Bioinformatisk analyse og genetisk modifisering av karbohydratmetabolismen i Pseudomonas fluorescens, med fokus på alginatsyntese fra glukose

Den sentrale karbohydratmetabolismen i Pseudomonas fluorescens, med vekt på opptak og metabolisme av glukose, ble forsøkt kartlagt ved litteraturstudier og bioinformatisk analyse av tilgjengelige genomsekvenser. Det ble konkludert med at P. fluorescens tar opp glukose fra omgivelsene via poriner i ytre membran og glukose ABC-transportere i indre membran, og at den sentrale glukosemetabolismen omfatter ED-sporet, det direkte oksidative sporet, pentosefosfatsporet, glukoneogenesen og nedre del av glykolysen. Ved glukosekatabolisme via det direkte oksidative sporet omdannes glukose til glukonat og 2-ketoglukonat i periplasma. Ketoglukonat og 2-ketoglukonat transporteres deretter over indre membran via transportere drevet av kjemiosmotiske gradienter. Genkandidater for alle enzymene og proteinene i de ovennevnte transportsystemene og metabolske sporene ble funnet. Biosyntese av alginat er koblet til den sentrale karbohydratmetabolismen ved at fruktose-6-fosfat er utgangspunktet for alginatsyntese. I forkant av alginatsyntese i P. fluorescens dannes sannsynligvis fruktose-6-fosfat fra glukose ved nedbryting til trioser via ED-sporet, etterfulgt av resirkulering til fruktose-6-fosfat via glukoneogensen. Konvertering av glukose til 6-fosfoglukonat, et viktig mellomprodukt i ED-sporet, kan gå via to spor; det direkte oksidative sporet og det fosforylative sporet. P. fluorescens har også genet for glukose-6-fosfat isomerase (pgi), som katalyserer isomerisering av glukose-6-fosfat til fruktose-6-fosfat. Det ble framsatt en hypotese om at å blokkere glukosemetabolisme via det direkte oksidative sporet eller det fosforylative sporet kunne kanalisere mer glukose mot direkte konvertering til fruktose-6-fosfat, fremfor nedbryting og resirkulering via ED-sporet og glukoneogenesen. Dette kunne tenkes å bedre utnyttelsen av glukose med hensyn på alginatsyntese. I den eksperimentelle delen av arbeidet ble P. fluorescens NCIMB 10525 og Pf201 benyttet. NCIMB 10525 produserer ikke alginat, mens Pf201 er en alginatproduserende NCIMB 10525-mutant. Med utgangspunkt i disse stammene ble det konstruert mutanter med en delesjon i genet for glukose dehydrogenase (gcd) eller glukose-6-fosfat dehydrogenase (zwf-1 eller zwf-2). Disse enzymene katalyserer tidlige trinn i henholdsvis det direkte oksidative sporet og det fosforylative sporet. Mutantenes evne til å vokse med glukose som karbonkilde, samt aktiviteten av glukose dehydrogenase og glukose-6-fosfat dehydrogenase, ble undersøkt. For mutantene avledet fra Pf201 ble også alginatproduksjon undersøkt. I tillegg ble pgi sekvensert og klonet inn i en vektor, da overuttrykk av dette genet også kunne tenkes å kanalisere mer glukose mot direkte konvertering til fruktose-6-fosfat. Overuttrykk ble imidlertid ikke gjennomført. De innførte mutasjonene så ikke ut til å forbedre alginatsyntese fra glukose. I Pf201Δgcd og Pf201Δzwf-2 lå alginatproduksjonen på samme nivå som i Pf201, mens Pf201Δzwf-1 ikke produserte alginat. Zwf-1 ser altså ut til å være nødvendig for alginatsyntese, men det er uvisst hvorfor. Alle de konstruerte mutantene var i stand til å vokse med glukose som eneste karbonkilde. Inaktivering av zwf-1 førte til kortere generasjonstid, mens inaktivering av zwf-2 ikke påvirket veksten. Dobbeltmutanten NCIMB 10525Δzwf-1Δzwf-2 hadde imidlertid både lengre lag-fase og lengre generasjonstid enn utgangsstammen. Inaktivering av gcd førte til lengre lagfase og lengre generasjonstid i Pf201-mutanten, men påvirket ikke vekst i mutanten avledet fra NCIMB 10525. Det er imidlertid usikkert om inaktivering av gcd lyktes i NCIMB 10525Δgcd. I både NCIMB 10525 og Pf201 ble det detektert høyest aktivitet av glukose dehydrogenase etter dyrking ved lave glukosekonsentrasjoner. I NCIMB 10525 og NCIMB 10525Δzwf-2 var aktivitet av glukose-6-fosfat dehydrogenase høyest etter dyrking ved lave glukosekonsentrasjoner, mens aktiviteten var lik ved begge konsentrasjonene i NCIMB 10525Δzwf-1. Pf201, Pf201Δzwf-1 og Pf201Δzwf-2 viste høyest aktivitet av glukose-6-fosfat dehydrogenase etter dyrking ved høye glukosekonsentrasjoner. Forskjellene i enzymaktivitet og påvirkning av vekst tyder på at det kan være forskjeller i glukosemetabolismen til NCIMB 10525 og Pf201

