- Volume 154, Issue 1, 2008
Volume 154, Issue 1, 2008
- Reviews
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Are all horizontal gene transfers created equal? Prospects for mechanism-based studies of HGT patterns
More LessDetecting patterns of horizontal gene transfer (HGT) in genomic sequences is an important problem, with implications for evolution, ecology, biotechnology and medicine. Extensive genetic, biochemical and genomic studies have provided a good understanding of sequence features that are associated with many (though not all) known mobile elements and mechanisms of gene transfer. This information, however, is not currently incorporated into automated methods for gene transfer detection in genomic data. In this review, we argue that automated annotation of sequence features associated with gene transfer mechanisms could be used both to build more sensitive, mechanism-specific compositional models for the detection of some types of HGT in genomic data, and to ask new questions about the classes of genes most frequently transferred by each mechanism. We then summarize the genes and sequence features associated with different mechanisms of horizontal transfer, emphasizing those that are most useful for distinguishing types of transfer when examining genomic data, and noting those classes of transfers that cannot be distinguished in genomic data using existing techniques. Finally, we describe software, databases and algorithms for identifying particular classes of mobile elements, and outline prospects for better detection of HGT based on specific mechanisms of transfer.
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The post-transcriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts
More LessThe importance of Csr post-transcriptional systems is gradually emerging; these systems control a variety of virulence-linked physiological traits in many pathogenic bacteria. This review focuses on the central role that Csr systems play in the pathogenesis of certain bacteria and in the establishment of successful infections in animal hosts. Csr systems appear to control the ‘switch’ between different physiological states in the infection process; for example switching pathogens from a colonization state to a persistence state. Csr systems are controlled by two-component sensor/regulator systems and by non-coding RNAs. In addition, recent findings suggest that the RNA chaperone Hfq may play an integral role in Csr-mediated bacterial adaptation to the host environment.
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- Cell And Developmental Biology
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Different patterns of integral membrane protein localization during cell division in Bacillus subtilis
More LessCell division in rod-shaped bacteria nearly always occurs exactly at mid-cell and is dependent on the formation of the cytokinetic FtsZ ring and its associated division proteins. Many thousands of copies of division, or septum-specific proteins assemble at this site and may lead to the exclusion of other integral membrane proteins that are normally able to diffuse freely throughout the cytoplasmic membrane. In this study we have investigated the localization of a series of integral membrane proteins in Bacillus subtilis and we show that the recruitment of division and septum-specific proteins does not necessarily preclude the diffusion of other integral membrane proteins. However, some proteins, namely ATP synthase and succinate dehydrogenase, are reduced/absent from the mid-cell region at the onset of cell division, which may reflect an association with lipid domains rich in phosphatidylglycerol that are thought to be present at diminished levels at sites of cell division.
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Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives
More LessThe Mycoplasma pneumoniae cluster is a clade of eight described species which all exhibit cellular polarity. Their polar attachment organelle is a hub of cellular activities including cytadherence and gliding motility, and its duplication in the species M. pneumoniae is coordinated with cell division and DNA replication. The attachment organelle houses a detergent-insoluble, electron-dense core whose presence is required for structural integrity. Although mutant analysis has led to the identification of attachment organelle proteins, the mechanistic basis for the activities of the attachment organelle remains poorly understood, with gliding motility attributed alternatively to the core or to the adhesins. In this study we investigated attachment organelle-associated phenotypes, including gliding motility characteristics and ultrastructural details, in seven species of the M. pneumoniae cluster under identical conditions, allowing direct comparison. We identified gliding ability in three species in which it has not previously been reported, Mycoplasma imitans, Mycoplasma pirum and Mycoplasma testudinis. Across species, ultrastructural features of attachment organelles and their cores do not correlate with gliding speed, and morphological features of cores are inconsistent with predictions about how these structures are involved in the gliding process, disfavouring a prominent, direct role for the electron-dense core in gliding. In addition, we found M. pneumoniae to be an outlier in terms of cell structure with respect to its close relatives, suggesting that it has acquired a special set of adaptations during its evolution.
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- Biochemistry And Molecular Biology
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The phagosomal nutrient transporter (Pht) family
More LessPhagosomal transporters (Phts), required for intracellular growth of Legionella pneumophila, comprise a novel family of multispanning α-helical proteins within the major facilitator superfamily (MFS). The members of this family derive exclusively from bacteria. Multiple paralogues are present in a restricted group of Alpha- and Gammaproteobacteria, but single members were also found in Chlamydia and Cyanobacteria. Their protein sequences were aligned, yielding a phylogenetic tree showing the relations of the proteins to each other. Topological analyses revealed a probable 12 α-helical transmembrane segment (TMS) topology. Motif identification and statistical analyses provided convincing evidence that these proteins arose from a six TMS precursor by intragenic duplication. The phylogenetic tree revealed some potential orthologous relationships, suggestive of common function. However, several probable examples of lateral transfer of the encoding genetic material between bacteria were identified and analysed. The Pht family most closely resembles a smaller MFS family (the UMF9 family) with no functionally characterized members. However, the UMF9 family occurs in a broader range of prokaryotic organism types, including Archaea. These two families differ in that organisms bearing members of the Pht family often have numerous paralogues, whereas organisms bearing members of the UMF9 family never have more than two. This work serves to characterize two novel families within the MFS and provides compelling evidence for horizontal transfer of some of the family members.
