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author:("Paul, sandia")
1.  Escherichia coli Isolate for Studying Colonization of the Mouse Intestine and Its Application to Two-Component Signaling Knockouts 
Journal of Bacteriology  2014;196(9):1723-1732.
The biology of Escherichia coli in its primary niche, the animal intestinal tract, is remarkably unexplored. Studies with the streptomycin-treated mouse model have produced important insights into the metabolic requirements for Escherichia coli to colonize mice. However, we still know relatively little about the physiology of this bacterium growing in the complex environment of an intestine that is permissive for the growth of competing flora. We have developed a system for studying colonization using an E. coli strain, MP1, isolated from a mouse. MP1 is genetically tractable and does not require continuous antibiotic treatment for stable colonization. As an application of this system, we separately knocked out each two-component system response regulator in MP1 and performed competitions against the wild-type strain. We found that only three response regulators, ArcA, CpxR, and RcsB, produce strong colonization defects, suggesting that in addition to anaerobiosis, adaptation to cell envelope stress is a critical requirement for E. coli colonization of the mouse intestine. We also show that the response regulator OmpR, which had previously been hypothesized to be important for adaptation between in vivo and ex vivo environments, is not required for MP1 colonization due to the presence of a third major porin.
PMCID: PMC3993324  PMID: 24563035
2.  Microbial Variome Database: Point Mutations, Adaptive or Not, in Bacterial Core Genomes 
Molecular Biology and Evolution  2013;30(6):1465-1470.
Analysis of genetic differences (gene presence/absence and nucleotide polymorphisms) among strains of a bacterial species is crucial to understanding molecular mechanisms of bacterial pathogenesis and selecting targets for novel antibacterial therapeutics. However, lack of genome-wide association studies on large and epidemiologically well-defined strain collections from the same species makes it difficult to identify the genes under positive selection and define adaptive polymorphisms in those genes. To address this need and to overcome existing limitations, we propose to create a “microbial variome"—a species-specific resource database of genomic variations based on molecular evolutionary analysis. Here, we present prototype variome databases of Escherichia coli and Salmonella enterica subspecies enterica (, last accessed March 26, 2013). The prototypes currently include the point mutations data of core protein-coding genes from completely sequenced genomes of 22 E. coli and 17 S. enterica strains. These publicly available databases allow for single- and multiple-field sorting, filtering, and searching of the gene variability data and the potential adaptive significance. Such resource databases would immensely help experimental research, clinical diagnostics, epidemiology, and environmental control of human pathogens.
PMCID: PMC3649677  PMID: 23493258
microbial variome; adaptive evolution; nucleotide polymorphisms; database
3.  Accelerated gene evolution via replication-transcription conflicts 
Nature  2013;495(7442):10.1038/nature11989.
Several mechanisms that increase the rate of mutagenesis across the entire genome have been identified; however, how the rate of evolution might be promoted in individual genes is unclear. A majority of the genes in bacteria are encoded on the leading strand of replication1–4. This presumably avoids the potentially detrimental head-on collisions that occur between the replication and transcription machineries when genes are encoded on the lagging strand1–4. We identified the ubiquitous (core) genes in Bacillus subtilis and determined that 17% of them are on the lagging strand. We found a higher rate of point mutations in the core genes on the lagging strand compared to those on the leading strand, with this difference being primarily in the amino acid changing (nonsynonymous) mutations. We determined that overall, the genes under strong negative selection against amino acid changing mutations tend to be on the leading strand, co-oriented with replication. In contrast, based on the rate of convergent mutations, genes under positive selection for amino acid changing mutations are more commonly found on the lagging strand, indicating faster adaptive evolution in many genes in the head-on orientation. Increased gene length and gene expression levels are positively correlated with the rate of accumulation of nonsynonymous mutations in the head-on genes, suggesting that the conflict between replication and transcription could be a driving force behind these mutations. Indeed, using reversion assays, we show that the difference in the rate of mutagenesis of genes in the two orientations is transcription-dependent. Altogether, our findings indicate that head-on replication-transcription conflicts are more mutagenic than co-directional conflicts and that these encounters can significantly increase adaptive structural variation in the coded proteins. We propose that bacteria, and potentially other organisms, promote faster evolution of specific genes through orientation-dependent encounters between DNA replication and transcription.
PMCID: PMC3807732  PMID: 23538833
4.  Role of Homologous Recombination in Adaptive Diversification of Extraintestinal Escherichia coli 
Journal of Bacteriology  2013;195(2):231-242.
