David L. Gutnick

Engineering bacterial biopolymers for the biosorption of heavy metals; new products and novel formulations

Abstract Bioremediation of heavy metal pollution remains a major challenge in environmental biotechnology. One of the approaches considered for application involves biosorption either to biomass or to isolated biopolymers. Many bacterial polysaccharides have been shown to bind heavy metals with varying degrees of specificity and affinity. While various approaches have been adopted to generate polysaccharide variants altered in both structure and activity, metal biosorption has not been examined. Polymer engineering has included structural modification through the introduction of heterologous genes of the biosynthetic pathway into specific mutants, leading either to alterations in polysaccharide backbone or side chains, or to sugar modification. In addition, novel formulations can be designed which enlarge the family of available bacterial biopolymers for metal-binding and subsequent recovery. An example discussed here is the use of amphipathic bioemulsifiers such as emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1, that forms stable, concentrated (70%), oil-in-water emulsions (emulsanosols). In this system metal ions bind primarily at the oil/water interface, enabling their recovery and concentration from relatively dilute solutions. In addition to the genetic modifications described above, a new approach to the generation of amphipathic bioemulsifying formulations is based on the interaction of native or recombinant esterase and its derivatives with emulsan and other water-soluble biopolymers. Cation-binding emulsions are generated from a variety of hydrophobic substrates. The features of these and other systems will be discussed, together with a brief consideration of possible applications.

Bioemulsifier production by Acinetobacter calcoaceticus strains

Nondialyzable bioemulsifiers were found in the extracellular fluid of 16 different strains ofAcinetobacter calcoaceticus following growth on ethanol-salts medium. The amount of emulsifying activity, its specific activity, and hydrocarbon substrate specificity varied from one strain to another. In general, strains that grew well on the ethanol medium (2.4–2.6 mg cell dry wt/ml) produced high emulsifying activities (88–239 units/ml), whereas strains that grew more poorly (1.0–1.7 mg cell dry wt/ml) also produced less emulsifying activity (14–52 units/ml). With one exception, hexadecane/2-methylnaphthalane mixtures were emulsified more efficiently than pure hexadecane or 2-ethylnaphthalane.

Emulsifier of Arthrobacter RAG-1: determination of emulsifier-bound fatty acids.

Growth of the hydrocarbon degrading bacterium, Arthrobacter sp. RAG-I, is accompanied by emulsification of the hydrocarbon substrate in the growth medium [ 11. Emulsification is due to the production of an extracellular lipopolysaccharide with av. mol. wt 9.8 X 10’ [2]. The major components of the polymeric emulsifier are D-galactosamine, an unidentified amino uranic acid, and fatty acids joined to the heteropolysaccharide backbone primarily through O-ester linkages [2]. The emulsifier-bound fatty acids appear to play an important role in the function of the polymer since: (i) Removal of part of the fatty acids by mild alkaline hydrolysis caused a decrease in emulsifying activity; (ii) Emulsifiers isolated from RAG-1 growing on hexadecane as the sole source of carbon and energy contained lower O-ester content and reduced specific activity [3]. In order for the Arthrobacter emulsifier to induce efficiently hydrocarbon in water emulsions, the hydrocarbon substrate must contain both aliphatic and cyclic components [4]. A better understanding of the structure-function relationships governing biologically produced emulsifiers requires a detailed characterization of the bioemulsifier. Here the major O-esters of the emulsifier are identified.

An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan.

ABSTRACT The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.

Involvement of a Protein Tyrosine Kinase in Production of the Polymeric Bioemulsifier Emulsan from the Oil-Degrading Strain Acinetobacter lwoffii RAG-1

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.

BACTERIAL COOPERATIVE ORGANIZATION UNDER ANTIBIOTIC STRESS

Bacteria have developed sophisticated modes of cooperative behavior to cope with unfavorable environmental conditions. Here we report the effect of antibiotic stress on the colonial development of Paenibacillus dendritiformis and P. vortex. We focus on the effect of co-trimoxazole on the colonial organization of P. dendritiformis. We find that the exposure to non-lethal concentrations of antibiotic leads to dramatic changes in the colonial growth patterns. Branching, tip-splitting patterns are affected by reduction in the colonial fractal dimension from Df=2.0 to 1.7, appearance of pronounced weak chirality and pronounced radial orientation of the growth. We combine the experimental observations with numerical studies of both discrete and continuous generic models to reveal the causes for the modifications in the patterns. We conclude that the bacteria adjust their chemotactic signaling together with variations in the bacteria length and increase in the metabolic load.

The Hydrocarbon-Oxidizing Bacteria

The chemical heterogeneity and water insolubility of hydrocarbons pose special problems for microorganisms that must use these substances as nutrients. In an attempt to define a petroleum-degrading bacterium, Gutnick and Rosenberg (1977) postulated three distinguishing traits or specificities: 1. Efficient hydrocarbon uptake system (special receptor sites for binding hydrocarbons and/or production of unique chemicals that assist in the emulsification and transport of hydrocarbons into the cell). 2. Group-specific oxygenases (genetic potential of a particular microorganism to introduce molecular oxygen into the hydrocarbon and, with relatively few reactions, generate intermediates that subsequently enter common energy-yielding catabolic pathways). 3. Inducer specificity (the positive response of the organism to petroleum and its constituents in inducing the first two systems; inducer specificity and substrate specificity for oxygenase need not coincide).

Paenibacillus dendritiformis sp. nov., proposal for a new pattern-forming species and its localization within a phylogenetic cluster

A new strain capable of forming distinctive patterns during colony development was identified by using a combination of phenotypic characterization, fatty acid analysis and analysis of the 16S rRNA gene sequence. The strain formed either a branched, tip-splitting colony morphology (referred to as the T morphotype) or a chiral pattern exhibiting thinner branches with distinctive curling patterns (referred to as the C morphotype). Isolates of the T morphotype exhibited sequence identities greater than 97% to Paenibacillus thiaminolyticus JCM 7540. Phylogenetic analysis placed the T morphotype within the Paenibacillus cluster on a phylogenetic tree. On the basis of unique colony morphology and distinctive phenotypic characteristics, it is proposed that the pattern-forming isolates should be placed within a new species of Paenibacillus, Paenibacillus dendritiformis sp. nov., the type strain of which is T168T (= 30A1T).

Energy conservation in membranes of mutants of Escherichia coli defective in oxidative phosphorylation

Abstract 1. 1. Energy conservation in membranes of Escherichia coli was measured using 9-amino-6-chloro-2-methoxyacridine fluorescence. 2. 2. Energy conservation in membranes, depleted of coupling factor, can be restored either by the isolated crude coupling factor or by N , N ′-dicyclohexylcarbodiimide (DCCD) but not by Dio-9 or oligomycin. 3. 3. Several mutants, defective in different components of the ATP-synthesizing complex, are described. 4. 4. Mutants possessing a DCCD-insensitive ATPase have been isolated which can or cannot be recoupled by DCCD.

Genome sequence of the pattern forming Paenibacillus vortex bacterium reveals potential for thriving in complex environments

BackgroundThe pattern-forming bacterium Paenibacillus vortex is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other Paenibacillus species (Paenibacillus sp. JDR-2 and Paenibacillus larvae) have been sequenced. However, no genomic data is available on the Paenibacillus species with pattern-forming and complex social motility. Here we report the de novo genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium.ResultsThe complete P. vortex genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the P. vortex genome revealed 6,437 open reading frames (ORFs) and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS) genes, transcription factors (TFs), transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes.ConclusionsThese findings suggest that P. vortex has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, P. vortex is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.