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Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU
Type IV pili are retractable protein fibres used by many bacterial pathogens for adherence, twitching motility, biofilm development and host colonization. In Pseudomonas aeruginosa, PilB and PilT are bipolar proteins belonging to the secretion NTPase superfamily, and power pilus extension and retraction, respectively, while the unipolar PilT paralogue PilU supports pilus retraction in an unknown manner. Assay of purified 6×His-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. Conserved residues in the Walker A (WA), Walker B (WB), Asp Box and His Box motifs characteristic of secretion NTPases were mutated, and complementation of twitching motility was tested. Mutation of conserved WA or WB residues in any of the three ATPases abrogated twitching motility, and for the WA mutant of PilT caused loss of polar localization. The requirement for three invariant acidic residues in the Asp Box motif, and for two invariant His residues in the His Box motif varied, with PilB being the least tolerant of changes. In all three proteins, the third acidic residue in the Asp Box and the second His of the His Box were crucial for function; mutation of these residues caused loss of PilT ATPase activity in vitro. Modelling of the effects of these mutations on the crystal structures of Aquifex aeolicus PilT and Vibrio cholerae EpsE (a PilB homologue) showed that the critical Asp Box and His Box residues contribute to a catalytic pocket that surrounds the ligand. These results provide experimental evidence differentiating widely conserved Asp and His Box residues that are essential for function from those whose roles are modulated by specific local environments.
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N-terminal residues of SipB are required for its surface localization on Salmonella enterica serovar Typhimurium
SipB, one of the invasion proteins encoded in Salmonella pathogenicity island 1 (SPI-1), is known to be secreted outside the cell, where it functions as a translocon by assembling into a host-cell plasma membrane-integral structure. Here, we confirmed that wild-type SipB could be localized to the bacterial outer membrane, and further showed that its localization was dependent on extracellular secretion, and was independent of the presence of the SipD protein. Proteinase K susceptibility and immunofluorescence assays indicated that SipB was not incorporated into the outer membrane, but rather was displayed on the bacterial surface. Finally, mutation studies revealed that the N-terminal 100–140 aa (especially amino acids 135–138) of SipB were required for its localization on the bacterial outer membrane.
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cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae
The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulatory effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene (norB) needs to be elucidated. To accomplish this, we analysed the functional regions of the norB upstream region. The promoter contains an extended −10 motif (TGNTACAAT) that is required for high-level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the −10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 bp inverted repeat sequence immediately downstream of the extended −10 motif gave high levels of aerobic expression of a norB : : lacZ fusion. Insertional inactivation of gonococcal nsrR, predicted to bind to this inverted repeat sequence, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In Escherichia coli, expression of a gonococcal nsrR gene repressed gonococcal norB; induction of norB occurred in the presence of exogenously added NO. NsrR also regulates aniA and dnrN, as well as its own expression. We also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homologue, a second repressor whose putative binding site overlaps the Fur binding site.
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EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis
More LessThe Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Araβ(1→2)Araα(1→5)] [Araβ(1→2)Araα(1→3)]Araα(1→5)Araα1→(Ara6) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P embA (Mtb) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P embA (Mtb) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P embA (Mtb) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.
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Bacterial sulfite dehydrogenases in organotrophic metabolism: separation and identification in Cupriavidus necator H16 and in Delftia acidovorans SPH-1
More LessThe utilization of organosulfonates as carbon sources by aerobic or nitrate-reducing bacteria usually involves a measurable, uncharacterized sulfite dehydrogenase. This is tacitly assumed to be sulfite : ferricytochrome-c oxidoreductase [EC 1.8.2.1], despite negligible interaction with (eukaryotic) cytochrome c: the enzyme is assayed at high specific activity with ferricyanide as electron acceptor. Purified periplasmic sulfite dehydrogenases (SorAB, SoxCD) are known from chemoautotrophic growth and are termed ‘sulfite oxidases’ by bioinformatic services. The catalytic unit (SorA, SoxC; termed ‘sulfite oxidases’ cd02114 and cd02113, respectively) binds a molybdenum-cofactor (Moco), and involves a cytochrome c (SorB, SoxD) as electron acceptor. The genomes of several bacteria that express a sulfite dehydrogenase during heterotrophic growth contain neither sorAB nor soxCD genes; others contain at least four paralogues, for example Cupriavidus necator H16, which is known to express an inducible sulfite dehydrogenase during growth with taurine (2-aminoethanesulfonate). This soluble enzyme was enriched 320-fold in four steps. The 40 kDa protein (denatured) had an N-terminal amino acid sequence which started at position 42 of the deduced sequence of H16_B0860 (termed ‘sulfite oxidase’ cd02114), which we named SorA. The neighbouring gene is an orthologue of sorB, and the sorAB genes were co-transcribed. Cell fractionation showed SorA to be periplasmic. The corresponding enzyme in Delftia acidovorans SPH-1 was enriched 270-fold, identified as Daci_0055 (termed ‘sulfite oxidase’ cd02110) and has a cytochrome c encoded downstream. We presume, from genomic data for bacteria and archaea, that there are several subgroups of sulfite dehydrogenases, which all contain a Moco, and transfer electrons to a specific cytochrome c.
