Partial and complete denitrification in Thermus thermophilus: lessons from genome drafts

We have obtained draft genomic sequences of PD (partial denitrificant) and CD (complete denitrificant) strains of Thermus thermophilus. Their genomes are similar in size to that of the aerobic strains sequenced to date and probably contain a similar megaplasmid. In the CD strain, the genes encoding a putative cytochrome cd1 Nir (nitrite reductase) and ancillary proteins were clustered with a cytochrome c-dependent Nor (nitric oxide reductase), and with genes that are probably implicated in their regulation. The Nar (nitrate reductase) and associated genes were also clustered and located 7?kb downstream of the genes coding for the Nir. The whole nar-nir-nor denitrification supercluster was identified as part of a variable region of a megaplasmid. No homologues of NosZ were found despite nitrogen balance supports the idea that such activity actually exists.     

Comparative genome analysis of Helicobacter pylori strains

DNA macroarrays were used to characterize 17 Helicobacter pylori strains isolated in four geographic regions of Russia (Moscow, St. Petersburg, Kazan, and Novosibirsk). Of all genes, 1272 (81%) proved to occur in all strains and to constitute a functional core of the genome, and 293 (18.7%) were strain-specific and greatly varied among the H. pylori strains. Most (71%) of the latter had unknown functions; the remainder included restriction-modification genes (3-9%), transposition genes (2-4%), and genes coding for outer membrane proteins (2-4%). The Russian H. pylori strains did not differ in genome organization or in the number and distribution of strain-specific genes from strains isolated in other countries.     

The genome of canarypox virus

Here we present the genomic sequence, with analysis, of a canarypox virus (CNPV). The 365-kbp CNPV genome contains 328 potential genes in a central region and in 6.5-kbp inverted terminal repeats. Comparison with the previously characterized fowlpox virus (FWPV) genome revealed avipoxvirus-specific genomic features, including large genomic rearrangements relative to other chordopoxviruses and novel cellular homologues and gene families. CNPV also contains many genomic differences with FWPV, including over 75 kbp of additional sequence, 39 genes lacking FWPV homologues, and an average of 47% amino acid divergence between homologues. Differences occur primarily in terminal and, notably, localized internal genomic regions and suggest significant genomic diversity among avipoxviruses. Divergent regions contain gene families, which overall comprise over 49% of the CNPV genome and include genes encoding 51 proteins containing ankyrin repeats, 26 N1R/p28-like proteins, and potential immunomodulatory proteins, including those similar to transforming growth factor beta and beta-nerve growth factor. CNPV genes lacking homologues in FWPV encode proteins similar to ubiquitin, interleukin-10-like proteins, tumor necrosis factor receptor, PIR1 RNA phosphatase, thioredoxin binding protein, MyD116 domain proteins, circovirus Rep proteins, and the nucleotide metabolism proteins thymidylate kinase and ribonucleotide reductase small subunit. These data reveal genomic differences likely affecting differences in avipoxvirus virulence and host range, and they will likely be useful for the design of improved vaccine vectors.     

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.     

Bacillus subtilis genome diversity

Microarray-based comparative genomic hybridization (M-CGH) is a powerful method for rapidly identifying regions of genome diversity among closely related organisms. We used M-CGH to examine the genome diversity of 17 strains belonging to the nonpathogenic species Bacillus subtilis. Our M-CGH results indicate that there is considerable genetic heterogeneity among members of this species; nearly one-third of Bsu168-specific genes exhibited variability, as measured by the microarray hybridization intensities. The variable loci include those encoding proteins involved in antibiotic production, cell wall synthesis, sporulation, and germination. The diversity in these genes may reflect this organism's ability to survive in diverse natural settings.     

Exploring Lactobacillus plantarum genome diversity by using microarrays

Lactobacillus plantarum is a versatile and flexible species that is encountered in a variety of niches and can utilize a broad range of fermentable carbon sources. To assess if this versatility is linked to a variable gene pool, microarrays containing a subset of small genomic fragments of L. plantarum strain WCFS1 were used to perform stringent genotyping of 20 strains of L. plantarum from various sources. The gene categories with the most genes conserved in all strains were those involved in biosynthesis or degradation of structural compounds like proteins, lipids, and DNA. Conversely, genes involved in sugar transport and catabolism were highly variable between strains. Moreover, besides the obvious regions of variance, like prophages, other regions varied between the strains, including regions encoding plantaricin biosynthesis, nonribosomal peptide biosynthesis, and exopolysaccharide biosynthesis. In many cases, these variable regions colocalized with regions of unusual base composition. Two large regions of flexibility were identified between 2.70 and 2.85 and 3.10 and 3.29 Mb of the WCFS1 chromosome, the latter being close to the origin of replication. The majority of genes encoded in these variable regions are involved in sugar metabolism. This functional overrepresentation and the unusual base composition of these regions led to the hypothesis that they represented lifestyle adaptation regions in L. plantarum. The present study consolidates this hypothesis by showing that there is a high degree of gene content variation among L. plantarum strains in genes located in these regions of the WCFS1 genome. Interestingly, based on our genotyping data L. plantarum strains clustered into two clearly distinguishable groups, which coincided with an earlier proposed subdivision of this species based on conventional methods.     

Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization

Two closely related strains of the Gram-positive, cellulolytic ruminal bacterium Ruminococcus flavefaciens were compared at the genomic level by suppressive subtractive hybridization. The two strains investigated in this study differ by 1.94% in their respective 16S rDNA genes. Three hundred and eighty-four PCR-amplified products were cloned and then screened for their strain identity by dot blot hybridization. Based on redundancy percentages of the clones sequenced, 9.5% of the genome of the R. flavefaciens FD-1 strain is not present in the JM1 strain. The majority of identities of individual cloned subtracted products (642 bp average length) bore no relation to deposited sequences in GenBank (42% of the subtracted library), whereas of those with putative assigned functions 7% are loosely associated with fibre-degradation, 6% with insertion elements, transposons and phage-like ORFs, 5% with cell membrane associated proteins and 3% with signal transduction. Subtracted sequences were then supplemented with the draft (2 x coverage) genome sequence of R. flavefaciens FD-1 to indicate potential regions of rearrangement within the genome, including a novel insertion sequence.     

Comparative genome analysis of Mycoplasma pneumoniae

Background

Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40 % of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England.

Results

Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99 % identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes.

Conclusions

These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1801-0) contains supplementary material, which is available to authorized users.     

Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) was found to induce the synthesis of mRNA encoding a basic protein with a 67% amino acid sequence homology to the known acidic pathogenesis-related (PR) proteins 1a, 1b and 1c. By Southern blot hybridization it was shown that the tobacco genome contains at least eight genes for acidic PR-1 proteins and a similar number of genes encoding the basic homologues. Clones corresponding to three of the genes for acidic PR-1 proteins were isolated from a genomic library of Samsun NN tobacco. The nucleotide sequence of these genes and their flanking sequences were determined. One clone was found to correspond to the PR-1a gene; the two other clones do not correspond to known TMV-induced PR-1 mRNA's and may represent silent genes. Compared to the PR-1a gene, these genes contain an insertion or deletion in the putative promoter region and mutations affecting the PR-1 reading frame.     

Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence

Background

The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents.

Methodology/Principal Findings

To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (d_A) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function.

Conclusions/Significance

Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics.     

A reference-quality,fully annotated genome from a Puerto Rican individual

Until 2019, the human genome was available in only one fully annotated version, GRCh38, which was the result of 18 years of continuous improvement and revision. Despite dramatic improvements in sequencing technology, no other genome was available as an annotated reference until 2019, when the genome of an Ashkenazi individual, Ash1, was released. In this study, we describe the assembly and annotation of a second individual genome, from a Puerto Rican individual whose DNA was collected as part of the Human Pangenome project. The new genome, called PR1, is the first true reference genome created from an individual of African descent. Due to recent improvements in both sequencing and assembly technology, and particularly to the use of the recently completed CHM13 human genome as a guide to assembly, PR1 is more complete and more contiguous than either GRCh38 or Ash1. Annotation revealed 37,755 genes (of which 19,999 are protein coding), including 12 additional gene copies that are present in PR1 and missing from CHM13. Fifty-seven genes have fewer copies in PR1 than in CHM13, 9 map only partially, and 3 genes (all noncoding) from CHM13 are entirely missing from PR1.     

Complete genome sequence of the marine, chemolithoautotrophic, ammonia-oxidizing bacterium Nitrosococcus oceani ATCC 19707

The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H(+)-dependent F(0)F(1) type, one Na(+)-dependent V type).     

Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain)

Sequences totalling 5472 nucleotides (nt) from four complementary DNA (cDNA) clones of the dengue virus type 2 (DEN-2) RNA (New Guinea strain, NGS-C) have been reported previously [Yaegashi et al., Gene 46 (1986) 257-267; Putnak et al., Virology 163 (1988) 93-103]. This report describes the complete nucleotide sequence, with the exception of about 7 nt at the 5'-noncoding region, of this RNA genome derived from several cDNA clones. It is 10,723 nt in length and contains a single long open reading frame of 10,173 nt, encoding a polyprotein of 3391 amino acids. The genomic organization is similar to that of other flaviviruses that have recently been reported. Among the three DEN-2 strains - the Jamaica genotype (DEN-2JAM), the DEN-2NGS-C, and the S1 candidate vaccine strain derived from Puerto Rico (PR)-159 isolate (DEN-2S1) - which have been sequenced to date, the amino acid sequences of the polyproteins bear 94%-99% similarity. When the amino acid sequences of DEN-2NGS-C are compared with those of the other two strains, the variations are greater in the DEN-2S1 than in the DEN-2JAM. When DEN-2 and DEN-4 are compared, the overall amino acid identities range from 30% to 80% in both the structural and nonstructural proteins; whereas between DEN-2 and DEN-1, they range from 68% to 79% in the region encoding the structural proteins and the nonstructural protein NS1.     

Genetic typing of Mycobacterium tuberculosis strains by spoligotyping and genome fingerprinting techniques

A total of 122 M. tuberculosis clinical drug-resistant strains isolated in Central Russia were studied by spoligotyping and genome fingerprinting techniques. According to spoligotyping results 77% of M. tuberculosis strains were distributed to 13 oligotypes, while 23% of these strains were found to form unique patterns. Most of them belonged to the families Beijing and Haarlem (43.4% and 13.9% respectively). The patterns of the strains of oligotype 12 (7F-7F-7E-0E-78-3E) were identical to those of the strains isolated in Brazil, France and the Netherlands. The strains of the spoligotype 22 (7F-1E-7F-7F-07-3E) had the patterns identical to those of the strains of group S13, also isolated in Brazil. According to genome fingerprinting 31.4% of the strains were found to belong to clusters with the similarity coefficient equal to 1. The strains belonging to genotypes W and A1 were found to prevail in the analyzed group.     

Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.

PR1 is a pathogenesis-related protein encoded in the parsley genome by a family of three genes (PR1-1, PR1-2 and PR1-3). Loss- and gain-of-function experiments in a transient expression system demonstrated the presence of two fungal elicitor responsive elements in each of the PR1-1 and PR1-2 promoters. These elements, W1, W2 and W3, contain the sequence (T)TGAC(C) and mutations that disrupt this sequence abolish function. Gel shift experiments demonstrated that W1, W2 and W3 are bound specifically by similar nuclear proteins. Three cDNA clones encoding sequence-specific DNA-binding proteins were isolated by South-Western screening and these proteins, designated WRKY1, 2 and 3, also bind specifically to W1, W2 and W3. WRKY1, 2 and 3 are members of the family of sequence-specific DNA-binding proteins, which we call the WRKY family. Treatment of parsley cells with the specific oligopeptide elicitor Pep25 induced a transient and extremely rapid increase in mRNA levels of WRKY1 and 3. WRKY2 mRNA levels in contrast showed a concomitant transient decrease. These rapid changes in WRKY mRNA levels in response to a defined signal molecule suggest that WRKY1, 2 and 3 play a key role in a signal transduction pathway that leads from elicitor perception to PR1 gene activation.     

Molecular evolution of the hepatitis B virus genome

The hepatitis B virus (HBV) has a circular DNA genome of about 3,200 base pairs. Economical use of the genome with overlapping reading frames may have led to severe constraints on nucleotide substitutions along the genome and to highly variable rates of substitution among nucleotide sites. Nucleotide sequences from 13 complete HBV genomes were compared to examine such variability of substitution rates among sites and to examine the phylogenetic relationships among the HBV variants. The maximum likelihood method was employed to fit models of DNA sequence evolution that can account for the complexity of the pattern of nucleotide substitution. Comparison of the models suggests that the rates of substitution are different in different genes and codon positions; for example, the third codon position changes at a rate over ten times higher than the second position. Furthermore, substantial variation of substitution rates was detected even after the effects of genes and codon positions were corrected; that is, rates are different at different sites of the same gene or at the same codon position. Such rates after the correction were also found to be positively correlated at adjacent sites, which indicated the existence of conserved and variable domains in the proteins encoded by the viral genome. A multiparameter model validates the earlier finding that the variation in nucleotide conservation is not random around the HBV genome. The test for the existence of a molecular clock suggests that substitution rates are more or less constant among lineages. The phylogenetic relationships among the viral variants were examined. Although the data do not seem to contain sufficient information to resolve the details of the phylogeny, it appears quite certain that the serotypes of the viral variants do not reflect their genetic relatedness. Correspondence to: Z. Yang     

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.