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17. Mennink-Kersten MA, Donnelly JP, Verweij PE: Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis 2004,4(6):349–357.PubMedCrossRef 18. Dagenais TR, Keller NP: Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clin Microbiol Rev 2009,22(3):447–465.PubMedCrossRef 19. Balloy V, Huerre M, Latge JP, Chignard M: Differences in patterns of infection Methocarbamol and inflammation for corticosteroid treatment and chemotherapy in experimental invasive pulmonary aspergillosis. Infect Immun 2005,73(1):494–503.PubMedCrossRef 20. Rementeria A, Lopez-Molina N, Ludwig A, Vivanco AB, Bikandi J, Ponton J, Garaizar J: Genes and molecules involved in Aspergillus fumigatus virulence. Rev Iberoam Micol 2005,22(1):1–23.PubMedCrossRef 21. Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J, Berriman M, Abe K, Archer DB, Bermejo C, et al.: Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 2005,438(7071):1151–1156.PubMedCrossRef 22. Galagan JE, Calvo SE, Cuomo C, Ma LJ, Wortman JR, Batzoglou S, Lee SI, Basturkmen M, Spevak CC, Clutterbuck J, et al.: Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae.

aureus adhesion to and invasion of human osteoblasts. MG-63 osteoblastic cells were infected for 2 h at approximately 50 bacteria/cell with S. aureus strain 8325-4, pre-treated or not (untreated control) with 1/2 MIC linezolid, oxacillin or rifampicin, and S. aureus strain DU5883 this website lacking

fnbA and fnbB (negative control). To enumerate cell-associated bacteria, infected cells were washed twice to discard unbound bacteria and analysed by osmotic shock in pure water, and then, suitable dilutions of the lysates were plated on agar. The same procedure was used to quantify intracellular bacteria, except that the cells were incubated for 1 h with 200 mg/L gentamicin before the lysis step to kill extracellular bacteria. Adherent bacteria were calculated by subtracting intracellular bacteria from cell-associated bacteria. The results were expressed as the means +/- standard deviation of the percentage of recovered internalised (a) or adherent (b) bacteria with respect to inoculated bacteria derived from four independent experiments performed in duplicate. Asterisk = significantly different from the control (corresponding isolate grown without antibiotic), with a P value

of 0.05 by one-way analysis of variance followed by a posteriori Dunnett’s test. Discussion Several Selleck Bafilomycin A1 major findings emerge from this investigation of the impact of sub-inhibitory concentrations of anti-staphylococcal drugs on S. aureus adhesion and invasion phenotypes. S. aureus binding to human fibronectin and the transcriptional levels of the fnbA/B genes encoding the fibronectin-binding proteins were differentially modulated by antimicrobial agents. Oxacillin, moxifloxacin and linezolid treatment led to the development of a hyper-adhesive phenotype, along with an increase in fnbA/B mRNA levels relative to the gyrB tetracosactide internal standard. The same hyper-adhesive phenotype was induced by clindamycin treatment, although no significant change in fnbA/B mRNA levels was observed. Rifampin was the only antimicrobial agent among

those tested that significantly inhibited S. aureus binding to fibronectin without affecting relative fnbA/B transcription profiles. Vancomycin and gentamicin induced no change in either the adhesion phenotype or the fnbA/B transcription. S. aureus adhesion to and invasion of live eukaryotic cells was also assessed after oxacillin, linezolid or rifampin treatment in an ex vivo infection model of cultured human osteoblasts. Oxacillin treatment significantly increased S. aureus adhesion but not invasion, while no significant change in adhesion or invasion levels was observed after linezolid or rifampin treatment. Several recent studies have focused on the influences of sub-inhibitory concentrations of antimicrobial agents on the expression of various virulence factors produced by S. aureus and on the various regulation mechanisms involved in this modulation [6, 8, 17].

PubMedCrossRef selleck inhibitor 22. Vihavainen EJ, Björkroth KJ: Diversity of Leuconostoc gasicomitatum associated with meat spoilage. Int J Food Microbiol 2009,136(1):32–36.PubMedCrossRef 23. Björkroth KJ, Geisen R, Schillinger U, Weiss N, De Vos P, Holzapfel WH, Korkeala HJ, Vandamme P: Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions. Appl Environ Microbiol 2000,66(9):3764–3772.PubMedCentralPubMedCrossRef 24. Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG: Multilocus sequence

typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 1998,95(6):3140–3145.PubMedCentralPubMedCrossRef 25.

Tanigawa K, Watanabe K: Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii . Microbiol 2011, 157:727–738.CrossRef 26. De Las RB, Marcobal A, Muñoz R: Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes. Appl Environ Microbiol 2004,70(12):7210–7219.CrossRef 27. Bilhère E, Lucas PM, Claisse O, Lonvaud-Funel A: Multilocus sequence typing of Oenococcus oeni : detection of two subpopulations Selleckchem LDE225 shaped by intergenic recombination. Appl Environ Microbiol 2009,75(5):1291–1300.PubMedCentralPubMedCrossRef 28. Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine

N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin Bay 11-7085 K, Lee JH, Díaz-Muñiz I, Dosti B, Smeianov V, Wechter W, Barabote R: Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A 2006,103(42):15611–15616.PubMedCentralPubMedCrossRef 29. Liang J, Ducatelle R, Pasmans F, Smet A, Haesebrouck F, Flahou B: Multilocus sequence typing of the porcine and human gastric pathogen Helicobacter suis . J Clin Microbiol 2013,51(3):920–926.PubMedCentralPubMedCrossRef 30. Baldo L, Dunning Hotopp JC, Jolley KA, Bordenstein SR, Biber SA, Choudhury RR, Hayashi C, Maiden MC, Tettelin H, Werren JH: Multilocus sequence typing system for the endosymbiont Wolbachia pipientis . Appl Environ Microbiol 2006,72(11):7098–7110.PubMedCentralPubMedCrossRef 31. Bisharat N, Cohen DI, Harding RM, Falush D, Crook DW, Peto T, Maiden MC: Hybrid Vibrio vulnificus. Emerg Infect Dis 2005,11(1):30–35.PubMedCentralPubMedCrossRef 32. Diancourt L, Passet V, Chervaux C, Garault P, Smokvina T, Brisse S: Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination. Appl Environ Microbiol 2007,73(20):6601–6611.PubMedCentralPubMedCrossRef 33. Madslien EH, Olsen JS, Granum PE, Blatny JM: Genotyping of B.

The ter region migrates from the new cell pole to the mid-cell position during chromosome replication MI-503 [8, 21]. This movement along the cell length occurs before ter replication (i.e., in cells with a single ter focus). Our results strongly support the view that the ter region migrates from the cell poles to mid-cell along the periphery of the nucleoid. This is also fully consistent with the notion that at least a part of the ter region connects the nucleoid edges via a peripheral link [12, 13]. It will be interesting to investigate if this particular behaviour of the ter

region is related to specific features of this region such as the presence of matP sites [16] or the action of the FtsK translocase. We used the T4 Ndd protein to interfere with chromosome organisation. Production of Ndd causes the centrally positioned nucleoid to move to the cell periphery by an unknown mechanism [24]. Following Ndd production and consequent nucleoid disruption, foci were detected as efficiently as in control cells

(Figure 4A), indicating that the delocalised DNA remained fully proficient for ParB binding and spreading over parS sites. Moreover, ParB binding to parS requires IHF, and IHF-ParB complexes strongly prefer supercoiled substrates [29]. Therefore, effective foci visualisation in our experiments involving rapid Ndd action indicates that DNA supercoiling Tigecycline order is not affected during Ndd-induced nucleoid delocalisation, consistent with previous observations during a slow Ndd disrupting process [24]. Ndd production reduced the number of foci per cell, particularly for the ori, right and NS-right loci (Additional file1, Figure S3). This effect was less pronounced for the ter locus indicating that it is not primarily due to a defect in the detection of foci. Following Ndd production, cell division is stopped more rapidly than chromosome replication [24], so the reduction in the number of foci per cell Phosphoglycerate kinase cannot

be due to a reduction of locus copy number. The smaller number of foci number may in part be due to the peripheral location of the chromosome in Ndd-treated cells. Indeed, the thickness of the peripheral DNA, as measured by DAPI staining, appeared to be in the same range as the optical resolution limit (about 200 nm, i.e., 3 pixels; see Additional file 1, Figure S2). Therefore, foci in close proximity inside disrupted nucleoids would appear as a single signal. Thus, the apparent reduction in the number of foci per cell strongly suggests that segregated sister loci are brought back together during nucleoid disruption. Chromosomal loci are therefore not completely free as they relocate toward the membrane during nucleoid disruption but conserve some positioning information.