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Analysis of the determinants of bba64 (P35) gene expression in Borrelia burgdorferi using a gfp reporter
More LessThe bba64 (P35) gene of Borrelia burgdorferi, the agent of Lyme disease, encodes a surface-exposed lipoprotein. The expression of bba64 in vitro is tightly regulated and dependent on several environmental factors. In nature, its expression is induced in the tick vector during feeding and maintained during infection of the vertebrate host. The pattern of expression of bba64 suggests that it imparts a critical function to the pathogen. A previous study has shown that the expression of bba64 is down-regulated in the absence of RpoS, suggesting that the alternative sigma factor may be involved in its expression. A DNA-binding protein has also been shown to specifically recognize a sequence in the 5′ regulatory region of the gene. Therefore, the contribution of these putative determinants to the differential expression of bba64 was investigated. The role of RpoS was critically evaluated by genetic complementation of the rpoS mutant using a chromosomally targeted copy of the wild-type gene. The results confirm that RpoS is indeed required for the expression of bba64. The role of the upstream DNA-binding site was examined using bba64 promoter–gfp transcriptional fusions in a shuttle vector. The DNA-binding site was studied by targeting mutations to an inverted repeat sequence (IRS), the most prominent feature within the binding site, as well as by deletion of the entire sequence upstream of the basal promoter. Quantitative assessment of gene expression demonstrated that neither the IRS nor the sequence upstream of the promoter was essential for expression. Moreover, the expression of the reporter (GFP) appeared to remain RpoS-dependent in all cases, based on the co-expression of GFP and OspC in a subpopulation of spirochaetes and the selective expression of GFP in the stationary phase. Collectively, the data indicate that RpoS is the sole determinant of differential bba64 expression in cultured spirochaetes.
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New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB
More LessThe expression of the bzd genes involved in the anaerobic degradation of benzoate in Azoarcus sp. CIB is controlled by the specific BzdR transcriptional repressor at the PN promoter. This catabolic promoter is also subject to catabolite repression by some organic acids. In vivo and in vitro experiments have shown that BzdR behaves as a repressor of the PR promoter by overlapping the transcription initiation site as well as the −35 and −10 boxes, benzoyl-CoA being the inducer molecule. In addition, by using a PN : : lacZ fusion both in Azoarcus sp. CIB and in an isogenic strain lacking the bzdR gene, we have shown that the succinate-dependent catabolite repression requires participation of the BzdR repressor.
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- Biodiversity And Evolution
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kinA mRNA is missing a stop codon in the undomesticated Bacillus subtilis strain ATCC 6051
More LessSeveral features distinguish laboratory and undomesticated strains of Bacillus subtilis. For example, unlike the laboratory strain 168, the undomesticated strain ATCC 6051 is deficient in sporulation in a rich sporulation medium, 2× SG. ATCC 6051 cannot induce transcription of the spoIIG operon, suggesting that this strain has a defect in initiation of sporulation. To determine the genetic difference between 168 and ATCC 6051, the DNA region responsible for the Spo− phenotype was transferred to strain 168. Genetic mapping and DNA sequencing analysis revealed that a stop codon (TAA) for kinA in 168 is replaced with Lys (TAT) in ATCC 6051, making the kinA open reading frame 201 bp longer in the undomesticated strain ATCC 6051. Introduction of kinA from strain 168 restored sporulation in ATCC 6051, indicating that the difference in kinA is responsible for the Spo− phenotype of ATCC 6051. A potential ρ-independent terminator is located upstream of a stop codon for the extended kinA open reading frame in ATCC 6051. Northern blot analysis showed that transcription of kinA terminated at this terminator, and kinA mRNA is missing a stop codon in ATCC 6051. Moreover, deletion of tmRNA suppresses the sporulation defect in ATCC 6051. These observations indicate that in ATCC 6051 the absence of a stop codon in kinA mRNA affects sporulation, probably by leading to rapid degradation of KinA via the trans-translation process. In ATCC 6051, the kinA mutation affects sporulation but not other Spo0A-dependent phenomena such as biofilm formation, which can be activated by a low level of Spo0A∼P. This is due to the fact that KinA activity is kept low during the exponential phase via transcriptional and post-translational regulation. Thus, the stop-codon-less kinA probably affects only sporulation. DNA sequencing of 30 B. subtilis strains revealed that another strain also produces stop-codon-less kinA mRNA. This observation suggests that the lack of a stop codon for kinA mRNA may give rise to a selective advantage under certain conditions.
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An analysis of the evolutionary relationships of integron integrases, with emphasis on the prevalence of class 1 integrons in Escherichia coli isolates from clinical and environmental origins
More LessIntegrons are genetic elements that allow the mobilization and expression of smaller elements called gene cassettes, and are considered to be key elements in the evolution of antibiotic resistance among enteric bacteria. Although in nature integrons appear to be abundant, the presence of class 1 integrons in Escherichia coli has been reported to be much less frequent among isolates of non-human origin than among clinical ones. Searching for integrons in a wide variety of E. coli isolates we found a steep decline in class 1 integron prevalence when going from clinical strains to environmental ones, from outdoor urban dust to the microbiota of wild animals. Attempting to assess the causes of this decline, we addressed the evolution of integron integrases, comparing the amino acid sequence of various of these enzymes, the key proteins in gene-cassette mobilization. We found that all integrases are homologues, but different classes have been recruited by enteric bacteria, supporting the notion that integrons can frequently be gained and lost. Additionally, we found that phylogenetically distant organisms that bear intI1, such as E. coli and other enteric bacteria, but also the Gram-positive corynebacteria, have a similar preferential genomic codon usage (CU), suggesting that CU might play an important role in the acquisition and/or maintenance of integrons. In fact, the CU of intI1 is more similar to the preferential genomic CU of non-enteric bacteria than it is to that of E. coli. CU has been proposed to be involved in the retention of horizontally transferred genes; integrons in E. coli are often plasmid-borne. This might explain the reduced prevalence of integrons in enteric bacteria when not under the selective pressure of antibiotics. Collectively, our results provide evidence that class 1 integrons are important gene mobilizers within E. coli, but are not acquired and/or stably maintained without selective pressure. Thus, although not effective to reduce the prevalence of resistance itself, decreasing the use of antibiotics could be useful to diminish the presence of gene-mobilization machineries.
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- Environmental Microbiology
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Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation
More LessIn aquatic environments, biofilms constitute an ecological niche where Legionella pneumophila persists as sessile cells. However, very little information on the sessile mode of life of L. pneumophila is currently available. We report here the development of a model biofilm of L. pneumophila strain Lens and the first transcriptome analysis of L. pneumophila biofilm cells. Global gene expression analysis of sessile cells as compared to two distinct populations of planktonic cells revealed that a substantial proportion of L. pneumophila genes is differentially expressed, as 2.3 % of the 2932 predicted genes exhibited at least a twofold change in gene expression. Comparison with previous results defining the gene expression profile of replicative- and transmissive-phase Legionella suggests that sessile cells resemble bacteria in the replicative phase. Further analysis of the most strongly regulated genes in sessile cells identified two induced gene clusters. One contains genes that encode alkyl hydroperoxide reductases known to act against oxidative stress. The second encodes proteins similar to PvcA and PvcB that are involved in siderophore biosynthesis in Pseudomonas aeruginosa. Since iron has been reported to modify biofilm formation in other species, we further focused on iron control of gene expression and biofilm formation. Among the genes showing the greatest differences in expression between planktonic cells and biofilm, only pvcA and pvcB were regulated by iron concentration. A ΔpvcA L. pneumophila mutant showed no changes in biofilm formation compared to the wild-type, suggesting that the pvcA product is not mandatory for biofilm formation. However, biofilm formation by L. pneumophila wild-type and a ΔpvcA strain was clearly inhibited in iron-rich conditions.
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Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract
More LessMembers of the genus Lactobacillus are common inhabitants of the proximal gastrointestinal tract of animals such as mice, rats, chickens and pigs, where they form epithelial biofilms. Little is known about the traits that facilitate biofilm formation and gut colonization. This study investigated the ecological role of a glucosyltransferase (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 and a fructosyltransferase (FtfA) of L. reuteri LTH5448. In vitro experiments using isogenic mutants revealed that GtfA was essential for sucrose-dependent autoaggregation of L. reuteri TMW1.106 cells under acidic conditions, while inactivation of Inu slowed the formation of cell aggregates. Experiments using an in vitro biofilm assay showed that GtfA and Inu contributed to biofilm formation of L. reuteri TMW1.106. Experiments using ex-Lactobacillus-free mice revealed that the ecological performance of the inu mutant, but not of the gtfA or ftfA mutant, was reduced in the gastrointestinal tract when in competition with the parental strain. In the absence of competition, the gtfA mutant showed delayed colonization of the murine gut relative to the wild-type. In addition, the gtfA mutant showed reduced ecological performance in competition experiments with Lactobacillus johnsonii #21. From the evidence provided in this study we conclude that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract.
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Degradation of ambient carbonyl sulfide by Mycobacterium spp. in soil
More LessThe ability to degrade carbonyl sulfide (COS) was confirmed in seven bacterial strains that were isolated from soil, without the addition of COS. Comparative 16S rRNA gene sequence analysis indicated that these isolates belonged to the genera Mycobacterium, Williamsia and Cupriavidus. For example, Mycobacterium sp. strain THI401, grown on PYG agar medium, was able to degrade an initial level of 30 parts per million by volume COS within 1 h, while 60 % of the initial COS was decreased by abiotic conversion in 30 h. Considering natural COS flux between soil and the atmosphere, COS degradation by these bacteria was confirmed at an ambient level of 500 parts per trillion by volume (p.p.t.v.), using sterilized soil to cultivate the bacterium. Autoclave sterilization of soil resulted in a small amount of COS emission, while Mycobacterium spp. degraded COS at a faster rate than it was emitted from the soil, and reduced the COS mixing ratio to a level that was lower than the ambient level: THI401 degraded COS from an initial level of 530 p.p.t.v. to a level of 330 p.p.t.v. in 30 h. These results provide experimental evidence of microbial activity in soil as a sink for atmospheric COS.
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- Genes And Genomes
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The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098
The coenzyme B12 production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B12 gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQOP, sirA, hemACBL, and cobUSC, hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B12 itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B12 pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B12 biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B12 biosynthesis and reuterin production, which might be implicated in Lb. reuteri's success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B12.
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Presence of a functional flagellar cluster Flag-2 and low-temperature expression of flagellar genes in Yersinia enterocolitica W22703
More LessTwelve Yersinia enterocolitica mutants carrying luxCDABE-transposon insertions in motility and chemotaxis genes were isolated on the basis of strong low-temperature induction. Two transposons were located within an 11.2 kb enteric flagellar cluster 2 (Flag-2) of Y. enterocolitica biotype 2, serotype O : 9 strain W22703. The Flag-2 gene cluster is absent from the corresponding genomic location of the sequenced strain Y. enterocolitica biotype 1B, serotype O : 8 strain 8081. Evidence for the functionality of the O : 9 Flag-2 genes, probably located within the plasticity zone of the genome, is provided by swarming assays. PCR analysis of 49 strains revealed the presence of Flag-2 genes in biotypes 2–5, but not in biotypes 1A or 1B. Bioluminescence, measured between 6 and 37 °C, showed that the expression of all genes located in Flag-2 and in the known flagellar cluster, Flag-1, was highest at approximately 20 °C, and that expression of two Flag-2 genes is FlhC-dependent. In a motility assay, a non-motile and a hyper-motile phenotype resulted from knockout mutations of the Flag-1 genes fliS1 and fliT, respectively. Complemented strains validated these results, confirming the regulatory role of FliT.
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Regulation of the expression of phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) genes in Corynebacterium glutamicum R
More LessThe phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses the transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. In Corynebacterium glutamicum R, the genes ptsH and ptsI encode general components of the PTS, and genes ptsF, ptsS and ptsG each encode fructose-, sucrose- and glucose-specific components of the PTS, respectively. In this study, we examined the mRNA levels of the pts genes in the presence or absence of PTS sugars. Glucose elevated the expression of ptsG, ptsH and ptsI genes, whereas fructose and sucrose induced the expression of all the pts genes examined, i.e. ptsF, -S, -G, -H and -I. We determined the transcriptional start sites of the pts genes and found that these promoters were activated in the presence of fructose. Disruption of fruR, which is a deoxyribonucleoside repressor (DeoR)-type transcriptional regulator co-transcribed with ptsF, resulted in enhanced induction of the fructose-pts operon, ptsI, and ptsH expression in response to fructose, indicating that FruR attenuates the induction of ptsI, ptsH and fructose-pts by fructose.
