Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage.

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed growth in minimal media. We speculate that this competitive advantage is contributing to the emergence of sublineage 2.2 in Malawi.

Comparative whole genome analysis reveals re-emergence of human Wa-like and DS-1-like G3 rotaviruses after Rotarix vaccine introduction in Malawi.

G3 rotaviruses rank among the most common rotavirus strains worldwide in humans and animals. However, despite a robust long-term rotavirus surveillance system from 1997 at Queen Elizabeth Central Hospital in Blantyre, Malawi, these strains were only detected from 1997 to 1999 and then disappeared and re-emerged in 2017, 5 years after the introduction of the Rotarix rotavirus vaccine. Here, we analysed representative twenty-seven whole genome sequences (G3P[4], n = 20; G3P[6], n = 1; and G3P[8], n = 6) randomly selected each month between November 2017 and August 2019 to understand how G3 strains re-emerged in Malawi. We found four genotype constellations that were associated with the emergent G3 strains and co-circulated in Malawi post-Rotarix vaccine introduction: G3P[4] and G3P[6] strains with the DS-1-like genetic backbone genes (G3-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2), G3P[8] strains with the Wa-like genetic backbone genes (G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1), and reassortant G3P[4] strains consisting of the DS-1-like genetic backbone genes and a Wa-like NSP2 (N1) gene (G3-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2). Time-resolved phylogenetic trees demonstrated that the most recent common ancestor for each ribonucleic acid (RNA) segment of the emergent G3 strains was between 1996 and 2012, possibly through introductions from outside the country due to the limited genetic similarity with G3 strains which circulated before their disappearance in the late 1990s. Further genomic analysis revealed that the reassortant DS-1-like G3P[4] strains acquired a Wa-like NSP2 genome segment (N1 genotype) through intergenogroup reassortment; an artiodactyl-like VP3 through intergenogroup interspecies reassortment; and VP6, NSP1, and NSP4 segments through intragenogroup reassortment likely before importation into Malawi. Additionally, the emergent G3 strains contain amino acid substitutions within the antigenic regions of the VP4 proteins which could potentially impact the binding of rotavirus vaccine-induced antibodies. Altogether, our findings show that multiple strains with either Wa-like or DS-1-like genotype constellations have driven the re-emergence of G3 strains. The findings also highlight the role of human mobility and genome reassortment events in the cross-border dissemination and evolution of rotavirus strains in Malawi necessitating the need for long-term genomic surveillance of rotavirus in high disease-burden settings to inform disease prevention and control.

Evolutionary changes between pre- and post-vaccine South African group A G2P[4] rotavirus strains, 2003-2017.

The transient upsurge of G2P[4] group A rotavirus (RVA) after Rotarix vaccine introduction in several countries has been a matter of concern. To gain insight into the diversity and evolution of G2P[4] strains in South Africa pre- and post-RVA vaccination introduction, whole-genome sequencing was performed for RVA positive faecal specimens collected between 2003 and 2017 and samples previously sequenced were obtained from GenBank (n=103; 56 pre- and 47 post-vaccine). Pre-vaccine G2 sequences predominantly clustered within sub-lineage IVa-1. In contrast, post-vaccine G2 sequences clustered mainly within sub-lineage IVa-3, whereby a radical amino acid (AA) substitution, S15F, was observed between the two sub-lineages. Pre-vaccine P[4] sequences predominantly segregated within sub-lineage IVa while post-vaccine sequences clustered mostly within sub-lineage IVb, with a radical AA substitution R162G. Both S15F and R162G occurred outside recognised antigenic sites. The AA residue at position 15 is found within the signal sequence domain of Viral Protein 7 (VP7) involved in translocation of VP7 into endoplasmic reticulum during infection process. The 162 AA residue lies within the hemagglutination domain of Viral Protein 4 (VP4) engaged in interaction with sialic acid-containing structure during attachment to the target cell. Free energy change analysis on VP7 indicated accumulation of stable point mutations in both antigenic and non-antigenic regions. The segregation of South African G2P[4] strains into pre- and post-vaccination sub-lineages is likely due to erstwhile hypothesized stepwise lineage/sub-lineage evolution of G2P[4] strains rather than RVA vaccine introduction. Our findings reinforce the need for continuous whole-genome RVA surveillance and investigation of contribution of AA substitutions in understanding the dynamic G2P[4] epidemiology.

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes.

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. Following the optimization of a logistics pipeline that involved shipping isolates as thermolysates in ambient conditions, the project assembled a diverse collection of 10,419 isolates from low- and middle-income countries. The genomes were sequenced using the LITE pipeline for library construction, with a total reagent cost of less than USD$10 per genome. Our method can be applied to other large bacterial collections to underpin global collaborations.

Factors Influencing Loss to Follow-up among Human Immunodeficiency Virus Exposed Infants in the Early Infant Diagnosis Program in Phalombe, Malawi.

