scholarly journals Significant Role for Microbial Autotrophy in the Sequestration of Soil Carbon

2012 ◽  
Vol 78 (7) ◽  
pp. 2328-2336 ◽  
Author(s):  
Hongzhao Yuan ◽  
Tida Ge ◽  
Caiyan Chen ◽  
Anthony G. O'Donnell ◽  
Jinshui Wu
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Fragment Length Polymorphism ◽  
Ralstonia Eutropha ◽  
Gene Diversity ◽  
Rhodopseudomonas Palustris ◽  
C Sequestration ◽  
Rubisco Activity ◽  
Bisphosphate Carboxylase ◽  
Content Type ◽  
Autotrophic Microorganisms

ABSTRACTSoils were incubated for 80 days in a continuously labeled14CO2atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of14C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 108km2, this represents a potential global sequestration of 0.6 to 4.9 Pg C year−1. Estimated global C sequestration rates suggest a “missing sink” for carbon of between 2 and 3 Pg C year−1. To determine whether14CO2incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO2-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominantcbbL-containing bacteria wereAzospirillum lipoferum,Rhodopseudomonas palustris,Bradyrhizobium japonicum,Ralstonia eutropha, andcbbL-containing chromophytic algae of the generaXanthophytaandBacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences incbbL-containing microbial communities between soils. Differences incbbLgene diversity were shown to be correlated with differences in SOC content. Bacterial and algalcbbLgene abundances were between 106and 108and 103to 105copies g−1soil, respectively. BacterialcbbLabundance was shown to be positively correlated with RubisCO activity (r= 0.853;P< 0.05), and bothcbbLabundance and RubisCO activity were significantly related to the synthesis rates of [14C]SOC (r= 0.967 and 0.946, respectively;P< 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling.

scholarly journals Differential Accumulation of Form I RubisCO in Rhodopseudomonas palustris CGA010 under Photoheterotrophic Growth Conditions with Reduced Carbon Sources

2009 ◽  
Vol 191 (13) ◽  
pp. 4243-4250 ◽  
Author(s):  
Gauri S. Joshi ◽  
Simona Romagnoli ◽  
Nathan C. VerBerkmoes ◽  
Robert L. Hettich ◽  
Dale Pelletier ◽  
...  
Keyword(s):  
Purple Bacteria ◽  
Rhodopseudomonas Palustris ◽  
Growth Conditions ◽  
Activity Levels ◽  
Two Component System ◽  
Rubisco Activity ◽  
Bisphosphate Carboxylase ◽  
Component System ◽  
Two Component ◽  
Photoheterotrophic Growth

ABSTRACT Rhodopseudomonas palustris is unique among characterized nonsulfur purple bacteria because of its capacity for anaerobic photoheterotrophic growth using aromatic acids. Like growth with other reduced electron donors, this growth typically requires the presence of bicarbonate/CO2 or some other added electron acceptor in the growth medium. Proteomic studies indicated that there was specific accumulation of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) subunit proteins (CbbL and CbbS), as well as the CbbX protein, in cells grown on benzoate without added bicarbonate; such cells used the small amounts of dissolved CO2 in the medium to support growth. These proteins were not observed in extracts from cells grown in the presence of high levels (10 mM) of added bicarbonate. To confirm the results of the proteomics studies, it was shown that the total RubisCO activity levels were significantly higher (five- to sevenfold higher) in wild-type (CGA010) cells grown on benzoate with a low level (0.5 mM) of added bicarbonate. Immunoblots indicated that the increase in RubisCO activity levels was due to a specific increase in the amount of form I RubisCO (CbbLS) and not in the amount of form II RubisCO (CbbM), which was constitutively expressed. Deletion of the main transcriptional regulator gene, cbbR, resulted in impaired growth on benzoate-containing low-bicarbonate media, and it was established that form I RubisCO synthesis was absolutely and specifically dependent on CbbR. To understand the regulatory role of the CbbRRS two-component system, strains with nonpolar deletions of the cbbRRS genes were grown on benzoate. Distinct from the results obtained with photoautotrophic growth conditions, the results of studies with various CbbRRS mutant strains indicated that this two-component system did not affect the observed enhanced synthesis of form I RubisCO under benzoate growth conditions. These studies indicate that diverse growth conditions differentially affect the ability of the CbbRRS two-component system to influence cbb transcription.

scholarly journals Calvin Cycle Flux, Pathway Constraints, and Substrate Oxidation State Together Determine the H2 Biofuel Yield in Photoheterotrophic Bacteria

mBio ◽  
2011 ◽  
Vol 2 (2) ◽  
Author(s):  
James B. McKinlay ◽  
Caroline S. Harwood
Keyword(s):  
Organic Compounds ◽  
Oxidation State ◽  
Rhodopseudomonas Palustris ◽  
Calvin Cycle ◽  
Substrate Oxidation ◽  
Hydrogen Gas ◽  
Anaerobic Growth ◽  
Anoxygenic Phototrophic Bacteria ◽  
Transportation Fuel ◽  
Content Type

ABSTRACTHydrogen gas (H2) is a possible future transportation fuel that can be produced by anoxygenic phototrophic bacteria via nitrogenase. The electrons for H2are usually derived from organic compounds. Thus, one would expect more H2to be produced when anoxygenic phototrophs are supplied with increasingly reduced (electron-rich) organic compounds. However, the H2yield does not always differ according to the substrate oxidation state. To understand other factors that influence the H2yield, we determined metabolic fluxes inRhodopseudomonas palustrisgrown on13C-labeled fumarate, succinate, acetate, and butyrate (in order from most oxidized to most reduced). The flux maps revealed that the H2yield was influenced by two main factors in addition to substrate oxidation state. The first factor was the route that a substrate took to biosynthetic precursors. For example, succinate took a different route to acetyl-coenzyme A (CoA) than acetate. As a result,R. palustrisgenerated similar amounts of reducing equivalents and similar amounts of H2from both succinate and acetate, even though succinate is more oxidized than acetate. The second factor affecting the H2yield was the amount of Calvin cycle flux competing for electrons. When nitrogenase was active, electrons were diverted away from the Calvin cycle towards H2, but to various extents, depending on the substrate. When Calvin cycle flux was blocked, the H2yield increased during growth on all substrates. In general, this increase in H2yield could be predicted from the initial Calvin cycle flux.IMPORTANCEPhotoheterotrophic bacteria, likeRhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H2as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H2. Despite repeated observations over the last 40 years, the reasons for this discrepancy have remained unclear. In this paper, we identified two metabolic factors that influence the H2yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO2-fixing Calvin cycle flux that competes against H2production for electrons. We show that the H2yield can be improved on all substrates by using a strain that is incapable of Calvin cycle flux. We also contributed quantitative knowledge to the long-standing question of why photoheterotrophs must produce H2or fix CO2even on relatively oxidized substrates.

scholarly journals Microbial Functional Gene Diversity with a Shift of Subsurface Redox Conditions duringIn SituUranium Reduction

2012 ◽  
Vol 78 (8) ◽  
pp. 2966-2972 ◽  
Author(s):  
Yuting Liang ◽  
Joy D. Van Nostrand ◽  
Lucie A. N′Guessan ◽  
Aaron D. Peacock ◽  
Ye Deng ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Field Experiments ◽  
Gene Diversity ◽  
Functional Gene ◽  
Redox Conditions ◽  
Functional Genes ◽  
Microbial Functional Diversity ◽  
Content Type ◽  
Microbial Functional Gene

ABSTRACTTo better understand the microbial functional diversity changes with subsurface redox conditions duringin situuranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance ofdsrABgenes (dissimilatory sulfite reductase genes) and methane generation-relatedmcrgenes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily fromGeobactersp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (Eh) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect thein situredox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.

scholarly journals Descriptions of Rhodopseudomonas parapalustris sp. nov., Rhodopseudomonas harwoodiae sp. nov. and Rhodopseudomonas pseudopalustris sp. nov., and emended description of Rhodopseudomonas palustris

2012 ◽  
Vol 62 (Pt_8) ◽  
pp. 1790-1798 ◽  
Author(s):  
V. Venkata Ramana ◽  
S. Kalyana Chakravarthy ◽  
P. Shalem Raj ◽  
B. Vinay Kumar ◽  
E. Shobha ◽  
...  
Keyword(s):  
Dna Hybridization ◽  
Type Species ◽  
Novel Species ◽  
Rhodopseudomonas Palustris ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Geographical Regions ◽  
Emended Description ◽  
Type Strains