The contribution of homologous exchange (recombination) of core genes in the adaptive evolution of bacterial pathogens is not well understood. To investigate this, we analyzed fully assembled genomes of two Escherichia coli strains from sequence type 131 (ST131), a clonal group that is both the leading cause of extraintestinal E. coli infections and the main source of fluoroquinolone-resistant E. coli. Although the sequences of each of the seven multilocus sequence typing genes were identical in the two ST131 isolates, the strains diverged from one another by homologous recombination that affected at least 9% of core genes. This was on a par with the contribution to genomic diversity of horizontal gene transfer and point gene mutation. The genomic positions of recombinant and mobile genetic regions were partially linked, suggesting their concurrent occurrence. One of the genes affected by homologous recombination was fimH, which encodes mannose-specific type 1 fimbrial adhesin, resulting in functionally distinct copies of the gene in ST131 strains. One strain, a uropathogenic isolate, had a pathoadaptive variant of fimH that was acquired by homologous replacement into the commensal strain background. Close examination of FimH structure and function in additional ST131 isolates revealed that recombination led to acquisition of several functionally distinct variants that, upon homologous exchange, were targeted by a variety of pathoadaptive mutations under strong positive selection. Different recombinant fimH strains also showed a strong clonal association with ST131 isolates that had distinct fluoroquinolone resistance profiles. Thus, homologous recombination of core genes plays a significant role in adaptive diversification of bacterial pathogens, especially at the level of clonally related groups of isolates.
PMCID: PMC3553836  PMID: 23123908
5.  Evolutionary Analysis Points to Divergent Physiological Roles of Type 1 Fimbriae in Salmonella and Escherichia coli 
mBio  2013;4(2):e00625-12.
Salmonella and Escherichia coli mannose-binding type 1 fimbriae exhibit highly similar receptor specificities, morphologies, and mechanisms of assembly but are nonorthologous in nature, i.e., not closely related evolutionarily. Their operons differ in chromosomal location, gene arrangement, and regulatory components. In the current study, we performed a comparative genetic and structural analysis of the major structural subunit, FimA, from Salmonella and E. coli and found that FimA pilins undergo diverse evolutionary adaptation in the different species. Whereas the E. coli fimA locus is characterized by high allelic diversity, frequent intragenic recombination, and horizontal movement, Salmonella fimA shows structural diversity that is more than 5-fold lower without strong evidence of gene shuffling or homologous recombination. In contrast to Salmonella FimA, the amino acid substitutions in the E. coli pilin heavily target the protein regions that are predicted to be exposed on the external surface of fimbriae. Altogether, our results suggest that E. coli, but not Salmonella, type 1 fimbriae display a high level of structural diversity consistent with a strong selection for antigenic variation under immune pressure. Thus, type 1 fimbriae in these closely related bacterial species appear to function in distinctly different physiological environments.
E. coli and Salmonella are enteric bacteria that are closely related from an evolutionary perspective. They are both notorious human pathogens, though with somewhat distinct ecologies and virulence mechanisms. Type 1 fimbriae are rod-shaped surface appendages found in most E. coli and Salmonella isolates. In both species, they mediate bacterial adhesion to mannose receptors on host cells and share essentially the same morphology and assembly mechanisms. Here we show that despite the strong resemblances in function and structure, they are exposed to very different natural selection environments. Sequence analysis indicates that E. coli, but not Salmonella, fimbriae are subjected to strong immune pressure, resulting in a high level of major fimbrial protein gene shuffling and interbacterial transfer. Thus, evolutionary analysis tools can provide evidence of divergent physiological roles of functionally similar traits in different bacterial species.
PMCID: PMC3604780  PMID: 23462115
6.  Convergent Molecular Evolution of Genomic Cores in Salmonella enterica and Escherichia coli 
Journal of Bacteriology  2012;194(18):5002-5011.
One of the strongest signals of adaptive molecular evolution of proteins is the occurrence of convergent hot spot mutations: repeated changes in the same amino acid positions. We performed a comparative genome-wide analysis of mutation-driven evolution of core (omnipresent) genes in 17 strains of Salmonella enterica subspecies I and 22 strains of Escherichia coli. More than 20% of core genes in both Salmonella and E. coli accumulated hot spot mutations, with a predominance of identical changes having recent evolutionary origin. There is a significant overlap in the functional categories of the adaptively evolving genes in both species, although mostly via separate molecular mechanisms. As a strong evidence of the link between adaptive mutations and virulence in Salmonella, two human-restricted serovars, Typhi and Paratyphi A, shared the highest number of genes with serovar-specific hot spot mutations. Many of the core genes affected by Typhi/Paratyphi A-specific mutations have known virulence functions. For each species, a list of nonrecombinant core genes (and the hot spot mutations therein) under positive selection is provided.
