The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N 2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions Actinobacteria, Acidobacteria, Proteobacteria, and Verrucomicrobia.It has been established that the genetic diversity of soil bacteria is high (7,34,48) and that soils contain many bacterial species of lineages for which no known cultivated isolates are available (14, 25). Many soil bacteria are referred to as uncultured or even nonculturable. A range of methods have been developed to study these organisms directly in their habitats (2, 10, 35). These methods are extremely useful for studying the ecology of microorganisms as parts of communities, but initial physiological and genetic studies of pure cultures should greatly facilitate such synecological studies. We believe that many of these bacteria are in fact culturable using relatively simple technologies. To test our hypothesis, we used a simple growth medium to investigate the culturability of soil bacteria. We reasoned that if we could increase the culturability of soil bacteria above the level of 5% that often appears to be the upper limit for cultivation studies, we should begin to cultivate bacteria that belong to some of the uncultured groups. MATERIALS AND METHODSSoil and sampling. The soil used in this study was collected from a rotationally grazed pasture dominated by Lolium perenne and Trifolium repens at the Dairy Research Institute, Ellinbank, Victoria, Australia. The soil is a krasnozem clay loam (Gn4.11 [32]; basaltic clay loam; Ferrosol [15]). The management regime, consisting of two cows per ha and 35 kg of...

The culturability of microorganisms in a 10 cm core of an Australian pasture soil was investigated using a minimal agar medium with xylan as the growth substrate. Culturability decreased with increasing depth, from a maximum of 19% of the total microscopically countable cells in the 0-2 cm section to 2.4% in the 8-10 cm section. Seventy-one isolates from the core were identified by comparative 16S rRNA gene sequence analysis. Many of these isolates belong to groups of globally distributed soil bacteria, including well-characterized families of the classes Alphaproteobacteria and Betaproteobacteria, and of the subclass Actinobacteridae. Other isolates belong to groups with few or no cultivated representatives: 10 isolates in two subdivisions of the phylum Acidobacteria, five isolates in a new order and nine isolates in a new family of the class Alphaproteobacteria, two isolates in a new order of the class Gammaproteobacteria, three isolates in two new families of the subclass Actinobacteridae, and two isolates in the subclass Rubrobacteridae. These new isolates represent the first laboratory cultures able to be assigned to some of these groups and greatly increase the number of cultivated strains known for others. This demonstrates that a minimal change in cultivation strategy (using a polymeric growth substrate and longer incubation times) can result in the isolation of globally distributed but previously uncultured phylogenetically novel soil bacteria.

The pH strongly influenced the development of colonies by members of subdivision 1 of the phylum Acidobacteria on solid laboratory media. Significantly more colonies of this group formed at pH 5.5 than at pH 7.0. At pH 5.5, 7 to 8% of colonies that formed on plates that were incubated for 4 months were formed by subdivision 1 acidobacteria. These colonies were formed by bacteria that spanned almost the entire phylogenetic breadth of the subdivision, and there was considerable congruence between the diversity of this group as determined by the cultivation-based method and by surveying 16S rRNA genes in the same soil. Members of subdivision 1 acidobacteria therefore appear to be readily culturable. An analysis of published libraries of 16S rRNAs or 16S rRNA genes showed a very strong correlation between the abundance of subdivision 1 acidobacteria in soil bacterial communities and the soil pH. Subdivision 1 acidobacteria were most abundant in libraries from soils with pHs of <6, but rare or absent in libraries from soils with pHs of >6.5. This, together with the selective cultivation of members of the group on lower-pH media, indicates that growth of many members of subdivision 1 acidobacteria is favored by slightly to moderately acidic growth conditions.Members of subdivision 1 of the phylum Acidobacteria (29) are widely distributed in soils and other habitats (2,3,5,17,19,28,29,31,34,43,45) and have been shown to be present as active cells (13,17,29,30). This subdivision should be regarded as a class-rank taxon (21). Kishimoto and Tano (23) cultured eight isolates from water, mud, and soil that were affected by acid mine runoff, one of which was subsequently described as Acidobacterium capsulatum (2). To date, this is still the only validly described species in this subdivision.Since the description of A. capsulatum, one isolate from the sediment of an acid mine drainage treatment system (18), four isolates from a soil in Michigan (42), and 48 isolates from a soil in Victoria, Australia (8,20,21,35) have been reported. Very little is known of the biology of this group (33), and no specific isolation methodologies have been developed to culture subdivision 1 acidobacteria. Detailed study of isolates that represent the phylogenetic breadth of the subdivision will allow a better understanding of their roles in soil. Stevenson et al. (42) monitored the appearance of colonies of members of the phylum Acidobacteria by PCR and found that acidobacteria formed colonies on solid media incubated under air enriched with CO 2 , but not on parallel media incubated under air. The four acidobacterial isolates obtained in that study were all members of subdivision 1. It was not clear if the isolation of acidobacteria in these experiments was the result of the presence of CO 2 or an associated drop in pH of the culture media due to the mildly acidic nature of dissolved CO 2 (42). We have investigated the effects of CO 2 and medium pH on the culturability of subdivision 1 acidobacteria and the phylogenetic diversity of isolates o...

BACKGROUND: Whole-genome sequencing of pathogens can improve resolution of outbreak clusters and define possible transmission networks. We applied high-throughput genome sequencing of SARS-CoV-2 to 75% of cases in the State of Victoria (population 6.24 million) in Australia. METHODS:Cases of SARS-CoV-2 infection were detected through active case finding and contact tracing. A dedicated SARS-CoV-2 multidisciplinary genomic response team was formed to enable rapid integration of epidemiological and genomic data. Phylodynamic analysis was performed to assess the putative impact of social restrictions. RESULTS:Between 25 January and 14 April 2020, 1,333 COVID-19 cases were reported in Victoria, with a peak in late March. After applying internal quality control parameters, 903 samples were included in genomic analyses. Sequenced samples from Australia were representative of the global diversity of SARS-CoV-2, consistent with epidemiological findings of multiple importations and limited onward transmission. In total, 76 distinct genomic clusters were identified; these included large clusters associated with social venues, healthcare facilities and cruise ships. Sequencing of sequential samples from 98 patients revealed minimal intra-patient SARS-CoV-2 genomic diversity.Phylodynamic modelling indicated a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after the implementation of travel restrictions and population-level physical distancing.