Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

Intrastrain and interstrain genetic variation within a paralogous gene family in Chlamydia pneumoniae

Background

Chlamydia pneumoniae causes human respiratory diseases and has recently been associated with atherosclerosis. Analysis of the three recently published C. pneumoniae genomes has led to the identification of a new gene family (the Cpn 1054 family) that consists of 11 predicted genes and gene fragments. Each member encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane.

Results

Comparative analysis of this gene family within the published genome sequences provided evidence that multiple levels of genetic variation are evident within this single collection of paralogous genes. Frameshift mutations are found that result in both truncated gene products and pseudogenes that vary among isolates. Several genes in this family contain polycytosine (polyC) tracts either upstream or within the terminal 5' end of the predicted coding sequence. The length of the polyC stretch varies between paralogous genes and within single genes in the three genomes. Sequence analysis of genomic DNA from a collection of 12 C. pneumoniae clinical isolates was used to determine the extent of the variation in the Cpn 1054 gene family.

Conclusions

These studies demonstrate that sequence variability is present both among strains and within strains at several of the loci. In particular, changes in the length of the polyC tract associated with the different Cpn 1054 gene family members are common within each tested C. pneumoniae isolate. The variability identified within this newly described gene family may modulate either phase or antigenic variation and subsequent physiologic diversity within a C. pneumoniae population.     

Effects of Streptococcus pneumoniae strain background on complement resistance

Background

Immunity to infections caused by Streptococcus pneumoniae is dependent on complement. There are wide variations in sensitivity to complement between S. pneumoniae strains that could affect their ability to cause invasive infections. Although capsular serotype is one important factor causing differences in complement resistance between strains, there is also considerable other genetic variation between S. pneumoniae strains that may affect complement-mediated immunity. We have therefore investigated whether genetically distinct S. pneumoniae strains with the same capsular serotype vary in their sensitivity to complement mediated immunity.

Methodology and Principal Findings

C3b/iC3b deposition and neutrophil association were measured using flow cytometry assays for S. pneumoniae strains with different genetic backgrounds for each of eight capsular serotypes. For some capsular serotypes there was marked variation in C3b/iC3b deposition between different strains that was independent of capsule thickness and correlated closely to susceptibility to neutrophil association. C3b/iC3b deposition results also correlated weakly with the degree of IgG binding to each strain. However, the binding of C1q (the first component of the classical pathway) correlated more closely with C3b/iC3b deposition, and large differences remained in complement sensitivity between strains with the same capsular serotype in sera in which IgG had been cleaved with IdeS.

Conclusions

These data demonstrate that bacterial factors independent of the capsule and recognition by IgG have strong effects on the susceptibility of S. pneumoniae to complement, and could therefore potentially account for some of the differences in virulence between strains.     

Genetic structure of duckweed population of Spirodela,Landoltia and Lemna from Lake Tai,China

Main conclusion

Presenting a basic framework for using MLST to characterize Spirodela, Landoltia and in particular Lemna strains at the species level, and to study population genetics and evolution history of natural duckweed populations.

Abstract

Duckweed is widely used in environmental biotechnology and has recently emerged as a potential feedstock for biofuels due to its high growth rate and starch content. The genetic diversity and composition of a natural duckweed population in genera Spirodela, Landoltia and Lemna from Lake Tai, China, were investigated using probabilistic analysis of multilocus sequence typing (MLST). The 78 strains were categorized into five lineages, among which strains representing L. aequinoctialis and S. polyrhiza were predominant. Among the five lineages, interlineage transfers of markers were infrequent and no recombination was statistically detected. Tajima’s D tests determined that all loci are subject to population bottlenecks, which is likely one of the main reasons for the low genetic diversity observed within the lineages. Interestingly, strains of L. turionifera are found to contain small admixture from L. minor, providing rare evidence of transfer of genetic materials in duckweed. This was discussed with respect to the hypothesis that a cross of these two gave rise to L. japonica. Moreover, the conventional maximum-likelihood phylogenetic analysis clearly recognized all the species in the three genera with high bootstrap supports. In conclusion, this work offers a basic framework for using MLST to characterize Spirodela, Landoltia and in particular Lemna strains at the species level, and to study population genetics and evolution history of natural duckweed populations.     

