Improvement in desulfurization of dibenzothiophene and dibenzothiophene sulfone by Paenibacillus strains using immobilization or nanoparticle coating.

AIMS: Biodesulfurization of fossil fuels is a promising technology for deep desulfurization. Previously, we have shown that Paenibacillus strains 32O-W and 32O-Y can desulfurize dibenzothiophene (DBT) and DBT sulfone (DBTS) effectively. In this work, improvements in DBT and DBTS desulfurization by these strains were investigated through immobilization and nanoparticle coating of cells. METHODS AND RESULTS: Paenibacillus strains 32O-W and 32O-Y immobilized in alginate gel beads or coated with Fe3 O4 magnetite nanoparticles were grown at various concentrations (0.1-2 mmol l-1 ) of DBT or DBTS for 96 h. The production of 2-hydroxybiphenyl (2-HBP) from the 4S pathway biotransformation of DBT or DBTS was measured. The highest amounts of 2-HBP production occurred at concentrations of 0.1 and 0.5 mmol l-1 . Compared to planktonic cultures maximum 2-HBP production increased by 54% for DBT and 90% for DBTS desulfurization with immobilized strains, and 44% for DBT and 66% for DBTS desulfurization by nanoparticle-coated strains. CONCLUSIONS: Nanoparticle-coated and immobilized cells may be of use in efforts to increase the efficiency of biodesulfurization. SIGNIFICANCE AND IMPACT OF THE STUDY: Alginate immobilization or nanoparticle coating of bacterial cells may be useful approaches for the enhancement of biodesulfurization for eventual use on an industrial scale.

Final Thoughts, Microorganisms Special Issue on Microbial Hemoglobins.

The year 2021 marked the thirty-fifth anniversary of the discovery of microbial hemoglobins by Dale Webster and his colleagues [...].

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

The Discovery of Vitreoscilla Hemoglobin and Early Studies on Its Biochemical Functions, the Control of Its Expression, and Its Use in Practical Applications.

In 1986, the surprising identification of a hemoglobin (VHb) in the bacterium Vitreoscilla greatly extended the range of taxa in which this oxygen binding protein functions. Elucidation of many of its biochemical properties and relation to overall cell physiology, as well as the sequence of the gene encoding it and aspects of control of its expression were determined in the following years. In addition, during the early years following its discovery, strategies were developed to use its expression in heterologous microbial hosts to enhance processes of practical usefulness. The VHb discovery also served as the foundation for what has become the fascinatingly rich field of bacterial hemoglobins. VHb's position as the first known bacterial hemoglobin and its extensive use in biotechnological applications, which continue today, make a review of the early studies of its properties and uses an appropriate and interesting topic thirty-five years after its discovery.

Complete Genome Sequence of Vitreoscilla sp. Strain C1, Source of the First Bacterial Hemoglobin.

Vitreoscilla sp. strain C1 is of historical importance as the source of the first prokaryotic hemoglobin identified. Vitreoscilla spp. rely on their hemoglobin and cytochrome oxidase to grow in microaerobic environments despite their aerobic nature. To help characterize this historically relevant strain, we sequenced the complete Vitreoscilla sp. strain C1 genome.

CARMIL family proteins as multidomain regulators of actin-based motility.

CARMILs are large multidomain proteins that regulate the actin-binding activity of capping protein (CP), a major capper of actin filament barbed ends in cells. CARMILs bind directly to CP and induce a conformational change that allosterically decreases but does not abolish its actin-capping activity. The CP-binding domain of CARMIL consists of the CP-interaction (CPI) and CARMIL-specific interaction (CSI) motifs, which are arranged in tandem. Many cellular functions of CARMILs require the interaction with CP; however, a more surprising result is that the cellular function of CP in cells appears to require binding to a CARMIL or another protein with a CPI motif, suggesting that CPI-motif proteins target CP and modulate its actin-capping activity. Vertebrates have three highly conserved genes and expressed isoforms of CARMIL with distinct and overlapping localizations and functions in cells. Various domains of these CARMIL isoforms interact with plasma membranes, vimentin intermediate filaments, SH3-containing class I myosins, the dual-GEF Trio, and other adaptors and signaling molecules. These biochemical properties suggest that CARMILs play a variety of membrane-associated functions related to actin assembly and signaling. CARMIL mutations and variants have been implicated in several human diseases. We focus on roles for CARMILs in signaling in addition to their function as regulators of CP and actin.

Repeated batch fermentation of immobilized E. coli expressing Vitreoscilla hemoglobin for long-term use.

This study describes an efficient and reusable process for ethanol production from medium containing whey powder, using alginate immobilized ethanologenic E. coli strains either expressing (TS3) or not expressing (FBR5) Vitreoscilla hemoglobin. Reuseabilities of the FBR5 and TS3 strains were investigated regarding their ethanol production capacities over the course of 15 successive 96-h batch fermentations. The ethanol production was fairly stable over the entire duration of the experiment, with strain TS3 maintaining a substantial advantage over strain FBR5. Storage of both strains in 2 different solutions for up to 60 d resulted in only a modest loss of ethanol production, with strain TS3 consistently outperforming strain FBR5 by a substantial amount. Strains stored for 15 or 30 d maintained their abilities to produce ethanol without dimunition over the course of 8 successive batch fermentations; again strain TS3 maintained a substantial advantage over strain FBR5 throughout the entire experiment. Thus, immobilization is a useful strategy to maintain the advantage in ethanol productivity afforded by expression of Vitreoscilla hemoglobin over long periods of time and large numbers of repeated batch fermentations, including, as in this case, using media with food processing wastes as the carbon source.

Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution.

In previous work from our laboratories a synthetic gene encoding a peptide ("Sulpeptide 1" or "S1") with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells' overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.

Effective ethanol production from whey powder through immobilized E. coli expressing Vitreoscilla hemoglobin.

Ethanol production from whey powder was investigated by using free as well as alginate immobilized E. coli and E. coli expressing Vitreoscilla hemoglobin (VHb) in both shake flask and fermenter cultures. Media with varying levels of whey (lactose contents of 3%, 5%, 8% or 15%) and yeast extract (0.3% or 0.5%) were evaluated with fermentation times of 48-96 h. Immobilization and VHb expression resulted in higher ethanol production with all media; the increases ranged from 2% to 89% for immobilization and from 2% to 182% for VHb expression. It was determined that growth medium containing 8% lactose with 0.5% yeast extract yielded the highest ethanol production for free or immobilized strains, with or without VHb expression, in both shake flask and fermenter cultures. Immobilization with alginate was found to be a promising process for ethanol production by VHb-expressing ethanologenic E. coli.

Recent trends in bioethanol production from food processing byproducts.

The widespread use of corn starch and sugarcane as sources of sugar for the production of ethanol via fermentation may negatively impact the use of farmland for production of food. Thus, alternative sources of fermentable sugars, particularly from lignocellulosic sources, have been extensively investigated. Another source of fermentable sugars with substantial potential for ethanol production is the waste from the food growing and processing industry. Reviewed here is the use of waste from potato processing, molasses from processing of sugar beets into sugar, whey from cheese production, byproducts of rice and coffee bean processing, and other food processing wastes as sugar sources for fermentation to ethanol. Specific topics discussed include the organisms used for fermentation, strategies, such as co-culturing and cell immobilization, used to improve the fermentation process, and the use of genetic engineering to improve the performance of ethanol producing fermenters.

Complete Genome Sequences of Two Interactive Moderate Thermophiles, Paenibacillus napthalenovorans 32O-Y and Paenibacillus sp. 32O-W.

Microorganisms with the capability to desulfurize petroleum are in high demand with escalating restrictions currently placed on fuel purity. Thermophilic desulfurizers are particularly valuable in high-temperature industrial applications. We report the whole-genome sequences of Paenibacillus napthalenovorans 32O-Y and Paenibacillus sp. 32O-W, which can and cannot, respectively, metabolize dibenzothiophene.

Differential expression of CARMIL-family genes during zebrafish development.

CARMILs are a conserved family of large multidomain proteins that regulate and target actin assembly by interacting with actin capping protein (CP). Vertebrates contain three highly conserved CARMIL isoforms encoded by three genes, whereas lower organisms contain only one isoform and gene. In order to investigate the functions of vertebrate CARMILs, we identified and characterized the three CARMIL genes in zebrafish (Danio rerio). We isolated and sequenced complete and partial cDNAs from embryos. The three genes display distinct spatial and temporal expression patterns during development. Sequence and phylogenetic analyses of cDNAs and predicted protein sequences reveal that the three zebrafish genes fall into the three conserved isoform groups previously defined for other vertebrates, which have isoform-specific and overlapping functions in human cultured cells. These results provide new tools and offer insight into understanding the role of the regulation of actin assembly dynamics during embryonic development and tissue morphogenesis.

Engineering of Nitrosomonas europaea to express Vitreoscilla hemoglobin enhances oxygen uptake and conversion of ammonia to nitrite.

Nitrosomonas europaea was transformed with a recombinant plasmid bearing the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla under control of the N. europaea amoC P1 promoter. Vgb was maintained stably and appeared to be expressed in the transformants at VHb levels of about 0.75 nmol/g wet weight. Expression of VHb in the N. europaea transformants was correlated with an approximately 2 fold increase in oxygen uptake rate by whole cells at oxygen concentrations in the range of 75-100% saturation, but no change in oxygen uptake rate at oxygen concentrations below 25% saturation. VHb expression was also correlated with an increase of as much as about 30% in conversion of ammonia to nitrite by growing cells. The results suggest that engineering of key aerobic wastewater bacteria to express bacterial hemoglobins may be a useful strategy to produce species with enhanced respiratory abilities.

Directed evolution to produce sludge communities with improved oxygen uptake abilities.

Two activated sludge cultures, seeded with activated sludge from the same source, were cultivated for 370 days in synthetic wastewater. Both cultures were transferred weekly to fresh medium; one culture was operated at high dissolved oxygen (DO) (near saturation) and the other at low DO (0.25 mg O2/L). There were significant changes in the abundances of bacterial species and phyla present in each culture throughout the 370-day operational period. In the low DO culture, over time, there was a continuously increasing proportion of cells of species known to encode truncated hemoglobins (Hbs). These are the types of Hbs which may enhance delivery of oxygen to the respiratory chain, to enhance ATP production, especially under low aeration conditions. The levels of heme b, the heme found in Vitreoscilla hemoglobin, increased in parallel to the increase in Hb-encoding species, to much higher levels in the low DO culture than in the high DO culture. Specific oxygen uptake rates increased by 3 % for the high DO culture near the end of the 370-day period, while those for the low DO culture increased steadily to a level 28 % higher than that of the starting culture. Thus, imposition of low DO conditions may, due to selection for Hb-expressing species, be useful in developing bacterial communities with enhanced ability to function efficiently in aerobic wastewater treatment, especially under low aeration conditions.

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability.

OBJECTIVE: To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. RESULTS: A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55 °C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 °C, with some activity up to at least 50 °C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 °C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 °C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 °C increased DBT metabolism 36-42 % from 40 to 45 °C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. CONCLUSION: The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

Efficient ethanol production from potato and corn processing industry waste using E. coli engineered to express Vitreoscilla haemoglobin.

Engineering of ethanologenic E. coli to express the haemoglobin (VHb) from the bacterium Vitreoscilla has been shown to enhance ethanol production by fermentation of pure sugars, sugars from hydrolysis of lignocellulose, components of whey, and sugars from wastewater produced during potato processing. Here, these studies were extended to see whether the same effect could be seen when a mixture of waste materials from processing of potatoes and corn into potato and corn chips were used as sugar sources. Consistent increases in ethanol production coincident with VHb expression were seen in shake flasks at both low aeration and high aeration conditions. The ethanol increases were due almost entirely to increases in the amount of ethanol produced per unit of cell mass. The VHb strategy for increasing fermentation to ethanol (and perhaps other valuable fermentation products) may be of general use, particularly regarding conversion of otherwise discarded materials into valuable commodities.

Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation.

Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5 years, the use of this "VHb technology" has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.

Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions.

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O2/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.

UCS protein Rng3p is essential for myosin-II motor activity during cytokinesis in fission yeast.

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.