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Introduction: This study aimed to identify and characterize novel siderophoreproducing organisms capable of secreting high quantities of the iron-binding compounds. In the course of this, two not yet reported halophilic strains designated ATCHAT and ATCH28T were isolated from hypersaline, alkaline surface waters of Salar de Llamará and Laguna Lejía, respectively. The alkaline environment limits iron bioavailability, suggesting that native organisms produce abundant siderophores to sequester iron. Methods: Both strains were characterized by polyphasic approach. Comparative analysis of the 16S rRNA gene sequences revealed their affiliation with the genus Halomonas. ATCHAT showed close similarity to Halomonas salicampi and Halomonas vilamensis, while ATCH28T was related closest to Halomonas ventosae and Halomonas salina. The ability of both strains to secrete siderophores was initially assessed using the chromeazurol S (CAS) liquid assay and subsequently further investigated through genomic analysis and NMR. Furthermore, the effect of various media components on the siderophore secretion by strain ATCH28T was explored. Results: The CAS assay confirmed the ability of both strains to produce iron-binding compounds. Genomic analysis of strain ATCHAT revealed the presence of a not yet reported NRPS-dependant gene cluster responsible for the secretion of siderophore. However, as only small amounts of siderophore were secreted, further investigations did not lie within the scope of this study. Via NMR and genomic analysis, strain ATCH28T has been determined to produce desferrioxamine E (DFOE). Although this siderophore is common in various terrestrial microorganisms, it has not yet been reported to occur within Halomonas, making strain ATCH28T the first member of the genus to produce a non-amphiphilic siderophore. By means of media optimization, the produced quantity of DFOE could be increased to more than 1000 µM. Discussion: Phenotypic and genotypic characteristics clearly differentiated both strains from other members of the genus Halomonas. Average nucleotide identity (ANI) values and DNA-DNA relatedness indicated that the strains represented two novel species. Therefore, both species should be added as new representatives of the genus Halomonas, for which the designations Halomonas llamarensis sp. nov. (type strain ATCHAT = DSM 114476 = LMG 32709) and Halomonas gemina sp. nov. (type strain ATCH28T = DSM 114418 = LMG 32708) are proposed. [ABSTRACT FROM AUTHOR]

Escherichia coli is one of the best studied micro-organisms and is the most widely used host in genetic engineering. The Gram-negative single cells are rod-shaped, and filaments are usually not found. Here, we describe the reproducible formation of elongated E. coli cells. During heterologous expression of the silent surface (S)-layer protein gene sllB from Lysinibacillus sphaericus JG-A12 in E. coli BL21(DE3), the cells were arranged as long chains which were surrounded by highly stable sheaths. These filaments had a length of >100 [micro]m. In the stationary growth phase, microscopic analyses demonstrated the formation of unusually long transparent tube-like structures which were enclosing separate single cells. The tube-like structures were isolated and analysed by SDS-PAGE, infrared-spectroscopy and different microscopic methods in order to identify their unusual composition and structure. The tube-like structures were found to be like outer membranes, containing high levels of proteins and to which the recombinant S-layer proteins were attached. Despite the entire structure being indicative of a disordered cell division, the bacterial cells were highly viable and stable. To our knowledge, this is the first time that the induction of drastic morphological changes in E. coli by the expression of a foreign protein has been reported. DOI 10.1099/mic.0.040808-0