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J Dent Educ 2003, 67:448–458.PubMed 18. Zero DT: Dentifrices, mouthwashes, and remineralization/caries arrestment strategies. BMC Oral Health 2006, 6:S9.CrossRefPubMed 19. Gregoire S, Singh AP, Vorsa N, Koo H: Influence of cranberry phenolics on glucan synthesis by glucosyltransferases and Streptococcus mutans acidogenicity. J Appl Microbiol 2007, 103:1960–1968.CrossRefPubMed 20. Koo H, Pearson SK, Scott-Anne K, Abranches J, Cury JA, Rosalen PL, Park YK, Marquis RE, Bowen WH: Effects of apigenin and tt- farnesol on glucosyltransferase activity, biofilm viability and caries development in rats. Oral Microbiol ABT-263 cost Immunol 2002, 17:337–343.CrossRefPubMed 21. Koo H, Hayacibara MF, Schobel LDK378 order BD, Cury JA, Rosalen PL, Park YK, Vacca-Smith AM, Bowen WH: Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt- farnesol.

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Microbiol 2008, 75:837–841.CrossRefPubMed 24. Hope CK, Wilson M: Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob Agents Chemother 2004, 48:1461–1468.CrossRefPubMed 25. Thurnheer T, Gmur R, Shapiro S, Guggenheim B: Mass transport of macromolecules within an in vitro model of supragingival plaque. Appl Environ Microbiol 2003, 69:1702–1709.CrossRefPubMed Protein kinase N1 26. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S: Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 2000, 146:2395–2407.PubMed 27. Duarte S, Klein MI, Aires CP, Cury JA, Bowen WH, Koo H: Influences of starch and sucrose on Streptococcus mutans biofilms. Oral Microbiol Immunol 2008, 23:206–212.CrossRefPubMed 28. Moore S, Stein WH: A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J Biol Chem 1954, 211:907–913.PubMed 29. Belli WA, Buckley DH, Marquis RE: Weak acid effects and fluoride inhibition of glycolysis by Streptococcus mutans GS-5. Can J Microbiol 1995, 41:785–791.CrossRefPubMed 30.

Sequencing

of the attB attP junction in this lysogen confirms the attP site of φX216 to be in the 3’ end of the predicted integrase gene corresponding to phage genome integration at tRNA-Phe (attB) [8]. Figure 2 φX216 genome annotation. Gene clusters and their predicted functions are indicated in different colors. Predicted capsid structural and assembly genes are shown in lime, host lysis proteins are shown in blue, genes required for phage tail structure and assembly are shown in cyan, and genes encoding proteins involved in lysogeny and DNA replication are shown in magenta. The phage attachment site (attP) is indicated by a yellow triangle. Sequence numbering is shown above Based on its genome sequence, φX216 is a P2-like member of the Myoviridae subgroup Proteasome inhibitor A. Its shares 99.8% pair-wise identity with φ52237 isolated from B. pseudomallei Pasteur 52237 (GenBank: DQ087285.2) [8]. There are 55 differences observed between φX216 and φ52237, which were independently

confirmed by both Illumina and Sanger sequencing. The majority of these differences, cluster within a six gene region predicted to be associated with tail structure and assembly although only 14 are missense mutations resulting in amino acid alterations. However, these mutations are of no biological Trichostatin A solubility dmso consequence since φ52237 and φX216 were found to have identical host ranges (see Additional file 1). Illumina sequencing also produced a second 1,141-bp contig independent of the φX216 genome contig. This contig has 100% pairwise identity with the highly active IS407a insertion element found in the B. mallei genome [11]. At present we do not know whether this contig is the result of IS407a insertion in a sub-population of φX216 virions during preparation of the B. mallei lysates used for Illumina sequencing or an integral part of φX216 DNA. However, since the IS407a insertion was absent from the genome sequence