Four strains (JA310T, JA531T, JA447 and JA490) of red to reddish brown pigmented, rod-shaped, motile and budding phototrophic bacteria were isolated from soil and freshwater sediment samples from different geographical regions of India. All strains contained bacteriochlorophyll a and carotenoids of the spirilloxanthin series. The major cellular fatty acid of strains JA310T and JA531T was C18 : 1ω7c, the quinone was Q-10 and polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an aminohopanoid and an unidentified aminolipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that all strains clustered with species of the genus Rhodopseudomonas in the class Alphaproteobacteria . Strains JA531T, JA447 and JA490 were genotypically (>80 % related based on DNA–DNA hybridization) and phenotypically closely related to each other and the three strains were distinct from strain JA310T (33 % related). Furthermore, all four strains had less than 48 % relatedness (DNA–DNA hybridization) with type strains of members of the genus Rhodopseudomonas , i.e. Rhodopseudomonas palustris ATCC 17001T, Rhodopseudomonas faecalis JCM 11668T and Rhodopseudomonas rhenobacensis DSM 12706T. The genomic DNA G+C contents of strains JA310T and JA531T were 63.8 and 62.4 mol%, respectively. On the basis of phenotypic, chemotaxonomic and molecular genetic evidence, it is proposed that strains JA310T ( = NBRC 106083T = KCTC 5839T) and JA531T ( = NBRC 107575T = KCTC 5841T) be classified as the type strains of two novel species of the genus Rhodopseudomonas , Rhodopseudomonas parapalustris sp. nov. and Rhodopseudomonas harwoodiae sp. nov., respectively. In addition, we propose that strain DSM 123T ( = NBRC 100419T) represents a novel species, Rhodopseudomonas pseudopalustris sp. nov., since this strain differs genotypically and phenotypically from R. palustris ATCC 17001T and other members of the genus Rhodopseudomonas . An emended description of R. palustris is also provided.

scholarly journals Estimating the Prevalence of Potential Enteropathogenic Escherichia coli and Intimin Gene Diversity in a Human Community by Monitoring Sanitary Sewage

2013 ◽  
Vol 80 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Kun Yang ◽  
Eulyn Pagaling ◽  
Tao Yan
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Gene Diversity ◽  
Content Type ◽  
E Coli ◽  
Enteric Diseases ◽  
Detection Frequency ◽  
Alternative Approach ◽  
Prevalent Subtype ◽  
Qpcr Quantification

ABSTRACTPresently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coli(EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of theeae,stx1, andstx2genes in sanitary sewage samples collected over a 13-month period detectedeaein all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) thanstx1andstx2, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio ofeaetouidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of theeaegene directly from the sewage samples covered the majority of theeaediversity in the sewage and detected 17 uniqueeaealleles belonging to 14 subtypes. Among them,eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmentalE. coliisolates were also obtained and used to determine the detection frequencies of the virulence genes as well aseaegenetic diversity for comparison.

Effect of cadmium on growth and oxidative metabolism of faba bean plants

2004 ◽  
Vol 52 (3) ◽  
pp. 269-276 ◽  
Author(s):  
H. R. Moussa
Keyword(s):  
Oxidative Stress ◽  
Faba Bean ◽  
Photosynthetic Efficiency ◽  
Nutrient Status ◽  
Significant Inhibition ◽  
Free Proline ◽  
Rubisco Activity ◽  
Bisphosphate Carboxylase ◽  
Inhibition Of Growth ◽  
Bean Plants

The effect of CdCl2(0-50 µM) on the growth, physiological parametersand leaf antioxidative enzymes of faba bean plants was studied in order toinvestigate the possible involvement of this metal in the generationof oxidative stress. In the roots and leaves of faba bean plants Cd produceda significant inhibition of growth, as well as a reduction inthe transpiration rate, photosynthetic efficiency (14CO2-fixation), ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) activity and leaf pigment content, and an alteration in the nutrient status in booth roots and leaves. an increased level of free proline was also detected. The results suggest thatthe treatment of faba bean plants with CdCl2 induced a concentration-dependentoxidative stress situation in the leaves, characterized by an accumulationof  H2O2, as a result of theinhibition of the antioxidant enzymes glutathione reductase (GR) and catalase (CAT). These results point to the possible inductionof leaf senescence by cadmium.

