Identification of the Polyhydroxybutyrate Granules in Mammalian Cultured Cells

Poly‐3‐hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long‐chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB‐accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short‐chain (or complexed) cPHB (10–100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous studies indicated that cPHB is localized in various subcellular compartments of the eukaryotic organisms. Here, we used fluorescent microscopy to directly investigate the localization of PHB in mammalian cells. PHB was visualized in cultured U87 cells using fluorescent probe BODIPY 493/503. Specificity of PHB staining was confirmed by markedly decreased fluorescence of samples treated with PHB‐specific depolymerase (PhaZ7). We found that PHB is associated with granules, and that these PHB‐enriched granules do not co‐localized with mitochondria, lysosomes, or endoplasmic reticulum. These results suggest that, in mammalian cells, PHB can accumulate in the cytoplasm in granules similar to ‘energy storage’ carbonosomes found in PHB‐accumulating bacteria.     

Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.     

Phylogenetic characterization of the blue filamentous bacterial community from an Icelandic geothermal spring

Molecular phylogenetic analysis of a blue filamentous community from an alkaline thermal spring (79-83 degrees C) in Iceland revealed that the blue filaments were affiliated with the Aquificales. The dominant sequence type, pIce1, was most closely related to a sequence (SRI-48) found in a white filamentous community from a separate Icelandic thermal spring and the pink filaments (EM17) from Yellowstone National Park. Fluorescent in situ hybridization with clone-specific oligonucleotide probes showed that the sample analyzed was essentially a monoculture of a single phylotype.     

Microbial Cometabolism and Polyhydroxyalkanoate Co-polymers

Polyhydroxyalkanoate (PHAs) are natural, biodegradable biopolymers, which can be produced from renewable materials. PHAs have potential to replace petroleum derived plastics. Quite a few bacteria can produce PHA under nutritional stress. They generally produce homopolymers of butyrate i.e., polyhydroxybutyrate (PHB), as a storage material. The biochemical characteristics of PHB such as brittleness, low strength, low elasticity, etc. make these unsuitable for commercial applications. Co-polymers of PHA, have high commercial value as they overcome the limitations of PHBs. Co-polymers can be produced by supplementing the feed with volatile fatty acids or through hydrolysates of different biowastes. In this review, we have listed the potential bacterial candidates and the substrates, which can be co-metabolized to produce PHA co-polymers.     

New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3‐hydroxybutyrate)

The metabolism of polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) has been investigated by many groups for about three decades, and good progress was obtained in understanding the mechanisms of biosynthesis and biodegradation of this class of storage molecules. However, the molecular events that happen at the onset of PHB synthesis and the details of the initiation of PHB/PHA granule formation, as well as the complex composition of the proteinaceous surface layer of PHB/PHA granules, have only recently come into the focus of research and were not reviewed yet. In this contribution, we summarize the progress in understanding the initiation and formation of the PHA granule complex at the example of Ralstonia eutropha H16 (model organism of PHB‐accumulating bacteria). Where appropriate, we include information on PHA granules of Pseudomonas putida as a representative species for medium‐chain‐length PHA‐accumulating bacteria. We suggest to replace the previous micelle mode of PHB granule formation by the Scaffold Model in which the PHB synthase initiation complex is bound to the bacterial nucleoid. In the second part, we highlight data on other forms of PHB: oligo‐PHB with ≈100 to 200 3‐hydroxybutyrate (3HB) units and covalently bound PHB (cPHB) are unrelated in function to storage PHB but are presumably present in all living organisms, and therefore must be of fundamental importance.     

Microbial Poly‐3‐Hydroxybutyrate (PHB) as a Feed Additive for Fishes and Piglets

The large‐scale use of petrochemical‐based plastics is damaging our environment. Discarded plastics are harmful to both marine and land animals, sometimes causing death when ingested. Biodegradable plastics have gained attentions from the public and the academia to reduce environmental burdens. Poly‐3‐hydroxybutyrate (PHB), the simplest and the best‐studied bioplastic member of the polyhydroxyalkanoate (PHA) family synthesized by many bacteria, has been studied as a feed additive for large yellow croaker fish and weaned piglets. The fish grow faster and gain more weight when 1% and 2% PHB is added as a feed additive, accompanied by increased survival rates. Weaned piglets are found to grow normally and showed no significant change in average daily weight gains, average daily feed intakes, feed efficiency, and organ developments when 0.5% PHB is added to the feed. It can therefore be concluded that biodegradable and biocompatible PHB is not harmful as a feed additive for marine large yellow croakers and sensitive weaned piglets. PHB therefore holds great promise as a plastic that combines biodegradability and biocompatibility with good tolerability as a feed supplement for animals.     

Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax

The bacterial storage polymer poly-β-hydroxybutyrate (PHB) has the potential to be used as an alternative anti-infective strategy for aquaculture rearing. In this research, the effects of (partially) replacing the feed of European sea bass juveniles with PHB were investigated. During a 6-week trial period, the PHB showed the ability to act as an energy source for the fish. This indicated that PHB was degraded and used during gastrointestinal passage. The gut pH decreased from 7.7 to 7.2 suggesting that the presence of PHB in the gut led to the increased production of (short-chain fatty) acids. The diets supplemented with 2% and 5% PHB (w/w) induced a gain of the initial fish weight with a factor 2.4 and 2.7, respectively, relative to a factor 2.2 in the normal feed treatment. Simultaneously, these treatments showed the highest bacterial range-weighted richness in the fish intestine. Based on molecular analysis, higher dietary PHB levels induced larger changes in the bacterial community composition. From our results, it seems that PHB can have a beneficial effect on fish growth performance and that the intestinal bacterial community structure may be closely related to this phenomenon.     

The role of PHB metabolism in the symbiosis of rhizobia with legumes

The carbon storage polymer poly-β-hydroxybutyrate (PHB) is a potential biodegradable alternative to plastics, which plays a key role in the cellular metabolism of many bacterial species. Most species of rhizobia synthesize PHB but not all species accumulate it during symbiosis with legumes; the reason for this remains unclear, although it was recently shown that a metabolic mutant of a nonaccumulating species retains the capacity to store PHB in symbiosis. Although the precise roles of PHB metabolism in these bacteria during infection, nodulation, and nitrogen fixation are not determined, the elucidation of these roles will influence our understanding of the metabolic nature of the symbiotic relationship. This review explores the progress that was made in determining the biochemistry and genetics of PHB metabolism. This includes the elucidation of the PHB cycle, variations in PHB metabolism among rhizobial species, and the implications of these variations, while proposing a model for the role of PHB metabolism and storage in symbiosis.     

Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization

A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively.     

A Highlights from MBoC Selection: Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology

The maintenance of rod-cell shape in many bacteria depends on actin-like MreB proteins and several membrane proteins that interact with MreB. Using superresolution microscopy, we show that at 50-nm resolution, Bacillus subtilis MreB forms filamentous structures of length up to 3.4 μm underneath the cell membrane, which run at angles diverging up to 40° relative to the cell circumference. MreB from Escherichia coli forms at least 1.4-μm-long filaments. MreB filaments move along various tracks with a maximal speed of 85 nm/s, and the loss of ATPase activity leads to the formation of extended and static filaments. Suboptimal growth conditions lead to formation of patch-like structures rather than extended filaments. Coexpression of wild-type MreB with MreB mutated in the subunit interface leads to formation of shorter MreB filaments and a strong effect on cell shape, revealing a link between filament length and cell morphology. Thus MreB has an extended-filament architecture with the potential to position membrane proteins over long distances, whose localization in turn may affect the shape of the cell wall.     

Substrate uptake tests and quantitative FISH show differences in kinetic growth of bulking and non-bulking activated sludge

The competition between filaments and floc formers in activated sludge has been historically described using kinetic selection. However, recent studies have suggested that bacterial storage may also be an important factor in microbial selection, since the dynamic nature of substrate flows into wastewater treatment plants elicit transient responses from microorganisms. Respirometry-based kinetic selection should thus be reevaluated by considering cell storage, and a more reliable method should be developed to include bacterial storage in the analysis of growth of filaments and floc formers in activated sludge. In this study, we applied substrate uptake tests combined with metabolic modeling to determine the growth rates, yields and maintenance coefficients of bulking and non-bulking activated sludge developed in lab scale reactors under feast and famine conditions. The results of quantitative fluorescence in situ hybridization (FISH) showed that the filaments Eikelboom Type 1851, Type 021N, and Thiothrix nivea were dominant in bulking sludge, comprising 42.0 % of mixed liquor volatile suspended solids (MLVSS), with 61.6% of the total filament length extending from flocs into bulk solution. Only low levels of Type 1851 filament length (4.9% of MLVSS) occurred in non-bulking sludge, 83.0% of which grew inside the flocs. The kinetic parameters determined from the substrate uptake tests were consistent with those from respirometry and showed that filamentous bulking sludge had lower growth rates and maintenance coefficients than non-bulking sludge. These results provide support for growth kinetic differences in explaining the competitive strategy of filamentous bacteria.     

Multiple approaches to assess filamentous bacterial growth in activated sludge under different carbon source conditions

Aims:  To compare molecular and microscopic approaches in determining which filamentous bacteria grow in activated sludge reactors when different carbon sources and different activated sludge mixed liquor inocula are used.
Methods and Results:  Microscopic and molecular (Denaturing Gradient Gel Electrophoresis and Fluorescent In Situ Hybridization) techniques were used to determine which filamentous bacteria became dominant in lab scale reactors treating wastewater composed of different carbon sources. Molecular analysis indicated the presence of Sphaerotilus natans and Thiothrix -related organisms . Microscopy indicated the presence of Nostocoida limicola in some reactors. Sludge volume index increased as filament abundance increased. The detection level of DGGE analysis increased when the abundance levels of the filaments were high.
Conclusions:  Simultaneous application of traditional and molecular methods was effective, and highlighted the advantages and limitations of each method. Readily biodegradable substances favoured the growth of specific filaments in a mixed liquor environment. The origin of inoculum influenced which specific filamentous bacteria grew.
Significance and Impact of the Study:  The study shows the potential problems when using particular techniques, and highlights the need for multiple approaches when studying filaments. The study also provides more information on which filaments will grow under different carbon source conditions for a given inoculum.     

Bloom of Filamentous Bacteria in a Mesotrophic Lake: Identity and Potential Controlling Mechanism

Ephemeral blooms of filamentous bacteria are a common phenomenon in the water column of oligo- to mesotrophic lakes. It is assumed that the appearance of such morphotypes is favored by selective predation of bacterivorous protists and that filter-feeding zooplankton plays a major role in suppressing these bacteria. The phylogenetic affiliation of the important bloom-forming filamentous bacteria in freshwaters is presently unknown. Here we report the identification of dominant members of a filamentous bacterial assemblage during a bloom of such morphotypes in a mesotrophic lake. By molecular cloning and fluorescence in situ hybridization with specific oligonucleotide probes, up to 98% of filamentous cells in lake water could be assigned to a clade of almost identical (99% similarity) 16S rRNA gene sequence types, the cosmopolitan freshwater LD2 cluster. For a period of less than 1 week, members of the LD2 clade constituted >40% of the total bacterial biomass, potentially favored by high grazing of planktivorous protists. This is probably the most pronounced case of dominance by a single bacterioplankton species ever observed in natural freshwaters. In enclosures artificially stocked with the metazoan filter feeder Daphnia, bacteria related to the LD2 clade formed a significantly larger fraction of filaments than in enclosures where Daphnia had been removed. However, in the presence of higher numbers of Daphnia individuals, the LD2 bacteria, like other filaments, were eventually eliminated both in enclosures and in the lake. This points at the potential importance of filter-feeding zooplankton in controlling the occurrence and species composition of filamentous bacterial morphotypes in freshwater plankton.     

Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.     

Using biopolymer bodies for encapsulation of hydrophobic products in bacterium

Producing some small hydrophobic molecules in microbes is challenging. Often these molecules cannot cross membranes, and thus their production may be limited by lack of storage space in the producing organism. This study reports a new technology for in vivo storage of valuable hydrophobic products in/on biopolymer bodies in Escherichia coli. A biodegradable and biocompatible polyester – poly (3-hydroxybutyrate) (PHB) – was selected as the intracellular storage vessel to encapsulate lycopene, which is a chromogenic model compound. The hydrophobic interaction between lycopene and PHB was verified by using in vitro binding test and sucrose density gradient centrifugation. Further in vivo characterization was performed by using Confocal Laser Scanning Microscopy (CLSM). The images validated the in vivo co-localization between PHB granules and lycopene. The images also showed that lycopene aggregated in bacteria that did not produce PHB, which may challenge the commonly accepted hypothesis that most lycopene molecules are stored in cell membranes of recombinant host. We also confirmed that producing PHB did not negatively affect lycopene biosynthesis in the E. coli strains and collected data suggesting that PHB titer and lycopene titer were positively correlated when the cells were engineered to co-produce them. The biopolymers that encapsulated hydrophobic molecules could have many useful applications, especially in controlled release because the polymers are biodegradable, and the encapsulated products would be released during the polymer degradation.     

