Parveen Sangwan

Laboratory Cultivation of Widespread and Previously Uncultured Soil Bacteria

ABSTRACT Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria. In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.

Detection and Cultivation of Soil Verrucomicrobia

ABSTRACT Only one isolate each of the class “Spartobacteria” (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class “Spartobacteria” and were related to “Chthoniobacter flavus.” Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class “Spartobacteria” were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class “Spartobacteria” were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class “Spartobacteria” was inhibited by other culturable bacteria.

Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes

BackgroundThe phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization.ResultsThe ultrastructure of cells of four members of phylum Verrucomicrobia – Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and strain Ellin514 – was examined using electron microscopy incorporating high-pressure freezing and cryosubstitution. These four members of phylum Verrucomicrobia, representing 3 class-level subdivisions within the phylum, were found to possess a compartmentalized cell plan analogous to that found in phylum Planctomycetes. Like all planctomycetes investigated, they possess a major pirellulosome compartment containing a condensed nucleoid and ribosomes surrounded by an intracytoplasmic membrane (ICM), as well as a ribosome-free paryphoplasm compartment between the ICM and cytoplasmic membrane.ConclusionA unique compartmentalized cell plan so far found among Domain Bacteria only within phylum Planctomycetes, and challenging our concept of prokaryote cell plans, has now been found in a second phylum of the Domain Bacteria, in members of phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two distinct phyla of the Bacteria, phyla which have been suggested from other evidence to be related phylogenetically in the proposed PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) superphylum. This planctomycete cell plan is present in at least 3 of 6 subdivisions of Verrucomicrobia, suggesting that the common ancestor of the verrucomicrobial phylum was also compartmentalized and possessed such a plan. The presence of this compartmentalized cell plan in both phylum Planctomycetes and phylum Verrucomicrobia suggest that the last common ancestor of these phyla was also compartmentalized.

Chthoniobacter flavus gen. nov., sp. nov., the First Pure-Culture Representative of Subdivision Two, Spartobacteria classis nov., of the Phylum Verrucomicrobia

ABSTRACT The phylum Verrucomicrobia is increasingly recognized as an environmentally significant group of bacteria, particularly in soil habitats. At least six subdivisions of the Verrucomicrobia are resolved by comparative analysis of 16S rRNA genes, mostly obtained directly from environmental samples. To date, only two of these subdivisions (1 and 4) have characterized pure-culture representatives. We have isolated and characterized the first known pure-culture representative of subdivision 2. Strain Ellin428 is an aerobic heterotrophic bacterium that is able to grow with many of the saccharide components of plant biomass but does not grow with amino acids or organic acids other than pyruvate. Cells are yellow, rod-shaped, nonmotile, and gram-stain negative, and they contain peptidoglycan with direct cross-linkages of the A1γ meso-Dpm type. The isolate grows well at 25°C on a variety of standard biological media, including some used in the routine cultivation of bacteria from soil. The pH range for growth is 4.0 to 7.0. Low levels of menaquinones MK-10 and MK-11 were detected. The major cellular fatty acids are C14:0, a-C15:0, C16:1ω7c, and/or 2OH i-C15:0, and C16:0. The G+C content of the genomic DNA is 61 mol%. We propose a new genus and species, Chthoniobacter flavus gen. nov., sp. nov., with isolate Ellin428 as the type strain, and a new class for the subdivision to which it belongs, Spartobacteria classis nov. Environmental sequences indicate that the class Spartobacteria is largely represented by globally distributed, abundant, and active soil bacteria.

Acidobacteria, Rubrobacteridae and Chloroflexi are abundant among very slow-growing and mini-colony-forming soil bacteria

Easily visible colonies of bacteria continued to form on plates inoculated with soil and incubated for 24 weeks. Using two different media, 13% and 29% of easily visible colonies appeared after more than 12 weeks. In addition, 10% and 18% of all colonies had diameters of 25-200 µm (mini-colonies), which could not be readily seen with the unaided eye. Members of soil bacterial groups that are only rarely cultured, such as members of the subclass Rubrobacteridae of the phylum Actinobacteria, members of subdivisions 1 and 2 of the phylum Acidobacteria and members of three subphyla of the phylum Chloroflexi, were more abundant among the easily visible colonies and mini-colonies that developed after > 12 weeks of incubation. Our results indicate that there is a hidden culturable diversity of soil bacteria that may require laboratory study at colony sizes and incubation periods outside those commonly anticipated by most microbiologists. Working at these scales increases the likelihood of obtaining cultures from groups of soil bacteria that have generally eluded laboratory study by cultivation methods.

New Insights into Polylactide Biodegradation from Molecular Ecological Techniques

Molecular ecological techniques for direct identification of microbes involved in PLA degradation under aerobic composting conditions are described. Gene sequences from genera Paecilomyces, Thermomonospora, and Thermopolyspora were most abundant in the compost samples. Members of these phylogenetic lineages are therefore likely to play an important role in PLA degradation. The use of molecular ecological techniques to design cultivation strategies may also provide a new tool for identification and investigation of biodegradation mechanisms and for future development of efficient biological treatment or recycling processes for PLA and other biodegradable polymers.

New Insight into Biodegradation of Polylactide (PLA)/Clay Nanocomposites Using Molecular Ecological Techniques

Novel molecular ecological techniques were used to study changes in microbial community structure and population during degradation of polylactide (PLA)/organically modified layered silicates (OMLS) nanocomposites. Cloned gene sequences belonging to members of the phyla Actinobacteria and Ascomycota comprized the most dominant groups of microorganisms during biodegradation of PLA/OMLS nanocomposites. Due to their numerical abundance, members of these microbial groups are likely to play an important role during biodegradation process. This paper presents new insights into the biodegradability of PLA/OMLS nanocomposites and highlights the importance of using novel molecular ecological techniques for in situ identification of new microorganisms involved in biodegradation of polymeric materials.

Genome sequence of Chthoniobacter flavus Ellin428, an aerobic heterotrophic soil bacterium

Chthoniobacter flavus Ellin428 is the first isolate from the class Spartobacteria of the bacterial phylum Verrucomicrobia. C. flavus Ellin428 can metabolize many of the saccharide components of plant biomass but is incapable of growth on amino acids or organic acids other than pyruvate.

Biodegradability of Poly-3-hydroxybutyrate/Bacterial Cellulose Composites under Aerobic Conditions, Measured via Evolution of Carbon Dioxide and Spectroscopic and Diffraction Methods.

Poly-3-hydroxybutyrate (PHB) and bacterial cellulose (BC) are both natural polymeric materials that have the potential to replace traditional, nonrenewable polymers. In particular, the nanofibrillar form of bacterial cellulose makes it an effective reinforcement for PHB. Neat PHB, bacterial cellulose, and a composite of PHB/BC produced with 10 wt % cellulose were composted under accelerated aerobic test conditions, with biodegradability measured by the carbon dioxide evolution method, in conjunction with spectroscopic and diffraction methods to assess crystallinity changes during the biodegradation process. The PHB/BC composite biodegraded at a greater rate and extent than that of PHB alone, reaching 80% degradation after 30 days, whereas PHB did not reach this level of degradation until close to 50 days of composting. The relative crystallinity of PHB and PHB in the PHB/BC composite was found to increase in the initial weeks of degradation, with degradation occurring primarily in the amorphous region of the material and some recrystallization of the amorphous PHB. Small angle X-ray scattering indicates that the change in PHB crystallinity is accompanied by a change in morphology of semicrystalline lamellae. The increased rate of biodegradability suggests that these materials could be applicable to single-use applications and could rapidly biodegrade in compost on disposal.

Phase Transition of Waxy and Normal Wheat Starch Granules during Gelatinization

The phase transition of waxy and normal wheat starches was systematically studied by light microscopy (LM) with a hot-stage, confocal laser scanning microscopy (CLSM) and differential scanning calorimetry (DSC). While being heated in water, waxy wheat starch showed a higher gelatinization enthalpy than that for the normal starch, which was also verified by the changes in birefringence. As confirmed by LM and CLSM, starch granules displayed an increased swelling degree with temperature increasing, and the gelatinization initially occurred at the hilum (botanical center) of the granules and then spread rapidly to the periphery. While the temperature range of birefringence was narrower than that of granule size change, the crystalline structure was melted at lower temperatures than those for the molecular orders. These results indicate that starch gelatinization was a complex process rather than a simple order-to-disorder granule transition.