PMCID: PMC3430314  PMID: 22797756
7.  Microbial Lifestyle and Genome Signatures 
Current Genomics  2012;13(2):153-162.
Microbes are known for their unique ability to adapt to varying lifestyle and environment, even to the extreme or adverse ones. The genomic architecture of a microbe may bear the signatures not only of its phylogenetic position, but also of the kind of lifestyle to which it is adapted. The present review aims to provide an account of the specific genome signatures observed in microbes acclimatized to distinct lifestyles or ecological niches. Niche-specific signatures identified at different levels of microbial genome organization like base composition, GC-skew, purine-pyrimidine ratio, dinucleotide abundance, codon bias, oligonucleotide composition etc. have been discussed. Among the specific cases highlighted in the review are the phenomena of genome shrinkage in obligatory host-restricted microbes, genome expansion in strictly intra-amoebal pathogens, strand-specific codon usage in intracellular species, acquisition of genome islands in pathogenic or symbiotic organisms, discriminatory genomic traits of marine microbes with distinct trophic strategies, and conspicuous sequence features of certain extremophiles like those adapted to high temperature or high salinity.
PMCID: PMC3308326  PMID: 23024607
Reductive genome evolution; Intra-amoeba pathogens; Strand-specific codon bias; Genome islands; Trophic strategies; Thermophiles; Halophiles.
8.  Distinct, ecotype-specific genome and proteome signatures in the marine cyanobacteria Prochlorococcus 
BMC Genomics  2010;11:103.
The marine cyanobacterium Prochlorococcus marinus, having multiple ecotypes of distinct genotypic/phenotypic traits and being the first documented example of genome shrinkage in free-living organisms, offers an ideal system for studying niche-driven molecular micro-diversity in closely related microbes. The present study, through an extensive comparative analysis of various genomic/proteomic features of 6 high light (HL) and 6 low light (LL) adapted strains, makes an attempt to identify molecular determinants associated with their vertical niche partitioning.
Pronounced strand-specific asymmetry in synonymous codon usage is observed exclusively in LL strains. Distinct dinucleotide abundance profiles are exhibited by 2 LL strains with larger genomes and G+C-content ≈ 50% (group LLa), 4 LL strains having reduced genomes and G+C-content ≈ 35-37% (group LLb), and 6 HL strains. Taking into account the emergence of LLa, LLb and HL strains (based on 16S rRNA phylogeny), a gradual increase in average aromaticity, pI values and beta- & coil-forming propensities and a decrease in mean hydrophobicity, instability indices and helix-forming propensities of core proteins are observed. Greater variations in orthologous gene repertoire are found between LLa and LLb strains, while higher number of positively selected genes exist between LL and HL strains.
Strains of different Prochlorococcus groups are characterized by distinct compositional, physicochemical and structural traits that are not mere remnants of a continuous genetic drift, but are potential outcomes of a grand scheme of niche-oriented stepwise diversification, that might have driven them chronologically towards greater stability/fidelity and invoked upon them a special ability to inhabit diverse oceanic environments.
PMCID: PMC2836286  PMID: 20146791
9.  Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes 
Genome Biology  2008;9(4):R70.
A comparative genomic and proteomic study of halophilic and non-halophilic prokaryotes identifies specific genomic and proteomic features typical of halophilic species that are independent from genomic GC-content and taxonomic position.
Halophilic prokaryotes are adapted to thrive in extreme conditions of salinity. Identification and analysis of distinct macromolecular characteristics of halophiles provide insight into the factors responsible for their adaptation to high-salt environments. The current report presents an extensive and systematic comparative analysis of genome and proteome composition of halophilic and non-halophilic microorganisms, with a view to identify such macromolecular signatures of haloadaptation.
Comparative analysis of the genomes and proteomes of halophiles and non-halophiles reveals some common trends in halophiles that transcend the boundary of phylogenetic relationship and the genomic GC-content of the species. At the protein level, halophilic species are characterized by low hydrophobicity, over-representation of acidic residues, especially Asp, under-representation of Cys, lower propensities for helix formation and higher propensities for coil structure. At the DNA level, the dinucleotide abundance profiles of halophilic genomes bear some common characteristics, which are quite distinct from those of non-halophiles, and hence may be regarded as specific genomic signatures for salt-adaptation. The synonymous codon usage in halophiles also exhibits similar patterns regardless of their long-term evolutionary history.
