Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host

Bacterial endosymbionts have been detected in some groups of plant‐parasitic nematodes, but few cases have been reported compared to other groups in the phylum Nematoda, such as animal‐parasitic or free‐living nematodes. This study was performed on a wide variety of plant‐parasitic nematode families and species from different host plants and nematode populations. A total of 124 nematode populations (previously identified morphologically and molecularly) were screened for the presence of potential bacterial endosymbionts using the partial 16S rRNA gene and fluorescence in situ hybridization (FISH) and confocal microscopy. Potential bacterial endosymbionts were only detected in nematode species belonging to the genus Xiphinema and specifically in the X. americanum group. Fifty‐seven partial 16S rRNA sequences were obtained from bacterial endosymbionts in this study. One group of sequences was closely related to the genus ‘Candidatus Xiphinematobacter’ (19 bacterial endosymbiont sequences were associated with seven nematode host species, including two that have already been described and three unknown bacterial endosymbionts). The second bacterial endosymbiont group (38 bacterial endosymbiont sequences associated with six nematode species) was related to the family Burkholderiaceae, which includes fungal and soil–plant bacterial endosymbionts. These endosymbionts were reported for the first time in the phylum Nematoda. Our findings suggest that there is a highly specific symbiotic relationship between nematode host and bacterial endosymbionts. Overall, these results were corroborated by a phylogeny of nematode host and bacterial endosymbionts that suggested that there was a high degree of phylogenetic congruence and long‐term evolutionary persistence between hosts and endosymbionts.     

Cell envelope architecture in the Chloroflexi: a shifting frontline in a phylogenetic turf war

It is important that attempts to understand bacterial phylogeny take into account fundamental bacterial characteristics such as cell envelope composition and organization. Several prominent phylogenetic studies have assumed that the cell envelopes of members of the phylum Chloroflexi are ‘Gram‐negative’ (diderm, i.e. defined by both an inner plasma membrane and an outer membrane) and some of these studies have placed the branch leading to the extant Chloroflexi near the root of the bacterial phylogenetic tree. This Correspondence summarizes the compelling evidence that the Chloroflexi are in fact monoderm, i.e. have only a single cellular membrane. The phylogenetic implications of this conclusion are discussed. The data reviewed also shed interesting light on the distribution of protein secretion systems in diderm bacteria.     

Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications

The study was envisaged to assess the extent of normally uncultivable endophytic bacteria in field papaya plants and in vitro established cultures adopting cultivation vs molecular analysis and microscopy. Surface‐sterilized axillary shoot‐buds of papaya ‘Arka Surya’ revealed high bacterial diversity as per 16S rRNA metagene amplicon sequencing (6 phyla, 10 classes, 21 families) with an abundance of Pseudomonas (Gammaproteobacteria), which also formed a common contaminant for in vitro cultured field explants. Molecular analysis of seedling shoot‐tip‐derived healthy proliferating cultures of three genotypes (‘Arka Surya’, ‘Arka Prabhath’, ‘Red Lady’) with regular monthly subculturing also displayed high bacterial diversity (11–16 phyla, >25 classes, >50 families, >200 genera) about 12–18 months after initial establishment. ‘Arka Surya’ and ‘Red Lady’ cultures bore predominantly Actinobacteria (75–78%) while ‘Arka Prabhath’ showed largely Alphaproteobacteria corroborating the slowly activated Methylobacterium sp. Bright‐field direct microscopy on tissue sections and tissue homogenate and epi‐fluorescence microscopy employing bacterial DNA probe SYTO‐9 revealed abundant intracellular bacteria embracing the next‐generation sequencing elucidated high taxonomic diversity. Phylogenetic investigation of communities by reconstruction of unobserved states‐ PICRUSt‐ functional annotation suggested significant operational roles for the bacterial‐biome. Metabolism, environmental information processing, and genetic information processing constituted major Kyoto Encyclopedia of Genes and Genomes KEGG attributes. Papaya stocks occasionally displayed bacterial growth on culture medium arising from the activation of originally uncultivable organisms to cultivation. The organisms included Bacillus (35%), Methylobacterium (15%), Pseudomonas (10%) and seven other genera (40%). This study reveals a hidden world of diverse and abundant conventionally uncultivable cellular‐colonizing endophytic bacteria in field shoots and micropropagating papaya stocks with high genotypic similarity and silent participation in various plant processes/pathways.     

