Wild-type Parent Strain Research Articles

In vitro and in vivo reduced fitness and virulence in ciprofloxacin-resistant Acinetobacter baumannii

Limited data on relative fitness and virulence of antimicrobialresistant Acinetobacter baumannii are known. We aimed to study the virulence and fitness cost of ciprofloxacin-resistance in A. baumannii (CipR) compared with the susceptible parental wild-type strain (CipS). Human lung epithelial cells were infected with CipS and CipR for 24 h. Competition fitness was monitored in vitro and in vivo in a murine peritoneal sepsis model. We showed that CipR induced less cell death than CipS and CipR growth was slow when in competition with CipS. Altogether, acquisition of ciprofloxacin resistance confers a biological fitness cost and reduces virulence in A. baumannii.

Pathway Profiling in Mycobacterium tuberculosis: ELUCIDATION OF CHOLESTEROL-DERIVED CATABOLITE AND ENZYMES THAT CATALYZE ITS METABOLISM

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-(14)C]cholesterol or [1,7,15,22,26-(13)C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2'-propanoate)-3aα-H-4α-(3'-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2'-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2'-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites.

OsmC proteins of Mycobacterium tuberculosis and Mycobacterium smegmatis protect against organic hydroperoxide stress

Bacterial antioxidants play a critical role in the detoxification of endogenously and host derived oxidative radicals during host-pathogen interactions. Recently, the osmotically induced bacterial protein C (OsmC) is included in the antioxidant category of enzymes as it shows structural and functional relationships with organic hydroperoxide reductase (Ohr) enzyme. A copy of the gene encoding OsmC is conserved across mycobacterial species, including Mycobacterium tuberculosis (Rv2923c) and Mycobacterium smegmatis (MSMEG2421), but its role in protecting these species against oxidative stress is unknown. To determine the role of OsmC in mycobacterial oxidative stress, we overexpressed and purified OsmCs of M. tuberculosis and M. smegmatis and assessed their ability to reduce peroxide substrates like hydrogen peroxide (H(2)O(2)), cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BHP) in Ferrous Ion Oxidation in Xylenol (FOX) assay. This revealed that OsmCs from both species were capable of reducing both inorganic (H(2)O(2)) and organic (CHP and t-BHP) peroxides. Further, an M. smegmatis mutant (MS∆osmC) deficient in OsmC exhibited reduced reduction of CHP and t-BHP than the parental wild type strain, indicating that OsmC protein contributes significantly for the total peroxide reductase activity of mycobacteria. The MS∆osmC strain was also sensitive to organic hydroperoxides, which could be reversed by complementing with a plasmid borne osmC. Plasmid borne osmC also increased the resistance of M. smegmatis wild type strain to isoniazid (INH) but at a relatively lower level than ahpC, an organic hydroperoxide reductase. These results suggest that OsmC plays an important role in peroxide metabolism and protecting mycobacteria against oxidative stress.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

Serine-Rich Repeat Proteins and Pili Promote Streptococcus agalactiae Colonization of the Vaginal Tract

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ΔpilA or Δsrr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment.

Characterization ofCpSte11, a MAPKKK gene ofCryphonectria parasitica, and initial evidence of its involvement in the pheromone response pathway

The gene CpSte11 of Cryphonectria parasitica, which encodes a yeast Ste11 homologue, was cloned and characterized. Gene replacement analysis revealed a high frequency of CpSte11 null mutants. When compared with the wild-type parent strain, CpSte11 null mutants showed no difference in terms of growth rate or pigmentation. However, CpSte11 null mutants showed a marked decrease in both the number and size of stromal pustules on chestnut twigs. The virulence test showed that, in comparison with those of the wild-type and virus-infected hypovirulent strains, CpSte11 null mutants produced necrotic areas of intermediate size. Disruption of the CpSte11 gene also resulted in defects in female fertility. Down-regulation of transcripts for the mating pheromone precursor gene, Mf2/2, and mating response transcription factors, such as cpst12 and pro1, was observed in CpSte11 null mutants. The down-regulation of Mf2/2, cpst12 and pro1 was also observed in the mutant phenotype of Cpmk2, a mating response Fus3-like mitogen-activated protein kinase (MAPK) gene, but not in the mutant of Cpmk1, a high-osmolarity glycerol Hog1-like MAPK gene. These results indicate that the cloned CpSte11 gene is functionally involved in the mating response pathway and acts through downstream targets, including Cpmk2, cpst12, pro1 and Mf2/2. However, the characteristics of the CpSte11 null mutant were fully phenocopied only in the cpst12 null mutant, but not in other studied null mutants of components of the putative mating response pathway.

