Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population

Abstract Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively. This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance. A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number. A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number. Received: 29 January 1999; Accepted: 29 April 1999     

猪链球菌2型srtBCD菌毛岛SSU2099基因的克隆表达与免疫学特性

通过同源性比较及生物信息学分析,发现猪链球菌2型srtBCD菌毛岛上菌毛辅助亚基编码基因SSU2099,PCR扩增目的片段,克隆入表达载体pET32a中原核表达,亲和层析法纯化重组蛋白。ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体,动物保护性实验表明该蛋白具有良好的免疫保护作用。提示菌毛亚基编码基因SSU2099是重要的疫苗候选分子,进一步研究该蛋白的定位和功能对于系统阐释srtBCD菌毛岛在猪链球菌2型致病机制中的作用有重要意义。     

Metagenomic retrieval of a ribosomal DNA repeat array from an uncultured marine alveolate

The aim of this study was to explore the use of large-scale sequencing to better describe the genome content of naturally occurring, uncultured protists. We constructed a metagenomic fosmid library from a picoplanktonic assemblage (0.2–3 μm size cells) collected at the Blanes Bay Microbial Observatory (Western Mediterranean). Seven clones contained a small-subunit ribosomal RNA gene (SSU rDNA) affiliating with prasinophytes and uncultured alveolates. One clone (FBB25; 35 kb in size) was completely sequenced and found to be a tandem repeat array (5.5 times) of the rDNA operon, including three rRNA genes (SSU, large-subunit and 5.8S rDNAs) and three spacer regions (internal transcribed spacers 1, 2 and intergenic spacer). The SSU rDNA of FBB25 affiliated with the marine alveolates group I, cluster 1, and was almost identical to sequences retrieved only in marine surveys from a wide geographic and ecological range. Phylogenetic trees using the different rRNA genes showed FBB25 as an independent branch among the main alveolate groups, but their closest affiliation varied between the SSU tree (dinoflagellates) and the large-subunit and 5.8S trees (perkinsids). The spacer regions of FBB25 were particularly short when compared with other eukaryotes, indicating a possible genome streamlining in this picoeukaryote. Finally, not a single polymorphism was found in the rDNA repeat array, suggesting that the high SSU rDNA variability typically found in molecular surveys derives from organismal and not intragenomic diversity. This first report on the rDNA genomic structure of an uncultured marine alveolate improves their phylogenetic position and helps interpreting data generated during picoeukaryotic molecular surveys.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Characterization of the ELPhiS prophage from Salmonella enterica serovar Enteritidis strain LK5

Phages are a primary driving force behind the evolution of bacterial pathogens by transferring a variety of virulence genes into their hosts. Similar to other bacterial genomes, the Salmonella enterica serovar Enteritidis LK5 genome contains several regions that are homologous to phages. Although genomic analysis demonstrated the presence of prophages, it was unable to confirm which phage elements within the genome were viable. Genetic markers were used to tag one of the prophages in the genome to allow monitoring of phage induction. Commonly used laboratory strains of Salmonella were resistant to phage infection, and therefore a rapid screen was developed to identify susceptible hosts. This approach showed that a genetically tagged prophage, ELPhiS (Enteritidis lysogenic phage S), was capable of infecting Salmonella serovars that are diverse in host range and virulence and has the potential to laterally transfer genes between these serovars via lysogenic conversion. The rapid screen approach is adaptable to any system with a large collection of isolates and may be used to test the viability of prophages found by sequencing the genomes of various bacterial pathogens.     

Expression of tandem gene fusions in transgenic tobacco plants.

We have studied the expression of four sets of tandem gene fusions in transgenic tobacco plants. This was to determine if the problem of between-transformant variability in expression of introduced genes could be overcome by using a linked reference gene as a co-ordinately expressed control. Tandem gene fusions containing identical 5' flanking regions (SSU301-ocs with either SSU301-cat or SSU301-SSU911) were not co-ordinately expressed in the transgenic tobacco plants whereas the tandem gene fusions containing similar but not identical 5' flanking regions (SSU301-ocs with SSU911-cat or SSU911-SSU301) were co-ordinately expressed. The lack of co-ordinate expression of some of the tandem gene fusions appears to be partially explained by absence of the corresponding genomic DNA segments in the transgenic plants.     

