Clade-Specific 16S Ribosomal DNA Oligonucleotides Reveal the Predominance of a Single Marine Synechococcus Clade throughout a Stratified Water Column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Quantitative Tracing, by Taq Nuclease Assays, of a Synechococcus Ecotype in a Highly Diversified Natural Population

Quantitative Taq nuclease assays (TNAs) (TaqMan PCR), nested PCR in combination with denaturing gradient gel electrophoresis (DGGE), and epifluorescence microscopy were used to analyze the autotrophic picoplankton (APP) of Lake Constance. Microscopic analysis revealed dominance of phycoerythrin (PE)-rich Synechococcus spp. in the pelagic zone of this lake. Cells passing a 3-μm-pore-size filter were collected during the growth period of the years 1999 and 2000. The diversity of PE-rich Synechococcus spp. was examined using DGGE to analyze GC-clamped amplicons of a noncoding section of the 16S-23S intergenic spacer in the ribosomal operon. In both years, genotypes represented by three closely related PE-rich Synechococcus strains of our culture collection dominated the population, while other isolates were traced sporadically or were not detected in their original habitat by this method. For TNAs, primer-probe combinations for two taxonomic levels were used, one to quantify genomes of all known Synechococcus-type cyanobacteria in the APP of Lake Constance and one to enumerate genomes of a single ecotype represented by the PE-rich isolate Synechococcus sp. strain BO 8807. During the growth period, genome numbers of known Synechococcus spp. varied by 2 orders of magnitude (2.9 × 10³ to 3.1 × 10⁵ genomes per ml). The ecotype Synechococcus sp. strain BO 8807 was detected in every sample at concentrations between 1.6 × 10¹ and 1.3 × 10⁴ genomes per ml, contributing 0.02 to 5.7% of the quantified cyanobacterial picoplankton. Although the quantitative approach taken in this study has disclosed several shortcomings in the sampling and detection methods, this study demonstrated for the first time the extensive internal dynamics that lie beneath the seemingly arbitrary variations of a population of microbial photoautotrophs in the pelagic habitat.     

A Suppression Subtractive Hybridization Approach Reveals Niche-Specific Genes That May Be Involved in Predator Avoidance in Marine Synechococcus Isolates

Picocyanobacteria of the genus Synechococcus are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains, Synechococcus sp. strain WH8103 (clade III) and Synechococcus sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.     

Characterization of Imcrop, a Mutator-like MITE family in the rice genome

Sequence comparisons of ammonium transporter 1?C2 genes (OsAMT1-2) in different rice accessions revealed a MITE insertion in the upstream region of the gene. The 391-bp MITE, classified as a Mutator superfamily member and named Imcrop, included terminal inverted repeat (TIR) and 9-bp target site duplication (TSD) sequences. We identified 151 Imcrop elements dispersed on 12 chromosomes of the japonica reference genome. Of these, 12.6% were found in genic regions and 33.1% were located within 1.5 kb of annotated rice genes. We constructed comparative insertion maps with 111 and 102 intact Imcrop elements in the japonica and indica reference genomes, respectively. The Imcrop elements showed relatively even distribution across all chromosomes although their frequency was higher on chromosomes 1, 3, and 4 in both genomes. Seventy seven Imcrop elements were detected in both subspecies, whereas 34 and 25 insertions were found only in the japonica or indica genome, respectively. We compared insertion polymorphisms of 19 Imcrop elements found inside genes in 48 Korean rice cultivars, consisting of 42 japonica and six Tongil-types (indica-japonica cross). Thirteen insertions were common to all cultivars indicating these elements were present before indica-japonica divergence. The six other elements showed insertion polymorphisms among accessions, showing their recent insertion history or no critical positive effect of their insertion on the rice genome.     

Repeated sequences including RS 1100 from Pseudomonas cepacia AC1100 function as IS elements

Summary Several lines of evidence were obtained that the previously identified, repeated sequence RS 1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences or a newly identified 3400 by repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.[/ab]Abbreviations aphc aminoglycoside phosphotransferase gene - BSM basal salts medium - chq chlorohydroxy hydroquinone degradative gene(s) - dCTP deoxycytidine triphosphate - IS insertion sequence - Tft 2,4,5-T degradative phenotype     

Searching Biomarkers in the Sequenced Genomes of <Emphasis Type="Italic">Staphylococcus</Emphasis> for their Rapid Identification

Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5–6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-016-0565-9) contains supplementary material, which is available to authorized users.     

Next-generation sequencing-based genome-wide mutation analysis of l-lysine-producing Corynebacterium glutamicum ATCC 21300 strain

In order to identify single nucleotide polymorphism and insertion/deletion mutations, we performed whole-genome re-sequencing of the enhanced l-lysine-producing Corynebacterium glutamicum ATCC 21300 strain. In total, 142 single nucleotide polymorphisms and 477 insertion/deletion mutations were identified in the ATCC 21300 strain when compared to 3,434 predicted genes of the wild-type C. glutamicum ATCC 13032 strain. Among them, 110 transitions and 29 transversions of single nucleotide polymorphisms were found from genes of the ATCC 21300 strain. In addition, 11 genes, involved in the L-lysine biosynthetic pathway and central carbohydrate metabolism, contained mutations including single nucleotide polymorphisms and insertions/deletions. Interestingly, RT-PCR analysis of these 11 genes indicated that they were normally expressed in the ATCC 21300 strain. This information of genome-wide gene-associated variations will be useful for genome breeding of C. glutamicum in order to develop an industrial amino acid-producing strain with minimal mutation.     

Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of sequence from the human genome and ~15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.     