Looking for the Big Picture:Genome-Based Approaches to Improve Alginate Production in Azotobacter vinelandii

Alginates are commercially important polysaccharides with a wide range of industrial and technological applications. Polymer chain length and monomer distribution greatly affect the material properties, which makes different alginate types ideal for different areas of use. All commercial alginate manufacture is currently based on extraction from brown algae, but the polymers are also produced by bacteria in the genera Pseudomonas and Azotobacter. Bacterial bioproduction is technically possible, but is not yet economically competitive with algal alginates. A. vinelandii is an attractive candidate for development of bacterial bioproduction strains due to its potential for producing homogenous alginates with tailored monomer compositions, and thus high market value. Successful development of strains and cultivation conditions for bioproduction is however dependent on extensive knowledge of the factors affecting the biosynthetic process in question. Along with the availability of complete genome sequences, a multitude of new opportunities has arised with regard to investigations of gene functions, metabolic and regulatory relationships, environmental adaptations etc. Part of the work presented in this thesis is a contribution to the manual curation and annotation of the first published A. vinelandii genome, which provided the basis for our subsequent genomebased analyses of factors affecting alginate production in this organism. The genome annotation and analysis carried out as part of this thesis was mainly concerned with carbohydrate metabolism genes, and has provided a foundation for further investigations of sugar uptake and utilization in this organism. A notable outcome of the genome analysis was the discovery of numerous highly similar and apparently conserved intra-genome homologs among A. vinelandii core carbohydrate metabolism genes. Investigations of 943 bacterial and archaeal genomes confirmed that the number of such homologs is indeed unusually large in A. vinelandii. We propose that the retention of multiple gene copies confers adaptive benefits via gene dosage and/or increased regulatory flexibility. Genes, and thus cellular processes, affecting alginate production in A. vinelandii were investigated by construction and screening of a transposon insertion library comprising 4000 mutant strains. Abolished or diminished alginate production was confirmed for ~70 transposon insertion mutants and the disrupted genes were identified by sequencing. The disrupted genes included structural and regulatory genes involved in alginate biosynthesis, as well as genes involved in iron uptake, peptidoglycan recycling, motility and synthesis of several cofactors and central metabolites. Based on these results the effect of various medium supplements on alginate production in wild type A. vinelandii was investigated, and addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate was shown to result in significantly increased alginate levels. The screening results also revealed two possible new regulators of alginate biosynthesis; the fructose phosphotransferase system protein FruA and an IclR family transcriptional regulator. Two mutants were confirmed to have gained an increase in alginate production. For one of these the disrupted gene was identified as mucA, encoding the main negative regulator of alginate biosynthesis. Global effects of inactivating MucA were investigated by phenotypic characterization and transcriptome analyses of fermentor-grown A. vinelandii wild type and mucA strains. The mucA mutant has a lowered growth rate, elevated alginate production and diminished respiration rate compared to the wild type strain. Both medium composition and MucA inactivation had profound effects on carbon source utilization. The transcriptome analyses revealed new roles for the key regulators MucA/AlgU with regard to control of alginate composition, cell mass production, respiration and possibly nitrogen fixation. The redirection of carbon utilization in the mucA mutant was also reflected in transcriptional changes in genes involved in gluconeogenesis/glycolysis and energy production. The results presented in this thesis will have importance for further work towards the long-term goal of establishing bacterial systems for commercial bioproduction of alginates

Safety in numbers: multiple occurrences of highly similar homologs among Azotobacter vinelandii carbohydrate metabolism proteins probably confer adaptive benefits