The surface layer (S-layer) protein genes of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 and of its relative B. sphaericus NCTC 9602 were analysed. The almost identical N-termini of the two S-layer proteins possess a unique structure, comprising three N-terminal S-layer homologous (SLH) domains. The central parts of the proteins share a high homology and are related to the S-layer proteins of B. sphaericus CCM 2177 and P-1. In contrast, the C-terminal parts of the S-layer proteins of JG-A12 and NCTC 9602 differ significantly between each other. Surprisingly, the C-terminal part of the S-layer protein of JG-A12 shares a high identity with that of the S-layer protein of B. sphaericus CCM 2177. In both JG-A12 and NCTC 9602 the chromosomal S-layer protein genes are followed by a newly identified putative insertion element comprising three ORFs, which encode a putative transposase, a putative integrase/recombinase and a putative protein containing a DNA binding helix-turn-helix motif, and the S-layer-protein-like gene copies sllA (9602) or sllB (JG-A12). Interestingly, both B. sphaericus strains studied were found to contain an additional, plasmid-located and silent S-layer protein gene with the same sequence as sllA and sflB. The primary structures of the corresponding putative proteins are almost identical in both strains. The N-terminal and central parts of these S-layer proteins share a high identity with those of the chromosomally encoded functional S-layer proteins. Their C-terminal parts, however, differ significantly. These results strongly suggest that the S-layer protein genes have evolved via horizontal transfer of genetic information followed by DNA rearrangements mediated by mobile elements.

To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.

Results indicate that drawbacks in the degradation of dichlororinatedtoluenes is due to the unspecific attack of tetrachlorobenzene dioxygenase leading to the production of dichlorobenzaldehydes or dichlorobenzoates. The metabolic fates of the dichloromethylcatechol intermediates in the total metabolism of dichlorotoluenes are discussed.

Research has been conducted on the cis-dienelactone enzymatic transformation. The results of the (super)1 H nuclear magnetic resonance assay analysis used to study this transformation have demonstrated that that product of the cis-dienelactone hydrolase reaction is maleylacetate.

Research has been conducted on the tecB gene located downstream of tecA and encoding tetrachlorobenzene dioxygenase in Ralstonia sp. strain PS12. The results of the tecB gene cloning into the Escherichia coli DH5alpha together with the tecA gene are described.

Abstract Background Escherichia coli is one of the best studied microorganisms and finds multiple applications especially as tool in the heterologous production of interesting proteins of other organisms. The heterologous expression of special surface (S-) layer proteins caused the formation of extremely long E. coli cells which leave transparent tubes when they divide into single E. coli cells. Such natural structures are of high value as bio-templates for the development of bio-inorganic composites for many applications. In this study we used genetically modified filamentous Escherichia coli cells as template for the design of polyelectrolyte tubes that can be used as carrier for functional molecules or particles. Diversity of structures of biogenic materials has the potential to be used to construct inorganic or polymeric superior hybrid materials that reflect the form of the bio-template. Such bio-inspired materials are of great interest in diverse scientific fields like Biology, Chemistry and Material Science and can find application for the construction of functional materials or the bio-inspired synthesis of inorganic nanoparticles. Results Genetically modified filamentous E. coli cells were fixed in 2% glutaraldehyde and coated with alternating six layers of the polyanion polyelectrolyte poly(sodium-4styrenesulfonate) (PSS) and polycation polyelectrolyte poly(allylamine-hydrochloride) (PAH). Afterwards we dissolved the E. coli cells with 1.2% sodium hypochlorite, thus obtaining hollow polyelectrolyte tubes of 0.7 μm in diameter and 5–50 μm in length. For functionalisation the polyelectrolyte tubes were coated with S-layer protein polymers followed by metallisation with Pd(0) particles. These assemblies were analysed with light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. Conclusion The thus constructed new material offers possibilities for diverse applications like novel catalysts or metal nanowires for electrical devices. The novelty of this work is the use of filamentous E. coli templates and the use of S-layer proteins in a new material construct.

Research has been conducted on Ralstonia sp. PS12 tetrachlorobenzene dioxygenase. The authors have investigated this dioxygenase which is involved in chlorinated toluene biodegradation, and report the results.

Investigates the degradation of monitoring key reactions of chloroaromatics by in situ ¹H nuclear magnetic resonance (&sup1H NMR). Use of ¹N HMR on the study of enzymatic transformation of cis-dienelactone; Maleylacetate as a product of the cis-dienelactone hydrolase reaction; Transformation of malyacetate.