The generality of molecular signatures for environmental adaptation of extreme salt-loving organisms, demonstrated in the present study, advocates the convergent evolution of halophilic species towards specific genome and amino acid composition, irrespective of their varying GC-bias and widely disparate taxonomic positions. The adapted features of halophiles seem to be related to physical principles governing DNA and protein stability, in response to the extreme environmental conditions under which they thrive.
PMCID: PMC2643941  PMID: 18397532
10.  Reverse Polarization in Amino acid and Nucleotide Substitution Patterns Between Human–Mouse Orthologs of Two Compositional Extrema 
Genome-wide analysis of sequence divergence patterns in 12 024 human–mouse orthologous pairs reveals, for the first time, that the trends in nucleotide and amino acid substitutions in orthologs of high and low GC composition are highly asymmetric and polarized to opposite directions. The entire dataset has been divided into three groups on the basis of the GC content at third codon sites of human genes: high, medium, and low. High-GC orthologs exhibit significant bias in favor of the replacements, Thr → Ala, Ser → Ala, Val → Ala, Lys → Arg, Asn → Ser, Ile → Val etc., from mouse to human, whereas in low-GC orthologs, the reverse trends prevail. In general, in the high-GC group, residues encoded by A/U-rich codons of mouse proteins tend to be replaced by the residues encoded by relatively G/C-rich codons in their human orthologs, whereas the opposite trend is observed among the low-GC orthologous pairs. The medium-GC group shares some trends with high-GC group and some with low-GC group. The only significant trend common in all groups of orthologs, irrespective of their GC bias, is (Asp)Mouse → (Glu)Human replacement. At the nucleotide level, high-GC orthologs have undergone a large excess of (A/T)Mouse → (G/C)Human substitutions over (G/C)Mouse → (A/T)Human at each codon position, whereas for low-GC orthologs, the reverse is true.
PMCID: PMC2533592  PMID: 17895298
high-GC orthologs; low-GC orthologs; amino acid replacement matrix; nucleotide replacement matrix; sequence divergence
11.  Analysis of Nanoarchaeum equitans genome and proteome composition: indications for hyperthermophilic and parasitic adaptation 
BMC Genomics  2006;7:186.
Nanoarchaeum equitans, the only known hyperthermophilic archaeon exhibiting parasitic life style, has raised some new questions about the evolution of the Archaea and provided a model of choice to study the genome landmarks correlated with thermo-parasitic adaptation. In this context, we have analyzed the genome and proteome composition of N. equitans and compared the same with those of other mesophiles, hyperthermophiles and obligatory host-associated organisms.
Analysis of nucleotide, codon and amino acid usage patterns in N. equitans indicates the presence of distinct selective constraints, probably due to its adaptation to a thermo-parasitic life-style. Among the conspicuous characteristics featuring its hyperthermophilic adaptation are overrepresentation of purine bases in protein coding sequences, higher GC-content in tRNA/rRNA sequences, distinct synonymous codon usage, enhanced usage of aromatic and positively charged residues, and decreased frequencies of polar uncharged residues, as compared to those in mesophilic organisms. Positively charged amino acid residues are relatively abundant in the encoded gene-products of N. equitans and other hyperthermophiles, which is reflected in their isoelectric point distribution. Pairwise comparison of 105 orthologous protein sequences shows a strong bias towards replacement of uncharged polar residues of mesophilic proteins by Lys/Arg, Tyr and some hydrophobic residues in their Nanoarchaeal orthologs. The traits potentially attributable to the symbiotic/parasitic life-style of the organism include the presence of apparently weak translational selection in synonymous codon usage and a marked heterogeneity in membrane-associated proteins, which may be important for N. equitans to interact with the host and hence, may help the organism to adapt to the strictly host-associated life style. Despite being strictly host-dependent, N. equitans follows cost minimization hypothesis.
The present study reveals that the genome and proteome composition of N. equitans are marked with the signatures of dual adaptation – one to high temperature and the other to obligatory parasitism. While the analysis of nucleotide/amino acid preferences in N. equitans offers an insight into the molecular strategies taken by the archaeon for thermo-parasitic adaptation, the comparative study of the compositional characteristics of mesophiles, hyperthermophiles and obligatory host-associated organisms demonstrates the generality of such strategies in the microbial world.
PMCID: PMC1574309  PMID: 16869956

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