Non-pathogenic Soil Bacterium Research Articles

Attachment of Extracellular Metabolic Products of Lysinibacillus sp . DRG3 on Sand Surface under Variable Flow Velocities and Bioprocesses

The efficacy of microbially mediated stabilization of soil mass depends on soil aggregation and further depends on the complex interplay of environmental parameters, microbial extracellular metabolic products, and surface characteristics of soil particles. Failures of flood control dikes or similar structures often culminate from minor erosion of soil particles initiated by groundwater seepage. Although the introduction of microbial metabolic products in controlling soil erosion has been studied by researchers, the influence of slow fluvial activities on the composition, characteristics, and their impacts on attachment mechanisms of extracellular polymeric substances (EPS) on soil surfaces have remained unexplored. Impacts of slow fluvial activities on the amount and chemical composition of EPS produced by Lysinibacillus sp. DRG3, a nonpathogenic soil bacterium, and the attachment mechanisms of the EPS produced under noncalcifying, nonureolytic, and ureolytic calcifying bioprocesses on the sand surfaces were investigated. DRG3-inoculated specimens were incubated in the presence of steady circulation of aqueous media containing minimal concentrations of minerals and carbon and nitrogen sources to simulate groundwater movements through soil. Quantity, compactness, continuity, and viscosity of EPS and the amounts of carbohydrate, protein, lipid, DNA, and RNA found in EPS increased with circulation velocity and incubation duration. EPS were found to attach to sand through electrostatic interaction and hydrogen bonding. Internally, EPS components interacted with each other through electrostatic interaction, hydrogen bonding, and hydrophobic interaction. Electrostatic interaction appeared to weaken with increasing media circulation intensity and alkalinity. In contrast, EPS production and hydrogen bonding intensified under increased media circulation. Results of this investigation suggest microbe-mediated soil aggregation becomes stronger and more efficient under slow media circulation and are expected to have implications on microbially mediated soil stabilization, particularly in addressing soil erosion. This study provides useful insights for successful field implementation of biomediated soil stabilization. Work presented herein also demonstrates a role for microbial activities found in subterranean environments in strengthening an existing sand deposit.

Physiological Responses of Ribosomal Protein S12 K43 Mutants of Corynebacterium glutamicum.

Bacterial resistance to streptomycin is often acquired as a consequence of mutations in rpsL, the gene encoding ribosomal protein S12. Corynebacterium glutamicum is a non-pathogenic Gram-positive soil bacterium that has been widely used in industry. In a previous study, we screened several streptomycin-resistant rpsL K43 mutants of C. glutamicum, and surprisingly found that two of them also confer chloramphenicol and/or kanamycin resistance. In order to understand whether or not a single mutation of rpsLK43 could confer resistance to multiple antibiotics, in this study we attempted to construct saturation mutagenesis of rpsL K43 by rational genetic manipulation. Despite many efforts had been made, only nine mutants were successfully constructed. They were indeed resistant to streptomycin, but not to other antibiotics. This suggested that other mutations should be acquired, contributing to multiple antibiotics in the screened strains. The growth and enhanced green fluorescent protein (eGFP) expression of these nine mutants were then investigated. The results showed that they grew differently in CGXII minimal medium, but not in BHI medium. When cultured in the absence of streptomycin, the expression of eGFP was positively proportional to the growth, approximately, while in the presence of streptomycin, the expression of eGFP was proportional to the ability of streptomycin resistance.

Acinetobacter baylyi ADP1-naturally competent for synthetic biology.

Acinetobacter baylyi ADP1 is a non-pathogenic soil bacterium known for its metabolic diversity and high natural transformation and recombination efficiency. For these features, A. baylyi ADP1 has been long exploited in studying bacterial genetics and metabolism. The large pool of information generated in the fundamental studies has facilitated the development of a broad range of sophisticated and robust tools for the genome and metabolic engineering of ADP1. This mini-review outlines and describes the recent advances in ADP1 engineering and tool development, exploited in, for example, pathway and enzyme evolution, genome reduction and stabilization, and for the production of native and non-native products in both pure and rationally designed multispecies cultures. The rapidly expanding toolbox together with the unique features of A. baylyi ADP1 provide a strong base for a microbial cell factory excelling in synthetic biology applications where evolution meets rational engineering.

Burkholderia ubonensis High-Level Tetracycline Resistance Is Due to Efflux Pump Synergy Involving a Novel TetA(64) Resistance Determinant.

Burkholderia ubonensis, a nonpathogenic soil bacterium belonging to the Burkholderia cepacia complex (Bcc), is highly resistant to some clinically significant antibiotics. The concern is that B. ubonensis may serve as a resistance reservoir for Bcc or B. pseudomallei complex (Bpc) organisms that are opportunistic human pathogens. Using a B. ubonensis strain highly resistant to tetracycline (MIC, ≥256 µg/ml), we identified and characterized tetA(64) that encodes a novel tetracycline-specific efflux pump of the major facilitator superfamily. TetA(64) and associated TetR(64) regulator expression are induced by tetracyclines. Although TetA(64) is the primary tetracycline and doxycycline resistance determinant, maximum tetracycline and doxycycline resistance requires synergy between TetA(64) and the nonspecific AmrAB-OprA resistance nodulation cell division efflux pump. TetA(64) does not efflux minocycline, tigecycline, and eravacycline. Comprehensive screening of genome sequences showed that TetA(64) is unequally distributed in the Bcc and absent from the Bpc. It is present in some major cystic fibrosis pathogens, like Burkholderia cenocepacia, but absent from others like Burkholderia multivorans The tetR(64)-tetA(64) genes are located in a region of chromosome 1 that is highly conserved in Burkholderia sp. Because there is no evidence for transposition, the tetR(64)-tetA(64) genes may have been acquired by homologous recombination after horizontal gene transfer. Although Burkholderia species contain a resident multicomponent efflux pump that allows them to respond to tetracyclines up to a certain concentration, the acquisition of the single-component TetA(64) by some species likely provides the synergy that these bacteria need to defend against high tetracycline concentrations in niche environments.

Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365

Paenibacillus polymyxa is a Gram-positive, non-pathogenic soil bacterium that has been extensively investigated for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers were restricted due to limited genetic accessibility of the organism up to now. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants were generated and compared in batch fermentations for the first time. Downstream processing was facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Spore formation was inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of P. polymyxa. Optimization of the carbon flux towards 2,3-butanediol was achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain showed 45 % increased productivity, reaching a final concentration of 43.8 g L−1 butanediol. A yield of 0.43 g g−1 glucose was achieved, accounting for 86 % of the theoretical maximum.

Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering of Pseudomonas putida

Genome engineering of non-conventional microorganisms calls for the development of dedicated synthetic biology tools. Pseudomonas putida is a Gram-negative, non-pathogenic soil bacterium widely used for metabolic engineering owing to its versatile metabolism and high levels of tolerance to different types of stress. Genome editing of P. putida largely relies on homologous recombination events, assisted by helper plasmid-based expression of genes encoding DNA modifying enzymes. Plasmid curing from selected isolates is the most tedious and time-consuming step of this procedure, and implementing commonly used methods to this end in P. putida (e.g. temperature-sensitive replicons) is often impractical. To tackle this issue, we have developed a toolbox for both target- and self-curing of plasmid DNA in Pseudomonas species. Our method enables plasmid-curing in a simple cultivation step by combining in vivo digestion of vectors by the I-SceI homing nuclease with synthetic control of plasmid replication, triggered by the addition of a cheap chemical inducer (3-methylbenzoate) to the medium. The system displays an efficiency of vector curing >90% and the screening of plasmid-free clones is greatly facilitated by the use of fluorescent markers that can be selected according to the application intended. Furthermore, quick genome engineering of P. putida using self-curing plasmids is demonstrated through genome reduction of the platform strain EM42 by eliminating all genes encoding β-lactamases, the catabolic ben gene cluster, and the pyoverdine synthesis machinery. Physiological characterization of the resulting streamlined strain, P. putida SEM10, revealed advantageous features that could be exploited for metabolic engineering.

Antibacterial Activity and Cytotoxicity of Silver Chloride/Silver Nanocomposite Synthesized by a Bacterium Isolated from Antarctic Soil

This study aims to describe the antibacterial and cytotoxic properties of a nanocomposite composed of silver chloride and silver nanoparticles (AgCl/Ag) biogenically synthesized using Arthrobacter sp., a non-pathogenic Antarctic soil bacterium. Spherical nanoparticles (hydrodynamic size of 42 nm) coated with biomolecules derived from Arthrobacter sp. were obtained. The antibacterial activity of the nanocomposite was demonstrated against Gram-positive and Gram-negative bacterial strains, including the multidrug-resistant Pseudomonas aeruginosa KPC 37. Furthermore, the cytotoxicity of AgCl/Ag nanocomposite was evaluated against the non-tumoral (Vero) and tumoral (SW-1353) cell lines. The nanocomposite was not cytotoxic to the Vero cells at concentrations below 150 μg/mL, whereas at concentrations above 25 μg/mL, a significant cytotoxic effect was observed for the SW-1353 cells. Interestingly, at 150 μg/mL, the nanocomposite demonstrated a potent antibacterial activity for all bacterial strains evaluated. To the best of our knowledge, this is the first report describing the green synthesis of AgCl/Ag nanocomposite by the bacterium Arthrobacter sp. isolated from Antarctic soil. The obtained nanocomposite may find important biomedical applications, with potent antibacterial and antitumoral effects and low toxicity to healthy cells.

Inhibition of Listeria monocytogenes using biofilms of non-pathogenic soil bacteria (Streptomyces spp.) on stainless steel under desiccated condition

Of the 1648 microbial isolates from 133 soil samples collected from 30 diverse locations in the Republic of Korea, two isolates exhibited strong antilisterial activity and ability to grow to high populations (>8.0 log CFU/ml) in Bennett's broth. Isolates were identified as Streptomyces lactacystinicus (strain Samnamu 5–15) and Streptomyces purpureus (strain Chamnamu-sup 4–15). Both isolates formed biofilms on the surface of stainless steel coupons (SSCs) immersed in Bennett's broth within 24 h at 25 °C. Cells retained antilisterial activity after biofilm formation and showed significantly (P ≤ 0.05) enhanced resistance against dry conditions (43% relative humidity [RH]) compared to the cells not in biofilm. An initial population (ca. 3.2 log CFU/cm2) of Listeria monocytogenes inoculated on SSCs lacking Streptomyces biofilm decreased to 1.4 log CFU/cm2 within 48 h at 25 °C and 43% RH. In contrast, L. monocytogenes (3.3 log CFU/cm2) inoculated on SSCs containing Streptomyces biofilm decreased to populations below the theoretical detection limit (0.5 log CFU/cm2) within 24 h. The results indicate that biofilms formed by the Streptomyces spp. inhibitory to L. monocytogenes showed enhanced resistance to desiccation condition (43% RH) and effectively inhibited the growth of L. monocytogenes on the surfaces of SSCs. Antilisterial biofilms developed in this study may be applicable on desiccated environmental surfaces in food related environments such as food storage, handling, and processing facilities to enhance the microbiological safety of foods.

Bacteria and Competing Herbivores Weaken Top-Down and Bottom-Up Aphid Suppression.

Herbivore suppression is mediated by both plant defenses and predators. In turn, plant defenses are impacted by soil fertility and interactions with soil bacteria. Measuring the relative importance of nutritional and microbial drivers of herbivore resistance has proven problematic, in part because it is difficult to manipulate soil-bacterial community composition. Here, we exploit variation in soil fertility and microbial biodiversity across 20 farms to untangle suppression of aphids (Brevicoryne brassicae) through bottom–up and top–down channels. We planted Brassica oleracea plants in soil from each farm, manipulated single and dual infestations of aphids alone or with caterpillars (Pieris rapae), and exposed aphids to parasitoid wasps (Diaeretiella rapae) in the open field. We then used multi-model inference to identify the strongest soil-based predictors of herbivore growth and parasitism. We found that densities of Bacillus spp., a genus known to include plant-growth-promoting rhizobacteria, negatively correlated with aphid suppression by specialist parasitoids. Aphid parasitism also was disrupted on plants that had caterpillar damage, compared to plants attacked only by aphids. Relative abundance of Pseudomonas spp. bacteria correlated with higher aphid growth, although this appeared to be a direct effect, as aphid parasitism was not associated with this group of bacteria. Non-pathogenic soil bacteria are often shown to deliver benefits to plants, improving plant nutrition and the deployment of anti-herbivore defenses. However, our results suggest that these plant growth-promoting bacteria may also indirectly weaken top–down aphid suppression by parasitoids and directly improve aphid performance. Against a background of varying soil fertility, microbial biodiversity, competing herbivores, and natural enemies, we found that effects of non-pathogenic soil microbes on aphid growth outweighed those of nutritional factors. Therefore, predictions about the strength of plant defenses along resource gradients must be expanded to include microbial associates.

Metabolic disharmony and sibling conflict mediated by T6SS

Bacteria in nature live in taxonomically complex communities where multitude of species and strains inhabit the same niches and compete for limited resources and space. Surviving in these competitive environments requires mechanisms to recognize and associate with kin and to discriminate against non-kin to increase reproductive success among close relatives. Some of the mechanisms bacteria use to address genetic differences are surface receptors, diffusible signals (e.g. quorum sensing) and toxin-immunity systems (e.g. type VI secretion system (T6SS)). Another way individuals vary within bacterial populations is their physiological states. This means that among clonal cells there is cell-to-cell variability in cells’ proteome, growth rates, age and cell damage loads caused by stochastic differences in gene expression/metabolism and variations in microenvironmental stimuli. While physiological heterogeneity benefits some bacteria by allowing populations to bet-hedge their survival odds in changing environments by expressing different phenotypes, it can also be harmful in cases where fitness depends on coordinated behaviors and synchronized actions by many cells; a function of particular importance to social bacteria. Myxococcus xanthus is a non-pathogenic soil bacterium known for its complex social and coordinated behaviors such as swarming, predation and formation of spore-filled fruiting bodies. These behaviors depend on M. xanthus ability to synchronize the actions of many cells within a population. Considering the collective nature of M. xanthus, we asked how do physiological differences affect cell-cell interactions in this species. To address this question, we investigated the interactions between two genetically related but physiologically distinct populations. We found that M. xanthus uses T6SS to eliminate less fit cells from their population and identified toxic effector and cognate immunity protein (TsxEI) that mediates this sibling antagonism.

Azospirillum brasilense: Laboratory Maintenance and Genetic Manipulation.

Bacteria of the genus Azospirillum, including the most comprehensively studied Azospirillum brasilense, are non-pathogenic soil bacteria that promote the growth of diverse plants, making them an attractive model to understand non-symbiotic, beneficial plant-bacteria associations. Research into the physiology and genetics of these organisms spans decades and a range of molecular tools and protocols have been developed for allelic exchange mutagenesis, in trans expression of genes, and fusions to reporter genes. © 2017 by John Wiley & Sons, Inc.

Bacterial Inoculant Treatment of Bermudagrass Alters Ovipositional Behavior, Larval and Pupal Weights of the Fall Armyworm (Lepidoptera: Noctuidae).

Nonpathogenic soil bacteria can colonize the rhizosphere and induce unique plant phenotypes that may influence plant-insect interactions. However, few studies have considered the influences of bacteria-plant interactions on insect feeding and oviposition. The objective of this study was to determine how rhizobacterial inoculation of bermudagrass affects larval development and ovipositional behaviors of the fall armyworm (Spodoptera frugiperda J.E. Smith). Eight blends of rhizobacteria known to induce root or shoot growth in grasses were applied weekly to hybrid bermudagrass for 5 wk. Oviposition was evaluated in two no-choice trials with bacteria-treated, fertilized, or nontreated grass. Grass blades from these treatments were extracted in polar and nonpolar solvents and assayed for oviposition responses. Another experiment compared the development of fall armyworm larvae on bermudagrass treated with each of the eight rhizobacterial blends for 5 wk to larvae fed nontreated bermudagrass. Females deposited more eggs on nontreated and fertilized grass and ≤34% of eggs on grass treated with rhizobacterial blends. Moths exposed to polar and nonpolar extracts were unable to reproduce these results. Larval and pupal weights at days 10 and 12 and the number of adults to eclose were lower for larvae fed some, but not all, bacteria-treated bermudagrass relative to controls. This is one of the few studies to investigate plant-microbe-insect interactions in an economically important system. Although the effects noted with fall armyworm are limited, induced changes in roots also reported for these bacteria may have greater utility than foliar changes for mediating interactions with biotic or abiotic stresses.

Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum

The genome-wide identification of transcription start sites, enabled by high-throughput sequencing of a cDNA library enriched for native 5′ transcript ends, is ideally suited for the analysis of promoters. Here, the transcriptome of Corynebacterium glutamicum, a non-pathogenic soil bacterium from the actinomycetes branch that is used in industry for the production of amino acids, was analysed by transcriptome sequencing of the 5′-ends of native transcripts. Total RNA samples were harvested from the exponential phase of growth, therefore the study mainly addressed promoters recognized by the main house-keeping sigma factor σA. The identification of 2454 transcription start sites (TSS) allowed the detailed analysis of most promoters recognized by σA and furthermore enabled us to form different promoter groups according to their location relative to protein-coding regions. These groups included leaderless transcripts (546 promoters), short-leadered (<500 bases) transcripts (917), and long-leadered (>500 bases) transcripts (173) as well as intragenic (557) and antisense transcripts (261). All promoters and the individual groups were searched for information, e.g. conserved residues and promoter motifs, and general design features as well as group-specific preferences were identified. A purine was found highly favored as TSS, whereas the −1 position was dominated by pyrimidines. The spacer between TSS and ‐10 region were consistently 6–7 bases and the −10 promoter motif was generally visible, whereas a recognizable −35 region was only occurring in a smaller fraction of promoters (7.5%) and enriched for leadered and antisense transcripts but depleted for leaderless transcripts. Promoters showing an extended −10 region were especially frequent in case of non-canonical −10 motifs (45.5%). Two bases downstream of the −10 core region, a G was conserved, exceeding 40% abundance in most groups. This fraction reached 74.6% for a group of putative σB-dependent promoters, thus giving a hint to a specific property of these promoters. In addition, the high number of promoters analysed allowed finding of subtle signals only showing up significantly with this large set. This included the observation of a periodically changing A+T-content with maxima spaced by a full turn of the DNA helix. This periodic structure includes the A+T-rich UP-element of bacterial promoters known before but was found to extend up to −100, indicating hitherto unknown constraints influencing promoter architecture and possibly also promoter function.

The Soil Bacterium Methylococcus capsulatus Bath Interacts with Human Dendritic Cells to Modulate Immune Function.

The prevalence of inflammatory bowel disease (IBD) has increased in Western countries during the course of the twentieth century, and is evolving to be a global disease. Recently we showed that a bacterial meal of a non-commensal, non-pathogenic methanotrophic soil bacterium, Methylococcus capsulatus Bath prevents experimentally induced colitis in a murine model of IBD. The mechanism behind the effect has this far not been identified. Here, for the first time we show that M. capsulatus, a soil bacterium adheres specifically to human dendritic cells, influencing DC maturation, cytokine production, and subsequent T cell activation, proliferation and differentiation. We characterize the immune modulatory properties of M. capsulatus and compare its immunological properties to those of another Gram-negative gammaproteobacterium, the commensal Escherichia coli K12, and the immune modulatory Gram-positive probiotic bacterium, Lactobacillus rhamnosus GG in vitro. M. capsulatus induces intermediate phenotypic and functional DC maturation. In a mixed lymphocyte reaction M. capsulatus-primed monocyte-derived dendritic cells (MoDCs) enhance T cell expression of CD25, the γ-chain of the high affinity IL-2 receptor, supports cell proliferation, and induce a T cell cytokine profile different from both E. coli K12 and Lactobacillus rhamnosus GG. M. capsulatus Bath thus interacts specifically with MoDC, affecting MoDC maturation, cytokine profile, and subsequent MoDC directed T cell polarization.

Antiviral Activity of Bacterial Extracellular Ribonuclease Against Single-, Double-Stranded RNA and DNA Containing Viruses in Cell Cultures

Binase, a small guanyl-preferring extracellular ribonuclease of Gram-positive non-pathogenic soil bacteria Bacillus pumilus. Binase is a well-known bacterial ribonuclease, and the most essential properties of the enzyme were characterized. Binase has demonstrated antiviral activity in various virus-infected animal models. Most experiments associated with binase treatment of virus-infected animals were performed using single stranded RNA viruses. It is still unclear, whether binase is able to inactivate the double stranded RNA virus. Moreover, the phenomenon of the antiviral activity of binase against the DNA containing virus in animal model is not explained. Here, we presented the experimental results reflecting binase effect towards eukaryotic cells infected with viruses containing different types of nucleic acids. The obtained data revealed the bacterial ribonuclease binase mode of action against single stranded RNA influenza A virus, double stranded RNA reovirus and DNA containing herpes virus to prove future application of new antiviral tool with a broad range of activity.