The efforts to prevent mother to child transmission of HIV in Malawi are impeded by the loss to follow-up of HIV-exposed infants (HEI) in care. Early infant diagnosis (EID) of HEI and linkage to care reduces morbidity and mortality. There has been limited attention to infants who are lost to follow up despite their mothers being compliant to the PMTCT program. This study explored factors that influence loss-to-follow up among HEI in the EID program whose mothers were retained in care for up to 24 months in Phalombe district, Malawi. We conducted a descriptive phenomenological qualitative study from May 2017 to July 2018. We purposively conducted 18 in-depth interviews among HIV positive mothers whose HEI were enrolled in the follow-up program and 7 key informant interviews among healthcare workers (HCW). All interviews were digitally recorded, transcribed, and translated verbatim. Data were analyzed manually using a thematic step-by-step approach. Results showed that retention in care is facilitated by aspirations to have a healthy infant and linkage to a nearer facility to a mother's place of residency. The barriers to retention were non-disclosure of HIV status, inadequate resources, and support, suboptimal guidelines for, a lack of privacy, and unsynchronized hospital visits between a mother and her baby. The study has shown that successful implementation of EID services requires concerted efforts from various contextualized stakeholders whilst focusing on family-centered care. To maximize retention in EID and innovative ways of reaching mothers and babies through flexible guidelines are urgently needed.

Stepwise evolution of Salmonella Typhimurium ST313 causing bloodstream infection in Africa.

Bloodstream infections caused by nontyphoidal Salmonella are a major public health concern in Africa, causing ~49,600 deaths every year. The most common Salmonella enterica pathovariant associated with invasive nontyphoidal Salmonella disease is Salmonella Typhimurium sequence type (ST)313. It has been proposed that antimicrobial resistance and genome degradation has contributed to the success of ST313 lineages in Africa, but the evolutionary trajectory of such changes was unclear. Here, to define the evolutionary dynamics of ST313, we sub-sampled from two comprehensive collections of Salmonella isolates from African patients with bloodstream infections, spanning 1966 to 2018. The resulting 680 genome sequences led to the discovery of a pan-susceptible ST313 lineage (ST313 L3), which emerged in Malawi in 2016 and is closely related to ST313 variants that cause gastrointestinal disease in the United Kingdom and Brazil. Genomic analysis revealed degradation events in important virulence genes in ST313 L3, which had not occurred in other ST313 lineages. Despite arising only recently in the clinic, ST313 L3 is a phylogenetic intermediate between ST313 L1 and L2, with a characteristic accessory genome. Our in-depth genotypic and phenotypic characterization identifies the crucial loss-of-function genetic events that occurred during the stepwise evolution of invasive S. Typhimurium across Africa.

Characterization of DNA methylation in Malawian Mycobacterium tuberculosis clinical isolates.

BACKGROUND: Although Mycobacterium tuberculosis (Mtb) strains exhibit genomic homology of >99%, there is considerable variation in the phenotype. The underlying mechanisms of phenotypic heterogeneity in Mtb are not well understood but epigenetic variation is thought to contribute. At present the methylome of Mtb has not been completely characterized. METHODS: We completed methylomes of 18 Mycobacterium tuberculosis (Mtb) clinical isolates from Malawi representing the largest number of Mtb genomes to be completed in a single study using Single Molecule Real Time (SMRT) sequencing to date. RESULTS: We replicate and confirm four methylation disrupting mutations in 4 lineages of Mtb. For the first time we report complete loss of methylation courtesy of C758T (S253L) mutation in the MamB gene of Indo-oceanic lineage of Mtb. Additionally, we report a novel missense mutation G454A (G152S) in the MamA gene of the Euro-American lineage which could potentially be attributed to total disruption of methylation in the CCCAG motif but partial loss in a partner motif. Through a genomic and methylome comparative analysis with a global sample of sixteen, we report previously unknown mutations affecting the pks15/1 locus in L6 isolates. We confirm that methylation in Mtb is lineage specific although some unresolved issues still remain.

Whole Genome In-Silico Analysis of South African G1P[8] Rotavirus Strains Before and After Vaccine Introduction Over A Period of 14 Years.

Rotavirus G1P[8] strains account for more than half of the group A rotavirus (RVA) infections in children under five years of age, globally. A total of 103 stool samples previously characterized as G1P[8] and collected seven years before and seven years after introducing the Rotarix® vaccine in South Africa were processed for whole-genome sequencing. All the strains analyzed had a Wa-like constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). South African pre- and post-vaccine G1 strains were clustered in G1 lineage-I and II while the majority (84.2%) of the P[8] strains were grouped in P[8] lineage-III. Several amino acid sites across ten gene segments with the exception of VP7 were under positive selective pressure. Except for the N147D substitution in the antigenic site of eight post-vaccine G1 strains when compared to both Rotarix® and pre-vaccine strains, most of the amino acid substitutions in the antigenic regions of post-vaccine G1P[8] strains were already present during the pre-vaccine period. Therefore, Rotarix® did not appear to have an impact on the amino acid differences in the antigenic regions of South African post-vaccine G1P[8] strains. However, continued whole-genome surveillance of RVA strains to decipher genetic changes in the post-vaccine period remains imperative.

Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.

Evolutionary Genomics at the Human-Environment Interface in Africa.

We report on the first meeting of SMBE in Africa. SMBE Malawi was initiated to bring together African and international researchers who use genetics or genomics to study natural systems impacted by human activities. The goals of this conference were 1) to reach a world-class standard of science with a large number of contributions from Africa, 2) to initiate exchange between African and international researchers, and 3) to identify challenges and opportunities for evolutionary genomics research in Africa. As repored, we think that we have achieved these goals and make suggestions on the way forward for African evolutionary genomics research.

Association of APOL1 renal disease risk alleles with Trypanosoma brucei rhodesiense infection outcomes in the northern part of Malawi.

Trypanosoma brucei (T.b.) rhodesiense is the cause of the acute form of human African trypanosomiasis (HAT) in eastern and southern African countries. There is some evidence that there is diversity in the disease progression of T.b. rhodesiense in different countries. HAT in Malawi is associated with a chronic haemo-lymphatic stage infection compared to other countries, such as Uganda, where the disease is acute with more marked neurological impairment. This has raised the question of the role of host genetic factors in infection outcomes. A candidate gene association study was conducted in the northern region of Malawi. This was a case-control study involving 202 subjects, 70 cases and 132 controls. All individuals were from one area; born in the area and had been exposed to the risk of infection since birth. Ninety-six markers were genotyped from 17 genes: IL10, IL8, IL4, HLA-G, TNFA, IL6, IFNG, MIF, APOL, HLA-A, IL1B, IL4R, IL12B, IL12R, HP, HPR, and CFH. There was a strong significant association with APOL1 G2 allele (p = 0.0000105, OR = 0.14, CI95 = [0.05-0.41], BONF = 0.00068) indicating that carriers of the G2 allele were protected against T.b. rhodesiense HAT. SNP rs2069845 in IL6 had raw p < 0.05, but did not remain significant after Bonferroni correction. There were no associations found with the other 15 candidate genes. Our finding confirms results from other studies that the G2 variant of APOL1 is associated with protection against T.b. rhodesiense HAT.

Brain swelling is independent of peripheral plasma cytokine levels in Malawian children with cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is often fatal, and severe brain swelling is a predictor of CM-related mortality. CM is characterized by elevated circulating pro-inflammatory cytokines TNF and IFN-γ and anti-inflammatory cytokine IL-10, however whether cytokine levels correlate with brain swelling severity is unknown. This study therefore was conducted to investigate the relationship between cytokine levels and brain swelling severity in children presenting with CM. METHODS: A total of 195 Malawian children presenting with CM were recruited and had the concentrations of plasma cytokines determined and compared to brain swelling severity, determined by MRI examination, and graded as severe, moderate, mild or none. RESULTS: Levels of IL-1ß, IL-6, IL-8 and IL-10 did not differ between CM patients with and without severe brain swelling. Compared to children without brain swelling, IL-12 levels were higher in children with severe swelling (p < 0.01, no swelling 1 pg/mL, IQR [1] vs. severe swelling 18.7 pg/mL, IQR [1-27]), whereas TNF concentrations were higher in children with moderate brain swelling compared to children with no swelling (p < 0.01, no swelling 3 pg/mL, IQR [1-20] vs. moderate swelling 24 pg/mL, IQR [8-58]. Multivariate analysis showed that no single cytokine independently predicted brain swelling. CONCLUSION: Severe brain swelling in paediatric CM was independent of tested blood pro-inflammatory and anti-inflammatory cytokines which are markers of systemic inflammation.

Emergence of Double- and Triple-Gene Reassortant G1P[8] Rotaviruses Possessing a DS-1-Like Backbone after Rotavirus Vaccine Introduction in Malawi.

To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced (n = 32) possessed a Wa-like genetic constellation, whereas postvaccine G1P[8] strains (n = 18) had a DS-1-like constellation. Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 × 10-4 to 4.1 × 10-3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation.IMPORTANCE The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine escape mutants. While multiple African countries have introduced a rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the postvaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998 to 2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting a limited effect on the recognition of G1-specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation.

The global distribution and diversity of protein vaccine candidate antigens in the highly virulent Streptococcus pnuemoniae serotype 1.

Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide coverage against 86% of the global population. SP0148, SP1912 and SP2108 were universally encoded and we further assessed their predicted amino acid, antigenic and structural variation. Multiple allelic variants of these proteins were identified, different allelic variants dominated in different continents; the observed variation was predicted to impact the antigenicity and structure of two SP0148 variants, one SP1912 variant and four SP2108 variants, however these variants were each only present in a small fraction of the global population (<2%). The vast majority of the observed variation was predicted to have no impact on the efficaciousness of a protein vaccine incorporating a single variant of SP0148, SP1912 and/or SP2108 from S. pneumoniae TIGR4. Our findings emphasise the importance of taking geographic differences into account when designing global vaccine interventions and support the continued development of SP0148, SP1912 and SP2108 as protein vaccine candidates against this important pneumococcal serotype.

Factors affecting the utilisation of electronic medical records system in Malawian central hospitals.

Background: In Malawi, paper-based medical record-keeping has been observed to exacerbate challenges related to accessing patient records and patient tracking. Despite the introduction of electronic medical record (EMR) systems in 2001, paper-based records continue to be in use. Some health workers prefer paper-based records to EMRs. This study assessed factors that affect the use of EMRs in Malawi, particularly at Queen Elizabeth and Kamuzu Central Hospitals. It further investigated the reasons why paper-based records are still in use despite the numerous associated disadvantages. The extent to which EMRs contribute to patient care was also analysed. Methods: In this cross-sectional study, 111 randomly selected health workers were interviewed, using a semistructured questionnaire, at the 2 largest central hospitals in Malawi, where EMRs were first introduced in the country. Focus group discussions were conducted to gather further information on factors identified during the individual interviews. Results and conclusions: Differences in age, gender, and previous computer experience were not associated with differences in EMR usage. However, education and employment levels has a positive association with EMR usage. Hardware and connectivity problems, as well as lack of training and managerial support negatively affected the use of EMRs. EMRs were found to improve data quality and efficiency in patient management.

Analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important Thermus species.

BACKGROUND: Bacteria of genus Thermus inhabit both man-made and natural thermal environments. Several Thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. Enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms. RESULTS: Using Meiothermus silvanus DSM 9946 as a reference genome, high level of coordinated rearrangements has been observed in extremely thermophilic Thermus that may imply existence of yet unknown evolutionary forces controlling adaptive re-organization of whole genomes of thermo-extremophiles. However, no remarkable differences were observed across species on distribution of functionally related genes on the chromosome suggesting constraints imposed by metabolic networks. The metabolic network exhibit evolutionary pressures similar to levels of rearrangements as measured by the cross-clustering index. Using stratigraphic analysis of donor-recipient, intensive gene exchanges were observed from Meiothermus species and some unknown sources to Thermus species confirming a well established DNA uptake mechanism as previously proposed. CONCLUSION: Global genome rearrangements were found to play an important role in the evolution of Thermus bacteria at both genomic and metabolic network levels. Relatively higher level of rearrangements was observed in extremely thermophilic Thermus strains in comparison to the thermo-tolerant Thermus scotoductus. Rearrangements did not significantly disrupt operons and functionally related genes. Thermus species appeared to have a developed capability for acquiring DNA through horizontal gene transfer as shown by the donor-recipient stratigraphic analysis.

Analysis of protein thermostability enhancing factors in industrially important thermus bacteria species.

Elucidation of evolutionary factors that enhance protein thermostability is a critical problem and was the focus of this work on Thermus species. Pairs of orthologous sequences of T. scotoductus SA-01 and T. thermophilus HB27, with the largest negative minimum folding energy (MFE) as predicted by the UNAFold algorithm, were statistically analyzed. Favored substitutions of amino acids residues and their properties were determined. Substitutions were analyzed in modeled protein structures to determine their locations and contribution to energy differences using PyMOL and FoldX programs respectively. Dominant trends in amino acid substitutions consistent with differences in thermostability between orthologous sequences were observed. T. thermophilus thermophilic proteins showed an increase in non-polar, tiny, and charged amino acids. An abundance of alanine substituted by serine and threonine, as well as arginine substituted by glutamine and lysine was observed in T. thermophilus HB27. Structural comparison showed that stabilizing mutations occurred on surfaces and loops in protein structures.

Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01.

BACKGROUND: Many strains of Thermus have been isolated from hot environments around the world. Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different Thermus thermophilus strains have been completed. This paper represents the completed genome from a second Thermus species - T. scotoductus. RESULTS: The genome of Thermus scotoductus SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the Thermus thermophilus genomes. The T. thermophilus megaplasmid genes are part of the T. scotoductus chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of T. scotoductus and T. thermophilus. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from Meiothermus ruber. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of Thermus scotoductus was confirmed experimentally as expected as most of the proteins of the natural transformation system of Thermus thermophilus are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle. CONCLUSIONS: The genome of Thermus scotoductus SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to Thermus thermophilus. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of Thermus scotoductus illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.