Analysis of the Pantoea ananatis pan-genome reveals factors underlying its ability to colonize and interact with plant,insect and vertebrate hosts

Background

Pantoea ananatis is found in a wide range of natural environments, including water, soil, as part of the epi- and endophytic flora of various plant hosts, and in the insect gut. Some strains have proven effective as biological control agents and plant-growth promoters, while other strains have been implicated in diseases of a broad range of plant hosts and humans. By analysing the pan-genome of eight sequenced P. ananatis strains isolated from different sources we identified factors potentially underlying its ability to colonize and interact with hosts in both the plant and animal Kingdoms.

Results

The pan-genome of the eight compared P. ananatis strains consisted of a core genome comprised of 3,876 protein coding sequences (CDSs) and a sizeable accessory genome consisting of 1,690 CDSs. We estimate that ~106 unique CDSs would be added to the pan-genome with each additional P. ananatis genome sequenced in the future. The accessory fraction is derived mainly from integrated prophages and codes mostly for proteins of unknown function. Comparison of the translated CDSs on the P. ananatis pan-genome with the proteins encoded on all sequenced bacterial genomes currently available revealed that P. ananatis carries a number of CDSs with orthologs restricted to bacteria associated with distinct hosts, namely plant-, animal- and insect-associated bacteria. These CDSs encode proteins with putative roles in transport and metabolism of carbohydrate and amino acid substrates, adherence to host tissues, protection against plant and animal defense mechanisms and the biosynthesis of potential pathogenicity determinants including insecticidal peptides, phytotoxins and type VI secretion system effectors.

Conclusions

P. ananatis has an ‘open’ pan-genome typical of bacterial species that colonize several different environments. The pan-genome incorporates a large number of genes encoding proteins that may enable P. ananatis to colonize, persist in and potentially cause disease symptoms in a wide range of plant and animal hosts.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-404) contains supplementary material, which is available to authorized users.     

Identification of Genes to Differentiate Closely Related Salmonella Lineages

Background

Salmonella are important human and animal pathogens. Though highly related, the Salmonella lineages may be strictly adapted to different hosts or cause different diseases, from mild local illness like gastroenteritis to fatal systemic infections like typhoid. Therefore, rapid and accurate identification of Salmonella is essential for timely and correct diagnosis of Salmonella infections. The current identification methods such as 16S rRNA sequencing and multilocus sequence typing are expensive and time consuming. Additionally, these methods often do not have sufficient distinguishing resolution among the Salmonella lineages.

Methodologies/Principal Findings

We compared 27 completely sequenced Salmonella genomes to identify possible genomic features that could be used for differentiation of individual lineages. We concatenated 2372 core genes in each of the 27 genomes and constructed a neighbor-joining tree. On the tree, strains of each serotype were clustered tightly together and different serotypes were unambiguously separated with clear genetic distances, demonstrating systematic genomic divergence among the Salmonella lineages. We made detailed comparisons among the 27 genomes and identified distinct sets of genomic differences, including nucleotide variations and genomic islands (GIs), among the Salmonella lineages. Two core genes STM4261 and entF together could unambiguously distinguish all Salmonella lineages compared in this study. Additionally, strains of a lineage have a common set of GIs and closely related lineages have similar sets of GIs.

Conclusions

Salmonella lineages have accumulated distinct sets of mutations and laterally acquired DNA (e.g., GIs) in evolution. Two genes entF and STM4261 have diverged sufficiently among the Salmonella lineages to be used for their differentiation. Further investigation of the distinct sets of mutations and GIs will lead to novel insights into genomic evolution of Salmonella and greatly facilitate the elucidation of pathogeneses of Salmonella infections.     

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

Background

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

Results

We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.

Conclusions

Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.     

Mycophenolate mofetil inhibits the development of Coxsackie B3-virus-induced myocarditis in mice

Background

Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.

Results

Observation of survival percentages over time and detection of LD₅₀s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.

Conclusions

These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.     