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for <Emphasis Type="Italic">Streptococcus</Emphasis>

The use of rrs (16S rRNA) gene is widely regarded as the “gold standard” for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4–7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0561-5) contains supplementary material, which is available to authorized users.     

Diversity of radA Genes from Cultured and Uncultured Archaea: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker

Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.     

SINE Retrotransposition During the Evolution of the Pecoran Ruminants

SINE retrotransposition events have proven their value as phylogenetic markers in several eukaryotic taxa at different taxonomic levels. The genomes of ruminants contain three related SINE elements, Bov-tA, Bov-A2, and Bov-B. To estimate the time points of retrotransposition of individual copies of these SINEs, we designed PCR primers on database sequences containing SINE insertions in cattle, sheep, or goat genomes and tested for the presence of these copies in the genomes of other ruminants. It was checked by sequencing whether length variation of the PCR products reflected a SINE retrotransposition. One Bov-B and nine Bov-tA insertions were shared by cattle, sheep, goat, and giraffe, indicating an early retrotransposition event before the radiation of the Pecora, while three other Bov-tA and two Bov-B elements were apparently inserted later. The ruminant α-lactalbumine gene contains a hotspot of early and more recent Bov-tA insertions, a Bov-tA replacement as well as a recent Bov-B insertion. Three Bov-A2 insertions were found to be shared only by the Bovidae, the Bovini, and the Bos and Bison species, respectively, indicating that most Bov-A2 insertions are relatively recent. The time elapsed since the retrotransposition was also reflected in the degeneration of the direct repeats that flank SINE inserts. We suggest that retrotransposition of SINEs may serve as phylogenetic markers in the ruminant families, subfamilies, and even tribes. In addition, sequencing of SINE insertions revealed several other unique deletions/insertions that also may be informative for phylogenetic reconstructions of ruminants. Received: 19 January 2001 / Accepted: 6 June 2001     

Contrasting evolutionary patterns and target specificities among three Tourist-like MITE families in the maize genome

Miniature inverted-repeat transposable elements (MITEs) are short, non autonomous DNA elements that are widespread and abundant in plant genomes. The high sequence and size conservation observed in many MITE families suggest that they have spread recently throughout their respective host genomes. Here we present a maize genome wide analysis of three Tourist-like MITE families, mPIF, and two previously uncharacterized families, ZmV1 and Zead8. We undertook a bioinformatic analysis of MITE insertion sites, developed methyl-sensitive transposon display (M-STD) assays to estimate the associated level of CpG methylation at MITE flanking regions, and conducted a population genetics approach to investigate MITE patterns of expansion. Our results reveal that the three MITE families insert into genomic regions that present specific molecular features: they are preferentially AT rich, present low level of cytosine methylation as compared to the LTR retrotransposon Grande, and target site duplications are flanked by large and conserved palindromic sequences. Moreover, the analysis of MITE distances from predicted genes shows that 73% of 263 copies are inserted at less than 5 kb from the nearest predicted gene, and copies from Zead8 family are significantly more abundant upstream of genes. By employing a population genetic approach we identified contrasting patterns of expansion among the three MITE families. All elements seem to have inserted roughly 1 million years ago but ZmV1 and Zead8 families present evidences for activity of several master copies within the last 0.4 Mya.     

ISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data

Background

Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data.

Results

ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots.

Conclusions

ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.     

Rearrangement of the Bacterial Chromosome Using Tn10 as a Region of Homology

The transposable tetracycline resistance element, Tn10, can serve as a region of homology to promote rec-dependent deletion, duplication and directed transposition of bacterial genes. Tn10 insertions in regions of the chromosome near the histidine operon (his) have been isolated and characterized in Salmonella typhimurium. When strains are constructed containing two Tn10 insertions flanking the his operon in the same orientation (Tn10-his-Tn10), recombination can occur between Tn10 sequences resulting in the deletion of the intervening his region. The sites of the Tn10 insertions determine the endpoints of the deletion. In crosses designed to construct strains carrying Tn10-his-Tn10, another class of unstable recombinants arises in which the his region exists in tandem duplication, with a Tn10 insertion joining the duplicated copies (his-Tn10-his). The sites of the parental Tn10 insertions mark the endpoints of the duplication. When a strain carrying Tn10-his-Tn10 is used as a donor of his⁺ in conjugation or P22-mediated transduction, recombinants can arise in which the his region has been transposed to the site of any Tn10 insertion, far from the normal location of his in the recipient chromosome. In this manner, the his operon has been moved to the site of a pyrB::Tn10 insertion and has been placed on F'' plasmids. At these new locations, the his⁺ character shows the rec-dependent deletion of his⁺ expected for a Tn10-his-Tn10 duplication. These methods should be generally useful for the manipulation of bacterial genes.     

Isolation and characterization of a novel violacein-like pigment producing psychrotrophic bacterial species Janthinobacterium svalbardensis sp. nov

A bacterial strain designated JA-1, related to Janthinobacterium lividum, was isolated from glacier ice samples from the island Spitsbergen in the Arctic. The strain was tested for phenotypic traits and the most prominent appeared to be the dark red brown to black pigmentation different from the violet pigment of Janthinobacterium, Chromobacterium and Iodobacter. Phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization tests showed that strain JA-1 belongs to the genus Janthinobacterium but represents a novel lineage distinct from the two known species of this genus, J. lividum and Janthinobacterium agaricidamnosum. The DNA G + C content of strain JA-1 was determined to be 62.3 mol %. The isolate is a psychrotrophic Gram negative bacterium, rod-shaped with rounded ends, containing intracellular inclusions and one polar flagellum. On the basis of the presented results strain JA-1 is proposed as the type strain of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium svalbardensis sp. nov. is proposed (JA-1^T = DSM 25734, ZIM B637).     

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.