Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.     

Variation among Genome Sequences of H37Rv Strains of Mycobacterium tuberculosis from Multiple Laboratories

The publication of the complete genome sequence for Mycobacterium tuberculosis H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS6110 transposition events were observed, supporting the theory that they play a significant role in plasticity of the M. tuberculosis genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous “in vitro evolution” experiment that demonstrates how the M. tuberculosis genome continues to evolve even in a controlled environment.Publication of the whole genome sequence of the H37Rv strain of Mycobacterium tuberculosis by Stewart Cole and colleagues in 1998 provided a breakthrough in tuberculosis (TB) research (8), leading to insights into the biology, metabolism, and evolution of this infectious pathogen. Large protein families related to fatty acid and polyketide biosynthesis, regulation (e.g., sigma factors and two-component sensor systems), drug efflux pumps and transporters, and the PE_PGRS proteins (a large duplicated family unique to the M. tuberculosis group of mycobacteria) were identified. In addition, transposons, prophage-like elements, and other repetitive and/or mobile genetic elements were identified (18). This genomic information has played an essential role in interpreting gene expression studies, modeling persistence, and identifying essential proteins as putative targets for drug discovery. However, to date the functions of only half of the genes (1,756/4,066) have been determined or predicted, and the rest remain annotated as “hypothetical proteins” (6).The H37Rv strain was initially selected for sequencing because it is a widely used laboratory strain that has retained its virulence. H37Rv was initially derived from a clinical isolate, H37, obtained from a patient with pulmonary tuberculosis in 1905. H37Rv falls in the T clade (5) and single-nucleotide polymorphism (SNP) cluster group SCG-6b (12). The virulence of H37Rv can be demonstrated in a number of animal models. For example, SCID mice infected with H37Rv typically have a mean time to death of 30 to 35 days, depending on the dose and route of inoculation (13).An avirulent strain, H37Ra, was also derived from H37 by culturing on solid egg medium and selecting for resistance to lysis (42). The strain was found not to cause disease in guinea pigs (43) or in mice (27). It has a colony morphology (smooth) different from that of H37Rv (rough) and several other phenotypic differences (14, 29). The H37Rv (ATCC 25618) and H37Ra (ATCC 25177) strains are maintained at the Trudeau Institute in New York (3), although unfortunately, the original H37 clinical isolate has been lost. Strain ATCC 27294 (TMC 102) is also frequently used as a representative of H37Rv in studies and treated equivalently in the literature. ATCC 25618 and ATCC 27294 were both isolated from the same patient in different years, and both are fully drug susceptible.The complete genome of H37Ra has been sequenced by Zheng et al. (48), who found 272 polymorphisms compared to the genome sequence determined by Cole et al. (8) for H37Rv. However, a subset of the polymorphic sites were found to match CDC1551, and upon resequencing of 85 such sites in H37Rv, 79 were determined to be errors in the H37Rv reference sequence. In addition, H37Ra has insertions of IS6110 at two novel sites and a loss of one, compared to the 16 sites in H37Rv. The 130 genuine H37Ra-specific polymorphisms found were divided into those in coding regions, those in upstream regulatory regions, and those in noncoding, nonregulatory intergenic regions in order to assess potential relevance to virulence. Polymorphisms in the promoter regions of sigC, nrdH (glutaredoxin-like electron transporter), and pabB (para-amino benzoate synthase), as well as nonsynonymous substitutions in mazG (regulator of stringent response), phoP (two-component sensor regulating biosynthesis of cell surface lipid antigens), pks12 (polyketide synthase involved in biosynthesis of mycoketides), and nrp (nonribosomal peptide synthetase potentially involved in phthiocerol dimycocerosate [PDIM] biosynthesis), were highlighted as possible causes of the loss of virulence. H37Ra does not synthesize a number of cell surface antigens, including sulfolipid-1, trehalose mycolates, and PDIM (7). The roles of mutations in phoP and sigM, both of which regulate expression of genes involved in biosynthesis of cell surface antigens, have been subsequently investigated, though neither seems to be singularly responsible for the avirulence of H37Ra (17, 35). Multiple mutations in PPE and PE_PGRS genes are also observed in H37Ra, and there has been speculation about the role of these genes in virulence (39). However, the RvD2 region (an 8-kb region present in H37Ra but deleted in H37Rv, including an IS6110 insertion element, mmpL14, and several hypothetical genes) is known not to be responsible for differences in virulence (25).Because of its importance as a model strain used in laboratory studies, it is essential to determine how consistent different stocks of H37Rv in different laboratories are with the reference genome sequence and with each other. Different stocks could accumulate independent polymorphisms over time, and such inconsistencies could potentially make results of studies obtained with H37Rv cultures from different labs difficult to compare, particularly if they affect virulence, drug tolerance, metabolism, cell wall constitution, etc. Furthermore, sequencing errors in the original genome sequence are possible. In order to evaluate differences among currently used variants of H37Rv, we resequenced the complete genomes of six extant H37Rv strains (two samples of ATCC 25618 and four of ATCC 27294) using Illumina sequencing technology. We compared differences among them and differences from the reference sequences for H37Rv and H37Ra available from GenBank. The results of this study identify a common set of 73 polymorphisms shared among all six sequenced strains relative to the H37Rv reference strain. Most (72) of these are shared with H37Ra and likely correspond to sequencing errors in the original H37Rv genome sequence. However, there are several sites where additional polymorphisms are shared among a subset of strains, and several strains have a small number of unique polymorphisms. Furthermore, examination of insertion sites of the IS6110 transposable element reveals several changes that have occurred among these strains. These results illustrate the ongoing evolution of this strain and divergence from the sequenced reference strain of H37Rv and highlight the importance of understanding the genetic differences unique to the stock used in each laboratory.     