Intraspecies comparison of Streptomyces pratensis genomes reveals high levels of recombination and gene conservation between strains of disparate geographic origin

Background

Streptomyces are widespread bacteria that contribute to the terrestrial carbon cycle and produce the majority of clinically useful antibiotics. While interspecific genomic diversity has been investigated among Streptomyces, information is lacking on intraspecific genomic diversity. Streptomyces pratensis has high rates of homologous recombination but the impact of such gene exchange on genome evolution and the evolution of natural product gene clusters remains uncharacterized.

Results

We report draft genome sequences of four S. pratensis strains and compare to the complete genome of Streptomyces flavogriseus IAF-45-CD (=ATCC 33331), a strain recently reclassified to S. pratensis. Despite disparate geographic origins, the genomes are highly similar with 85.9% of genes present in the core genome and conservation of all natural product gene clusters. Natural products include a novel combination of carbapenem and beta-lactamase inhibitor gene clusters. While high intraspecies recombination rates abolish the phylogenetic signal across the genome, intraspecies recombination is suppressed in two genomic regions. The first region is centered on an insertion/deletion polymorphism and the second on a hybrid NRPS-PKS gene. Finally, two gene families accounted for over 25% of the divergent genes in the core genome. The first includes homologs of bldB (required for spore development and antibiotic production) while the second includes homologs of an uncharacterized protein with a helix-turn-helix motif (hpb). Genes from these families co-occur with fifteen pairs spread across the genome. These genes have evidence for co-evolution of co-localized pairs, supporting previous assertions that these genes may function akin to a toxin-antitoxin system.

Conclusions

S. pratensis genomes are highly similar with exceptional levels of recombination which erase phylogenetic signal among strains of the species. This species has a large core genome and variable terminal regions that are smaller than those found in interspecies comparisons. There is no geographic differentiation between these strains, but there is evidence for local linkage disequilibrium affecting two genomic regions. We have also shown further observational evidence that the DUF397-HTH (bldB and hpb) are a novel toxin-antitoxin pair.     

Serotypes and Patterns of Antibiotic Resistance in Strains Causing Invasive Pneumococcal Disease in Children Less than 5 Years of Age

Objective

The serotypes and patterns of antibiotic resistance of Streptococcus pneumoniae (S. pneumoniae) strains that cause invasive pneumococcal disease (IPD) in infants were analyzed to provide guidance for clinical disease prevention and treatment.

Methods

The clinical features of confirmed IPD were evaluated in 61 patients, less than 5 years of age, who were admitted to our hospital between January 2009 and December 2011. The serotypes and antibiotic resistance of strains of S.pneumoniae were determined using the capsular swelling method and the E-test.

Results

A total of 61 invasive strains were isolated. The serotype distribution of those isolates were 19A (41.0%), 14 (19.7%), 19F (11.5%), 23F (9.8%), 8 (4.9%), 9V (4.9%), 1 (3.3%), and 4, 6B, and 20 (each 1.6%). The percentage of S. pneumoniae strains resistant to erythromycin, clindamycin, and cotrimoxazole were 100%, 86.9%, and 100%, respectively. The percentage of S. pneumoniae strains resistant to penicillin, amoxicillin/clavulanic acid, cefuroxime, ceftriaxone, cefotaxime, cefepime, and meropenem were 42.6%, 18.0%, 82.0%, 18.0%, 13.1%, 13.1%, and 36.1%, respectively. The percentage of multidrug-resistant strains was 95.6%. Strains of all serotypes isolated in this study were highly resistant to erythromycin, cotrimoxazole, and clindamycin. Strains with serotype 19A had the highest rates of resistance.

Conclusions

Serotype 19A strains were most frequently isolated from children with IPD treated in our hospital. The strains causing IPD are highly resistant to antibiotics.     

