Ecological Significance of Microdiversity: Coexistence Among Casing Soil Bacterial Strains Through Allocation of Nutritional Resource

A combination of cultivation-based methods with a molecular biological approach was employed to investigate whether bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of eight bacterial strains wherein three were Pseudomonas putida and rest were Acinetobacter calcoaceticus, were isolated from casing soils community by conventional plating. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction. Each strain utilized a specific combination of 154 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences. It is worthwhile approach to explore prokaryotic diversity in different ecological niches.     

Recovering microbial genomes from metagenomes in hypersaline environments: The Good,the Bad and the Ugly

Current metagenomic tools allow the recovery of microbial genomes directly from the environment. This can be accomplished by binning metagenomic contigs according to their coverage and tetranucleotide frequency, followed by an estimation of the bin quality. The public availability of bioinformatics tools, together with the decreasing cost of next generation sequencing, are democratizing this powerful approach that is spreading from specialized research groups to the general public. Using metagenomes from hypersaline environments, as well as mock metagenomes composed of Archaea and Bacteria frequently found in these systems, we have analyzed the advantages and difficulties of the binning process in these extreme environments to tackle microbial population diversity. These extreme systems harbor relatively low species diversity but high intraspecific diversity, which can compromise metagenome assembly and therefore the whole binning process. The main goal is to compare the output of the binning process with what is previously known from the analyzed samples, based on years of study using different approaches. Several scenarios have been analyzed in detail: (i) a good quality bin from a species highly abundant in the environment; (ii) an intermediate quality bin with incongruences that can be solved by further analyses and manual curation, and (iii) a low-quality bin to investigate the failure to recover a very abundant microbial genome as well as some possible solutions. The latter can be considered the “great metagenomics anomaly” and is mainly due to assembly problems derived from the microdiversity of naturally co-existing populations in nature.     

Paraburkholderia gardini sp. nov. and Paraburkholderia saeva sp. nov.: Novel aromatic compound degrading bacteria isolated from garden and forest soil samples

Three forest and four botanical garden top soil isolates with unique MALDI-TOF mass spectra were identified as Paraburkholderia strains closely related to Paraburkholderia sartisoli through recA gene sequence analysis. OrthoANIu, digital DNA-DNA hybridization analyses and phylogenomic analyses demonstrated that the five strains represented two new Paraburkholderia species closely related to P. sartisoli. The genome of strain LMG 31841^T had a cumulative size of 6.3 Mb and a G + C content of 62.64 mol%; strain LMG 32171^T had a genome size of 5.8 Mb and a G + C content of 62.91 mol%. Hemolysis on horse blood agar, beta-galactosidase and phosphoamidase activity, and assimilation of adipic acid and trisodium citrate allowed phenotypic differentiation of strains LMG 31841^T, LMG 32171^T and P. sartisoli LMG 24000^T. An analysis of the genomic potential for aromatic compound degradation yielded additional differences among strains representing these three species, but also highlighted some discrepancies between the presence of genes and pathways, and the phenotype revealed through growth experiments using a mineral salts medium supplemented with single aromatic compounds as carbon sources. We propose to classify all isolates from the present study into two novel Paraburkholderia species, for which we propose the names Paraburkholderia gardini with LMG 32171^T (=CECT 30344^T) as the type strain, and Paraburkholderia saeva with LMG 31841^T (=CECT 30338^T) as the type strain.     

The phylogenetic and ecological context of cultured and whole genome-sequenced planktonic bacteria from the coastal NW Mediterranean Sea

Microbial isolates are useful models for physiological and ecological studies and can also be used to reassemble genomes from metagenomic analyses. However, the phylogenetic diversity that can be found among cultured marine bacteria may vary significantly depending on the isolation. Therefore, this study describes a set of 136 bacterial isolates obtained by traditional isolation techniques from the Blanes Bay Microbial Observatory, of which seven strains have had the whole genome sequenced. The complete set was compared to a series of environmental sequences obtained by culture-independent techniques (60 DGGE sequences and 303 clone library sequences) previously obtained by molecular methods. In this way, each isolate was placed in both its “ecological” (time of year, nutrient limitation, chlorophyll and temperature values) context or setting, and its “phylogenetic” landscape (i.e. similar organisms that were found by culture-independent techniques, when they were relevant, and when they appeared). Nearly all isolates belonged to the Gammaproteobacteria, Alphaproteobacteria, or the Bacteroidetes (70, 40 and 20 isolates, respectively). Rarefaction analyses showed similar diversity patterns for sequences from isolates and molecular approaches, except for Alphaproteobacteria where cultivation retrieved a higher diversity per unit effort. Approximately 30% of the environmental clones and isolates formed microdiversity clusters constrained at 99% 16S rRNA gene sequence identity, but the pattern was different in Bacteroidetes (less microdiversity) than in the other main groups. Seventeen cases (12.5%) of nearly complete (98–100%) rRNA sequence identity between isolates and environmental sequences were found: nine in the Alphaproteobacteria, five in the Gammaproteobacteria, and three in the Bacteroidetes, indicating that cultivation could be used to obtain at least some organisms representative of the various taxa detected by molecular methods. Collectively, these results illustrated the largely unexplored potential of culturing on standard media for complementing the study of microbial diversity by culture-independent techniques and for obtaining phylogenetically distinct model organisms from natural seawater.     

Gammaproteobacteria occurrence and microdiversity in Tyrrhenian Sea sediments as revealed by cultivation-dependent and -independent approaches

Bacterial diversity in Tyrrhenian Sea sediments was assessed using cultivation-dependent and -independent approaches. Samples collected from the different sediment layers (up to 30 cm) relative to four seamount and non-seamount stations, at depths from 3425 to 3580 m, were subjected to DNA extraction and 16S rRNA amplification targeting the V3 region. Denaturing gradient gel electrophoresis (DGGE) showed several heterogeneous profiles and 27 single bands were excised and sequenced. Sequence analysis revealed the presence of Firmicutes, Actinobacteria and Chloroflexi in 26% of the DGGE bands and a predominance of sequences affiliated to cultivable and uncultivable clones of Gammaproteobacteria (55%). To corroborate these findings, cultivation attempts were performed that allowed the isolation of 87 strains assigned to the proteobacterial classes. Identification was achieved by means of automated ribosomal intergenic spacer analysis (ARISA) and by 16S rDNA sequencing. The isolates were related to the gamma, alpha and beta subclasses of Proteobacteria with respective percentages of 77, 17 and 6%. The most predominant Gammaproteobacteria isolates, assigned to the Psychrobacter marincola and P. submarinus clade (n = 53) and to Halomonas aquamarina (n = 14), showed a huge intraspecific diversity with 29 distinct ARISA haplotypes. The detection by both approaches of these psychrophilic and moderately halophilic species and their extensive microdiversity indicated their predominance in Tyrrhenian Sea sediments where they constituted the indigenous microflora.