Motility and chemotaxis of filamentous cells of Escherichia coli

Filamentous cells of Escherichia coli can be produced by treatment with the antibiotic cephalexin, which blocks cell division but allows cell growth. To explore the effect of cell size on chemotactic activity, we studied the motility and chemotaxis of filamentous cells. The filaments, up to 50 times the length of normal E. coli organisms, were motile and had flagella along their entire lengths. Despite their increased size, the motility and chemotaxis of filaments were very similar to those properties of normal-sized cells. Unstimulated filaments of chemotactically normal bacteria ran and stopped repeatedly (while normal-sized bacteria run and tumble repeatedly). Filaments responded to attractants by prolonged running (like normal-sized bacteria) and to repellents by prolonged stopping (unlike normal-sized bacteria, which tumble), until adaptation restored unstimulated behavior (as occurs with normal-sized cells). Chemotaxis mutants that always ran when they were normal sized always ran when they were filament sized, and those mutants that always tumbled when they were normal sized always stopped when they were filament sized. Chemoreceptors in filaments were localized to regions both at the poles and at intervals along the filament. We suggest that the location of the chemoreceptors enables the chemotactic responses observed in filaments. The implications of this work with regard to the cytoplasmic diffusion of chemotaxis components in normal-sized and filamentous E. coli are discussed.     

Bloom of filamentous bacteria in a mesotrophic lake: identity and potential controlling mechanism

Ephemeral blooms of filamentous bacteria are a common phenomenon in the water column of oligo- to mesotrophic lakes. It is assumed that the appearance of such morphotypes is favored by selective predation of bacterivorous protists and that filter-feeding zooplankton plays a major role in suppressing these bacteria. The phylogenetic affiliation of the important bloom-forming filamentous bacteria in freshwaters is presently unknown. Here we report the identification of dominant members of a filamentous bacterial assemblage during a bloom of such morphotypes in a mesotrophic lake. By molecular cloning and fluorescence in situ hybridization with specific oligonucleotide probes, up to 98% of filamentous cells in lake water could be assigned to a clade of almost identical (99% similarity) 16S rRNA gene sequence types, the cosmopolitan freshwater LD2 cluster. For a period of less than 1 week, members of the LD2 clade constituted >40% of the total bacterial biomass, potentially favored by high grazing of planktivorous protists. This is probably the most pronounced case of dominance by a single bacterioplankton species ever observed in natural freshwaters. In enclosures artificially stocked with the metazoan filter feeder Daphnia, bacteria related to the LD2 clade formed a significantly larger fraction of filaments than in enclosures where Daphnia had been removed. However, in the presence of higher numbers of Daphnia individuals, the LD2 bacteria, like other filaments, were eventually eliminated both in enclosures and in the lake. This points at the potential importance of filter-feeding zooplankton in controlling the occurrence and species composition of filamentous bacterial morphotypes in freshwater plankton.     

Bacteriology of activated sludge,in particular the filamentous bacteria

Microscopic examination of bulking activated sludge samples showed the presence of a variety of filamentous microorganisms, some of which have not yet been described in the literature. A method was developed to obtain pure cultures of these threaded bacteria. To date, five clearly different groups of filamentous bacteria may be distinguished by the determination of a few morphological and physiological characteristics of the isolates. A variety of sheathed bacteria are included in Group I. Group II includes non-motile, gram-negative, orange- or yellow-pigmented filamentous bacteria. These microorganisms are thought to be related to some species of the genusFlavobacterium. The gram-negative, threaded bacteria of Group III show a more or less distinct gliding movement and form red colonies on rich agar media. These bacteria may apparently be related to species described in the generaMicroscilla andFlexibacter. The filamentous bacteria of Group IV structurally resemble someCyanophyceae, but do not contain photosynthetic pigments. They are gram-positive and non-motile. A number of unknown, non-motile bacteria which stain gram-positive with a variable number of gram-negative autolyzed cells in the filaments, are assigned to Group V. The properties of the isolated bacteria are described briefly and their occurrence in bulking activated sludge is discussed.     

Quantitation of poly-β-hydroxybutyrate by fluorescence of bacteria and granules stained with Nile blue A

Summary Fluorescence from poly--hydroxybutyrate (PHB) inclusions inside Azotobacter vinelandii UWD cells stained with Nile blue A was shown to be proportional to PHB concentration. The intensity of the fluorescence was greatest in native, fluid inclusions and was the least in extracted, crystallized granules. However, isolated air-dried PHB granules also were proportionally stained with Nile blue A. The results show that Nile blue A can be used in the quantitative determination of PHB in a variety of cells.     

rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.