Phylogenetic diversity and metagenomics of candidate division OP3

Except for environmental 16S rRNA gene sequences, no information is available for members of the candidate division OP3. These bacteria appear to thrive in anoxic environments, such as marine sediments, hypersaline deep sea, freshwater lakes, aquifers, flooded paddy soils and methanogenic bioreactors. The 16S rRNA phylogeny suggests that OP3 belongs to the Planctomycetes/Verrucomicrobia/Chlamydiae (PVC) superphylum. Metagenomic fosmid libraries were constructed from flooded paddy soil and screened for 16S rRNA gene‐containing fragments affiliated with the PVC superphylum. The screening of 63 000 clones resulted in 23 assay‐positive fosmids, of which three clones were affiliated with OP3. The 16S rRNA gene sequence divergence between the fragments OP3/1, OP3/2 and OP3/3 ranges from 18% to 25%, indicating that they belong to different OP3 subdivisions. The 23S rRNA phylogeny confirmed the membership of OP3 in the PVC superphylum. Sequencing the OP3 fragments resulted in a total of 105 kb of genomic information and 90 ORFs, of which 47 could be assigned a putative function and 11 were conserved hypothetical. Using BLASTP searches, a high proportion of ORFs had best matches to homologues from Deltaproteobacteria, rather than to those of members of the PVC superphylum. On the fragment OP3/3, a cluster of nine ORFs was predicted to encode the bacterial NADH dehydrogenase I. Given the high proportion of homologues present in deltaproteobacteria and anoxic conditions in the natural environment of OP3 bacteria, the detection of NADH dehydrogenase I may suggest an anaerobic respiration mode. Oligonucleotide frequencies calculated for OP3/1, OP3/2 and OP/3 show high intraphylum correlations. This novel sequence information could therefore be used to identify OP3‐related fragments in large metagenomic data sets using marker gene‐independent procedures in the future. In addition to the OP3 fragments, a single metagenomic fragment affiliated with the candidate division BRC1 was obtained and analysed.     

Karst pools in subsurface environments: collectors of microbial diversity or temporary residence between habitat types

We studied bacterial diversity and community composition in three shallow pools of a Swiss karst cave system with contrasting hydrological and hydrochemical properties. The microbial assemblages in the pools were remarkably different, and only one operational taxonomic unit of 16S rRNA genes (OTU, 97% similarity) was shared between the three of them (total OTU number in all pools: 150). Unexpectedly high microbial phylotype richness was found even in the two pools without groundwater contact and with low concentrations of organic carbon and total cell numbers (< 10⁴ ml^?1). One of these seepage water fed systems harboured 15 distinct OTUs from several deeply branching lineages of the candidate phylum OP3, whereas representatives of this group were not detected in the other two pools. A tentative phylogeographic analysis of available OP3‐related sequences in the context of our data set revealed that there was generally little agreement between the habitats of origin of closely related sequence types. Two bacterial clades affiliated with the obligate methylamine utilizer Methylotenera mobilis were only found in the pool that was exposed to repeated flooding events. These bacteria formed relatively stable populations of up to 6% of total cell counts over periods of several months irrespective of inundation by groundwater. This suggests that karst water may provide a means of transport for these bacteria from terrestrial to freshwater habitats.     

The Succession of Bacterial Community Structure in Groundwater from a 250-m Gallery in the Horonobe Underground Research Laboratory

We investigated the change in bacterial community structure after drilling boreholes, 09-V250-M02 and 09-V250-M03, in the 250-m deep research gallery of the Horonobe Underground Research Laboratory. In the 09-V250-M02 borehole, ?-Proteobacteria were predominantly detected in the clone library analyses of the groundwater samples conducted immediately after drilling. All the ?-Proteobacteria clones were closely related to Arcobacter spp., which are known to be sulfide-oxidizing chemoautotrophic bacteria. After 4 years, the microbial structure drastically changed, and most detected operational taxonomic units were uncultured species such as candidate division OP9 and Chloroflexi relatives, which are frequently detected in deep sea sediments. The results indicated that the microbial community structure was drastically affected by borehole drilling and was concomitant with oxidation perturbation. However, these disturbed microbial communities changed within a few years to a microbial community composed of uncultivated species such as OP9 and Chloroflexi.     

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria , Acidobacteria , Chloroflexi , Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria , Firmicutes , Thermus/Deinococcus , Actinobacteria and Bacteroidetes , as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria , Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.     

