Review and re-analysis of domain-specific 16S primers

The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the ''universal'' primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective 'universal' Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.

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Review and re-analysis of domain-specific 16S primers (Review article)

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30°C up to 95°C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C4 to C16) examined was p-nitrophenyl caproate (C6), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

New Thermophilic and Thermostable Esterase with Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned from a Metagenomic Library

The prokaryotic diversity in the Bor Khlueng hot spring in Ratchaburi province, Thailand was investigated by a culture-independent molecular approach. This hydrothermal pool is located in the central part of Thailand and contains sulfide-rich mineral water that is believed to relieve muscle ache and pain. The water flow year-round with temperature ranging between 50 – 57 °C. Community DNA was extracted directly from sediment samples by coring to depth of ~20 – 30 cm. Small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the domains Archaea and Bacteria. The PCR products were cloned and sequenced. For the bacterial rDNA clone library, 200 clones were randomly selected for further analyses. After restriction fragment length polymorphism (RFLP) analysis of rDNA clones and exclusion of chimeric sequences 36 phylotypes were obtained. The Bor Khlueng phylotypes spanned a wide range within the domain Bacteria, occupying eleven major lineages (phyla). Almost a quarter (23%) of the clones were classifed as Acidobacteria. The other clones were grouped into the Bacteriodetes (19%), Nitrospirae (13%), Proteobacteria (12%), Deinococcus-Thermus lineage (11%), planctomycetes (6%), and Verrucomicrobia (5%). The four remaining phyla, 5% each, were assigned to Actinobacteria, Chloroflexi, Cyanobacteria, and the candidate division “OP10”. For the archaeal 16S rRNA gene sequence library, 25 distinct phylotypes were obtained, 17 clones were found to be associated with Crenarahaeota and 8 clones were associated with Euryarachaeota. The findings of the molecular survey of this so far not investigated site showed that Bor Khlueng hot spring is a potential rich source of unique bacterial and archaeal species. The great majority (~80%) of the prokaryotic sequences detected in Bor Khlueng were unknown.

Diversity and abundance of bacteria and archaea in the Bor Khlueng hot spring in Thailand

Despite detailed study of selected thermophilic taxa, overall community diversity of bacteria in thermophilic mats remains relatively poorly understood. A sequence-based survey of bacterial communities from several hot spring locations in central Tibet was undertaken. Diversity and frequency of occurrence for 140 unique 16S rRNA gene phylotypes were identified in clone libraries constructed from environmental samples. A lineage-per-time plot revealed that individual locations have evolved to support relatively large numbers of phylogenetically closely related phylotypes. Application of the F
 ST
 statistic and P test to community data was used to demonstrate that phylogenetic divergence between locations was significant, thus emphasizing the status of hot springs as isolated habitats. Among phylotypes, only the Chlorobi were ubiquitous to all mats, other phototrophs (Cyanobacteria and Chloroflexi) occurred in most but not all samples and generally accounted for a large number of recovered phylotypes. Phylogenetic analyses of phototrophic phylotypes revealed support for location-specific lineages. The alpha, beta and gamma proteobacteria were also frequently recovered phyla, suggesting they may be abundant phylotypes in mats, a hitherto unappreciated aspect of thermophilic mat biodiversity. Samples from one location indicated that where phototrophic bacteria were rare or absent due to niche disturbance, the relative frequency of proteobacterial phylotypes increased.

Bacterial community composition in thermophilic microbial mats from five hot springs in central Tibet

We report the first attempts to describe thermophilic bacterial communities in Indonesia's thermal springs using molecular phylogenetic analyses. 16S rRNA genes from laboratory cultures and DNA directly amplified from three hot springs in West Java were sequenced. The 22 sequences obtained were assignable to the taxa Proteobacteria, Bacillus and Flavobacterium, including a number of clades not normally associated with thermophily.

Adrian R. Suharto

Papers

Bacterial community analysis of Indonesian hot springs.