Complete genome sequence of Salmonella bacteriophage SPN3US

Salmonella bacteriophage SPN3US was isolated from a chicken fecal sample. It is a virulent phage belonging to the Myoviridae family and showing effective inhibition of Salmonella enterica and a few Escherichia coli O157:H7 strains. Here we announce the completely sequenced first genome of a Salmonella phage using flagella as receptors. It is the largest genome among Salmonella phages sequenced to date, and major findings from its annotation are described.     

Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants.

The petunia rbcS gene SSU301 was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. The time at which rbcS expression was maximal after transfer of the tobacco plants to the greenhouse was determined. The expression level of the SSU301 gene varied up to 9 fold between individual tobacco plants which had been standardized physiologically as much as possible. The presence of adjacent pUC plasmid sequences did not affect the expression of the SSU301 gene. In an attempt to reduce the between-transformant variability in expression, the SSU301 gene was introduced into tobacco surrounded by 10kb of 5' and 13 kb of 3' DNA sequences which normally flank SSU301 in petunia. The longer flanking regions did not reduce the between-transformant variability of SSU301 gene expression.     

Genome plasticity and ori-ter rebalancing in Salmonella typhi

Genome plasticity resulting from frequent rearrangement of the bacterial genome is a fascinating but poorly understood phenomenon. First reported in Salmonella typhi, it has been observed only in a small number of Salmonella serovars, although the over 2,500 known Salmonella serovars are all very closely related. To gain insights into this phenomenon and elucidate its roles in bacterial evolution, especially those involved in the formation of particular pathogens, we systematically analyzed the genomes of 127 wild-type S. typhi strains isolated from many places of the world and compared them with the two sequenced strains, Ty2 and CT18, attempting to find possible associations between genome rearrangement and other significant genomic features. Like other host-adapted Salmonella serovars, S. typhi contained large genome insertions, including the 134 kb Salmonella pathogenicity island, SPI7. Our analyses showed that SPI7 disrupted the physical balance of the bacterial genome between the replication origin (ori) and terminus (ter) when this DNA segment was inserted into the genome, and rearrangement in individual strains further changed the genome balance status, with a general tendency toward a better balanced genome structure. In a given S. typhi strain, genome diversification occurred and resulted in different structures among cells in the culture. Under a stressed condition, bacterial cells with better balanced genome structures were selected to greatly increase in proportion; in such cases, bacteria with better balanced genomes formed larger colonies and grew with shorter generation times. Our results support the hypothesis that genome plasticity as a result of frequent rearrangement provides the opportunity for the bacterial genome to adopt a better balanced structure and thus eventually stabilizes the genome during evolution.     

Sequences 5'' to translation start regulate expression of petunia rbcS genes.

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes.     

The Mitochondrial Genome of Chlorogonium elongatum Inferred from the Complete Sequence

The 22,704-bp circular mitochondrial DNA (mtDNA) of the chlamydomonad alga Chlorogonium elongatum was completely cloned and sequenced. The genome encodes seven proteins of the respiratory electron transport chain, subunit 1 of the cytochrome oxidase complex (cox1), apocytochrome b (cob), five subunits of the NADH dehydrogenase complex (nad1, nad2, nad4, nad5, and nad6), a set of three tRNAs (Q, W, M), and the large (LSU)- and small (SSU)-subunit ribosomal RNAs. Six group-I introns were found, two each in the cox1, cob, and nad5 genes. In each intron an open reading frame (ORF) related to maturases or endonucleases was identified. Both the LSU and the SSU rRNA genes are split into fragments intermingled with each other and with other genes. Although the average A + T content is 62.2%, GC-rich clusters were detected in intergenic regions, in variable domains of the rRNA genes, and in introns and intron-encoded ORFs. A comparison of the genome maps reveals that C. elongatum and Chlamydomonas eugametos mtDNAs are more closely related to one another than either is to Chlamydomonas reinhardtii mtDNA. Received: 3 November 1997 / Accepted: 12 January 1998     

Chromosomal location of genes for Rubisco small subunit and Rubisco-binding protein in common wheat

Summary The genes coding for the Rubisco small subunit (SSU) and for the -subunit of the Rubisco-binding protein were located to chromosome arms of common wheat. HindIII-digested total DNA from the hexaploid cultivar Chinese Spring and from ditelosomic and nullisomic-tetrasomic lines was probed with these two genes, whose chromosomal location was deduced from the disappearance of or from changes in the relative intensity of the relevant band(s). The Rubisco SSU pattern consisted of 14 bands, containing at least 21 different types of DNA fragments, which were allocated to two homoeologous groups: 15 to the short arm of group 2 chromosomes (4 to 2AS, 7 to 2BS, and 4 to 2DS) and 6 to the long arm of group 5 chromosomes (2 on each of arms 5AL, 5BL, and 5DL). The pattern of the Rubisco-binding protein consisted of three bands, each containing one type of fragment. These fragments were located to be on the short arm of group 2 chromosomes. The restriction fragment length polymorphism (RFLP) patterns of several hexaploid and tetraploid lines were highly conserved, whereas the patterns of several of their diploid progenitors were more variable. The variations found in the polyploid species were mainly confined to the B genome. The patterns of the diploids T. monococcum var. urartu and Ae. squarrosa were similar to those of the A and D genome, respectively, in polyploid wheats. The pattern of T. monococcum var. boeoticum was different from the patterns of the A genome, and the patterns of the diploids Ae. speltoides, Ae. longissima, and Ae. Searsii differed from that of the B genome.     

Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea,bacteria, eukaryotes,mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets

The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa (), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.     

Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).     

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.     

Quantitative metagenomic analyses based on average genome size normalization

Over the past quarter-century, microbiologists have used DNA sequence information to aid in the characterization of microbial communities. During the last decade, this has expanded from single genes to microbial community genomics, or metagenomics, in which the gene content of an environment can provide not just a census of the community members but direct information on metabolic capabilities and potential interactions among community members. Here we introduce a method for the quantitative characterization and comparison of microbial communities based on the normalization of metagenomic data by estimating average genome sizes. This normalization can relieve comparative biases introduced by differences in community structure, number of sequencing reads, and sequencing read lengths between different metagenomes. We demonstrate the utility of this approach by comparing metagenomes from two different marine sources using both conventional small-subunit (SSU) rRNA gene analyses and our quantitative method to calculate the proportion of genomes in each sample that are capable of a particular metabolic trait. With both environments, to determine what proportion of each community they make up and how differences in environment affect their abundances, we characterize three different types of autotrophic organisms: aerobic, photosynthetic carbon fixers (the Cyanobacteria); anaerobic, photosynthetic carbon fixers (the Chlorobi); and anaerobic, nonphotosynthetic carbon fixers (the Desulfobacteraceae). These analyses demonstrate how genome proportionality compares to SSU rRNA gene relative abundance and how factors such as average genome size and SSU rRNA gene copy number affect sampling probability and therefore both types of community analysis.     

Theoretical and Practical Approaches to Evaluate Suitable Primer Sets for the Analysis of Soil Fungal Communities

Six published fungal specific primer sets (NS1/NS2, SSU‐0817/SSU11‐96, SSU‐0817/SSU‐1536, EF4/EF3, EF4/fung5 and FR1/FF390) were examined for their applicability to the analysis of soil fungal communities using bioinformatic tools as well as real PCR systems. Virtual primer matching for EF4/EF3 and EF4/fung5 revealed good matching with zygomycetous, ascomycetous and basidiomycetous 18S rDNA database entries. Whereas primer EF4/EF3 had no cross matches in the rDNA databases for plant and invertebrate, primer EF4/fung5 gave one signal with the corresponding database. Similar results were obtained for the primer set SSU‐0817/SSU‐1536. Two matches with plant rDNAs and 22 or 12 matches with the invertebrate database could be identified for the primer sets SSU‐0817/SSU‐1196 and FR1/FF390, respectively. Primer pair NS1/NS2 showed only a 70% match with fungal 18S rDNA sequences, but a 75% to 90% match with non‐fungal sequences. Alignments of 2000 eukaryotic sequences using “ARB” confirmed that PCR fragments obtained by the primer sets EF4/EF3, EF4/fung5, SSU‐0817/SSU‐1536 and FR1/FF390 were supposed to include hypervariable regions (V4, V7, V9), whereas the others included regions which were more phylogenetically conserved. Practical PCR approaches affirmed fungal specificity as predicted by virtual primer matching for EF4/EF3, EF4/fung5 and FR1/FF390. However FR1/FF390 amplified only 60% of the fungal samples under investigation. All other primer sets amplified fungal as well as non‐fungal samples.