Biocontrol by plant growth promoting rhizobacteria against black scurf and stem canker disease of potato caused by Rhizoctonia solani

Non-pathogenic soil bacteria living in association with roots of higher plants enhance their adaptive potential and thus could be beneficial for their growth. Here, we present the current status of the use of Bacillus subtilis in biocontrol. Rhizobacteria are found in the rhizosphere. Plant growth promoting rhizobacteria (PGPR) strains, such as Bacillus and Pseudomonas, were isolated by using Nutreint dextrose Agar medium or Potato Dextrose Agar medium. The selection of PGPR strains was done by duel culture methods against the potato pathogens. The interaction of PGPR (Bacillus) with potato seeds or vegetative parts show promising antagonism by virtue of producing siderophore and antibiotics against black scurf and stem canker diseases of potato caused by Rhizoctonia solani, thereby resulting in increase of potato yield. The effectiveness of PGPR strain (Bacillus spp.) in improving the yield of potato in greenhouse conditions and in the field was observed.

ROS production during symbiotic infection suppresses pathogenesis-related gene expression

Leguminous plants have exclusive ability to form symbiotic relationship with soil bacteria of the genus Rhizobium. Symbiosis is a complex process that involves multiple molecular signaling activities, such as calcium fluxes, production of reactive oxygen species (ROS) and synthesis of nodulation genes. We analyzed the role of ROS in defense gene expression in Medicago truncatula during symbiosis and pathogenesis. Studies in Arabidopsis thaliana showed that the induction of pathogenesis-related (PR) genes during systemic acquired resistance (SAR) is regulated by NPR1 protein, which resides in the cytoplasm as an oligomer. After oxidative burst and return of reducing conditions, the NPR1 undergoes monomerization and becomes translocated to the nucleus, where it functions in PR genes induction. We show that ROS production is both stronger and longer during symbiotic interactions than during interactions with pathogenic, nonhost or common nonpathogenic soil bacteria. Moreover, root cells inoculated with Sinorhizobium meliloti accumulated ROS in the cytosol but not in vacuoles, as opposed to Pseudomonas putida inoculation or salt stress treatment. Furthermore, increased ROS accumulation by addition of H2O2 reduced the PR gene expression, while catalase had an opposite effect, establishing that the PR gene expression is opposite to the level of cytoplasmic ROS. In addition, we show that salicylic acid pretreatment significantly reduced ROS production in root cells during symbiotic interaction.

The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase

The non-pathogenic Gram-positive soil bacterium Streptomyces davawensis synthesizes the riboflavin (vitamin B2) analogs roseoflavin (RoF) and 8-demethyl-8-amino-riboflavin (AF). Both compounds are antibiotics. Notably, a number of other riboflavin analogs are currently under investigation with regard to the development of novel antiinfectives. As a first step towards understanding the metabolism of riboflavin analogs in humans, the key enzymes flavokinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2) were studied. Human flavokinase efficiently converted RoF and AF to roseoflavin mononucleotide (RoFMN) and 8-demethyl-8-amino-riboflavin mononucleotide (AFMN), respectively. Human FAD synthetase accepted RoFMN but not AFMN as a substrate. Consequently, roseoflavin adenine dinucleotide (RoFAD) was synthesized by the latter enzyme but not 8-demethyl-8-amino-riboflavin adenine dinucleotide (AFAD). The cofactor analogs RoFMN, AFMN and RoFAD have different physicochemical properties as compared to FMN and FAD. Thus, the cofactor analogs have the potential to render flavoenzymes inactive, which may negatively affect human metabolism. RoF, but not AF, was found to inhibit human flavokinase. In summary, we suggest that AF has a lower toxic potential and may be better suited as a lead structure to develop antimicrobial compounds.

Control of heme homeostasis in Corynebacterium glutamicum by the two-component system HrrSA.

The response regulator HrrA of the HrrSA two-component system (previously named CgtSR11) was recently found to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Here, we provide evidence that HrrA mediates heme-dependent gene regulation in this nonpathogenic soil bacterium. Growth experiments and DNA microarray analysis revealed that C. glutamicum is able to use hemin as an alternative iron source and emphasize the involvement of the putative hemin ABC transporter HmuTUV and heme oxygenase (HmuO) in heme utilization. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of an hrrA deletion mutant and C. glutamicum wild-type strain in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase. Based on our results, it can be furthermore deduced that HrrA activates the expression of heme-containing components of the respiratory chain, namely, ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa(3) oxidase and three subunits of the cytochrome bc(1) complex. In addition, HrrA was found to repress almost all genes involved in heme biosynthesis, including those for glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Growth experiments with an hrrA deletion mutant showed that this strain is significantly impaired in heme utilization. In summary, our results provide evidence for a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.

Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.