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- Pathogens And Pathogenicity
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Investigation of the algT operon sequence in mucoid and non-mucoid Pseudomonas aeruginosa isolates from 115 Scandinavian patients with cystic fibrosis and in 88 in vitro non-mucoid revertants
More LessPseudomonas aeruginosa is the dominant pathogen causing chronic lung infections in patients with cystic fibrosis (CF). After an initial phase characterized by intermittent colonizations, a chronic infection is established upon conversion of P. aeruginosa from the non-mucoid to the mucoid, alginate-overproducing phenotype. During the chronic infection the isolation of both mucoid and non-mucoid isolates in CF sputum samples is very common. The purpose of the present study was to establish, by sequence analysis, the types of mutations present in the algTmucABD operon in a large number of mucoid and non-mucoid P. aeruginosa isolates from Scandinavian CF patients and in in vitro-derived non-mucoid revertants. Mucoid (83) and non-mucoid isolates (103) from 91 Scandinavian patients with chronic P. aeruginosa infection and 24 non-mucoid isolates from intermittently colonized CF patients were investigated. In addition, 88 spontaneous non-mucoid revertants obtained in vitro from nine mucoid CF isolates were also included in the study. Mutations in mucA were found in 92 % of the mucoid and in up to 70 % of the non-mucoid isolates from chronically infected patients, indicating that the majority of non-mucoid isolates are revertants. None of the non-mucoid isolates from intermittently colonized CF patients harboured mucA mutations. Although algT has been considered an important gene for secondary-site mutations responsible for reversion to non-mucoidy, only 30 % of the mucA-mutated non-mucoid CF isolates had mutations in algT. In contrast, 83 % of the in vitro-derived spontaneous non-mucoid revertants had mutations in algT, showing that in the CF lung there is a selection for non-mucoid revertants with secondary-site mutations in genes other than algT. In addition, we report, to our knowledge for the first time, loss-of-function mutations in the negative regulators mucB and mucD in CF clinical isolates. In some of the CF isolates these mutations are associated with moderate alginate production. In conclusion, most non-mucoid isolates from chronically infected CF patients are revertants and the mechanism of revertance is algT-independent in the CF lung.
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The ferric yersiniabactin uptake receptor FyuA is required for efficient biofilm formation by urinary tract infectious Escherichia coli in human urine
More LessUrinary tract infection (UTI) is the most common infection in patients with indwelling urinary catheters, and bacterial biofilm formation is a major problem in this type of infection. Escherichia coli is responsible for the large majority of UTIs. Free iron is strictly limited in the human urinary tract and there is fierce competition between the host and infectious bacteria for this essential metal. Urinary tract infectious E. coli have highly efficient mechanisms of iron acquisition, one of which is the yersiniabactin system. The fyuA gene, encoding the yersiniabactin receptor, is one of the most upregulated genes in biofilm; it was upregulated 63-fold in the E. coli UTI strain VR50. FyuA was found to be highly important for biofilm formation in iron-poor environments such as human urine. Mutants in fyuA show aberrant biofilm formation and the cells become filamentous; a VR50fyuA mutant showed a 92 % reduction in biofilm formation in urine flow-cell chambers compared with the wild-type. The FyuA/yersiniabactin system is known to be important for virulence. Here we demonstrate a direct link between FyuA and biofilm formation in iron-poor environments. We also show that the availability of iron greatly influences UTI strains' ability to form biofilm.
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Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C
Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx2 variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx2-vhb variant, which was frequently associated with stx2 , stx2-vha or stx2c . Levels of stx2 and stx2-related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx2 variant than in the remaining strains of seropathotype C. The stx2-vhb genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx2 -related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx2 -related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx2 gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.
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Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains
More LessThe conjugative plasmid pOLA52, which confers resistance to olaquindox and other antimicrobial agents through a multidrug efflux pump, was investigated for its ability to promote biofilm formation in Escherichia coli. Screening of a transposon-mutagenized pOLA52 clone library revealed several biofilm-deficient mutants, which all mapped within a putative operon with high homology to the mrkABCDF operon of Klebsiella pneumoniae, where these genes are responsible for type 3 fimbriae expression, attachment to surfaces and biofilm formation. Biofilm formation in microtitre plates and in urinary catheters of clones containing pOLA52 with a disrupted putative mrk operon was reduced by more than 100-fold and 2-fold, respectively, compared to mutants with an intact mrk operon. The conjugative transfer rate of pOLA52 was also significantly lower when the mrk operon was disrupted. Through reverse transcriptase analysis, it was demonstrated that the genes contained in the putative mrk operon were linked and likely to be expressed as a single operon. Immunoblotting with type 3 fimbriae (MrkA)-specific antibodies further verified expression of type 3 fimbriae. When transferred to other, potentially pathogenic, members of the family Enterobacteriaceae, including Klebsiella pneumoniae, Salmonella Typhimurium, Kluyvera sp. and Enterobacter aerogenes, pOLA52 facilitated increased biofilm formation. pOLA52 is believed to represent the first example of a conjugative plasmid encoding type 3 fimbriae, resulting in enhanced conjugation frequencies and biofilm formation of the plasmid-harbouring strain.