obtained these by Sanger sequencing it is unlikely an indigenous part of the φX216 genome. Burkholderia P2-like prophage distribution and correlation with ϕX216 host range Although φX216 has a broad B. pseudomallei host range it fails to form plaques on approximately 22% of the strains tested in this study. We sought to determine if this was perhaps due to infection immunity conferred by the presence of related prophages. To that end, we designed a series of multiplex and individual PCR probes based on six isolated or predicted Burkholderia P2-like phages from Ronning et al. [8]. These included three subgroup A (φE202, φK96243 and φ52237/φX216) and three subgroup B (φE12-2, GI15, PI-E264-2) P2-like phages (see Additional file 2) [8]. PCR probes were designed to identify candidate P2-like prophages with increasing levels of relatedness to φX216/φ52237. The P2-like 1 and P2-like 2 probes amplify regions in the capsid gene (gene #6; for gene numbers see GenBank: JX681814) and Fels-2 gene (gene #29) and are conserved in both P2-like A and B subgroups.

To make the transcriptional fusion of gtsA (PP1015) with lacZ reporter gene, we used the promoter probe plasmid p9TTBlacZ.

The 980-bp-long gtsA promoter region was amplified from P. putida PaW85 chromosome see more using oligonucleotides PP1014kesk (5′-GCTGTCGACGCCAATACGCT) and PP1015alg (5′-GCATCTAGACGAAGCGTGGAATTCATC). The PCR-amplified DNA fragment was cleaved with HincII and XbaI and ligated into SmaI-XbaI-opened p9TTBlacZ, yielding p9TT1015. β-galactosidase assay β-galactosidase activities were measured either from solid or liquid medium-grown bacteria. For the analysis of gtsA promoter, total enzyme activity was measured using permeabilized cells as described elsewhere [33]. Cell lysis assay To evaluate the cell lysis of the colR mutant, we have previously used so-called unmasked β-galactosidase assay which relies on the detection of a cytoplasmic enzyme β-galactosidase leaked out from the cells [25, 34]. In this assay we measured the β-galactosidase activity in suspension of cells permeabilized Protein Tyrosine Kinase inhibitor with SDS and chloroform (total activity), and also in intact, non-permeabilized cells. The percentage of unmasked β-galactosidase activity was calculated from equation: xn/xp × 100%, where xp is β-galactosidase

activity measured in SDS and chloroform-treated cells, and xn is β-galactosidase activity measured in non-permeabilized cells. We have shown earlier that in case of ColR-deficiency-dependent cell lysis, unmasked β-galactosidase values are above 5% [25]. As a source of β-galactosidase, the plasmid pKTlacZS/C containing the lacZ gene, was used [35]. Bacteria were grown for 24, 48, or 72 hours on glucose (0.2, 0.4, or 0.8%) or gluconate (0.2%) M9 minimal media. To enhance lysis, 1 mM phenol was added to the growth medium in some experiments. Bacteria were scraped off the agar plate using plastic Cobimetinib chemical structure swabs and suspended in M9 solution. Optical density of the cell suspension was determined

at 580 nm and β-galactosidase activity was measured [34]. Isolation of outer membrane proteins For the isolation of outer membrane proteins (OMPs) bacteria were grown for 24 hours on two Petri plates. Bacteria were scraped off the agar and suspended in 3 ml of 10 mM HEPES buffer (pH 7.4). For the analyses of peripheral and central subpopulations, bacteria were grown on agar plate in sectors as pictured in Results. To collect enough cells from the sectors, five to ten plates were used, i.e., cells from 15 to 30 sectors per strain were collected and suspended in 3 ml of 10 mM HEPES buffer (pH 7.4). Cells were disrupted by ultrasonication and the cell debris was pelleted by centrifugation at 10 000 g at 4°C for 10 minutes. The supernatant was then centrifuged at 100 000 g at 4°C for 1 hour to pellet membrane proteins.

alleles Simpson’s Unique SNP SNP/allele (average) Mutations per allele (average) dN/dS    

