High Confidence Prediction of Essential Genes in Burkholderia Cenocepacia

BACKGROUND: Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that essential genes may be highly conserved within a group of evolutionary closely related organisms. Using a bioinformatics approach we determined that the core genome of the order Burkholderiales consists of 649 genes. All but two of these identified genes were located on chromosome 1 of Burkholderia cenocepacia. Although many of the 649 core genes of Burkholderiales have been shown to be essential in other bacteria, we were also able to identify a number of novel essential genes present mainly, or exclusively, within this order. The essentiality of some of the core genes, including the known essential genes infB, gyrB, ubiB, and valS, as well as the so far uncharacterized genes BCAL1882, BCAL2769, BCAL3142 and BCAL3369 has been confirmed experimentally in B. cenocepacia. CONCLUSIONS/SIGNIFICANCE: We report on the identification of essential genes using a novel bioinformatics strategy and provide bioinformatics and experimental evidence that the large majority of the identified genes are indeed essential. The essential genes identified here may represent valuable targets for the development of novel antimicrobials and their detailed study may shed new light on the functions required to support life

Comparative functional genomics of amino acid metabolism of lactic acid bacteria

The amino acid metabolism of lactic acid bacteria used as starters in industrial fermentations has profound effects on the quality of the fermented foods. The work described in this PhD thesis was initiated to use genomics technologies and a comparative approach to link the gene content of some well-known lactic acid bacteria to flavor formation and to increase our general knowledge in the area of amino acid metabolism. The three well-known lactic acid bacteria that were used in these studies were Streptococcus thermophilus, Lactococcus lactis and Lactobacillus plantarum. The complete genomes of all these model bacteria have been sequenced and annotated in detail. Comparative experimental and in silico studies of Streptococcus thermophilus with the other two lactic acid bacteria, revealed the low degree of amino acid auxotrophies of this species; it only needs two amino acids for (minimal) growth and this strain is able to produce a varied amount of flavors. Lactococcus lactis and Lactobacillus plantarum require more amino acids and produce fewer flavors than S. thermophilus. Furthermore, S. thermophilus has a simple primary metabolism; homolactic growth is the only possible route under anaerobic conditions and, remarkably, it does not have a complete pentose phosphate pathway in contrast to the other two studied bacteria. This latter property has important consequences for the redox metabolism of S. thermophilus and particularly its ability to produce NADPH. A genome-scale metabolic model was developed and predicted that amino acid metabolism, and especially glutamate degradation, and citrate metabolism are the most obvious alternatives for NADPH generation. Several of these predictions were confirmed by constructing a glutamate dehydrogenase mutant of S. thermophilus. This mutant revealed theimportance of the citrate pathway (and other amino acid degradation pathways) in NADPH generation. A comparative and functional genomics study of the three lactic acid bacteria showed that amino acid depletion not only affects amino acid metabolism, but also flavor formation and overall growth. The comparative genomics approach presented in this thesis can be used to understand the amino acid metabolism of different lactic acid bacteria and their potential to produce flavors under different conditions. Finally, it can be applied for optimization of industrial fermentation

Detailed genomic and antimicrobial resistance comparison of UK Streptococcus agalactiae isolates from adults to those of diverse global origins

Invasive group B Streptococcus (GBS), a leading cause of illness and death among infants in the first week of life is also an important infectious agent able to cause invasive infections in adults. Serious life-threatening invasive GBS infections are increasingly recognized in the elderly and individuals compromised by underlying diseases such as diabetes, cirrhosis, and cancer. The significance of GBS as a cause of severe infections among adults is not widely appreciated. In adults, the modes of transmission and acquisition are less identified. Penicillin is the antibiotic of choice for treatment of GBS infection however, resistance to multiple antibiotics is increasing in Europe and worldwide in these organisms, making them increasingly difficult to treat, but also making them a potential danger as a silent resource for donating resistance genes to more aggressive pathogens. To date, there are no guidelines for the prevention of adult GBS disease; vaccines in development may hold promise. GBS emerged rapidly all over the world during the 1970s to become the leading cause of neonatal sepsis. However, several reports in recent years suggest that the incidence of GBS disease is also increasing among adults. The driving force behind this change has not been fully explained, and recent trends in disease incidence in adults have not been characterized in any systematic reviews due to inadequate data available on adult GBS disease.The aim of the work presented in this thesis was to characterize the population structure of human source isolated invasive and non-invasive GBS from adults in United Kingdom, and to evaluate the genetic diversity of isolates recovered from invasive disease in adult patients of Brazil and from vaginal and rectal colonization in Australian pregnant women with ≤22 weeks gestations to contribute to the global epidemiology of GBS and our understanding of GBS population biology. For this a set of conventional techniques were employed including antimicrobial susceptibility testing using disc diffusion test, serotyping using software Geneious (Biomatters ltd., New Zealand) and published primers for ten different GBS capsular types, multilocus sequence typing (MLST) and surface protein gene profiling using SRST2 v2.2. In combination, these methods allowed the identification of the main genetic lineages circulating in UK, Brazil, and Australia, providing the means for an appropriate comparison of Brazilian and Australian GBS to UK GBS population. In addition, analysis of WGS to determine GBS potential to switch capsule, antimicrobial resistance (AMR) genes associated with mobile genetic elements (MGEs), examining relatedness of the GBS strains using whole genome phylogeny and correlate serotyping, patient age group to AMR genes and pan genome wide association study (pan-GWAS) on UK, Brazil, and publicly available genomes of GBS isolates from Canada and the United States was performed. These analyses revealed a reasonable number of isolates with potential capsular switch including some cases switched from current vaccine (under trail) covered capsular type to non-vaccine covered serotypes. Further a diverse group of MGEs were identified with a capacity to disseminate the resistance phenotype, the association between strains clustered in a group based on patients age group they isolated from and the AMR genes they carry were studied. An intense pan-GWAS was performed to discover Clonal Complex (CC) specific genes that may play role in increased colonization, invasiveness, pathogenicity, and better survival of GBS in the host cell. The thesis work started with investigating 193 clinical GBS strains isolated from adults submitted to the UK national reference laboratory (179 invasive; 13 non-invasive; 1 with no information provided) for capsule type, MLST, presence of virulence factors, antimicrobial resistance genes, phylogeny, and genetic recombination. The genetic lineages defined by MLST identified very diverse populations but consistent in terms of serotypes prevalence and clonal structure identified previously in GBS invasive disease in United Kingdom. The prevalence of serotype III in this population, regardless of age, highlighted the importance of this serotype in GBS pathogenesis as a leading cause of invasive infections in adults. Macrolide resistance is disseminated in UK by both a multiclonal mechanism resulting from the spread of resistance genes throughout most serotypes and genetic backgrounds, as well as by clonal expansion of specific lineages, such as the serotype V ST1/alp3. Attachment and invasion of host cells are key steps in GBS pathogenesis, strong associations were identified between serotypes and virulence genes, such as serotype V/alp3, serotype II and III/bca+cba, serotype Ia/bibA predominantly clustered in CC1, CC8/CC10 and CC23, respectively, whereas serotype III/rib clustered in CC17 and CC19 demonstrating GBS strains belonging to a particular CC differ in their abilities to attach and invade to host cell types and express key virulence genes that are relevant to the disease process. A major finding includes a high number of capsular serotype-CC mismatches (14/179, 7.8%) iGBS identified with a concerning recombination of hypervirulent hvgA core genome expressing a non-vaccine covered serotype IV capsule. The mechanism for these genetic transfer events involved the replacement of the whole capsular locus instead of the previously proposed genetic transfer of only the serotype specific genes. The consequent analysis of MGEs carrying multidrug resistance genes in 41/193 GBS isolates