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Reduced DNA binding and uptake in the absence of DsbA1 and DsbA2 of Neisseria meningitidis due to inefficient folding of the outer-membrane secretin PilQ
More LessDsbA ensures the correct folding of many exported bacterial proteins by forming intramolecular disulphide bonds in the bacterial periplasm. The pathogen Neisseria meningitidis is unusual in its possession of three different dsbA genes (dsbA1, dsbA2 and dsbA3), encoding two membrane-anchored (DsbA1 and DsbA2) and one periplasmic (DsbA3) thiol-disulphide oxidoreductase enzymes. In this study, the involvement of DsbA1 and DsbA2 in natural competence was confirmed and attributed to events in the early stages of the transformation process. Strains lacking both DsbA1 and DsbA2 were reduced in competence as a result of decreased DNA binding and uptake. Overexpression of DsbA3 could not overcome this defect, suggesting differences in substrate specificity and protein-folding abilities between the DsbA homologues. Competence in Neisseria is dependent on the expression of type IV pili, which are extruded and retracted through the outer-membrane secretin PilQ. Both DsbA1 and DsbA2 were able to specifically bind PilQ in solid-phase overlay assays. Consistent with this, deletion of both dsbA1 and dsbA2 resulted in reduced levels of PilQ, confirming inefficient folding of PilQ, while pilus expression was apparently unaffected. The secretin PilQ is involved in DNA binding and transport as well as pilus biogenesis, and the defect in PilQ folding resulting from the absence of DsbA1 and DsbA2 is revealed in the observed decreased DNA binding and uptake.
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- Physiology
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Multiple phenotypic alterations caused by a c-type cytochrome maturation ccmC gene mutation in Pseudomonas aeruginosa
More LessIn some Proteobacteria biogenesis of c-type cytochromes depends on the products of the ccmABCDEFG(H) genes, which encode inner-membrane proteins. Inactivation of some ccm genes, in particular ccmC, has an impact on other processes as well, including siderophore production and utilization. Non-polar insertions were generated in the Pseudomonas aeruginosa ccmA, ccmC, ccmE, ccmF and ccmH genes, and their impacts on different phenotypes were compared. Only in the case of the ccmC mutant was cytochrome c production totally abrogated. The ccmC mutant, and to a lesser extent the ccmF mutant, showed a range of other phenotypic changes. The production of the siderophore pyoverdine was very low and growth under the condition of iron limitation was severely restricted, but production of the second siderophore, pyochelin, was increased. Interestingly, other traits were also strongly affected by the ccmC mutation, including the production of pyocyanin, swarming and twitching motility, and rhamnolipid production. The production of N-acyl homoserine lactones or the Pseudomonas quinolone signal (PQS) was, however, not affected in the ccmC and ccmF mutants. The ccmC mutant was also found to accumulate porphyrins, and catalase production was undetectable, consistent with the increased sensitivity to hydrogen peroxide. Finally, reduction in the content of [Fe–S] clusters was evidenced in both ccmC and ccmF mutants. Wild-type phenotypes were restored by complementation with a ccmC gene from Pseudomonas fluorescens ATCC 17400. In conclusion, we have demonstrated that CcmC is a key determinant for cytochrome c biogenesis, pyoverdine maturation, and expression of some quorum sensing-regulated traits.
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Involvement of BmoR and BmoG in n-alkane metabolism in ‘Pseudomonas butanovora’
More Less‘Pseudomonas butanovora’ uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C2–C9 n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative σ 54-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C2–C5 n-alkanes and showed little to no growth on C6–C8 n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the ‘P. butanovora’ bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C2–C8 carbon sources. In acetate- or lactate-grown cells, C2–C8 n-alcohols failed to induce β-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced ∼45 % of the β-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C2–C5 n-alcohols induced β-galactosidase activity, whereas C7 and C8 n-alcohols did not. BmoR may act as a σ 54-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. ≤C6). The bmoG-deficient mutant did not grow on C2–C8 n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.
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Low temperature (23 °C) increases expression of biofilm-, cold-shock- and RpoS-dependent genes in Escherichia coli K-12
Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 °C compared to 37 °C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 °C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 °C. Another category of genes more highly expressed at 23 °C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 °C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 °C. Increased expression at 23 °C compared to 37 °C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 °C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 °C and viability at 4 °C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 °C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 °C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 °C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response – the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.
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Identification of proteins involved in formaldehyde metabolism by Rhodobacter sphaeroides
More LessFormaldehyde is an intermediate formed during the metabolism of methanol or other methylated compounds. Many Gram-negative bacteria generate formaldehyde from methanol via a periplasmic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase in which the α subunit of an α 2 β 2 tetramer has catalytic activity. The genome of the facultative formaldehyde-oxidizing bacterium Rhodobacter sphaeroides encodes XoxF, a homologue of the catalytic subunit of a proposed PQQ-containing dehydrogenase of Paracoccus denitrificans. R. sphaeroides xoxF is part of a gene cluster that encodes periplasmic c-type cytochromes, including CycI, isocytochrome c 2 and CycB (a cyt c 553i homologue), as well as adhI, a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), and gfa, a homologue of a glutathione–formaldehyde activating enzyme (Gfa). To test the roles of XoxF, CycB and Gfa in formaldehyde metabolism by R. sphaeroides, we monitored photosynthetic growth with methanol as a source of formaldehyde and whole-cell methanol-dependent oxygen uptake. Our data show that R. sphaeroides cells lacking XoxF or CycB do not exhibit methanol-dependent oxygen uptake and lack the capacity to utilize methanol as a sole photosynthetic carbon source. These results suggest that both proteins are required for formaldehyde metabolism. R. sphaeroides Gfa is not essential to activate formaldehyde, as cells lacking gfa are capable of both methanol-dependent oxygen uptake and growth with methanol as a photosynthetic carbon source.