An evolutionary strategy for isobutanol production strain development in Escherichia coli

Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σ s-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production.

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

Role of pH-regulated antigen 1 of Candida albicans in the fungal recognition and antifungal response of human neutrophils

Candida albicans is an opportunistic human-pathogenic fungus, which can cause superficial but also life-threatening invasive infections. The pH-regulated antigen 1 (Pra1) of C. albicans is a surface-associated and secreted protein highly expressed in the hyphal form. Pra1 can bind to complement receptor 3 (CD11b/CD18) and can mediate adhesion to and migration of human phagocytes. Here, we investigated the role of Pra1 in the activation of human neutrophils. A C. albicans mutant strain lacking Pra1 (pra1Δ) supported neutrophil migration to a lower extent than did the parental wild-type strain. A Pra1-overexpressing C. albicans strain enhanced neutrophil migration and adherence. While inactivated hyphae of the Pra1-overexpressing mutant with surface-associated Pra1 enhanced the production and release of reactive oxygen species, myeloperoxidase, lactoferrin, and interleukin 8 by neutrophils, such responses were reduced when stimulated with inactivated hyphae of the pra1Δ strain. However, Pra1-overexpressing living hyphae, which secrete large amounts of Pra1, also caused a reduced neutrophil activation, indicating that the release of extracellular Pra1 can inhibit the activation of these innate immune cells. Similarly, soluble recombinant Pra1 inhibited the neutrophil responses elicited by cell-wall bound Pra1. Finally, fungal cells lacking Pra1 were more efficiently killed by neutrophils. In conclusion, surface-exposed Pra1 plays a role in the recognition of C. albicans, especially hyphal cells, by human neutrophils and enhances neutrophil antimicrobial responses. However, the fungus can counteract some of these defense mechanisms by releasing soluble Pra1.

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns

IntroductionWe investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains. Materials and methodsWe used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37°C for 150min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA). ResultsIL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p<0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p<0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p<0.05) and 50% (p<0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p<0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p<0.05). ConclusionSusceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.

Inactivation of tesA Reduces Cell Wall Lipid Production and Increases Drug Susceptibility in Mycobacteria

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

Differential Sensitivity to Prepubertal Xenoestrogen Disruption in C57 and CD10 Mice.

Mice are routinely used as model organisms to study the effects of endocrine disrupting chemicals. Different strains of mice differ in their sensitivity to endocrine disruption and the difference in sensitivity may be life stage and or tissue dependent. Previous work has shown that C57BL/6 (C57) and CD-1 mouse strains have differential sensitivity to prepubertal xenoestrogen disruption of the testis. In order to identify mechanisms of differential strain sensitivity to prepubertal xenoestrogen exposure C57 or CD10 mice were backcrossed > seven times to Estrogen Receptor action INdicator (ERIN) mice that contain a transgene with an estrogen responsive promoter linked to the reporter gene beta-galactosidase. At weaning mice were group housed and exposed to ethinyl estradiol [(EE2) 1, 10, or 100 micro-g/kg body weight)] via their drinking water (vehicle control = 0.1% ethanol). Mice were maintained on treated drinking water until collection at 48 days of age. Body, testes, preputial gland, seminal vesicle, and coagulating gland weights were obtained at necropsy. The presence of preputial separation, which is a marker of puberty, and semen in the seminal vesicles was also noted at necropsy. We confirmed that the backcrossed ERIN mice were very similar to their respective wildtype parent strains and exhibited the same differential sensitivity to prepubertal xenoestrogen exposure. Overall, the C57 mice were more sensitive to prepubertal xenoestrogen disruption of the male reproductive tract. The effect on testis weight and preputial separation were the most striking, where C57 mice were over 33-times more sensitive than CD10 mice (IC50 was not reached in CD10 mice). Testes from C57 mice exposed to 1, 10, or 100 micro-g/kg EE2 were 12%, 79%, and 89% smaller, respectively, than those from vehicle exposed mice, whereas testes from CD10 mice were 3%, 11% and 13% smaller than those from vehicle exposed mice. The differential sensitivity of preputial separation was striking at the dose of 10 micro-g/kg EE2, where none of the C57 mice displayed preputial separation while all of the CD10 mice did. Even at the highest EE2 dose, 80% of the CD10 mice still showed preputial separation. This indicates that C57 mice were over 33-times more sensitive to inhibition of puberty than CD10 mice. The preputial glands, seminal vesicles, and coagulating glands decreased in weight following prepubertal xenoestrogen exposure. These tissues were 8-11 times more sensitive to disruption by EE2 in C57 mice than CD10 mice. Preputial gland weight was the most sensitive marker for prepubertal xenoestrogen disruption (IC50 of 1 and 10 micro-g/kg for C57 and CD10 mice, respectively). In summary, C57 mice were much more sensitive to testis disruption by prepubertal xenoestrogen exposure than CD10 mice. The differential sensitivity to estrogen disruption between C57 and CD10 mice, appears to be life stage dependent, however, as we have not found differences in sensitivity to disruption from adult xenoestrogen exposures. Furthermore, the ERIN transgenic mice backcrosses responded very similarly to wildtype mice following prepubertal EE2 exposure and will be used to examine the mechanisms of differential prepubertal disruption by xenoestrogens. (poster)