    ID CI (95%)     Silent Conserv. Missense Nonsense Average St. dev.   blaZ 54 11 79.2 69.6-88.8 41 11.4 5.3 1.7 3.7 0.1 0.21 0.11 MRSA blaI 27 7 82.1 74.6-89.5 10 3.9 2.9 0 1.0 0 0.11 0.05   blaR1 31 10 88.8 83.2-94.4 60 24.4 9.7 5.3 8.0 0 0.24 0.11   blaZ 24 9 76.1 61.3-90.9 35 14.7 7.1 1.9 4.6 0 0.17 0.04 MSSA blaI 20 6 74.2 60.5-87.9 9 2.5 1.5 0.2 0.8 0 0.08 0.03   blaR1 17 8 88.2 81.2-95.3 61 24.6 10.4 5.5 7.8 0 0.24 0.10   blaZ 78 13 81.1 75.0-87.3 43 12.4 5.8 1.8 4.0 0.1 0.20 0.10 All blaI 47 9 78.4 71.0-85.9 13 3.4 2.3 0.1 1.0 0 0.10 0.04   blaR1 OTX015 mouse 48 12 88.5 84.0-93.0 65 24.8 10.2 5.3 8.1 0 0.25 0.10 ID, index of diversity; CI, confidence interval; SNP, single-nucleotide polymorphism; Conserv., Apoptosis Compound Library mw conservative; St. dev., standard deviation Figure 1 blaZ allotype frequency per MRSA lineage as defined

by MLST clonal cluster. Figure 2 Cluster tree of blaZ gene allotypes found in the MRSA and MSSA collections. The tree was constructed with the neighbor-joining (NJ) method. In each branch is shown the corresponding bootstrap NJ values, taken over 1000 replicates, which assigns confidence values for the groupings in the tree. For each allele, it is indicated the collection(s) (MRSA or MSSA) and genetic lineage (clonal cluster) where it was found. The BlaZ variability in the MRSA and MSSA strains at the protein level was evaluated by comparison of the deduced amino acid sequence of all alleles against the deduced amino acid sequence for the BlaZ of Tn552. Overall, the deduced amino acid sequences of blaZ alleles from the MRSA and MSSA strains revealed on average 5.8 silent mutations, 1.8 conservative missense mutations and 4 non-conservative missense mutations per allotype (see Tables 3 and 4). For MRSA strain HAR40, a nonsense mutation at Gln76 was detected which presumably originates a non-functional truncated BlaZ protein. As this strain was positive for the nitrocefin test, the DNA extraction and the blaZ sequencing were repeated and the nonsense mutation was confirmed.

No frameshift mutations were found in blaZ allotypes. Allelic variability of blaZ regulatory genes Based on the blaZ variability analysis, we selected 51 representative strains to further characterize the variability in the blaZ selleck inhibitor regulatory genes, blaI and blaR1. Some of these strains failed in the amplification of one of the blaZ regulatory genes (see Tables 1 and 2). Within the length of blaI region analyzed (351 nucleotides), we detected 13 unique SNP, which account for the nine blaI allotypes detected (see Tables 3 and 4).

e) Theoretical molecular weight and pI. f) MASCOT score of MS/MS. g) Number of peptides identified by MS/MS. h) Functional classification using KEGG database. i) the ratio of ratoon cane soil (RS)

to control soil (CK). j) the ratio of ratoon cane soil (RS) to plant cane soil (NS). Among the plant-originating differentially expressed proteins, the largest functional group found was of the proteins involved in carbohydrate and energy metabolism (constituting 47.37%), followed by those associated with stress/defense response (constituting 15.79%) (Figure 5). Furthermore, most of plant proteins related to carbohydrate/energy metabolism (including spot 12, succinate dehydrogenase; spot 13, phosphofructokinase; spots 16 and 35, glyceraldehyde-3-phosphate dehydrogenase; spot 28, NADP dependent malic enzyme and spot 32, fumarate hydratase 1) and amino acid metabolism (i.e. GDC-0068 spot 25, betaine aldehyde hydrogenase) were found up-regulated in the ratoon cane soil, compared to the plant cane and control soils (Table 4). These up-regulated plant proteins involved in carbohydrate and amino acid metabolism probably provide the energy necessary Olaparib and precursor materials for plant root secretion and rhizodeposition process, which serve as a nutrient source for root-associated microbes. Several proteins (including spot 4, catalase; spot 23, PrMC3 and spot 27, heat