Here,there and everywhere: Ecology and biology of the Dependentiae phylum

Our view of bacterial diversity has been dramatically impacted by cultivation-independent approaches such as metagenomics and 16S rRNA gene sequencing. Consequently, most bacterial phyla known to date are only documented by the presence of DNA sequences in databases and lack cultivated representatives. This bacterial majority that is yet-to-be cultivated, is forming the ‘Microbial Dark Matter’, (MDM) a consortium, whose ecology and biology remain largely unexplored. The Candidatus Dependentiae stands as one of many phyla within this MDM, found worldwide in various environments. Genomic evidence suggests ancestral, unusual adaptations of all Ca. Dependentiae to a host dependent lifestyle. In line with this, protists appear to be important for Ca. Dependentiae biology, as revealed by few recent studies, which enabled their growth in laboratory through host cultivation. However, the Ca. Dependentiae still remain to this day a poorly documented phylum. The present review aims to summarize the current knowledge accumulated on this often found, but rarely highlighted, bacterial phylum.     

Insights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions

DNA‐SIP (stable isotope probing) was conducted on anaerobic municipal solid waste samples incubated with ¹³C‐cellulose, ¹³C‐glucose and ¹³C‐acetate under mesophilic conditions. A total of 567 full‐length bacterial and 448 1100‐bp‐length archaeal 16S rRNA gene sequences were analysed. In the clone libraries derived from ‘heavy’ DNA fractions, the most abundant sequences were affiliated with the phyla Firmicutes, Bacteroidetes, the gamma‐subclass of Proteobacteria and methanogenic orders Methanomicrobiales and Methanosarcinales. Sequences related to the genus Acetivibrio (phylum Firmicutes) were recovered only in the ‘heavy’ DNA fraction derived from the ¹³C‐cellulose incubation. An oligonucleotide probe (UCL284) targeting specifically Acetivibrio was designed and used for fluorescent in situ hybridization (FISH) experiments. Interestingly, hybridization of the probe was detected in microorganisms aggregated around cellulose fibres, strengthening the conclusion that these microorganisms were major cellulose degraders. Sequences related to genus Clostridium (phylum Firmicutes) and to the family Porphyromonadaceae (phylum Bacteroidetes) were retrieved in large numbers from the ‘heavy’ DNA library of ¹³C‐Glucose incubation, suggesting their involvement in saccharide fermentation. Design and hybridization of specific FISH‐probes confirmed the abundant representation of Clostridium (CLO401, CLO1248) and Porphyromonadaceae (BAC1040), which were mostly observed in the planktonic phase. Surprisingly, in the ¹³C‐acetate experiment, the ‘heavy’ DNA archaeal library was dominated by sequences related to the strictly hydrogenotrophic methanogenic genus Methanoculleus. One single operational taxonomic unit containing 70 sequences, affiliated to the gamma‐subclass of Proteobacteria, was retrieved in the corresponding bacterial library. FISH observations with a newly designed specific probe (UGA64) confirmed the dominance of this bacterial group. Our results show that combination of DNA‐SIP and FISH applied with a series of functionally connected substrates can shed light on the networks of uncultured microbes catalysing the methanization of the most abundant chemical renewable energy source on Earth.     

Candidate OP Phyla: Importance,Ecology and Cultivation Prospects

OP phyla were created in the domain bacteria, based on the group of 16S rRNA gene sequences recovered from the Obsidian Pool. However, due to the lack of cultured representative it is referred to as candidate phyla. Wider ecological occurrence was predicted for the OP phyla, especially OP3, OP10 and OP11. Recently, members of phylum OP5 and OP10 were cultured, providing clues to their cultivation prospects. At last the bioprospecting potentials of the OP members are discussed herein.     

Metagenomic Investigation of Bacterial and Archaeal Diversity of Hammam Essalihine Hot Spring from Khenchela,Algeria