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

差显技术分析结核杆菌H37Rv与H37Ra差异表达的基因

采用差异显示技术比较了结核分支杆菌强毒株H37Rv和弱毒株H37Ra在体外培养条件下的基因表达差异。通过20种引物组合进行mRNA差异显示,克隆到了两菌株间的20余个差异表达基因,经序列分析及杂交鉴定发现其中2个基因仅在H37Rv中表达。其中Rv1894c基因编码的可能是H37Rv中的一个新蛋白。而在H37Ra的基因组中含有这2个基因的编码序列,但均未检测到基因的表达。     

利用抑制消减杂交技术研究结核分支杆菌强毒株H37Rv和弱毒株H37Ra的基因差异

为了解结核病的致病分子机理和筛选结核病致病菌的毒力基因,利用抑制消减杂交（SSH）技术分析了结核分支杆菌强毒株H37Rv和弱毒株H37Ra间的基因组DNA间差异。通过Southern杂交验证及序列分析得到仅在强毒株H37Rv基因组中有的DNA片段8个,其中一个编码已知的毒力因子mce蛋白,1个编码PE家族蛋白,1个编码purC合成酶,和4个潜在蛋白,另一个为非编码区片段。其中有2个基因经PCR方法已证实在强毒株H37Rv和临床分离的强毒株中存在,而在H37Ra和临床弱毒株中无;仅在弱毒株H37Ra基因组中的DNA片段3个,其中2个为新基因片段,已被GenBank收录。     

tRNA genes in mycobacteria: organization and molecular cloning.

DNAs from nine mycobacteria cleaved with restriction endonucleases were hybridized with cDNA probes synthesized to tRNAs from Mycobacterium tuberculosis and Mycobacterium smegmatis. The tRNA genes are conserved, but their gross genomic organization has diverged in six of the nine species examined. Organisms of the M. tuberculosis H37Ra and H37Rv-M. bovis BCG complex appeared to have identical tRNA gene organization and were indistinguishable from each other. M. tuberculosis and M. smegmatis tRNA-derived cDNA probes hybridized differentially to tRNA-coding DNA segments in five of the species examined, suggesting the existence of qualitatively different tRNA pools in these slow- and fast-growing species. Mycobacterial DNAs hybridized with cDNA synthesized to 23S plus 16S rRNAs from Escherichia coli, and the data suggested that the tRNA genes map close to the rRNA genes. A gene bank of M. tuberculosis H37Rv DNA was constructed, and a recombinant plasmid, pSB2, coding for tRNA(s) and rRNA(s) was partially characterized. Plasmid pSB2 recognized a SalI restriction fragment length polymorphism (RFLP) in M. tuberculosis H37Rv and H37Ra; however, the RFLP is not linked to the tRNA-coding region. To the best of our knowledge, this is the first report of an RFLP which distinguishes the pathogenic strain M. tuberculosis H37Rv from its avirulent derivative H37Ra.     

Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H37Rv and H37Ra reveal novel and interesting differences with implications in virulence

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H₃₇Rv and H₃₇Ra, derived from the same parental strain M. tuberculosis H₃₇, show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H₃₇Ra and virulent H₃₇Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with 5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H₃₇Rv and H₃₇Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H₃₇Rv and H₃₇Ra can be correlated to differences in pathogenesis and virulence of the two strains.     