scholarly journals Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

2015 ◽  
Vol 82 (3) ◽  
pp. 910-921 ◽  
Author(s):  
Leonie Wenning ◽  
Nadine Stöveken ◽  
Jan Hendrik Wübbeler ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Metal Chelate ◽  
Sulfinic Acid ◽  
Cysteine Sulfinic Acid ◽  
Content Type ◽  
Apparent Homogeneity ◽  
Metal Cofactor ◽  
Metal Chelate Affinity Chromatography

ABSTRACTCysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. InRalstonia eutrophaH16, two Cdo homologues (CdoA and CdoB) have been identified previously.In vivostudies showed thatEscherichia colicells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing anin vitrocharacterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos fromR. eutrophaH16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis inR. eutrophaH16, deletion ofcdoAmight enable increased synthesis of PTEs.

Thioalkalivibrio sulfidiphilus sp. nov., a haloalkaliphilic, sulfur-oxidizing gammaproteobacterium from alkaline habitats

2012 ◽  
Vol 62 (Pt_8) ◽  
pp. 1884-1889 ◽  
Author(s):  
Dimitry Y. Sorokin ◽  
Maria S. Muntyan ◽  
Anzhela N. Panteleeva ◽  
Gerard Muyzer
Keyword(s):  
Soda Lake ◽  
16S Rrna Gene Sequencing ◽  
Salinity Range ◽  
Rrna Gene ◽  
Type I ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Content Type ◽  
Link Type ◽  
Sulfur Oxidizing

A moderately salt-tolerant and obligately alkaliphilic, chemolithoautotrophic sulfur-oxidizing bacterium, strain HL-EbGr7T, was isolated from a full-scale bioreactor removing H2S from biogas under oxygen-limited conditions. Another strain, ALJ17, closely related to HL-EbGr7T, was isolated from a Kenyan soda lake. Cells of the isolates were relatively long, slender rods, motile by a polar flagellum. Although both strains were obligately aerobic, micro-oxic conditions were preferred, especially at the beginning of growth. Chemolithoautotrophic growth was observed with sulfide and thiosulfate in a pH range of 8.0–10.5 (optimum at pH 10.0) and a salinity range of 0.2–1.5 M total Na+ (optimum at 0.4 M). The genome sequence of strain HL-EbGr7T demonstrated the presence of genes encoding the reverse Dsr pathway and a truncated Sox pathway for sulfur oxidation and enzymes of the Calvin–Benson cycle of autotrophic CO2 assimilation with ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) type I. The dominant cellular fatty acids were C18 : 1ω7, C16 : 0 and C19 : 0 cyclo. Based on 16S rRNA gene sequencing, the two strains belonged to a single phylotype within the genus Thioalkalivibrio in the Gammaproteobacteria . Despite being related most closely to Thioalkalivibrio denitrificans , the isolates were unable to grow by denitrification. On the basis of phenotypic and phylogenetic analysis, the novel isolates are proposed to represent a novel species, Thioalkalivibrio sulfidiphilus sp. nov., with the type strain HL-EbGr7T ( = NCCB 100376T  = UNIQEM U246T).

scholarly journals Genetic diversity in wild and hatchery populations of stinging catfish (Heteropneustes fossilis Bloch) revealed by RAPD analysis

2012 ◽  
Vol 19 ◽  
pp. 81-87
Author(s):  
Md Nazrul Islam ◽  
Abhishak Basak ◽  
Dr Ashrafullah ◽  
Md Samsul Alam
Keyword(s):  
Genetic Diversity ◽  
Genetic Variation ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Gene Diversity ◽  
Similarity Index ◽  
Heteropneustes Fossilis ◽  
Wild Populations ◽  
Polymorphic Loci

Context: DNA fingerprinting using genetic markers such as Random Amplification of Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), microsatellite (Simple sequence repeat), Amplified Fragment Length Polymorphism (AFLP) etc. can be successfully used to reveal genetic variation within and among different populations. Objective: The aim of the present study was to assess genetic diversity in two wild and one hatchery populations of stinging catfish Heteropneustes fossilis by RAPD fingerprinting. Materials and Methods: A total of 90 live fish (H. fossilis), 30 from each source, were collected from a beel in Patuakhali, a beel in Jessore and Rupali Hatchery, Mymensingh. Genomic DNA was extracted from fin tissues. The concentration of DNA was estimated using a spectrophotometer. Fifteen decamer primers of random sequence from three kits (six from kit A, seven from kit B and two from kit C) (Operon technologies, Inc., Alameda, CA, USA) were screened on sub-samples of one randomly chosen H. fossilis DNA sample from the each population to test their suitability for amplifying RAPDs. The amplified products from each sample were separated by electrophoresis on 1.4% agarose gel containing ethidium bromide. The sizes of the bands were calculated using the software DNAFRAG and the sizes in base pair (bp) were used for identification of the bands (RAPD markers). The similarity index values (SI) between the RAPD fingerprint of any two individuals on the same gel were calculated from RAPD band sharing. Results: A total of 28 RAPD bands were obtained using four decamer random primers, among which 21 bands were polymorphic. The percentage of polymorphic loci, intra-population similarity indices and Nei's gene diversity values were 85.71%, 78.75 and 0.304±0.183 for Jessore population, 83.71%, 82.62 and 0.280±0.159 for Patuakhali population, 82.14%, 85.25 and 0.271±0.165 for Rupali hatchery population, respectively. The overall gene flow (Nm) among the populations was 5.755. The highest inter-similarity (Sij) was found between Patuakhali - Rupali hatchery populations. Among the three populations, the highest genetic distance (0.069) was found between Jessore and Patuakhali population. Considering polymorphic loci, intrapopulation similarity index and gene diversity the genetic variation in the Jessore population was higher than the other two populations. The genetic variation of the hatchery population was found to be lower than the two wild populations. Conclusion: The result of the present study can be used as baseline information regarding the genetic variation and population structure before undertaking any breeding programme. Study indicated that the genetic variation in the hatchery populations were slightly lower than those of the wild populations. DOI: http://dx.doi.org/10.3329/jbs.v19i0.13005 J. bio-sci. 19 81-87, 2011

scholarly journals Biotin Synthesis in Ralstonia eutropha H16 Utilizes Pimeloyl Coenzyme A and Can Be Regulated by the Amount of Acceptor Protein

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Jessica Eggers ◽  
Carl Simon Strittmatter ◽  
Kira Küsters ◽  
Emre Biller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Acyl Carrier Protein ◽  
Value Added ◽  
Auxotrophic Mutant ◽  
Bioinformatic Analysis ◽  
Biotechnological Production ◽  
Content Type ◽  
Homologous Expression ◽  
Regulator Protein ◽  
Eukaryotic Microbes

ABSTRACT The biotin metabolism of the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha (syn. Cupriavidus necator), which is used for biopolymer production in industry, was investigated. A biotin auxotroph mutant lacking bioF was generated, and biotin depletion in the cells and the minimal biotin demand of a biotin-auxotrophic R. eutropha strain were determined. Three consecutive cultivations in biotin-free medium were necessary to prevent growth of the auxotrophic mutant, and 40 ng/ml biotin was sufficient to promote cell growth. Nevertheless, 200 ng/ml biotin was necessary to ensure growth comparable to that of the wild type, which is similar to the demand of biotin-auxotrophic mutants among other prokaryotic and eukaryotic microbes. A phenotypic complementation of the R. eutropha ΔbioF mutant was only achieved by homologous expression of bioF of R. eutropha or heterologous expression of bioF of Bacillus subtilis but not by bioF of Escherichia coli. Together with the results from bioinformatic analysis of BioFs, this leads to the assumption that the intermediate of biotin synthesis in R. eutropha is pimeloyl-CoA instead of pimeloyl-acyl carrier protein (ACP) like in the Gram-positive B. subtilis. Internal biotin content was enhanced by homologous expression of accB, whereas homologous expression of accB and accC2 in combination led to decreased biotin concentrations in the cells. Although a DNA-binding domain of the regulator protein BirA is missing, biotin synthesis seemed to be influenced by the amount of acceptor protein present. IMPORTANCE Ralstonia eutropha is applied in industry for the production of biopolymers and serves as a research platform for the production of diverse fine chemicals. Due to its ability to grow on hydrogen and carbon dioxide as the sole carbon and energy source, R. eutropha is often utilized for metabolic engineering to convert inexpensive resources into value-added products. The understanding of the metabolic pathways in this bacterium is mandatory for further bioengineering of the strain and for the development of new strategies for biotechnological production.

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