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Nitrogen status and heat-stress-dependent differential expression of the cpn60 chaperonin gene influences thermotolerance in the cyanobacterium Anabaena
More LessHeat stress caused rapid and severe inhibition of photosynthesis and nitrate reduction in nitrate-supplemented cultures of the cyanobacterium Anabaena sp. strain L-31, compared to nitrogen-fixing cultures. Anabaena strains harbour two hsp60 family genes, groEL and cpn60, respectively encoding the 59 kDa GroEL and 61 kDa Cpn60 chaperonin proteins. Of these two Hsp60 chaperonins, GroEL was strongly induced during heat stress, irrespective of the nitrogen status of the cultures, but Cpn60 was rapidly repressed and degraded in heat-stressed nitrate or ammonium-supplemented cultures. The recovery of photosynthesis, nitrate assimilation and growth in heat-stressed, nitrate-supplemented cultures were preceded by resynthesis and restoration of cellular Cpn60 levels. Glutamine synthetase activity, although adversely affected by prolonged heat stress, was not dependent on either the nitrogen status or Cpn60 levels during heat stress. Overexpression of the Cpn60 protein in the closely related Anabaena sp. strain PCC7120 conferred significant protection from heat stress to growth, photosynthesis and nitrate reduction in the recombinant strain. The data favour a role for Cpn60 in carbon and nitrogen assimilation in Anabaena.
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Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: a chemostat study
Saccharomyces cerevisiae SFP1 promotes transcription of a large cluster of genes involved in ribosome biogenesis. During growth in shake flasks, a mutant deleted for SFP1 shows a small size phenotype and a reduced growth rate. We characterized the behaviour of an sfp1Δ mutant compared to an isogenic reference strain growing in chemostat cultures at the same specific growth rate. By studying glucose (anaerobic)- and ethanol (aerobic)-limited cultures we focused specifically on nutrient-dependent effects. Major differences in the genome-wide transcriptional profiles were observed during glucose-limited growth. In particular, Sfp1 appeared to be involved in the control of ribosome biogenesis but not of ribosomal protein gene expression. Flow cytometric analyses revealed size defects for the mutant under both growth conditions. Our results suggest that Sfp1 plays a role in transcriptional and cell size control, operating at two different levels of the regulatory network linking growth, metabolism and cell size.
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- Plant-Microbe Interactions
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Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit
More LessFusarium verticillioides produces the mycotoxin fumonisin B1 (FB1) on maize kernels. In this study, we identified a putative protein phosphatase gene CPP1 in F. verticillioides, and investigated its role in FB1 regulation. Previous work has shown that CPP1 expression is elevated in an FB1-suppressing genetic background. Thus, we hypothesized that CPP1 is negatively associated with FB1 production. To test this hypothesis, we generated a CPP1 knockout mutant, PP179, and studied the effects of gene deletion on FB1 biosynthesis and fungal development. PP179 showed elevated expression of FUM genes, and in turn produced higher levels of FB1 than the wild-type progenitor. Other significant mutant phenotypes included reduced radial growth on agar plates, reduced conidia germination rates, significantly increased macroconidia formation, and hyphal swelling. To verify that these phenotypes were directly due to CPP1 deletion, we complemented PP179 with the wild-type CPP1 gene. The complemented strain PPC4 showed FUM1 expression and FB1 production similar to that of the wild-type, providing evidence that CPP1 is negatively associated with FB1 biosynthesis. Other PP179 phenotypes, such as macroconidiation and hyphal swelling, were also restored to that of wild-type progenitor. Furthermore, we complemented F. verticillioides PP179 strain with Neurospora crassa wild-type ppe-1 gene, demonstrating that Cpp1 and PPE-1 proteins are functionally conserved. Pleiotropic effects of CPP1 deletion led us to hypothesize that CPP1 is associated with multiple downstream signalling pathways in F. verticillioides. Identification and functional characterization of downstream Cpp1-interacting proteins are necessary to better understand the complex regulatory mechanisms associated with Cpp1.
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Volumes and issues
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Volume 170 (2024)
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Volume 169 (2023)
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Volume 168 (2022)
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Volume 167 (2021)
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Volume 166 (2020)
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Volume 165 (2019)
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