Halothiobacillus diazotrophicus sp. nov., a chemolithoautotrophic sulphur-oxidizing and nitrogen-fixing bacterium isolated from freshwater.

A sulphur-oxidizing and nitrogen-fixing bacterium, designated strain LS2T, was isolated from freshwater collected from the Pearl River in Guangzhou, PR China. The strain was an obligate chemolithoautotroph, utilizing reduced sulphur compounds (sulphide, sulphite, elemental sulphur, thiosulphate and tetrathionate) as energy sources and electron donors. Diazotrophic growth of strain LS2T was observed at 15-40 °C, pH 5-9, with a NaCl concentration range of 0-0.68 mol l-1 and with oxygen content higher than 21 %. The major cellular fatty acids were summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The DNA G+C content of the complete genome sequence was 60.7 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain LS2T formed a lineage within the family Halothiobacillaceae, showing gene sequence identity of 96.8 % with its closest relative Halothiobacillus neapolitanus c2. The genome of strain LS2T contains multiple genes encoding sulphur-oxidizing enzymes that catalyse the oxidation of reduced sulphur compounds and an nif complex encoding enzymes for nitrogen fixation. In addition, the genome contains genes encoding cbb3-type cytochrome c oxidase, aa3-type cytochrome c oxidase, bd-type quinol oxidase and cytochrome o oxidase, which enable the survival strain LS2T under oxic and microaerophilic conditions. On the basis of phenotypic, genotypic and phylogenetic data, strain LS2T is considered to represent a novel species of the genus Halothiobacillus, for which the name Halothiobacillus diazotrophicus sp. nov. is proposed. The type strain is LS2T (=GDMCC 1.4095T=JCM 39442T).

Structure and assembly of cargo Rubisco in two native α-carboxysomes.

Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions.

Decoding the Absolute Stoichiometric Composition and Structural Plasticity of α-Carboxysomes.

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO2-fixing enzymes, Rubisco, and carbonic anhydrase using a polyhedral protein shell that is constructed by hundreds of shell protein paralogs. The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. In this study, we adopted a QconCAT-based quantitative mass spectrometry approach to determine the stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native α-carboxysomes and their recombinant counterparts heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications. IMPORTANCE A wide range of bacteria use special protein-based organelles, termed bacterial microcompartments, to encase enzymes and reactions to increase the efficiency of biological processes. As a model bacterial microcompartment, the carboxysome contains a protein shell filled with the primary carbon fixation enzyme Rubisco. The self-assembling organelle is generated by hundreds of proteins and plays important roles in converting carbon dioxide to sugar, a process known as carbon fixation. In this study, we uncovered the exact stoichiometry of all building components and the structural plasticity of the functional α-carboxysome, using newly developed quantitative mass spectrometry together with biochemistry, electron microscopy, and enzymatic assay. The study advances our understanding of the architecture and modularity of natural carboxysomes. The knowledge learned from natural carboxysomes will suggest feasible ways to produce functional carboxysomes in other hosts, such as crop plants, with the overwhelming goal of boosting cell metabolism and crop yields.

The McdAB system positions α-carboxysomes in proteobacteria.

Carboxysomes are protein-based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is used to equally space out ß-carboxysomes across cell lengths by a two-component system (McdAB) in the model cyanobacterium Synechococcus elongatus PCC 7942. More recently, we found that McdAB systems are widespread among ß-cyanobacteria, which possess ß-carboxysomes, but are absent in α-cyanobacteria, which possess structurally and phyletically distinct α-carboxysomes. Cyanobacterial α-carboxysomes are thought to have arisen in proteobacteria and then horizontally transferred into cyanobacteria, which suggests that α-carboxysomes in proteobacteria may also lack the McdAB system. Here, using the model chemoautotrophic proteobacterium Halothiobacillus neapolitanus, we show that a McdAB system distinct from that of ß-cyanobacteria operates to position α-carboxysomes across cell lengths. We further show that this system is widespread among α-carboxysome-containing proteobacteria and that cyanobacteria likely inherited an α-carboxysome operon from a proteobacterium lacking the mcdAB locus. These results demonstrate that McdAB is a cross-phylum two-component system necessary for positioning both α- and ß-carboxysomes. The findings have further implications for understanding the positioning of other protein-based bacterial organelles involved in diverse metabolic processes. PLAIN LANGUAGE SUMMARY: Cyanobacteria are well known to fix atmospheric CO2 into sugars using the enzyme Rubisco. Less appreciated are the carbon-fixing abilities of proteobacteria with diverse metabolisms. Bacterial Rubisco is housed within organelles called carboxysomes that increase enzymatic efficiency. Here we show that proteobacterial carboxysomes are distributed in the cell by two proteins, McdA and McdB. McdA on the nucleoid interacts with McdB on carboxysomes to equidistantly space carboxysomes from one another, ensuring metabolic homeostasis and a proper inheritance of carboxysomes following cell division. This study illuminates how widespread carboxysome positioning systems are among diverse bacteria. Carboxysomes significantly contribute to global carbon fixation; therefore, understanding the spatial organization mechanism shared across the bacterial world is of great interest.

Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production.

Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli.

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.

Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation.

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

DABs are inorganic carbon pumps found throughout prokaryotic phyla.

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative ß-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.

Heterologous expression of the Halothiobacillus neapolitanus carboxysomal gene cluster in Corynebacterium glutamicum.

Compartmentalization represents a ubiquitous principle used by living organisms to optimize metabolic flux and to avoid detrimental interactions within the cytoplasm. Proteinaceous bacterial microcompartments (BMCs) have therefore created strong interest for the encapsulation of heterologous pathways in microbial model organisms. However, attempts were so far mostly restricted to Escherichia coli. Here, we introduced the carboxysomal gene cluster of Halothiobacillus neapolitanus into the biotechnological platform species Corynebacterium gluta-micum. Transmission electron microscopy, fluorescence microscopy and single molecule localization microscopy suggested the formation of BMC-like structures in cells expressing the complete carboxysome operon or only the shell proteins. Purified carboxysomes consisted of the expected protein components as verified by mass spectrometry. Enzymatic assays revealed the functional production of RuBisCO in C. glutamicum both in the presence and absence of carboxysomal shell proteins. Furthermore, we could show that eYFP is targeted to the carboxysomes by fusion to the large RuBisCO subunit. Overall, this study represents the first transfer of an α-carboxysomal gene cluster into a Gram-positive model species supporting the modularity and orthogonality of these microcompartments, but also identified important challenges which need to be addressed on the way towards biotechnological application.

Enriching acid rock drainage related microbial communities from surface-deposited oil sands tailings.

Little is known about the microbial communities native to surface-deposited pyritic oil sands tailings, an environment where acid rock drainage (ARD) could occur. The goal of this study was to enrich sulfur-oxidizing organisms from these tailings and determine whether different populations exist at pH levels 7, 4.5, and 2.5. Using growth-based methods provides model organisms for use in the future to predict potential activities and limitations of these organisms and to develop possible control methods. Thiosulfate-fed enrichment cultures were monitored for approximately 1 year. The results showed that the enrichments at pH 4.5 and 7 were established quicker than at pH 2.5. Different microbial community structures were found among the 3 pH environments. The sulfur-oxidizing microorganisms identified were most closely related to Halothiobacillus neapolitanus, Achromobacter spp., and Curtobacterium spp. While microorganisms related to Chitinophagaceae and Acidocella spp. were identified as the only possible iron-oxidizing and -reducing microbes. These results contribute to the general knowledge of the relatively understudied microbial communities that exist in pyritic oil sands tailings and indicate these communities may have a potential role in ARD generation, which may have implications for future tailings management.

Response of cbb gene transcription levels of four typical sulfur-oxidizing bacteria to the CO2 concentration and its effect on their carbon fixation efficiency during sulfur oxidation.

The variability in carbon fixation capability of four sulfur-oxidizing bacteria (Thiobacillus thioparus DSM 505, Halothiobacillus neapolitanus DSM 15147, Starkeya novella DSM 506, and Thiomonas intermedia DSM 18155) during sulfur oxidation was studied at low and high concentrations of CO2. The mechanism underlying the variability in carbon fixation was clarified by analyzing the transcription of the cbb gene, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. DSM 15147 and DSM 505 fixed carbon more efficiently during sulfur oxidation than DSM 506 and DSM 18155 at 0.5% and 10% CO2, which was mainly because their cbb gene transcription levels were much higher than those of DSM 506 and DSM 18155. A high CO2 concentration significantly stimulated the carbon fixation efficiency of DSM 505 by greatly increasing the cbb gene transcription efficiency. Moreover, the influence of the CO2 concentration on the carbon fixation efficiency of the four strains differed greatly during sulfur oxidation.