revealed a diverse group of MGES used three different insertion sites (rumA, rplL and rpsI) to disseminate phenotypic resistance in GBS isolated from adult patients of United Kingdom. Out of 41 isolates, only one isolate carried the macrolide resistance (ermT) gene was on a plasmid, while for 4 isolates fluoroquinolone resistance was mediated by double point somatic mutation in parC and gyrA; for all other isolates ARGs were acquired by MGEs including five novel MGEs identified in this study, including ICESag84 and ICESag100414 carrying ermA alone, ICESag662 containing ermB, tetS, ant(6-Ia) and aph(3'-III), ICESag71 carrying ermB and tetO, and ICESag139 containing ermA and the high gentamicin level resistance gene aac(6')-aph(2"). The Tn916 and Tn5801 belonging to Tn916/Tn1545 family harboured majority of tetM genes (88%, 154/175) found in UK tetracycline resistant GBS isolates and were significantly associated to CC1, CC8/10, CC19 and CC17 and CC23 isolates, respectively suggesting these ICEs are clonally related, acquired through limited and rare insertion events and led to expansion of these lineages, also supporting the earlier interpretations [1]. In addition, the Tn916/Tn1545 family ICE identified in this study were mostly integrated at two insertion sites, adjacent to two target genes (TGs) – rumA for Tn916 and guaA for Tn5801 demonstrating their integration preferences at hot spot regions. The high prevalence of MGEs carrying ARGs in UK adults GBS isolates implied that in GBS, these MGEs probably act like a reservoir of ARGs, and play a central role in the dissemination of resistance genes via horizontal gene transfer. Antibiotic susceptibility testing (AST) of GBS isolates recovered from UK (n=193) and Brazil (n=26) adults, and from vaginal and rectal sites of Australian pregnant women (n=171) revealed that all tested GBS populations were sensitive to ampicillin, vancomycin, and gentamicin, except a single UK strain that conferred high level gentamicin resistance through aminoglycoside modifying enzyme encoding gene aac(6')-aph(2"). Further all UK and Brazil GBS isolates carried five penicillin binding protein (PBPs) types with amino acid substitutions that did not appear to be associated with decreased β-lactam susceptibility suggesting penicillin as the first choice and vancomycin as the second choice of drug for GBS disease treatment as currently recommended by Centers for Disease Control and Prevention (CDC) and Royal College of Obstetricians and Gynaecologists (RCOG) guidelines [2, 3]. Accelerating resistance rates to erythromycin and clindamycin were observed in UK, which in comparison found less in Australian pregnant women colonized GBS and Brazilian GBS populations suggesting routine susceptibility testing of erythromycin and clindamycin for penicillin allergic patients to ensure effective treatment. A very few UK and Australian GBS strains were found resistant to chloramphenicol and/or levofloxacin while all Brazilian GBS were susceptible to these two antibiotics. High resistance rate to tetracycline was detected in UK, Brazilian and Australian GBS isolates and the tetM gene was found widespread and carried predominantly by Tn916/Tn1545 family elements. Three distinctive variants of Tn916 were observed in UK GBS strains, respectively including two Tn916 variants carrying tetM as a single resistance gene with additional 9 orfs in conjugation module, while the third Tn916 variant carried tetracycline efflux MFS transporter (tetL) gene near the tetM gene. The pangenome wide association study (pan-GWAS) was conducted on 447 GBS genomes, including this study sequenced UK (n=193) and Brazil (n=26) GBS strains, in addition to deliberately selected serotype V and III publicly available genomes from Canada (n=134) and the United States (n=94) to study genes specific to CC and their role in pathogenicity and invasiveness, since these two serotypes are universally found highly associated with adults and neonatal diseases, respectively. Each CC was characterized by specific genes that provides selective advantage to GBS for improved colonization, invasion, virulence, and survival within host. This analysis identified 97 CC-specific genes associated (excluding hypothetical proteins) with virulence, metabolism, and regulation of cellular mechanisms that may explain the differential virulence potential of the CCs. Among CC17 and CC23 GBS isolates, micronutrient uptake proteins (iron and manganese), two component systems, accessory secondary proteins, pilus and quorum-sensing genes were identified which were absent in less invasive lineages (CC1, CC8 and CC19). Metal resistance genes (arsenic, cadmium, and copper) and CRISPR associated genes (cas1/cas2) were confined to CC8 whereas the type IV secretory protein (VirD4) was significantly associated to CC19. Collectively this analysis underlines the lineage-specific basis of GBS niche adaptation and virulence. In summary, in this thesis GBS shown to have an evident stable clonal structure both temporally and geographically. Interestingly, capsular switching occurred across multiple serotypes and among strains with dissimilar genomic backgrounds in high numbers demonstrated ongoing GBS diversification due to recombination and highlights the importance of ongoing surveillance of GBS and may have implications for vaccine development strategies. Regardless of increasing information on invasive disease and maternal colonization, a thorough understanding of colonization in adults and natural reservoirs of GBS is required for the appropriate management of the GBS infections