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.     

Chromosomal-level genome assembly of the semi-dwarf rice Taichung Native 1, an initiator of Green Revolution

Here we report the 409.5 Mb chromosome-level assembly of the first bred semi-dwarf rice, the Taichung Native 1 (TN1), which served as the template for the development of the Green Revolution (GR) cultivar IR8 “miracle rice”. We sequenced the TN1 genome utilizing multiple platforms and produced PacBio long reads, Illumina paired-end reads, Illumina mate-pair reads and 10x Genomics linked reads. We used a hybrid approach to assemble the 226× coverage of sequences by a combination of de novo and reference-guided approaches. The assembled TN1 genome has an N50 scaffold size of 33.1 Mb with the longest measuring 45.5 Mb. We annotated 37,526 genes, in which 24,102 (64.23%) were assigned Blast2GO annotations. The genome has 4672 or 95.4% complete BUSCOs and a repeat content of 51.52%. We developed our own method of creating a GR pangenome using the orthologous relationships of the proteins of TN1, IR8, MH63 and IR64, identifying 16,999 core orthologue groups of Green Revolution. From the pangenome, we identified a set of shared and unique gene ontology terms for the accessory clusters, characterizing TN1, IR8, MH63 and IR64. This TN1 genome assembly and GR pangenome will be a resource for new genomic discoveries about Green Revolution, and for improving the disease and insect resistances and the yield of rice.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.     

Identification of unique genetic markers in Rv0927c among Mycobacterium tuberculosis W-Beijing strains

Fifty-six clinical isolates of Mycobacterium tuberculosis were analyzed by spoligotyping to determine the prevalence of W-Beijing strains. Forty-nine of the 56 isolates belonged to W-Beijing strains and 7 isolates were non-Beijing strains. Comparative two-dimensional gel electrophoresis analysis of protein patterns between the W-Beijing and non-Beijing strains identified a unique protein Rv0927c that is absent in the former but present in the latter and the reference strain M. tuberculosis H37Rv. Compared with 7 non-Beijing clinical isolates and H37Rv, all 49 W-Beijing strains had two characteristic mutations, a deletion of AGC at nucleotide position 421 of Rv0927c gene encoding a putative short dehydrogenase/reductase, causing deletion of serine codon at amino acid position 141 and a -127 G-->A mutation in Rv0927c-pstS3 intergenic region, resulting in failure to express Rv0927c. Western blot analysis indicated that polyclonal antibody raised against H37Rv Rv0927c overexpressed in Escherichia coli reacted with non-Beijing strains and H37Rv but not W-Beijing strains. Characteristic mutations of Rv0927c that are present in W-Beijing strains can be used as a novel genetic marker for rapid molecular typing of M. tuberculosis W-Beijing strains.     

Comparative pangenomic analyses and biotechnological potential of cocoa-related Acetobacter senegalensis strains

Acetobacter senegalensis belongs to the group of acetic acid bacteria (AAB) that present potential biotechnological applications, for production of D-gluconate, cellulose and acetic acid. AAB can overcome heat and acid stresses by using strategies involving the overexpression of heat-shock proteins and enzymes from the complex pyrroquinoline-ADH, besides alcohol dehydrogenases (ADH). Nonetheless, the isolation of A. senegalensis and other AAB from food may be challenging due to presence of viable but non-culturable (VBNC) cells and due to uncertainties about nutritional requirements. To contribute for a better understanding of the ecology of AAB, this paper reports on the pangenome analysis of five strains of A. senegalensis recently isolated from a Brazilian spontaneous cocoa fermentation. The results showed biosynthetic clusters exclusively found in some cocoa-related AAB, such as those related to terpene pathways, which are important for flavour development. Genes related to oxidative stress were conserved in all the genomes, with multiple clusters. Moreover, there were genes coding for ADH and putative ABC transporters distributed in core, shell and cloud genomes, while chaperonin-encoding genes were present only in the core and soft-core genomes. Regarding quorum sensing, a response regulator gene was in the shell genome, and the gene encoding for acyl-homoserine lactone efflux protein was in the soft-core genome. There were quorum quenching-related genes, mainly encoding for lactonases, but also for acylases. Moreover, A. senegalensis did not have determinants of virulence or antibiotic resistance, which are good traits for strains intended to be applied in food fermentation.