Background Gene duplication and horizontal gene transfer are common processes in bacterial and archaeal genomes, and are generally assumed to result in either diversification or loss of the redundant gene copies. However, a recent analysis of the genome of the soil bacterium Azotobacter vinelandii DJ revealed an abundance of highly similar homologs among carbohydrate metabolism genes. In many cases these multiple genes did not appear to be the result of recent duplications, or to function only as a means of stimulating expression by increasing gene dosage, as the homologs were located in varying functional genetic contexts. Based on these initial findings we here report in-depth bioinformatic analyses focusing specifically on highly similar intra-genome homologs, or synologs, among carbohydrate metabolism genes, as well as an analysis of the general occurrence of very similar synologs in prokaryotes. Results Approximately 900 bacterial and archaeal genomes were analysed for the occurrence of synologs, both in general and among carbohydrate metabolism genes specifically. This showed that large numbers of highly similar synologs among carbohydrate metabolism genes are very rare in bacterial and archaeal genomes, and that the A. vinelandii DJ genome contains an unusually large amount of such synologs. The majority of these synologs were found to be non-tandemly organized and localized in varying but metabolically relevant genomic contexts. The same observation was made for other genomes harbouring high levels of such synologs. It was also shown that highly similar synologs generally constitute a very small fraction of the protein-coding genes in prokaryotic genomes. The overall synolog fraction of the A. vinelandii DJ genome was well above the data set average, but not nearly as remarkable as the levels observed when only carbohydrate metabolism synologs were considered. Conclusions Large numbers of highly similar synologs are rare in bacterial and archaeal genomes, both in general and among carbohydrate metabolism genes. However, A. vinelandii and several other soil bacteria harbour large numbers of highly similar carbohydrate metabolism synologs which seem not to result from recent duplication or transfer events. These genes may confer adaptive benefits with respect to certain lifestyles and environmental factors, most likely due to increased regulatory flexibility and/or increased gene dosage

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner

Benzoic Acid-Inducible Gene Expression in Mycobacteria

<div><p>Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the <i>Pseudomonas putida</i>-derived positively regulated XylS/<i>Pm</i> expression system to control inducible gene expression in <i>Mycobacterium smegmatis</i> and <i>Mycobacterium tuberculosis</i>, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/<i>Pm-</i>regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer <i>m</i>-toluate, we achieve a robust, time- and dose-dependent reversible induction of <i>Pm</i>-mediated expression in mycobacteria, with low background expression levels. XylS/<i>Pm</i> is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.</p></div

Induction of <i>Pm</i> with <i>m</i>-toluate is robust, time- and dose-dependent.

<p><i>Msmeg</i> transformed with the expression vectors pMDX-luc or the empty vector pMDX (no reporter gene) treated with increasing concentrations of <i>m</i>-toluate (induced) or ethanol carrier (uninduced). Cells were incubated at 30°C, and luciferase expression was determined at 2.5, 5.5, 11, 23, 31 and 49 hours after addition of <i>m</i>-toluate. (A) Fold induction of RLU in induced samples compared to uninduced samples. (B) Growth of uninduced and induced samples of pMDX-luc corresponding to samples in (A). (C) Time course of luciferase induction from pMDX-luc with 1.5 mM <i>m</i>-toluate or ethanol carrier. (D) Maximal induction of pMH109 and pMDX-luc-transformed <i>Msmeg</i> induced with 2 mM <i>m</i>-toluate. (E) Amount of luciferase produced as determined by the activity of known luciferase concentrations (0, 0.1, 0.2, 0.4 0.6, 0.8 and 1 μg/ml luciferase) in mid log phase or (F) stationary phase. Luciferase fraction of total bacterial protein shown in brackets. RLUs were normalized to the OD₆₀₀ of the samples before luciferase assay. All results are representative of two or more independent experiments.</p

<i>Pm</i>–mediated basal expression is low and compares favorably to <i>Ptet-</i>mediated basal expression in <i>Mtb</i>.

<p>(A-B) <i>Mtb</i> transformed with pMDX-luc or pUV15tetORm::luciferase was diluted to OD₆₀₀ 0.005 in the presence or absence of m-toluate (1.5 mM) or atc (200 ng/ml), respectively. Samples were grown in triplicates and monitored for 7 days registering OD₆₀₀ at 2, 4 and 7 days. Basal expression from <i>Pm</i> and <i>Ptet</i> is presented by level of luciferase produced by <i>Mtb</i> pMDX-luc and <i>Mtb</i> pUV15tetORm::luciferase. (A) shows the luciferase expression over time in induced sample and (B) shows the basal expression from uninduced samples over time. The results represent two independent experiments.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p