Terry J. Beveridge

Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria

ABSTRACT Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane. Cationic peptides from different structural classes were used in experiments withStaphylococcus aureus and other medically important gram-positive bacteria to gain insight into the mechanism of action. The membrane potential-sensitive fluorophore dipropylthiacarbocyanine was used to assess the interactions of selected antimicrobial peptides with the cytoplasmic membrane of S. aureus. Study of the kinetics of killing and membrane depolarization showed that, at early time points, membrane depolarization was incomplete, even when 90% or more of the bacteria had been killed. CP26, a 26-amino-acid α-helical peptide with a high MIC against S. aureus, still had the ability to permeabilize the membrane. Cytoplasmic-membrane permeabilization was a widespread ability and an action that may be necessary for reaching an intracellular target but in itself did not appear to be the killing mechanism. Transmission electron microscopy of S. aureus andStaphylococcus epidermidis treated with CP29 (a 26-amino-acid α-helical peptide), CP11CN (a 13-amino-acid, proline- and tryptophan-rich peptide), and Bac2A-NH2 (a linearized version of the 12-amino-acid loop peptide bactenecin) showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus, whereas cell wall effects and mesosomes were seen with S. epidermidis. Nuclear condensation and abherrent septation were occasionally seen in S. epidermidis. Our experiments indicated that these peptides vary in their mechanisms of action and that the mechanism of action likely does not solely involve cytoplasmic-membrane permeabilization.

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

ABSTRACT A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter−1 and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed aKm of 23 mg liter−1 (437 μM) and a Vmax of 0.98 mg of Cr h−1 mg of protein−1 (317 nmol min−1 mg of protein−1). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.

Membrane Vesicles: an Overlooked Component of the Matrices of Biofilms

The matrix helps define the architecture and infrastructure of biofilms and also contributes to their resilient nature. Although many studies continue to define the properties of both gram-positive and gram-negative bacterial biofilms, there is still much to learn, especially about how structural characteristics help bridge the gap between the chemistry and physical aspects of the matrix. Here, we show that membrane vesicles (MVs), structures derived from the outer membrane of gram-negative bacteria, are a common particulate feature of the matrix of Pseudomonas aeruginosa biofilms. Biofilms grown using different model systems and growth conditions were shown to contain MVs when thin sectioned for transmission electron microscopy, and mechanically disrupted biofilms revealed MVs in association with intercellular material. MVs were also isolated from biofilms by employing techniques for matrix isolation and a modified MV isolation protocol. Together these observations verified the presence and frequency of MVs and indicated that MVs were a definite component of the matrix. Characterization of planktonic and biofilm-derived MVs revealed quantitative and qualitative differences between the two and indicated functional roles, such as proteolytic activity and binding of antibiotics. The ubiquity of MVs was supported by observations of biofilms from a variety of natural environments outside the laboratory and established MVs as common biofilm constituents. MVs appear to be important and relatively unacknowledged particulate components of the matrix of gram-negative or mixed bacterial biofilms.

Structural and Spectral Features of Selenium Nanospheres Produced by Se-Respiring Bacteria

ABSTRACT Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ∼300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H2Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.

Elucidation of Functional Groups on Gram-Positive and Gram-Negative Bacterial Surfaces Using Infrared Spectroscopy

Surface functional group chemistry of intact Gram-positive and Gram-negative bacterial cells and their isolated cell walls was examined as a function of pH, growth phase, and growth media (for intact cells only) using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Infrared spectra of aqueous model organic molecules, representatives of the common functional groups found in bacterial cell walls (i.e., hydroxyl, carboxyl, phosphoryl, and amide groups), were also examined in order to assist the interpretation of the infrared spectra of bacterial samples. The surface sensitivity of the ATR-FTIR spectroscopic technique was evaluated using diatom cells, which possess a several-nanometers-thick layer of glycoprotein on their silica shells. The ATR-FTIR spectra of bacterial surfaces exhibit carboxyl, amide, phosphate, and carbohydrate related features, and these are identical for both Gram-positive and Gram-negative cells. These results provide direct evidence to the previously held conviction that the negative charge of bacterial surfaces is derived from the deprotonation of both carboxylates and phosphates. Variation in solution pH has only a minor effect on the secondary structure of the cell wall proteins. The cell surface functional group chemistry is altered neither by the growth phase nor by the growth medium of bacteria. This study reveals the universality of the functional group chemistry of bacterial cell surfaces.

Cryo-Transmission Electron Microscopy of Frozen-Hydrated Sections of Escherichia coli and Pseudomonas aeruginosa

High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the periplasmic space was compressed during cryosectioning, a finding which provided supporting evidence that this space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the space between them (although still part of the periplasmic space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the periplasmic space. It is possible that the protoplast has some capacity to float freely within the periplasm.

MINERALIZATION OF BACTERIAL SURFACES

Bacteria are very small (∼ 1.5 μm3), but have the largest surface area to volume ratio of any life form. As a result, by providing interfaces for sorption of metal cations, bacteria are efficient scavengers of dilute metals and can concentrate them from the surrounding aqueous environment. This is mainly due to the overall anionic charge of bacterial surfaces imparted by the macromolecules which make up their fabric. Once metal ions have interacted with the electronegative sites on these molecules, they nucleate the formation of fine-grained minerals using anions from the external milieu as counter-ions for additional metal complexation. It is not unusual for bacteria to precipitate an amount of metal equal to, or exceeding, their cellular weight. Diverse mineral types, including hydroxides /oxides, carbonates, sulfates/ sulfides, and phosphates are common and can be in. amorphous or crystalline phases depending on the stage of mineral development. Since bacteria are ubiquitous in the natural environment, they can potentially play a significant role in the development of fine-grain minerals in soils and sediments. It is also possible that their capacity for metal immobilization can be used to counteract the disastrous effects of acid mine leachates.

Bacteria as nucleation sites for authigenic minerals in a metal-contaminated lake sediment

Abstract Sediment samples contaminated with metals arising from mine tailings drainage were obtained from Lower Moose Lake in the Onaping region near Sudbury, Ontario, Canada. The samples were examined by electron microscopy, selected-area electron diffraction and energy-dispersive X-ray spectroscopy. Individual bacterial cells and their remains were prominent as nucleation sites for both metal sulfides and a complex polymorphic (Fe,Al)-silicate. The principal metal sulfide species associated with the bacteria were amorphous mackinawite (FeS 1 - x ) and microcrystalline millerite (NiS). Trace amounts of Cu and Zu were also detected in some of the sulfide precipitates. At least two structural forms of the (Fe,Al)-silicate were present, and energy-dispersive X-ray spectroscopy point analyses revealed corresponding differences in chemical composition. Poorly ordered limonitic clay-type phases had a granular morphology and contained less Fe than well-developed crystalline material which generated hexagonal diffraction patterns with reflections ( d = 4.60 and 2.55 Ȧ) characteristic of an interstratified chamositic clay.

Metallic ion binding by Bacillus subtilis: Implications for the fossilization of microorganisms

The silicification of bacterial cells has been followed in a laboratory simulation by electron microscopy and energy-dispersive X-ray spectroscopy. Structural degradation of the bacteria was not influenced by silica availability, but the remains of degraded cells did promote the precipitation of silica. In contrast, cells reacted with ferric iron before aging remained intact, and structures conforming to the original morphology of the bacteria were preserved by silicification. The binding of metallic ions by microbial cells, in particular the retention of iron, is therefore considered to be an important contributing factor to the fossilization of microorganisms.

Cryo‐electron microscopy reveals native polymeric cell wall structure in Bacillus subtilis 168 and the existence of a periplasmic space

Ultrarapid freezing of bacteria (i.e. vitrification) results in optimal preservation of native structure. In this study, cryo‐transmission electron microscopy of frozen‐hydrated sections was used to gain insight into the organization of the Bacillus subtilis 168 cell envelope. A bipartite structure was seen above the plasma membrane consisting of a low‐density 22 nm region above which a higher‐density 33 nm region or outer wall zone (OWZ) resided. The interface between these two regions appeared to possess the most mass. In intact and in teichoic acid‐extracted wall fragments, only a single region was seen but the mass distribution varied from being dense on the inside to less dense on the outside (i.e. similar to the OWZ). In plasmolysed cells, the inner wall zone (IWZ)s thickness expanded in size but the OWZs thickness remained constant. As the IWZ expanded it became filled with plasma membrane vesicles indicating that the IWZ had little substance and was empty of the walls polymeric network of peptidoglycan and teichoic acid. Together these results strongly suggest that the inner zone actually represents a periplasmic space confined between the plasma membrane and the wall matrix and that the OWZ is the peptidoglycan‐teichoic acid polymeric network of the wall.