Abstract

Hot springs are natural environments where hot groundwater comes out from the earth. Exploring the microbial diversity present in hot springs is important first to determine the microorganisms able to proliferate there and to understand their role in biogeochemical cycles. In Algeria, research concerning microbial populations in those ecosystems is limited. This study describes bacterial and archaeal diversity of the ‘Hammam Essalihine’ hot spring in Khenchela province in north-east Algeria using a culture-independent approach. This is the first microbial diversity investigation in the ‘Hammam Essalihine’ hot spring using next-generation sequencing techniques to assess the species classification of thermophilic microorganisms. Genomic DNA was extracted from water samples and the V4–V5 region of 16S rRNA gene were amplified, sequenced, and analyzed. The average temperature of water varies from 68 to 70?°C. High-throughput sequencing analysis revealed the presence of 21 bacterial phyla, including an unknown phylum and distributed across 42 families and 39 genera. The majority of the sequences were observed to belong to the kingdom Bacteria. The bacterial community from this hot spring is dominated by Proteobacteria (41.52%), Chloroflexi (7.62%), and Bacteroidetes (7.62%), whereas the community of Archaea is scarcely present in the study site and the two identified operational taxonomic units (OTUs) are far from what is known in the GenBank database. The study shows several uncharacterized sequences, indicating that the water of ‘Hammam Essalihine’ hot spring contains undescribed microorganisms. This study is thought to add to the understanding of thermophile diversity and ecology of ‘Hammam Essalihine’ hot spring.     

Distinct bacterial communities across a gradient of vegetation from a preserved Brazilian Cerrado

The Cerrado biome in the Sete Cidades National Park, an Ecological Reserve in Northeastern Brazil, has conserved its native biodiversity and presents a variety of plants found in other savannas in Brazil. Despite this finding the soil microbial diversity and community structure are poorly understood. Therefore, we described soil bacterial diversity and distribution along a savanna vegetation gradient taking into account the prevailing environmental factors. The bacterial composition was retrieved by sequencing a fragment of the 16S ribosomal RNA gene. The bacterial operational taxonomic units (OTUs) were assigned to 37 different phyla, 96 classes, and 83 genera. At the phylum level, a core comprised by Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, Verrucomicrobia and Planctomycetes, was detected in all areas of Cerrado. ‘Cerrado stricto sensu’ and ‘Cerradao’ share more similarities between edaphic properties and vegetation and also present more similar bacterial communities, while ‘Floresta decidual’ and ‘Campo graminoide’ show the largest environmental differences and also more distinct bacterial communities. Proteobacteria (26%), Acidobacteria (21%) and Actinobacteria (21%) were the most abundant phyla within the four areas. All the samples present similar bacteria richness (alpha diversity) and the observed differences among them (beta diversity) were more related to the abundance of specific taxon OTUs compared to their presence or absence. Total organic C, N and P are the main abiotic factors structuring the bacterial communities. In summary, our findings show the bacterial community structure was clearly different across the Cerrado gradient, but that these environments share a bacterial phylum-core comprising Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia and Planctomycetes with other Brazilian savannas.     

Cultivation‐independent characterization of ‘Candidatus Magnetobacterium bavaricum’ via ultrastructural,geochemical, ecological and metagenomic methods

‘Candidatus Magnetobacterium bavaricum’ is unusual among magnetotactic bacteria (MTB) in terms of cell size (8–10 µm long, 1.5–2 µm in diameter), cell architecture, magnetotactic behaviour and its distinct phylogenetic position in the deep‐branching Nitrospira phylum. In the present study, improved magnetic enrichment techniques permitted high‐resolution scanning electron microscopy and energy dispersive X‐ray analysis, which revealed the intracellular organization of the magnetosome chains. Sulfur globule accumulation in the cytoplasm point towards a sulfur‐oxidizing metabolism of ‘Candidatus M. bavaricum’. Detailed analysis of ‘Candidatus M. bavaricum’ microhabitats revealed more complex distribution patterns than previously reported, with cells predominantly found in low oxygen concentration. No correlation to other geochemical parameters could be observed. In addition, the analysis of a metagenomic fosmid library revealed a 34 kb genomic fragment, which contains 33 genes, among them the complete rRNA gene operon of ‘Candidatus M. bavaricum’ as well as a gene encoding a putative type IV RubisCO large subunit.     

Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri

Aims: To monitor variations in the bacterial community and fermentation products of maize silage within and between bunker silos. Methods and Results: Silage samples were collected in 2008 and 2009 from three dairy farms, wherein the farmers arranged for a contractor to produce maize silage using bunker silos. Silage was prepared using a lactic acid bacteria (LAB) inoculant consisting of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Eight samples were collected from each bunker silo; 4 ‘outer’ and 4 ‘inner’ samples were collected from near the top and the bottom of the silo. The dry matter, lactic acid, acetic acid, ethanol, 1‐propanol and 1,2‐propanediol contents differed between bunker silos in both sampling years. Higher acetic acid, 1‐propanol and 1,2‐propanediol contents were found in the bottom than the top layers in the 2008 samples, and higher lactic acid content was found in the top than the bottom layers in the 2009 samples. The bacterial community varied more between bunker silos than within a bunker silo in the 2008 samples, whereas differences between the top and the bottom layers were seen across bunker silos in the 2009 samples. The inoculated LAB were uniformly distributed, while several nonconventional silage bacteria were also detected. Lactobacillus acetotolerans, Lactobacillus panis and Acetobacter pasteurianus were detected in both years. Stenotrophomonas maltophilia was detected in the 2008 samples, and Lactobacillus reuteri, Acinetobacter sp. and Rahnella sp. were detected in the 2009 samples. Conclusions: Although differences were seen within and between bunker silos, the bacterial community may indicate a different relationship between bunker silos and sampling locations within a bunker silo from that indicated by the fermentation products. Significance and Impact of the Study: Analysis of bacterial community can help understand how diverse non‐LAB and LAB species are involved in the ensiling process of bunker‐made maize silage.     

Ecological and genomic analyses of candidate phylum WPS-2 bacteria in an unvegetated soil

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron–sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 10⁸ cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.     

Genomics and phylogeny of the proposed phylum ‘Candidatus Poribacteria’ associated with the excavating sponge Thoosa mismalolli

Members of the proposed phylum ‘Candidatus Poribacteria’ are among the most abundant microorganisms in the highly diverse microbiome of the sponge mesohyl. Genomic and phylogenetic characteristics of this proposed phylum are barely known. In this study, we analyzed metagenome-assembled genomes (MAGs) obtained from the coral reef excavating sponge Thoosa mismalolli from the Mexican Pacific Ocean. Two MAGs were extracted and analyzed together with 32 MAGs and single-amplified genomes (SAGs) obtained from NCBI. The phylogenetic tree based on the sequences of 139 single-copy genes (SCG) showed two clades. Clade A (23 genomes) represented 67.7% of the total of the genomes, while clade B (11 genomes) comprised 32.3% of the genomes. The Average Nucleotide Identity (ANI) showed values between 66 and 99% for the genomes of the proposed phylum, and the pangenome of genomes revealed a total of 37,234 genes that included 1722 core gene. The number of genes used in the phylogenetic analysis increased from 28 (previous studies) to 139 (this study), which allowed a better resolution of the phylogeny of the proposed phylum. The results supported the two previously described classes, ‘Candidatus Entoporibacteria’ and ‘Candidatus Pelagiporibacteria’, and the genomes SB0101 and SB0202 obtained in this study belong to two new species of the class ‘Candidatus Entoporibacteria’. This is the first comparative study that includes MAGs from a non-sponge host (Porites lutea) to elucidate the taxonomy of the poorly known Candidatus phylum in a polyphasic approach. Finally, our study also contributes to the sponge microbiome project by reporting the first MAGs of the proposed phylum ‘Candidatus Poribacteria’ isolated from the excavating sponge T. mismalolli.

     

Single‐cell genomics of uncultivated deep‐branching magnetotactic bacteria reveals a conserved set of magnetosome genes

While magnetosome biosynthesis within the magnetotactic Proteobacteria is increasingly well understood, much less is known about the genetic control within deep‐branching phyla, which have a unique ultrastructure and biosynthesize up to several hundreds of bullet‐shaped magnetite magnetosomes arranged in multiple bundles of chains, but have no cultured representatives. Recent metagenomic analysis identified magnetosome genes in the genus ‘Candidatus Magnetobacterium’ homologous to those in Proteobacteria. However, metagenomic analysis has been limited to highly abundant members of the community, and therefore only little is known about the magnetosome biosynthesis, ecophysiology and metabolic capacity in deep‐branching MTB. Here we report the analysis of single‐cell derived draft genomes of three deep‐branching uncultivated MTB. Single‐cell sorting followed by whole genome amplification generated draft genomes of Candidatus Magnetobacterium bavaricum and Candidatus Magnetoovum chiemensis CS‐04 of the Nitrospirae phylum. Furthermore, we present the first, nearly complete draft genome of a magnetotactic representative from the candidate phylum Omnitrophica, tentatively named Candidatus Omnitrophus magneticus SKK‐01. Besides key metabolic features consistent with a common chemolithoautotrophic lifestyle, we identified numerous, partly novel genes most likely involved in magnetosome biosynthesis of bullet‐shaped magnetosomes and their arrangement in multiple bundles of chains.