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Marine biofouling poses significant challenges to the economy, safety, and reliability of marine infrastructure. While antimicrobial peptides (AMPs) have emerged as green and efficient antifouling agents, their single functionality and the complexity of preparing antifouling surfaces remain key challenges. This study introduces a multifunctional AMP with combined adhesion and antimicrobial properties, derived from 3,4-dihydroxy-l-phenylalanine (DOPA) and IP12 (sequence IRLRWRWKWPWP). The directional recombination of AMP was guided by theoretical calculations. Density functional theory (DFT) simulations identify that the hydroxyl groups of DOPA were the main activating groups that react with aluminum alloy. Coarse-grained molecular dynamics (CG MD) and all-atom molecular dynamics (AA MD) simulations revealed that amino acid residues near the N-terminal of the IP12 could induce cell membrane bending and rupture. The AMP surfaces were fabricated to validate the accurate calibration of the simulations and performance of multifunctional AMP. Atomic force microscopy, Fourier transform infrared, and X-ray photoelectron spectroscopy results confirm the successful construction of AMP surfaces through adhesion function. Antifouling evaluations demonstrated the antifouling properties of AMP surfaces against Escherichia coli (Gram-negative) and Bacillus sp. (Gram-positive), achieving antifouling rates of 85.8 and 82.4%, respectively. This study provides valuable insights into the design of multifunctional AMPs.

In lead optimization, open, solvent-exposed protein pockets are often disregarded as prospective binding sites. Because of bulk-solvent proximity, researchers are instead enticed to attach charged polar groups at inhibitor scaffolds to improve solubility and pharmacokinetic properties. It is rarely considered that solvent effects from water reorganization in the first hydration shell of protein-ligand complexes can have a significant impact on binding. We investigate the thermodynamic fingerprint of thermolysin inhibitors featuring terminal charged ammonium groups that are gradually pulled from a distal, solvent-exposed position into the flat, bowl-shaped S 2 ' pocket. Even for the most remote attachment, costs for partial desolvation of the polar group next to the protein-solvent interface are difficult to compensate by interactions with the protein or surrounding water molecules. Through direct comparison with hydrophobic analogues, a significant 180-fold affinity loss was recorded, which questions popular strategies to attach polar ligand-solubilizing groups at the exposed terminus of substituents accommodated in flat open pockets.

Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry.

The biodegradation efficiency of beta-cypermethrin (β-CY) is low especially at high concentrations mainly due to poor contact between this hydrophobic pesticide and microbial cells. In this study, the effects of two biodegradable surfactants (Tween-80 and Brij-35) and β-cyclodextrin (β-CD) on the growth and cell surface hydrophobicity (CSH) of Bacillus licheniformis B-1 were studied. Furthermore, their effects on the solubility, biosorption, and degradation of β-CY were investigated. The results showed that Tween-80 could slightly promote the growth of the strain while Brij-35 and β-CD exhibited little effect on its growth. The CSH of strain B-1 and the solubility of β-CY were obviously changed by using Tween-80 and Brij-35. The surfactants and β-CD could enhance β-CY biosorption and degradation by the strain, and the highest degradation was obtained in the presence of Brij-35. When the surfactant or β-CD concentration was 2.4 g/L, the degradation rate of β-CY in Brij-35, Tween-80, and β-CD treatments was 89.4%, 50.5%, and 48.1%, respectively. The half-life of β-CY by using Brij-35 was shortened by 69.1 h. Beta-CY content in the soil with both strain B-1 and Brij-35 decreased from 22.29 mg/kg to 4.41 mg/kg after incubation for 22 d. This work can provide a promising approach for the efficient degradation of pyrethroid pesticides by microorganisms.