Development of computational, mathematical and instrumental methods for the analysis of physiological processes in microbial populations

Die Fortschritte der modernen Mikrobiologie und die Verfügbarkeit neuer Hochdurchsatzmethoden liefern eine Fülle von Daten und Informationen. Durch geeignete Methoden und Modelle prozessiert, schaffen diese eine Basis für ein besseres Verstehen physiologischer Prozesse in mikrobiellen Populationen. Im ersten Teil dieser Arbeit wurde die Software eSOMet entwickelt. Sie ist ein Werkzeug zur Untersuchung metabolischer Profile mit dem Ziel diese ähnlichkeitsbasiert zu clustern. Da verschiedene Cluster unterschiedliche physiologische Zustände beschreiben, lassen sich damit diejenigen Metabolite identifizieren, deren Abundanzänderung für den Wechsel zwischen zwei Zuständen charakteristisch ist. Zum hochqualitativen Clustern wurde ein auf einer Hauptkomponentenanalyse basierter Rauschfilter und die Methode der emergenten selbstorganisierenden Karten implementiert. Am Beispiel verschiedener Kultivierungen von Corynbacterium glutamicum über mehrere Wachstumsphasen, konnte die Software erfolgreich evaluiert werden. Im zweiten Teil wurde ein spieltheoretisches Modell zur Untersuchung der Kulturheterogenität in mikrobiellen Populationen entwickelt. Als Heterogenitätskriterium wurde die Anzahl von Distribusomen definiert. Dies sind Zellbestandteile, deren Abundanz einen quantitativen Einfluss auf die Fitness der Subpopulation haben und die durch Zellteilung stochastisch weiterverteilt werden. Unter der Annahme, dass der Photosyntheseapparat von Dinoroseobacter shibae in solchen Distribusomen organisiert ist, konnte theoretisch gezeigt werden, dass sich unter fluktuierenden Umweltbedingungen oszillierend-stabile Subpopulationen ausbilden, die einen evolutionären Vorteil prägen. Um die theoretischen Ergebnisse des zweiten Teils experimentell zu verifizieren, wurde ein Kultivierungsverfahren im Mikrotitermaßstab entwickelt, welches vollautomatisiert durchgeführt eine in vivo Quantifizierung von Bacteriochlorophyll-a unter fluktuierenden Lichtbedingungen zuließ.The progress of modern microbiology and availability of novel high-throughput methods deliver a wealth of data and information. Processed by appropriate methods and models, they provide a basis for a better understanding of physiological processes in microbial populations. In the first part of this work the software eSOMet was developed. It is a tool for the investigation of metabolic profiles aiming to cluster those based on their similarity. Since distinct clusters are descriptors for different physiological states, this method allows the identification of metabolites, which changes of abundance are characteristic for the switch between two states. For high-quality clustering a principle component analysis based noise filter and the method of emergent selforganizing maps were implemented. The software could be successfully evaluated, using the example of different cultivations of Corynebacterium glutamicum over several growth phases. In the second part of this work, a game-theoretical model for the investigation of culture heterogeneity in microbial populations was developed. As heterogeneity criterion the number of distribusomes was defined. These are intracellular particles, which abundance influences the fitness of each subpopulation and which are distributed stochastically during cell division. Under the hypotheses, that the photosynthetic apparatus of Dinoroseobacter shibae is organized in such distribusomes it was shown in theory, that under fluctuating environmental conditions oscillating-stable fractions of subpopulations emerge, exhibiting an evolutionary advantage. In order to verify the theoretical results of the second part experimentally, a cultivation technique was developed at microtiter scale, which can be operated in a fully automated manner and allows for the in vivo quantification of Bchl-a under fluctuating light conditions