Molecular and morphological characterization of free-floating filamentous cyanobacterial mats from geothermal springs in the Philippines

A novel cyanobacterial mat type is characterized from near-neutral pH, low sulphide geothermal springs of 45-60 °C in the Philippines. Mats were free floating, several metres in diameter and several cm in thickness. The upper surface of mats was covered in a waxy scytonemin-like layer, solvent extracts of which absorbed light strongly at 384nm. Light microscopy revealed mats to posses highly ordered layers of air spaces at both the macroscopic and microscopic level, apparently as an adaptation to buoyancy. Morphospecies composition was exclusively filamentous, with Fischerella-like and Oscillatoria-like taxa closely associated throughout mats. Abundant heterocystous cells were observed in Fischerella filaments, suggesting nitrogen fixation occurs in these mats. Morphological structure did not vary among mats from pools of different temperature, but several 16S rDNA-defined genotypes were resolved by DGGE with some displaying greater thermophily than others. Sequencing of fourteen DGGE bands (Genbank accession numbers: AY236467-AY236480) yielded nine novel Fischerella sequences, whilst the five Oscillatoria sequences showed high similarity to other thermophilic Oscillatoria sequences. These data are relevant to astrobiology in that they expand our knowledge of oxygenic photosynthetic community diversity in geothermal environments, which serve as modern analogues for early life on Earth and other planets. Acknowledgements The authors are extremely grateful to the Philippine Institute of Volcanology and Seismology (PHIVOLCS) for advice and assistance with fieldwork. This work was supported by grants awarded by The University of Hong Kong CRCG Seed Funding for Basic Research and Small Projects programmes.published_or_final_versio

Cyanobacteria and chloroflexi-dominated hypolithic colonization of quartz at the hyper-arid core of the Atacama Desert, Chile

Quartz stones are ubiquitous in deserts and are a substrate for hypoliths, microbial colonists of the underside of such stones. These hypoliths thrive where extreme temperature and moisture stress limit the occurrence of higher plant and animal life. Several studies have reported the occurrence of green hypolithic colonization dominated by cyanobacteria. Here, we describe a novel red hypolithic colonization from Yungay, at the hyper-arid core of the Atacama Desert in Chile. Comparative analysis of green and red hypoliths from this site revealed markedly different microbial community structure as revealed by 16S rRNA gene clone libraries. Green hypoliths were dominated by cyanobacteria (Chroococcidiopsis and Nostocales phylotypes), whilst the red hypolith was dominated by a taxonomically diverse group of chloroflexi. Heterotrophic phylotypes common to all hypoliths were affiliated largely to desiccation-tolerant taxa within the Actinobacteria and Deinococci. Alphaproteobacterial phylotypes that affiliated with nitrogen-fixing taxa were unique to green hypoliths, whilst Gemmatimonadetes phylotypes occurred only on red hypolithon. Other heterotrophic phyla recovered with very low frequency were assumed to represent functionally relatively unimportant taxa. © 2010 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

Low-diversity fungal assemblage in an Antarctic Dry Valleys soil

The McMurdo Dry Valleys of Antarctica present extreme environmental challenges. Life is restricted to patchy occurrence of lichens, mosses and invertebrates, plus soil microbial communities. Fungi have been described in lichen symbioses but relatively little is known about the occurrence of free-living soil fungi in the Dry Valleys. A challenge in estimating fungal species richness has been the extent to which estimates based on either cultivation or environmental DNA reflect the active assemblage in cold-arid soils. Here, we describe analysis for inland Dry Valleys soil of environmental DNA and RNA (cDNA) to infer total and putative metabolically active assemblages, respectively, plus cultivation approaches using a variety of laboratory growth conditions. Environmental sequences indicated a highly restricted assemblage of just seven phylotypes that affiliated phylogenetically within two known genera, Helicodendron and Zalerion, plus previously unidentified fungal phylotypes. None of the commonly encountered molds and mitosporic genera recorded from maritime Antarctic locations were encountered. A striking difference was observed in the frequency of recovery for phylotypes between libraries. This suggests that both species richness and beta diversity estimates based on DNA libraries have the potential to misinform putatively active assemblages. Cultivation yielded a cold-tolerant Zalerion strain that affiliated with DNA and RNA library clones, and a psychrotrophic yeast (Debaryomyces hansenii), which was not detected using either culture-independent approach. © 2011 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