Bacterial spores can survive for long periods without nutrients and in harsh environmental conditions. This survival is influenced by the structure of the spore, the presence of protective compounds, and water retention. These compounds, and the physical state of water in particular, allow some species of bacterial spores to survive sterilization schemes with hydrogen peroxide and UV light. The chemical nature of the spore core and its water has been a subject of some contention and the chemical environment of the water impacts resistance paradigms. Either the spore has a glassy core, where water is immobilized along with other core components, or the core is gel-like with mobile water diffusion. These properties affect the movement of peroxide and radical species, and hence resistance. Deuterium solid-state NMR experiments are useful for examining the nature of the water inside the spore. Previous work in our lab with spores of Bacillus subtilis indicate that, for spores, the core water is in a more immobilized state than expected for the gel-like core theory, suggesting a glassy core environment. Here, we report deuterium solid-state NMR observations of the water within UV- and peroxide-resistant spores from Bacillus pumilus SAFR-032. Variable-temperature NMR experiments indicate no change in the line shape after heating to 50 °C, but an overall decrease in signal after heating to 100 °C. These results show glass-like core dynamics within B. pumilus SAFR-032 that may be the potential source of its known UV-resistance properties. The observed NMR traits can be attributed to the presence of an exosporium containing additional labile deuterons that can aid in the deactivation of sterilizing agents.

A wide range of oligo-p-phenylene ethynylenes has been shown to exhibit good biocidal activity against both Gram-negative and Gram-positive bacteria. While cell death may occur in the dark, these biocidal compounds are far more effective in the light as a result of their ability to sensitize the production of cell-damaging reactive oxygen species. In these studies, the interactions of a specific cationic oligo-p-phenylene ethynylene with spore-forming Bacillus atrophaeus and Bacillus anthracis Sterne have been investigated. Flow cytometry assays are used to rapidly monitor cell death as well as spore germination. This compound effectively killed Bacillus anthracis Sterne vegetative cells (over 4 log reduction), presumably by severe perturbations of the bacterial cell wall and cytoplasmic membrane, while also acting as an effective spore germinant in the dark. While 2 log reduction of B. anthracis Sterne spores was observed, it is hypothesized that further killing could be achieved through enhanced germination.

Thiopeptides are post-translationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery's tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogues. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched hydrophobic residues are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogues were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

Accidental release of biological agents from a bioweapon facility may contaminate large areas, possibly causing disastrous environmental consequences. To address this issue, novel halogen-containing reactive materials are being designed with the added capability to inactivate viable airborne microorganisms. This study determined the efficiency of combustion products of such materials to inactivate aerosolized bacteria and viruses. Spores of Bacillus atrophaeus and MS2 viruses dispersed in dry air were exposed for subsecond time intervals to hydrocarbon flames seeded with different reactive powders so that bioaerosol particles interacted with the combustion products in a controlled high-temperature environment. The experiments were designed to quantify differences in the biocidal effects of different reactive material powders including Al and Mg, a B•Ti nanocomposite, an 8Al•MoO(3) nanothermite, and a novel Al•I(2) nanocomposite. Compared to pure hydrocarbon flame, powder-seeded flame (with no iodine) produced about an order of magnitude greater inactivation of bacterial spores. The iodine-containing material increased the spore inactivation by additional 2 orders of magnitude. The aerosolized MS2 viruses (generally not as stress-resistant as spores) were fully inactivated when exposed to combustion of either the iodinated or noniodinated powders. Overall, the study suggests a great biocidal potential of combustion products generated by novel iodine-containing nanocomposite materials.

Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 μM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis., (© 2012 American Chemical Society)

The cytotoxicity of Al(2)O(3) nanoparticles (NP) at very low exposure levels (1 μg/mL and less) to a dominant bacterial isolate from freshwater (lake water), Bacillus licheniformis, was examined. Sterile lake water was directly used as a test medium or matrix to simulate the freshwater environment. Exposure to 1 μg/mL Al(2)O(3) NP for 2 h caused a 17% decrease in cell viability (as determined by plate count and MTT assay). During the test period, the particles were found to be stable against aggregation in the matrix and exerted a nano-size effect on the exposed test organisms. The decrease in cell viability was proven not to be due to the release of Al(3+) ions from the nanoparticles in the dispersion. The zeta potential and FT-IR analyses suggested that the surface charge based attachment of nanoparticles on to the bacterial cell wall was responsible for flocculation leading to toxicity. The cell wall damage confirmed through SEM and the lipid peroxidation assay also contributed toward toxicity. This study warns of possible ecotoxicity of nanoparticles even at environmentally relevant concentrations. However, detailed studies need to be carried out to establish probable mechanistic aspects of this low concentration toxicity phenomenon.