Interplay between the cell envelope and mobile genetic elements shapes gene flow in populations of the nosocomial pathogen Klebsiella pneumoniae

Mobile genetic elements (MGEs) drive genetic transfers between bacteria using mechanisms that require a physical interaction with the cellular envelope. In the high-priority multidrug-resistant nosocomial pathogens (ESKAPE), the first point of contact between the cell and virions or conjugative pili is the capsule. While the capsule can be a barrier to MGEs, it also evolves rapidly by horizontal gene transfer (HGT). Here, we aim at understanding this apparent contradiction by studying the covariation between the repertoire of capsule genes and MGEs in approximately 4,000 genomes of Klebsiella pneumoniae (Kpn). We show that capsules drive phage-mediated gene flow between closely related serotypes. Such serotype-specific phage predation also explains the frequent inactivation of capsule genes, observed in more than 3% of the genomes. Inactivation is strongly epistatic, recapitulating the capsule biosynthetic pathway. We show that conjugative plasmids are acquired at higher rates in natural isolates lacking a functional capsular locus and confirmed experimentally this result in capsule mutants. This suggests that capsule inactivation by phage pressure facilitates its subsequent reacquisition by conjugation. Accordingly, capsule reacquisition leaves long recombination tracts around the capsular locus. The loss and regain process rewires gene flow toward other lineages whenever it leads to serotype swaps. Such changes happen preferentially between chemically related serotypes, hinting that the fitness of serotype-swapped strains depends on the host genetic background. These results enlighten the bases of trade-offs between the evolution of virulence and multidrug resistance and caution that some alternatives to antibiotics by selecting for capsule inactivation may facilitate the acquisition of antibiotic resistance genes (ARGs).

A study of how the complex interaction between capsules and mobile genetic elements shapes gene flow in populations of Klebsiella pneumoniae reveals that capsule inactivation by phage pressure facilitates its subsequent re-acquisition by conjugation, and this loss and re-gain process influences the gene flow towards other lineages whenever it leads to serotype changes.     

The Oral Commensal Streptococcus mitis Shows a Mixed Memory Th Cell Signature That Is Similar to and Cross-Reactive with Streptococcus pneumoniae

Background

Carriage of and infection with Streptococcus pneumoniae is known to predominantly induce T helper 17 (Th17) responses in humans, but the types of Th cells showing reactivity towards commensal streptococci with low pathogenic potential, such as the oral commensals S. mitis and S. salivarius, remain uncharacterized.

Methods

Memory CD4⁺ T helper (Th) cell subsets were isolated from healthy human blood donors according to differential expression of chemokine receptors, expanded in vitro using polyclonal stimuli and characterized for reactivity against different streptococcal strains.

Results

Th cells responding to S. mitis, S. salivarius and S. pneumoniae were predominantly in a CCR6⁺CXCR3⁺ subset and produced IFN-γ, and in a CCR6⁺CCR4⁺ subset and produced IL-17 and IL-22. Frequencies of S. pneumoniae-reactive Th cells were higher than frequencies of S. mitis- and S. salivarius-specific Th cells. S. mitis and S. pneumoniae isogenic capsule knock-out mutants and a S. mitis mutant expressing the serotype 4 capsule of S. pneumoniae showed no different Th cell responses as compared to wild type strains. S. mitis-specific Th17 cells showed cross-reactivity with S. pneumoniae.

Conclusions

As Th17 cells partly control clearance of S. pneumoniae, cross-reactive Th17 cells that may be induced by commensal bacterial species may influence the immune response, independent of capsule expression.     

Comparative genomics of the Bifidobacterium breve taxon

Background

Bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, where their presence is positively correlated with the host’s health status. In this study, we assessed the genomes of thirteen representatives of Bifidobacterium breve, which is not only a frequently encountered component of the (adult and infant) human gut microbiota, but can also be isolated from human milk and vagina.

Results

In silico analysis of genome sequences from thirteen B. breve strains isolated from different environments (infant and adult faeces, human milk, human vagina) shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. These latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign DNA (i.e. CRISPR loci and restriction/modification systems), and genes that encode enzymes responsible for carbohydrate fermentation. Gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol.

Conclusions

Genome analysis of thirteen representatives of the B. breve species revealed that the deduced pan-genome exhibits an essentially close trend. For this reason our analyses suggest that this number of B. breve representatives is sufficient to fully describe the pan-genome of this species. Comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of B. breve.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-170) contains supplementary material, which is available to authorized users.     

