.

A patch coating method for preparing biocatalytic films of Escherichia coli

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.

Effect of Pathogen Isolate, Potato Cultivar, and Antagonist Strain on Potato Scab Severity and Biological Control

Antibiotic-producing Streptomyces spp. have shown potential in biocontrol of potato scab caused by Streptomyces scabies. However, results have been inconsistent among field trials. Pathogen isolate, antagonist strain and potato cultivar were investigated as potential sources of variation in the efficacy of potato scab biocontrol. Biocontrol success varied significantly among pathogen isolates and was not correlated with in vitro sensitivity to antibiotic inhibition. Antagonists also varied in their effectiveness as biocontrol agents, and the relative effectiveness of different antagonists varied among growing seasons. Finally, biocontrol varied among potato cultivars in the field. The diverse origins of significant variation in potato scab biocontrol suggest that consistent control in the field is likely to be difficult to achieve.

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery.

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T(m)), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T(m) from 8.4+/-1.7 degrees C (in undried control samples) to 16.5+/-1.3 degrees C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T(m), which began approximately 1 day after initial rehydration and plateaued at 12.5+/-1.8 degrees C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T(m) and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.

Isolation and characterization of an antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII

An antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII has been isolated and partially characterized. The antibiotic is produced throughout culture growth, with maximum amounts accumulating in the broth when the culture is in the early stationary phase of growth. The activity declines within about 30 h after the culture enters stationary phase. Purification techniques included chromatography on Amberlite XAD-2, DEAE Sephadex and SP Sephadex in addition to C18 HPLC with an average yield of 75%. This antibiotic only inhibits pathogenic strains of S. scabies that cause scab disease on potato and other tuberous vegetables and does not affect S. griseus, S. venezuelae, Actinomyces bovis, Nocardia asteroides, Clostridium perfringens, Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, Micrococcus luteus, Serratia marcescens and Escherichia coli. The antibiotic has a molecular weight of 500 or less, and is stable for weeks at acidic pH but is very labile at alkaline pH conditions.

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli

The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course of Escherichia coli culture growth. mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle. The half-lives of cat mRNA and total mRNA also increase three- to fourfold during amino acid starvation when compared to exponential culture growth. Even though the stability of the cat message changes about fourfold during culture growth, the amount of cat mRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes in cat gene transcription. Translation of cat mRNA also changes during culture growth. In exponential phase, the rate of cat translation is about 14-fold higher than when the culture is in stationary phase. This is in contrast to the fourfold increase in stability of cat mRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability. When a stationary-phase culture was diluted into fresh medium, there was a five- to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin. These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited.

Mercuric reductase enzymes from Streptomyces species and group B Streptococcus

Mercury volatilization (Hg2+ reductase) activity has been found with Hg2+-resistant isolates of three Streptomyces species and with three Hg2+-resistant strains of group B Streptococcus from clinical sources in Japan. Hg2+ reductase activities in crude cell extracts showed the temperature sensitivity, the requirement for an added thiol compound and the characteristic dependence on NAD(P)H cofactors of similar enzymes isolated from other bacteria.