shock 70 kDa protein) related to plant stress defense were up-regulated MRIP in the ratoon cane soil (Table 4). Figure 5 The functional category distribution of differentially expressed proteins originated from the plants (a) and the microbes (b). Among the microbe-originating

differentially expressed proteins, most of them were associated with the carbohydrate/energy metabolism (22.22%) and signal transduction (22.22%) (Figure 5). Several microbial proteins were found related to the root-colonizing ability of microorganisms (including spot 30, two-component system sensor kinase) and the utilization of root exudates (including spot 2, sugar ABC transporter and spot 5, ABC transporter ATP-binding subunit) were up-regulated in the ratoon cane soil, as compared to the plant cane and control soil (Table 4), which might be a response of microbes to the rhizodeposition of ratoon cane. Furthermore, most of proteins originated from fungi (including spot 3, mitochondrial N-glycosylase/DNA lyase; spot 7, ORP1; spot 20, kinesin-like protein and spot 34, isocitrate dehydrogenase) were up-regulated in the ratoon cane soil (Table 4). Besides, one cytoskeleton protein (spot 38, i.e. tubulin gamma) originated from the fauna was identified as well. Therefore, sugarcane ratooning induced the alteration of the expression of soil proteins from the plants, microbes and fauna. Discussion The consecutive monocultures for many medicinal plants and crop plants, such as Rehmannia glutinosa[22] and soybea [23], etc., result in a significant reduction in the yield and quality of the harvest.

brevicompactum mpaF (type IMPDH-B). There are 30 residues known to be important for catalytic function and these are completely conserved in all IMPDHs identified at present [1]. All of the 30 residues, except for the one corresponding to position 415 (numbering follows MpaFp), were also conserved in IMPDH-B from both P. chrysogenum and P. brevicompactum. The residue at position 415 is part of the active site and was found to be phenylalanine in both IMPDH-B sequences (Figure 4); whereas

this position is featured by tyrosine in all IMPDH-A type proteins. In addition, when comparing IMPDH-A and IMPDH-B sequences, the so-called IMPDH click here “”flap-region”" [1] is variable including a five-residue-long gap in the two IMPDH-Bs (Figure 4). Although these sequence differences may seem significant, they are not obvious candidates for conferring MPA resistance. The substitution

at position 415 is not in close proximity to the MPA binding site and the sequence of the “”flap-region”" is known to be highly variable and has so far not been linked to MPA sensitivity [16]. Furthermore, P. chrysogenum is not a MPA producer and it is therefore not self-evident that the IMPDH-B from this fungus is resistant. Additional IMPDH sequences from MPA producers and non-producers will be see more useful in the search for the functionally critical residues. Moreover, comparative biochemical characterization of IMPDH-A and IMPDH-B, as well as of mutant derivatives, will be necessary to quantify the degree of resistance,

and to pinpoint the residues important for MPA resistance. Such biochemical characterization, together with the measurement of expression levels of IMPDH-A and IMPDH-B in MPA producers, will help in dissecting the relative contribution of each type to MPA self-resistance. Figure 4 Multiple sequence alignment of selected fungal IMPDHs. The region ROS1 including the amino acid residue at position 415 and part of the flap-region (flap-region being spanned by residues 412 – 467) is presented in the figure. The position 415 is tyrosine in all IMPDHs identified prior to this work [1]. Note that the flap region is very variable, with only residue 415Y and key catalytic residues 441R and 442Y completely conserved in all IMPDHs identified prior to this work [1]. Residues conserved among all nine sequences are highlighted in grey. P. brevicompactum IMPDH-B (encoded by mpaF) is used as a reference while referring to position numbers. P, Penicillium; A, Aspergillus. IMPDH-B has possibly emerged through gene duplication IMPDHs are highly conserved enzymes, which points to their important role in fitness. A high level of conservation was also observed for the sequences obtained from the six Penicillium strains investigated in our study.