Background The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.published_or_final_versio

Community structure of free-floating filamentous cyanobacterial mats from the Wonder Lake geothermal springs in the Philippines

Cyanobacterial mats were characterized from pools of 45-60 °C in near-neutral pH, low-sulphide geothermal springs in the Philippines. Mat structure did not vary with temperature. All mats possessed highly ordered layers of airspaces at both the macroscopic and microscopic level, and these appear to be an adaptation to a free-floating growth habit. Upper mat layers supported biomass with elevated carotenoid:chlorophyll a ratios and an as yet uncharacterized waxy layer on the dorsal surface. Microscopic examination revealed mats comprised a single Fischerella morphotype, with abundant heterocysts throughout mats at all temperatures. Molecular analysis of mat community structure only partly matched morphological identification. All samples supported greater 16S rDNA-defined diversity than morphology suggested, with a progressive loss in the number of genotypes with increasing temperature. Fischerella-like sequences were recovered from mats occurring at all temperatures, but some mats also yielded Oscillatoria-like sequences, although corresponding phenotypes were not observed. Phylogenetic analysis revealed that Fischerella-like sequences were most closely affiliated with Fischerella major and the Oscillatoria-like sequences with Oscillatoria amphigranulata. © 2005 NRC Canada.published_or_final_versio

Multilocus sequence analysis of phylogroup 1 and 2 oral treponeme strains

More than 75 ‘species-level' phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet uncultivated taxa, or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly-conserved 16S rRNA, pyrH, recA and flaA genes. We utilize this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n=71) of diverse geographical origins. This comprises phylogroup 1 (n=23) and phylogroup 2 (n=48) treponeme strains; including all relevant ATCC reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450 bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074 nt), recA (1,377 nt) and pyrH (696 nt) gene sequence datasets. Our data confirmed the species differentiation between Treponema denticola (n=41) and Treponema putidum (n=7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into 5 distinct ‘species-level' phylotypes. These respectively corresponded to ‘Treponema vincentii' (n=11), Treponema medium (n=1); ‘Treponema sinensis' (T. sp. IA; n=4); Treponema sp. IB (n=3); and Treponema sp. IC (n=4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence.published_or_final_versio

Complete Genome Sequence of the Oral Spirochete Bacterium Treponema putidum Strain OMZ 758T (ATCC 700334T)

The oral spirochete bacterium Treponema putidum inhabits human periodontal niches. The complete genome sequence of the OMZ 758(T) (ATCC 700334(T)) strain of this species was determined, revealing a 2,796,913-bp chromosome, with a G+C content of 37.30% and a single plasmid (pTPu1; 3,649 bp) identical to pTS1 from Treponema denticola

Complete Genome Sequence for Treponema sp. OMZ 838 (ATCC 700772, DSM 16789), Isolated from a Necrotizing Ulcerative Gingivitis Lesion

The oral treponeme bacterium Treponema sp. OMZ 838 was originally isolated from a human necrotizing ulcerative gingivitis (NUG) lesion. Its taxonomic status remains uncertain. The complete genome sequence length was determined to be 2,708,067 bp, with a G+C content of 44.58%, and 2,236 predicted coding DNA sequences (CDS)

Gene expression of bacterial collagenolytic proteases in root caries

Objective: It is unknown whether bacteria play a role in the collagen matrix degradation that occurs during caries progression. Our aim was to characterize the expression level of genes involved in bacterial collagenolytic proteases in root biofilms with and without caries. Method: we collected samples from active cavitated root caries lesions (RC, n = 30) and from sound root surfaces (SRS, n = 10). Total microbial RNA was isolated and cDNA sequenced on the Illumina Hi-Seq2500. Reads were mapped to 162 oral bacterial reference genomes. Genes encoding putative bacterial collagenolytic proteases were identified. Normalization and differential expression analysis was performed on all metatranscriptomes (FDR8) but none in SRS were Pseudoramibacter alactolyticus [HMPREF0721_RS02020; HMPREF0721_RS04640], Scardovia inopinata [SCIP_RS02440] and Olsenella uli DSM7084 [OLSU_RS02990]. Conclusion: Our findings suggest that the U32 proteases may be related to carious dentine. The contribution of a small number of species to dentine degradation should be further investigated. These proteases may have potential in future biotechnological and medical applications, serving as targets for the development of therapeutic agents