We demonstrate herein a method for chemically modifying cotton fibers and cotton-containing fabric with a light-activated, cationic phenylene-ethynylene (PPE-DABCO) conjugated polyelectrolyte biocide. When challenged with Pseudomonas aeruginosa and Bacillus atropheaus vegetative cells from liquid suspension, light-activated PPE-DABCO effects 1.2 and 8 log, respectively, losses in viability of the exposed bacteria. These results suggest that conjugated polyelectrolytes retain their activity when grafted to fabrics, showing promise for use in settings where antimicrobial textiles are needed.

The indanomycin biosynthetic gene (idm) cluster was recently identified from Streptomyces antibioticus NRRL 8167. The disruption of one of these genes, idmH, and the increased production of a previously unreported metabolite in this mutant is reported. The structure of this compound was elucidated and was shown to possess a linear tetraene. This metabolite is not a logical biosynthetic intermediate of indanomycin but instead is likely an alternate product of the pathway.

As a result of the extensive number of applications of silver nanoparticles (AgNPs), their potential impacts, once released into the environment, are of concern. The toxicity of AgNPs was reported to be dependent on various factors such as particle size, shape and capping agent. Although these factors may play a role in AgNPs toxicity, the results presented herein suggest that surface charge is one of the most important factors that govern the toxicity of AgNPs. In the current study, the toxicity of four AgNPs representing various surface charging scenarios ranging from highly negative to highly positive was investigated. These AgNPs were (1) uncoated H(2)-AgNPs, (2) citrate coated AgNPs (Citrate-AgNPs), (3) polyvinylpyrrolidone coated AgNPs (PVP-AgNPs), and (4) branched polyethyleneimine coated AgNPs (BPEI-AgNPs). Our results clearly demonstrate that the AgNPs exhibited surface charge-dependent toxicity on the bacillus species investigated. Furthermore, ultrafiltration membranes were utilized to purify the AgNPs suspensions from residual impurities prior to the introduction to the microbes. This step was crucial in determining the true AgNPs toxicity and is either missing or not explicitly mentioned in most of the reported toxicity studies.

Winter savory Satureja montana is a medicinal herb used in traditional gastronomy for seasoning meats and salads. This study reports a comparison between conventional (hydrodistillation, HD, and Soxhlet extraction, SE) and alternative (supercritical fluid extraction, SFE) extraction methods to assess the best option to obtain bioactive compounds. Two different types of extracts were tested, the volatile (SFE-90 bar, second separator vs HD) and the nonvolatile fractions (SFE-250 bar, first and second separator vs SE). The inhibitory activity over acetyl- and butyrylcholinesterase by S. montana extracts was assessed as a potential indicator for the control of Alzheimer's disease. The supercritical nonvolatile fractions, which showed the highest content of (+)-catechin, chlorogenic, vanillic, and protocatechuic acids, also inhibited selectively and significantly butyrylcholinesterase, whereas the nonvolatile conventional extract did not affect this enzyme. Microbial susceptibility tests revealed the great potential of S. montana volatile supercritical fluid extract for the growth control and inactivation of Bacillus subtilis and Bacillus cereus, showing some activity against Botrytis spp. and Pyricularia oryzae. Although some studies were carried out on S. montana, the phytochemical analysis together with the biological properties, namely, the anticholinesterase and antimicrobial activities of the plant nonvolatile and volatile supercritical fluid extracts, are described herein for the first time.

Manganese oxides are widespread in the environment and their surface reactivity has the potential to modifythe geochemical behavior of uranium. We have investigated the effect of different concentrations of U and Mn on the coupled biogeochemical oxidation-reduction reactions of U and Mn. Experiments conducted in the presence of Mn(II)-oxidizing spores from Bacillus sp. strain SG-1 and 5% headspace oxygen show that the Mn oxides produced by these spores can rapidly oxidize UO2. Thirty to fifty times more UO2 is oxidized in the presence of Mn oxides compared to Mn oxide free controls. As a consequence of this U02 oxidation, Mn oxides are reduced to soluble Mn(II) that can be reoxidized by SG-1 spores. SG-1 spores cannot directly oxidize U02, but U02 oxidation proceeds rapidly with Mn(II) concentrations of <5 microM. The rate of UO2 oxidation is equal to the rate of MnO2 reduction with UO2 oxidation controlled by the initial concentrations of UO2, dissolved Mn(II) (in systems with spores), or Mn(IV) oxides (in systems containing preformed MnO2). U(VI) and UO2 decrease the Mn(II) oxidation rate in different ways by inhibiting the Mn(II)-oxidizing enzyme or decreasing the available Mn(II). These results emphasize the need to consider the impact of Mn(II)-oxidizing bacteria when predicting the potential for U02 oxidation in the subsurface.

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifluoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the "TFA inactivation protocol" was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDI-TOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactivation of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.

Bleach solutions containing the active ingredient hypochlorite (OCl-) serve as powerful biological disinfectants but are highly caustic and present a significant compatibility issue when applied to contaminated equipment or terrain. A neutral, bicarbonate-buffered aqueous solution of Oxone (2K2HSO5.KHSO4.K2SO4) and sodium chloride that rapidly generates hypochlorite and hypochlorous acid (HOCl) in situ was evaluated as a new alternative to bleach for the inactivation of biological agents. The solution produced a free chlorine (HOCl + OCl-) concentration of 3.3 g/L and achieved > or =5.8-log inactivation of spores of Bacillus atrophaeus, Bacillus thuringiensis, Aspergillus niger, and Escherichia coli vegetative cells in 1 min at 22 degrees C. Seawaterwas an effective substitute for solid sodium chloride and inactivated 5 to 8 logs of each organism in 10 min over temperatures ranging from -5 degrees C to 55 degrees C. Sporicidal effectiveness increased as free chlorine concentrations shifted from OCl- to HOCl. Neutrally buffered Oxone-chloride and Oxone-seawater solutions are mitigation alternatives for biologically contaminated equipment and environments that would otherwise be decontaminated using caustic bleach solutions.

The search for the best quantitative structure-activity relationship (QSAR) models in ecotoxicology is an ever-topical research activity. Hence, the development of new descriptors and applying them successfully in QSAR studies seems demanding in ecotoxicology. In the present work, group philicities are utilized for the first time in QSAR analysis for ecotoxicological studies on chlorophenols (CPs). It is important to point out that group philicities are capable of providing the best QSAR model for the toxicity of CPs against Daphnia magna. Furthermore, global electrophilicity and group philicities together give the best QSAR models for Brachydanio rerio and Bacillus with the maximum value of coefficient of determination and high internal predictive ability. The developed QSAR models clearly show the importance of the selected density functional reactivity indices as descriptors in ecotoxicological studies.

Poly-gamma-D-glutamic acid from Bacillus licheniformis is a water-soluble, nontoxic, nonimmunogenic exopolymer. Using synthetic linkers, the alpha-carboxylate side chains of PGA were conjugated to an exposed thiol side chain of an antibody F(ab') fragment, Mc109F4. Analysis of the PGA-Mc109F4 conjugate by gel filtration HPLC revealed a mixture of multivalent conjugates. The PGA-Mc109F4 conjugate retained biological activity, but showed a lower binding affinity to target BCL3B3 cells than free Mc109F4 F(ab')(2) by flow cytometry, and a lower efficacy for BCL3B3 growth inhibition than free Mc109F4 F(ab')(2). PGA was also conjugated with the free amino group of glycopeptide antibiotic vancomycin. The PGA-vancomycin conjugate showed slightly lower antibacterial activity than free vancomycin versus susceptible Bacillus subtilis, but slightly higher activity versus intrinsically resistant Leuconostoc mesenteroides.

Three spices, chili, black pepper, and turmeric, were tested for the effect of their aqueous extracts on the sensitivity of three bacteria, Escherichia coli, Bacillus megaterium, and Bacillus pumilusspores, to gamma-radiation. It was found that the extracts of the three spices offered protection to these organisms against inactivation by gamma-radiation. These spice extracts were also tested for their protection of naked plasmid DNA. Radiation-induced degradation of plasmid pUC18 DNA was reduced in the presence of the spice extracts. The maximum protection was offered by the chili extract followed by that of black pepper and turmeric. The two known antioxidants, curcumin and piperine from turmeric and black pepper, respectively, were shown to protect the plasmid DNA from the degradation by gamma-radiation. Experiments with the plasmid pUC18 DNA indicated that the spice extracts probably protected microorganisms by protecting their DNA. These studies indicated the importance of spices among ingredients in food as dose-modifying factors during radiation processing.

Curcumin, the yellow color pigment of turmeric, is produced industrially from turmeric oleoresin. The mother liquor after isolation of curcumin from oleoresin contains approximately 40% oil. The oil was extracted from the mother liquor using hexane at 60 degrees C, and the hexane extract was separated into three fractions using silica gel column chromatography. These fractions were tested for antibacterial activity by pour plate method against Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Fraction II eluted with 5% ethyl acetate in hexane was found to be most active fraction. The turmeric oil, fraction I, and fraction II were analyzed by GC and GC-MS. ar-Turmerone, turmerone, and curlone were found to be the major compounds present in these fractions along with other oxygenated compounds.

The palladium-catalyzed coupling of 3- and 4-(trialkylstannyl)pyridines with 7-bromo or 7-chloro 1-substituted 1,4-dihydro-4-oxo-3-quinolinecarboxylates has provided access to the corresponding 1-substituted 1,4-dihydro-4-oxo-7-pyridinyl-3-quinolinecarboxylic acids. The antibacterial activity of these derivatives was studied with the finding that the optimal 1- and 7-position substituents for Gram positive activity are cyclopropyl and 4-(2,6-dimethylpyridinyl), respectively. We find that for the fluorine-substituted derivatives studied, the position of the fluorine on the quinolone nucleus or the number of fluorine atoms does not seem to be important for good Gram positive activity. For 1-cyclopropyl 7-(2,6-dimethyl-4-pyridinyl) derivatives, the 6-fluoro 4a, 8-fluoro 10d, 6,8-difluoro 10b, and 5,6,8-trifluoro 8, all provided equal antibacterial activity against Staphylococcus aureus ATCC 29213. There is also a correlation between the substitution on the 7-(4-pyridinyl) group and the Gram positive activity, particularly for S. aureus, clearly indicating that the 2,6-dimethylpyridinyl group is optimal. The MIC50 value for the most potent agents in this study against S. aureus ATCC 29213 is 0.008 microgram/mL. By comparison, ciprofloxacin and aminopyrrolidine 28 gave values of 0.25 and 0.015 microgram/mL, respectively, against this organism.

Lys-234 has been postulated to participate in beta-lactamase catalysis by acting as an electrostatic anchor for the C3 carboxylate of penicillins [Herzberg, O., & Moult, J. (1987) Science 236, 694-701]. To test this hypothesis, site-directed mutagenesis was used to convert the Lys-234 in Bacillus licheniformis beta-lactamase into Glu-234 or Ala-234. The wild-type, Glu-234, and Ala-234 beta-lactamases have been expressed in Bacillus subtilis and purified to homogeneity. The wild-type, K234E, and K234A enzymes have virtually identical circular dichroism and fluorescence spectra, similar thermal stabilities at neutral pH, and the same susceptibilities to proteolysis, indicating the lack of significant structural perturbation caused by the mutation. At acidic and basic pH the mutant enzymes have the same native circular dichroism as the wild-type enzyme but the thermal stability is significantly different. The mutations cause perturbations of the pK values of the ionizing groups responsible for the pH dependence of the catalytic reaction in both the free enzyme and the E.S complex. As expected, conversion of Lys-234 to Ala or Glu decreased substrate binding (Km) by 1-2 orders of magnitude for several penicillin and cephalosporin substrates at neutral and higher pH. However, at low pH, Km is essentially the same for the K234E and K234A enzymes as for the wild-type enzyme. Furthermore, decreases of 2-3 orders of magnitude in kcat were also observed, indicating substantial effects on the transition-state binding, as well as on ground-state binding. Surprisingly, changing the C3 carboxylate of phenoxymethylpenicillin to a hydroxymethyl group led to little difference in kinetic properties with the K234E or K234A enzyme. The results of this investigation indicate the Lys-234 is an important active-site residue involved in both ground-state and transition-state binding.

Leinamycin is a recently discovered antitumor antibiotic with an unusual 1,3-dioxo-1,2-dithiolane structure. It preferentially inhibits the incorporation of [3H]thymidine into the acid-insoluble fraction of Bacillus subtilis. In vitro, leinamycin causes single-strand cleavage of supercoiled double-helical pBR322 DNA in the presence of thiol cofactors. Scavengers of oxygen radical did not supress the DNA-cleaving activity. Thiol-activated leinamycin binds calf thymus DNA at 4 degrees C and thermal treatment of the leinamycin-DNA adduct released a chemically modified leinamycin from the complex. The lack of cytotoxicity and DNA-cleaving activity for S-deoxyleinamycin indicates that the 1,3-dioxo-1,2-dithiolane moiety is essential for the activity of leinamycin. Thus, the primary cellular target of leinamycin appears to be DNA. It binds DNA and causes single-strand break at low concentrations, which may account for the potent antitumor activity.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.

Succinyl-CoA:tetrahydrodipicolinate-N-succinyltransferase is a key enzyme in the biosynthesis of diaminopimelic acid (DAP), a component of the cell wall peptidoglycan of nearly all bacteria. This enzyme converts the cyclic precursor tetrahydrodipicolinic acid (THDPA) to a succinylated acyclic product. L-2-Aminopimelic acid (L-1), an acyclic analogue of THDPA, was found to be a good substrate for this enzyme and was shown to cause a buildup of THDPA in a cell-free enzyme system but was devoid of antibacterial activity. Incorporation of 1 into a di- or tripeptide yielded derivatives that exhibited antibacterial activity against a range of Gram-negative organisms. Of the five peptide derivatives tested, (L-2-aminopimelyl)-L-alanine (6) was the most potent. These peptides were shown to inhibit DAP production in intact resting cells. High levels (30 mM) of 2-aminopimelic acid were achieved in the cytoplasm of bacteria as a result of efficient uptake of the peptide derivatives through specific peptide transport systems followed, presumably, by cleavage by intracellular peptidases. Finally, the antibacterial activity of these peptides could be reversed by DAP or a DAP-containing peptide. These results demonstrate that the peptides containing L-2-aminopimelic acid exert their antibacterial action by inhibition of diaminopimelic acid biosynthesis.

A ribosome-independent synthesis of guanosine 5',3'-polyphosphates has been found in the soluble fraction of Bacillus brevis (ATCC 8185) extracts. The partially purified enzyme catalyzes the formation of both guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate, does not require 20% methanol to stimulate the rate of reaction, and is not stimulated by complexing with ribosomes of either Escherichia coli or B. brevis. The B. brevis enzyme system is not inhibited by RNase A or thiostrepton, and is only slightly inhibited by tetracycline. The pyrophosphoryl donor specificity of the B. brevis enzyme is similar to that of the E. coli ribosome-stringent factor system.