Programmed Ribosomal Frameshifting Mediates Expression of the α-Carboxysome.

Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic analysis of the assembly process of this complex (more than 200MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a molecular function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent molecular weight incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess -1 frameshifting elements that lead to the production of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes.

Engineering a thermostable Halothermothrix orenii ß-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining ß-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a ß-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ.

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. We show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.

Isolation and characterization of an obligately chemolithoautotrophic Halothiobacillus strain capable of growth on thiocyanate as an energy source.

Molecular and microbiological analysis of a laboratory bioreactor biomass oxidizing thiocyanate at autotrophic conditions and at 1 M NaCl showed a domination of a single chemolithoautotrophic sulfur-oxidizing bacterium (SOB) capable of using thiocyanate as an energy source. The bacterium was isolated in pure cultures and identified as a member of the Halothiobacillus halophilus/hydrothermalis clade. This clade includes moderately halophilic chemolithoautotrophic SOB from marine and hypersaline habitats for which the ability to utilize thiocyanate as an electron donor has not been previously demonstrated. Halothiobacillus sp. strain SCN-R1 grew with thiocyanate as the sole energy and nitrogen source oxidizing it to sulfate and ammonium via the cyanate pathway. The pH range for thiocyanate oxidation was within a neutral region between 7 and 8 and the range of salinity was from 0.2 to 1.5 M NaCl, with an optimum at 0.5 M. Despite the close phylogenetic relatedness, none of the tested type strains and other isolates from the H. halophilus/hydrothermalis group exhibited thiocyanate-oxidizing capacity.

Phylogenetic assessment of culture collection strains of Thiobacillus thioparus, and definitive 16S rRNA gene sequences for T. thioparus, T. denitrificans, and Halothiobacillus neapolitanus.

The 16S rRNA gene sequences of 12 strains of Thiobacillus thioparus held by different culture collections have been compared. A definitive sequence for the reference type strain (Starkey; ATCC 8158(T)) was obtained. The sequences for four examples of the Starkey type strain were essentially identical, confirming their sustained identity after passage through different laboratories. One strain (NCIMB 8454) was reassigned as a strain of Halothiobacillus neapolitanus, and a second (NCIMB 8349) was a species of Thermithiobacillus. These two strains have been renamed in their catalog by the National Collection of Industrial and Marine Bacteria. The 16S rRNA gene sequence of the type strain of Halothiobacillus neapolitanus (NCIMB 8539(T)) was determined and used to confirm the identity of other culture collection strains of this species. The reference sequences for the type strains of Thiobacillus thioparus and Halothiobacillus neapolitanus have been added to the online List of Prokaryotic Names with Standing in Nomenclature. Comparison of the 16S rRNA gene sequences available for strains of Thiobacillus denitrificans indicated that the sequence for the type strain (NCIMB 9548(T)) should always be used as the reference sequence for new and existing isolates.

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme.

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.

Comparative analysis of carboxysome shell proteins.

Carboxysomes are metabolic modules for CO(2) fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysome's icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO(2) fixation in other organisms or creating novel biological nanostructures.

Thiosulphate oxidation by Thiobacillus thioparus and Halothiobacillus neapolitanus strains isolated from the petrochemical industry

Sulphur Oxidizing Bacteria (SOB) is a group of microorganisms widely used for the biofiltration of Total Reduced Sulphur compounds (TRS). TRS are bad smelling compounds with neurotoxic activity which are produced by different industries (cellulose, petrochemical). Thiobacillus thioparus has the capability to oxidize organic TRS, and strains of this bacterium are commonly used for TRS biofiltration technology. In this study, two thiosulphate oxidizing strains were isolated from a petrochemical plant (ENAP BioBio, Chile). They were subjected to molecular analysis by real time PCR using specific primers for T. thioparus. rDNA16S were sequenced using universal primers and their corresponding thiosulphate activities were compared with the reference strain T. thioparus ATCC 10801 in batch standard conditions. Real time PCR and 16S rDNA sequencing showed that one of the isolated strains belonged to the Thiobacillus branch. This strain degrades thiosulphate with a similar activity profile to that shown by the ATCC 10801 strain, but with less growth, making it useful in biofiltration.

The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier.

BACKGROUND: Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO(2) fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected. CONCLUSIONS/SIGNIFICANCE: In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO(2) and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome.