Os conhecimentos sobre os mecanismos regulatórios responsáveis pelo crescimento dos fungos filamentosos apresentam lacunas e sua compreensão é necessária para o desenvolvimento de uma terapêutica antifúngica mais adequada, assim como para incrementar a síntese de produtos de interesse comercial. Assim sendo, estudar o envolvimento do Ca2+ na resposta de um fungo modelo como A. nidulans sob fontes de carbono diferentes constitui um meio de gerar conhecimentos sobre as características de crescimento dos fungos filamentosos, de sua resposta a adaptação ambiental e dos mecanismos que controlam essa resposta. Analisou-se na linhagem A26 e na AP27, esta última com ruptura do gene da plcA, o gradiente de Ca2+ citosólico, a morfologia das hifas, a germinação e o ciclo de divisão nuclear quando as linhagens tinham calcineurina ou calmodulina inibidas e quando os canais de Ca2+ estavam bloqueados ou abertos. Os níveis de Ca2+ citosólico na linhagem A26, crescendo em presença de glicose, foram maiores que os detectados em meio suplementado com pectina. O ciclo de germinação e divisão celular no AP27, independentemente da fonte de carbono, mostrou-se mais lento se comparado com a linhagem A26, provavelmente devido ao fato de seus estoques intracelulares de Ca2+, tanto em nível vesicular quanto citosólico, serem menores. A linhagem AP27 apresentou ramificações dicotômicas nas pontas das hifas e nas hifas laterais em ambas as fontes de carbono nas quais foi cultivada, o que não se observou na linhagem A26. Quando calcineurina foi inibida por ciclosporina A, as hifas das duas linhagens, em ambas condições de cultivo, alongaram-se menos e apresentaram-se mais ramificadas, no entanto este efeito foi mais pronunciado em presença de glicose, e entre as duas linhagens pode-se dizer que foi mais intenso na linhagem AP27, demonstrando a importância dos níveis de cálcio na atividade desta enzima e conseqüentemente no desenvolvimento normal das hifas. A abertura dos canais de Ca2+, por ionóforo, produziu hiperramificação em ambas as linhagens, mas principalmente quando cresciam em pectina e ao contrário do efeito observado em presença de verapamil, que bloqueia os canais de Ca2+, não promoveram hifas laterais e nem pontas dicotômicas. No entanto o outro bloqueador dos canais de Ca2+ testado, ácido caurenóico, apresentou efeito morfológico diferente, pois as hifas tornaram-se curvas o que indica perda de polaridade. O inibidor da calmodulina (TFP) retardou a germinação, principalmente no mutante AP27, quando crescendo em presença de glicose. Lembrando que o complexo Ca2+/CaM ativa a calcineurina e que o mutante apresenta menores níveis de cálcio, esse resultado é justificável. A ruptura do gene plcA não impediu o crescimento e desenvolvimento do mutante, provavelmente porque a função desta enzima poder ser provida por outras partes do genoma, mas comprometeu os níveis intracelulares de cálcio e conseqüentemente a sua morfologia. Este estudo mostra a importância da fosfolipase C, para manutenção dos níveis intracelulares de Ca2+, no desenvolvimento normal das hifas de A. nidulans e, pela primeira vez, demonstra que esses níveis são diferentes quando o fungo cresce em presença de uma fonte de carbono, prontamente metabolizável ou não. Esses resultados conferem ao cálcio um papel modulador nessas condições de cultivo. / The knowledge about regulatory mechanisms responsible for filamentous fungi growth presents lacks and its understanding is important to develop adequate antifungal therapy either to contribute the synthesis of interestingly commercial products. By this way, study Ca2+ relationship to a fungal model as A. nidulans about different carbon sources, constitute knowledge about the filamentous fungi growing characteristics, environment adaptation and its control mechanisms. The strains A26 and AP27 was analyzed, this last one with disruption plcA gene, cytosolic Ca2+ gradient hyphal morfology, germination and nuclear division cycle when these strains had calcineurin or calmodulin inhibition and Ca2+ channel were blocked or opened. Cytosolic Ca2+ levels in A26 strain, growing in the presence of glucose was higher than supplemented media with pectin. AP27 strain, independently of carbon source, demonstrated lower germination and cell division than A26 strains, probably due to the fact that intracellular Ca2+ stocks either vesicular as cytosolic levels were lower. AP27 strain presented dichotomous branching at tip-high and lateral hyphae, at both carbon source that was grown, didnt observed at A26 linkage. When calcineurin was inhibited by cyclosporin A, hyphae from both strains, in both growth conditions, had less elongated and showed more branching, however this effect was more pronounced in presence of glucose, and between both strains was more intense at AP27 strain, indicating the importance of Ca2+ levels at this enzymatic activity and therefore the normal development of hyphae. The opening of Ca2+ channel by ionophore, produced hyperbranching in both strains, even when growth in pectin and in contrast of effect observed in the presence of verapamil, that blocks Ca2+ channels, didnt promote lateral or tip high dichotomous branching. However kaurenoic acid, an another Ca2+ channel blocker tested, presented different morphological effect, because hyphae became curved, indicating loss of polarity. Calmodulin inhibitor (TFP) delayed germination mainly at mutant AP27, when growing in the presence of glucose. Remembering that Ca2+/CaM complex activate calcineurin and the mutant exhibit lower Ca2+ levels, justifying this results. The rupture of plcA gene didnt affected growth and development of mutant, probably because the function of this enzyme can be provided by another parts of genoma, damaged the Ca2+ intracellular levels and consequently its morphology. This study shows the importance of fosfolipase C to maintaining the intracellular Ca2+ levels, at the normal hyphae development of A. nidulans and for the first time, demonstrating that this levels are different when fungi are grown in the presence of carbon source, promptly metabolizable or not. This results gives to Ca2+ as modulator at growth conditions.

A transformação de Aspergillus nidulans, com RNA de macrófagos de ratos, resultou na linhagem RT2, produtora de uma glicoproteína antiinflamatória. Nosso objetivo foi avaliar esta linhagem genenomicamente e caracterizar esta glicoproteína quanto à natureza bioquímica e sua atividade. Para tal, foi realizado RAPD e análise fenotípica desta linhagem. A Nandina foi purificada e submetida à espectrometria de massa para sequenciamento e identificação dos carboidratos. Testes da atividade antiinflamatória in vivo foram realizados em peritonite e edema de pata e inibição dos receptores de glicocorticóides. Os testes in vitro, sobre a produção das COXs e de PGE2, foram realizados em cultura de macrófagos. Os resultados mostraram que a linhagem RT2 é resultante da UT448, mas contém diferenças em seu genoma. A proteína purificada possui 40KDa. A espectrometria de massa caracterizou dois fragmentos da proteína e sua glicosilação. Os testes in vivo mostraram que a proteína inibe o edema e o influxo leucocitário e que esta atividade não é dependente de glicocorticóides, mas sim da inibição in vitro de COX-2, mas não de COX-1 e nem de PGE2. / Aspergillus nidulans transformation with rat macrophage RNA results on RT2 strain, producer of an antiinflammatory glycoprotein. Our objective was to evaluate this strain genomically and characterize biochemically and activity of its glycoprotein. To this, RAPD and fenotipical analysis were performed. The Nandin was purified and mass spectrometry analyzed to sequencing and carbohydrates analysis. Antiinflammatory activity testes in vivo in peritonitis and edema, and glucocorticoid receptors inhibition were performed. The in vitro testes, over expression and activity of COXs and PGE2, were performed in macrophage culture. The results show that RT2 strain came from UT448, but have genomics differences. The purified glycoprotein has been 40KDa. The mass spectrometry sequenced two protein fragments and showed that glycosylation. The in vivo testes showed that the glycoprotein has antiinflammatory activity inhibiting the edema and leukocyte influx. The RU38486 experiments evidenced that activity is not glucocorticoid receptors dependent, but in vivo inhibition of COX-2, but not COX-1 neither its product PGE2.

"May 2004" / Addendum inside back page. / Bibliography: p. 99-114. / xii, 114 p. : ill. (some col.), photos (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the cloning and analysis of creD, and the characterization of the acrB gene, two components of a regulatory network controlling carbon source utilization in the filamentous fungus Aspergillus nidulans that involves ubiquitination and deubiquitination. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Genetics, 2004

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.

Die Regulation des sekundären Metabolismus und der Entwicklung in Aspergillus nidulans sind zwei miteinander verbundene Prozesse. Ihre Regulation geschieht in Abhängigkeit von Licht. Die molekulare Grundlage dieser Verbindung ist ein Komplex bestehend aus den beiden Velvet-Proteinen VeA, VelB und der putativen Methyltransferase LaeA. Ein weiteres Protein der Velvet-Familie ist VosA, dass in Verbindung mit VelB eine essenzielle Rolle in der Sporogenese und Biosynthese von Trehalose spielt. Bisheriges Wissen über die generelle Funktion der einzelnen Proteine basiert überwiegend auf in vivo Analysen. Unbekannt hingegen ist die molekulare Funktionsweise sowie Struktur dieser Proteine. Zu diesem Zweck wurde in der vorliegenden Arbeit versucht dieses Defizit durch eine röntgenkristallographische Strukturanalyse der Proteine aufzuheben.

The cell wall is essential for fungal survival in natural environments. Galactofuranose (Galf) decorates certain carbohydrates and lipids of Aspergillus cell wall, is absent in humans and appears to play a role in fungal cell wall maturation. Previous studies in our lab showed that deletion of any of three sequential-acting genes (ugeA, ugmA, and ugtA) of Galf pathway caused substantially reduced growth and spore production. Two genes upstream of the Galf pathway, galD and galE are essential for galactose metabolism in many systems including the budding yeast, Saccharomyces cerevisiae. Interestingly, characterization of galD and galE in A. nidulans using cell and molecular techniques showed that unlike yeast, neither of these genes was essential for growth at physiological pH 7.5. Nevertheless for each case, their expressions were up-regulated by growth on galactose, revealing the relative complexity of galactose metabolism in A. nidulans. Our study also showed that repression of the three sequentially acting Galf pathway genes by conditional promoters phenocopied previously characterized deletion morphology. Using anti-Galf (L10) we also showed that deletion and repression of these genes caused no Galf in the hyphal wall. Gene deletion or repression also increased sensitivity to the wall-targeting drug, caspofungin. Related results from qPCR showed that deletion or repression of ugmA increased gene expression of α-glucan synthase agsB and decreased that of β-glucan synthase fksA. Therefore, Galf is non-essential but important for many aspects of Aspergillus growth, sporulation, and wall maturation. Aspergillosis, the most common airborne systemic fungal disease, is typically caused by Aspergillus fumigatus. Several A. fumigatus UgmA (AfUgmA) mutants with altered enzyme activity due to single amino acid changes were used to assess their effect on growth and wall composition in A. nidulans. Wild type AfugmA complemented the phenotypic defects in an A. nidulans ugmAΔ strain, consistent with these two genes being homologous. The AfUgmA crystal structure has been solved, and the in vitro enzymatic effects of specific mutations in the enzyme active site have been published. AfUgmA mutated strains with reduced activity in vitro impaired A. nidulans growth in a manner substantially similar to gene deletion and gene down-regulation. Site directed mutagenesis showed that AfUgmA residues R182 and R327 were critical for Galf generation both in vivo and in vitro. This supports previous results showing that UgmA is essential for Galf biosynthesis. Using fluorescent latex beads, we showed that reduction of wall Galf increased hyphal surface adhesion. Consistent with qPCR studies, immunofluorescence and ELISA results showed that loss or absence of Galf increased wall α-glucan but reduced wall β -glucan. Galf is important for wall surface integrity and for maintaining dynamic co-ordination with other pathways. To begin to assess this dynamic co-ordination, Tandem Affinity Purification (TAP) tagging combined with LC-MS/MS was used to identify the interacting partners of UgmA. Our results showed that UgmA interacted with proteins that are involved in cytoskeleton generation, osmotic adaptation, and cell signalling pathway. Further study will help us to understand the dynamic coordination of Galf biosynthesis pathway with other wall carbohydrate polymers for Aspergillus wall formation. In summary, my thesis results have clearly shown that Galf plays important roles in Aspergillus growth, and wall surface integrity. We also showed that Galf deficient strains are hypersensitive to wall-targeting drugs, indicating that Galf biosynthesis pathway could be potential target for combination therapy. The Galf pathway also maintained a dynamic co-ordination with alpha-glucan and beta-glucan carbohydrate pathways. Future study may include developing an inhibitor against UgmA and exploring the relationship of Galf pathway with alpha-glucan and beta-glucan carbohydrate pathways.

Orientador: Sandra Regina Pombeiro Sponchiado / Banca: Carlos Renato Corso / Banca: Meuris Gurgel Carlos da Silva / Resumo: Os metais terras-raras (TRs) apresentam um alto valor agregado, o qual não provém de sua baixa ocorrência mineral, mas sim da dificuldade na separação destes pelos métodos clássicos, uma vez que os elementos desta série apresentam semelhantes comportamentos físico-químicos. Desta maneira, altos custos são despendidos anualmente para a obtenção das TRs na forma pura, sendo esta necessária para as suas aplicações tecnológicas. Uma alternativa a esta problemática é o emprego da biossorção, a qual apresenta potencial aplicação para a extração e separação de metais valiosos, por ter como vantagem uma alta eficiência agregada a um baixo custo. Alguns requisitos são necessários para que o processo de biossorção possa ter competitividade técnica e econômica, tais como, o material biológico empregado deve apresentar um baixo custo, sendo assim necessário estabelecer as condições ótimas de cultivo do micro-organismo para sua produção em larga escala, e também a utilização do biossorvente na forma imobilizada possibilita seu uso como um adsorvente convencional, o qual apresenta tamanho desejado, alta porosidade e um bom desempenho físicoquímico. Por esta razão, o presente trabalho teve como principal objetivo avaliar a potencialidade de utilização da biomassa melanizada (inativa) obtida da linhagem mutante MEL 1 do fungo Aspergillus nidulans, na sua forma livre e imobilizada, no processo de biossorção dos metais terras-raras neodímio, lantânio e cério em soluções monometálicas. Com os resultados obtidos foi possível estabelecer a condição ótima de cultivo da linhagem MEL 1 do A. nidulans que proporcionou uma biomassa mais melanizada, a qual apresentou uma maior capacidade biossortiva. Para tal condição, a massa micelial obtida após o crescimento do fungo por três dias, sob agitação à... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The rare-earth metals (TRs) have a high added value, which does not come from its low mineral occurrence, but the difficulty in separating these by classical methods, since the elements of this series show similar physical and chemical behavior. Thus, high costs are spent annually to obtain the TRs in pure form, which is necessary for their technological applications. An alternative to this problem is the use of biosorption, which has potential application for extraction and separation of valuable metals, having the advantage of a high aggregate efficiency at a low cost of production. Some requirements are necessary for the biosorption process may have technical and economic competitiveness, such as the biological material used must be low cost and it is necessary to establish the optimum conditions for cultivation of the microorganism to its large scale production, and also the use of biosorbent immobilized in the form enables its use as a conventional adsorbent, which has the desired size, high porosity and good physical and chemical performance. For this reason, this study aimed to evaluate the potential use of biomass melanized inactive mutant of the fungus Aspergillus nidulans MEL1 in its free form and immobilized in calcium alginate, in the process of biosorption of neodymium rare-earth metals, lanthanum and cerium. The results concerning the kinetics of biosorption showed that the ability of TRs biosorptive varied between different forms of biomass, and this capacity was higher compared to free biomass immobilized. Regarding the maximum biosorption capacity (Qmax), we observed that the immobilized biomass caused a reduction in Qmax of lanthanum and neodymium in approximately 60 and 33% respectively, have kept their Ce Qmax for both types of biomass (open and immobilized). The affinity of biomass... (Complete abstract click electronic access below) / Mestre

The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.

Membrane proteins play an integral role in the control of ion transport across the cell membrane in biological systems. However, due to experimental constraints, structural and functional data available for these proteins is limited, especially considering their importance. In this study, two membrane proteins which transport nitrate and nitrate into the model filamentous ascomycete Aspergillus nidulans were investigated. Work on the twelve trans-membrane domain nitrate transport protein NrtA is well established. As a member of the major facilitator super family (MFS) the role of signature sequences characteristic of this family have previously been studied. Here, a series of point mutations were made to facilitate an understanding of key residues in the nitrate binding domain, the first nitrate signature motif and residues of the unique fungal central-loop domain. Using an expanded alignment package, the proposed secondary structure of NrtA was enhanced and used as a starting point for mutagenesis. Alanine scanning mutagenesis showed that glycine residues in the conserved nitrate nitrite porter (NNP) motif were critical for NrtA function. Two asparagines in the NNP were investigated; N160 and N168. N168 was found to be critical for NrtA function as all mutants were devoid of growth on nitrate solid agar medium though they expressed in the membrane to varying degrees. The nitrate binding site has been studied previously, revealing the interaction of conserved arginine residues with the anion as it traverses the bilayer. Though it was thought that mutations of residue T83 to a small, charge neutral, amino acid would substitute for no alteration to enzyme kinetics in mutant T83S was found when using ¹³NO₃⁻. Another major part of this thesis examined NitA which is part of a distinct nitrite transport family to NrtA (the Formate Nitrite Transporters, FNT). A mutagenesis approach targeted NitA residues conserved amongst homologous proteins. Residues in position D88 in an alignment of homologues were conserved in terms of charge. Mutagenesis of D88 revealed that maintaining charge at this position was essential for NitA function, likely due to a role in salt-bridge formation during conformational changes. Mutations to asparagine, glutamine, serine and valine showed reduced growth on agar though the protein was expressed to approximately wild-type levels. Nitrite uptake assays using a ¹³NO₂⁻ tracer were performed on D88N, D88E and D88Q and all showed wild-type Km and Vmax. Finally, the role of conserved asparagine residues found throughout NitA was investigated by mutagenesis. Expression studies revealed that mutants created in N122 and N246, changed to aspartic acid, lysine, glutamine and serine were generally not present in the membrane and thus did not grow on nitrite agar. However, mutations in N173 (in Tm 4) and N214 (in Tm 5), which are conserved in > 95 % of NitA homologues, showed varying degrees of growth and expression. Both of these residues are located in FNT signature motifs, so it is likely that they are involved with conformational changes or protein dynamics.