Biofilm adaptation to iron availability in the presence of biotite and consequences for chemical weathering.

Bacteria in nature often live within biofilms, exopolymeric matrices that provide a favorable environment that can differ markedly from their surroundings. Biofilms have been found growing on mineral surfaces and are expected to play a role in weathering those surfaces, but a clear understanding of how environmental factors, such as trace-nutrient limitation, influence this role is lacking. Here, we examine biofilm development by Pseudomonas putida in media either deficient or sufficient in Fe during growth on biotite, an Fe rich mineral, or on glass. We hypothesized that the bacteria would respond to Fe deficiency by enhancing biotite dissolution and by the formation of binding sites to inhibit Fe leaching from the system. Glass coupons acted as a no-Fe control to investigate whether biofilm response depended on the presence of Fe in the supporting solid. Biofilms grown on biotite, as compared to glass, had significantly greater biofilm biomass, specific numbers of viable cells (SNVC), and biofilm cation concentrations of K, Mg, and Fe, and these differences were greater when Fe was deficient in the medium. Scanning electron microscopy (SEM) confirmed that biofilm growth altered the biotite surface, smoothing the rough, jagged edges of channels scratched by hand on the biotite, and dissolving away small, easy-to-access particles scattered across the planar surface. High-resolution magic angle spinning proton nuclear magnetic resonance (HRMAS 1 H NMR) spectroscopy showed that, in the Fe-deficient medium, the relative amount of polysaccharide nearly doubled relative to that in biofilms grown in the medium amended with Fe. The results imply that the bacteria responded to the Fe deficiency by obtaining Fe from biotite and used the biofilm matrix to enhance weathering and as a sink for released cation nutrients. These results demonstrate one mechanism by which biofilms may help soil microbes overcome nutrient deficiencies in oligotrophic systems.

Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme-ligand complexes in solution, we have isolated the 26-kDa N-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate-3-phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N-terminal domain upon S3P binding.

Antifungal compounds from idioblast cells isolated from avocado fruits.

(E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene was isolated from avocado, Persea americana Mill., idioblast cells. It inhibited spore germination of the fungal pathogen Colletotrichum gloeosporioides. Full characterization is also reported for two additional compounds that have been described and partially characterized previously.

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.

5-O-(alpha-D-galactopyranosyl)-D-glycero-pent-2-enono-1,4-lactone: characterization in the oxalate-producing fungus, Sclerotinia sclerotiorum.

Extracts of sclerotia from Sclerotinia sclerotiorum, a fungal phytopathogen, contain two electrochemically-active constituents, D-glycero-pent-2-enono-1,4-lactone (trivial name: D-erythroascorbic acid), and a previously unidentified compound, here characterized as 5-O-(alpha-D-galactopyranosyl)-D-glycero-pent-2-enono-1,4-lactone on the basis of its physical and chemical properties and its two hydrolytic products, D-galactose and D-erythroascorbic acid. Treatment of this galactoside with alkaline hydrogen peroxide produces oxalic acid as observed earlier with erythroascorbic acid.

L-735,334, a novel sesquiterpenoid potassium channel-agonist from Trichoderma virens.

A novel oleic acid ester of the carotane sesquiterpene 14-hydroxy CAF-603 was isolated from Trichoderma virens grown in a solid brown rice-based medium, a solid millet-based medium, or a mannitol-based liquid medium. Its structure was determined on the basis of ms and nmr analysis. It retains distinct biological activity on the high conductance calcium-activated potassium channel, unlike its analogues 14-hydroxy CAF-603, CAF-603 3-oleate, or CAF-603 3-linoleate.

15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis.

A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [13C]bicarbonate and/or sodium [15N]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [13C,15N]nisin A using H[13C]MQC, H[13C]MBC, 2D INADEQUATE, and H[15N]MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H[13C]MQC to unlabeled and [13C]leucocin A afforded the complete 1H and 13C assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.