Monitoring the long-term molecular epidemiology of the pneumococcus and detection of potential 'vaccine escape' strains

Background

While the pneumococcal protein conjugate vaccines reduce the incidence in invasive pneumococcal disease (IPD), serotype replacement remains a major concern. Thus, serotype-independent protection with vaccines targeting virulence genes, such as PspA, have been pursued. PspA is comprised of diverse clades that arose through recombination. Therefore, multi-locus sequence typing (MLST)-defined clones could conceivably include strains from multiple PspA clades. As a result, a method is needed which can both monitor the long-term epidemiology of the pneumococcus among a large number of isolates, and analyze vaccine-candidate genes, such as pspA, for mutations and recombination events that could result in ‘vaccine escape’ strains.

Methodology

We developed a resequencing array consisting of five conserved and six variable genes to characterize 72 pneumococcal strains. The phylogenetic analysis of the 11 concatenated genes was performed with the MrBayes program, the single nucleotide polymorphism (SNP) analysis with the DNA Sequence Polymorphism program (DnaSP), and the recombination event analysis with the recombination detection package (RDP).

Results

The phylogenetic analysis correlated with MLST, and identified clonal strains with unique PspA clades. The DnaSP analysis correlated with the serotype-specific diversity detected using MLST. Serotypes associated with more than one ST complex had a larger degree of sequence polymorphism than a serotype associated with one ST complex. The RDP analysis confirmed the high frequency of recombination events in the pspA gene.

Conclusions

The phylogenetic tree correlated with MLST, and detected multiple PspA clades among clonal strains. The genetic diversity of the strains and the frequency of recombination events in the mosaic gene, pspA were accurately assessed using the DnaSP and RDP programs, respectively. These data provide proof-of-concept that resequencing arrays could play an important role within research and clinical laboratories in both monitoring the molecular epidemiology of the pneumococcus and detecting ‘vaccine escape’ strains among vaccine-candidate genes.     

Pan-genomics of Ochrobactrum species from clinical and environmental origins reveals distinct populations and possible links

Ochrobactrum genus is comprised of soil-dwelling Gram-negative bacteria mainly reported for bioremediation of toxic compounds. Since last few years, mainly two species of this genus, O. intermedium and O. anthropi were documented for causing infections mostly in the immunocompromised patients. Despite such ubiquitous presence, study of adaptation in various niches is still lacking. Thus, to gain insights into the niche adaptation strategies, pan-genome analysis was carried out by comparing 67 genome sequences belonging to Ochrobactrum species. Pan-genome analysis revealed it is an open pan-genome indicative of the continuously evolving nature of the genus. The presence/absence of gene clusters also illustrated the unique presence of antibiotic efflux transporter genes and type IV secretion system genes in the clinical strains while the genes of solvent resistance and exporter pumps in the environmental strains. A phylogenomic investigation based on 75 core genes depicted better and robust phylogenetic resolution and topology than the 16S rRNA gene. To support the pan-genome analysis, individual genomes were also investigated for the mobile genetic elements (MGE), antibiotic resistance genes (ARG), metal resistance genes (MRG) and virulence factors (VF). The analysis revealed the presence of MGE, ARG, and MRG in all the strains which play an important role in the species evolution which is in agreement with the pan-genome analysis. The average nucleotide identity (ANI) based on the genetic relatedness between the Ochrobactrum species indicated a distinction between individual species. Interestingly, the ANI tool was able to classify the Ochrobactrum genomes to the species level which were assigned till the genus level on the NCBI database.     

The biochemical differentiation of Enterobacter sakazakii genotypes

Background

Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome.

Results

Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.

Conclusion

This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.     

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.     

Conserved features of cohesin binding along fission yeast chromosomes

Background

Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.

Results

Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.

Conclusions

P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.     

Mito-nuclear genetic comparison in a Wolbachia infected weevil: insights on reproductive mode, infection age and evolutionary forces shaping genetic variation

Background

The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.

Results

In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages. Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7.

Conclusions

The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies.