Metal-Binding Characteristics of the Gamma-Glutamyl Capsular Polymer of Bacillus licheniformis ATCC 9945

Abstract

The metal-binding affinity of the anionic poly-y-D-glutamyl capsule of Bacillus licheniformis was investigated by using Na+, Mg2+, Al3+, Ca2+, Cr3+, Mn2+, Fe3+, Ni2+, and Cu2+. Purified capsule was suspended in various concentrations of the chloride salts of the various metals, and after dialysis the bound metals were analyzed either by graphite furnace atomic absorption spectroscopy or by inductively coupled plasma-mass spectrometry. Exposure of purified capsule to excess concentrations of Na+ revealed it to contain 8.2 ,umol of anionic sites per mg on the basis of Na binding. This was confirmed by titration of the capsule with HCI and NaOH. Other metal ions were then added in ionic concentrations equivalent to 25, 50, 75, 100, 200, and 400% of the available anionic sites. The binding characteristics varied with the metal being investigated. Addition of Cu2+, Al3+, Cr3+, or Fe3+ induced flocculation. These metal ions showed the greatest affinity for B. licheniformis capsule in competitive-binding experiments. Flocculation was not seen with the addition of other metal ions. With the exception of Ni2+ and Fe3+ all capsule-metal-binding sites readily saturated. Ni2+ had low affinity for the polymer, and its binding was increased at high metal concentrations. Fe3+ binding resulted in the development of rust-colored ferrihydrite which itself could bind additional metal. Metal-binding characteristics of B. licheniformis capsule appear to be influenced by the chemical and physical properties of both the capsule and the metal ions.

The alphabet of intrinsic disorder

“…144 Furthermore, PGA in capsules of these bacteria consists of either a mixture of L-and D-enantiomers (S. epidermidis) 144 or solely D-enantiomer (B. anthracis), 145 which makes them particularly non-immunogenic. 135 The released form of PGA is used by the producing organism for rather different purposes, starting from the sequestration of toxic metal ions that increases the resistance of some soil bacteria to harsh conditions, 146 to serving as a source of glutamate for bacteria in a starvation state during late stationary phase, 147 to playing a role in decrease of the high local salt concentrations that helps extremophilic bacteria and archaea to survive in a hostile environment, 148,149 and in Hydra, to control explosion of the special stringing cells, nematocysts, that are used to capture prey, for locomotion and for defense. 150 In addition to have multiple functional roles, bacterially produced PGA has found its way to serve as an important biodegradable component 151 with multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields.…”

Interactions of chromium with microorganisms and plants

“…CrO 23 4 ) and thus cannot be trapped by the anionic components of bacterial envelopes [26]. However, cationic Cr(III) derivatives bind tightly to Salmonella lipopolysaccharides [27], Bacillus subtilis and E. coli cell walls [28], and capsular polymers of Bacillus licheniformis [29].…”

A Heavy Metal Biotrap for Wastewater Remediation Using Poly-γ-Glutamic Acid

“…Trace levels of contamination due to proteins (0.81 µg/mg), phospholipids (9.5 nmol organic phosphate/mg), and uronic acids (0.63 µg/mg) were detected in the purified γ-PGA polymer. Overall, the purity of the γ-PGA was much higher (at least by a factor of 10) than a capsule preparation of γ-PGA obtained previously (19). The purified biopolymer preparation contained only trace amounts of copper (8 µg/mg) and zinc (3.9 µg/mg), likely arising from background metal sorption from the growth medium.…”

“…γ-PGA (potassium salt) produced from Bacillus licheniformis ATCC 9945 was obtained through the WPI Bioprocess Technology Laboratory (Worcester, MA). Production and purification of the potassium salt of γ-PGA have been described (18) with modification (19). In a final preparation step, the polymer was purified using a 100 kDa membrane in a tangential flow filtration apparatus, thereby removing any small polymer fragments (<30 kDa) that might bind Cu 2+ ions when used in the experiments outlined in this study.…”

“…The Bradford reagent is most sensitive to arginine and aromatic residues but insensitive to glutamic acid and therefore also the γ-PGA polymer (21). Purified γ-PGA was assayed for organic phosphate content, indicative of probable contamination by phospholipids (i.e., cell membrane components, nucleic acids, and teichoic acids) (19), using a spectrophotometry based protocol (22). Purified γ-PGA was assayed for teichuronic acid contamination using the colorimetric assay (23).…”