Candida albicans adhesin Als3p is dispensable for virulence in the mouse model of disseminated candidiasis

The presence of specific proteins, including Ece1p, Hwp1p and Als3p, distinguishes the Candida albicans hyphal cell wall from that of yeast-form cells. These proteins are thought to be important for the ability of C. albicans cells to adhere to living and non-living surfaces and for the cell-to-cell adhesion necessary for biofilm formation, and also to be pivotal in mediating C. albicans interactions with endothelial cells. Using an in vitro flow adhesion assay, we previously observed that yeast cells bind in greater numbers to human microvascular endothelial cells than do hyphal or pseudohyphal cells. This is consistent with previous observations that, in a murine model of disseminated candidiasis, cells locked in the yeast form can efficiently escape the bloodstream and invade host tissues. To more precisely explore the role of Als3p in adhesion and virulence, we deleted both copies of ALS3 in a wild-type C. albicans strain. In agreement with previous studies, our als3Δ null strain formed hyphae normally but was defective in biofilm formation. Whilst ALS3 was not expressed in our null strain, hypha-specific genes such as ECE1 and HWP1 were still induced appropriately. Both the yeast form and the hyphal form of the als3Δ strain adhered to microvascular endothelial cells to the same extent as a wild-type strain under conditions of flow, indicating that Als3p is not a significant mediator of the initial interaction between fungal cells and the endothelium. Finally, in a murine model of haematogenously disseminated candidiasis the mutant als3Δ remained as virulent as the wild-type parent strain.

Ex Vivo Bioluminescence Detection of Alcelaphine Herpesvirus 1 Infection during Malignant Catarrhal Fever

Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and is caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and nonlymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain, and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to rabbits infected with the parental wild-type strain, with hyperthermia and increases of both CD8(+) T cell frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver, and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in nonlymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF.

The lmo0501 gene coding for a putative transcription activator protein in Listeria monocytogenes promotes growth under cold, osmotic and acid stress conditions

In Listeria monocytogenes EGDe, the lmo0501 gene locus encodes a protein similar to the mannitol transcription regulator (MltR) protein in Bacillus subtilis and Bacillus stearothermophilus. In this study we investigated its functional role in L. monocytogenes EGDe cells in view of growth under different stress conditions. Increased lmo0501 gene expression at mRNA level was detected in response to cold, osmotic and organic acid stress exposure. An EGDe Δlmo0501 mutant strain was diminished in growth compared to the wild type strain in minimal defined medium containing either glucose or fructose, as carbon sources. Growth of the lmo0501 null mutant was retarded growth under cold (4 °C), salt (NaCl) and organic acid stress conditions relative to the parental wild type strain. Our results confirm the role of the lmo0501 gene in adaptation of L. monocytogenes cells to food preservation stress conditions as well as to the efficient utilization of glucose and fructose as carbon sources.

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase ( hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM ®-T-Easy cloning vector (pGEM ®-T- hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM ®-T- hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X- hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H 2/mol glucose, 1.68 fold higher than the wild type parent strain.

Genotype and Phenotypes of an Intestine-Adapted Escherichia coli K-12 Mutant Selected by Animal Passage for Superior Colonization

We previously isolated a spontaneous mutant of Escherichia coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, that has colonization traits superior to the wild-type parent strain (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). This intestine-adapted strain (E. coli MG1655*) grew faster on several different carbon sources than the wild type and was nonmotile due to deletion of the flhD gene. We now report the results of several high-throughput genomic analysis approaches to further characterize E. coli MG1655*. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655*. Microarray analysis revealed modest yet significant induction of catabolic gene systems across the genome in both E. coli MG1655* and an isogenic flhD mutant constructed in the laboratory. Catabolome analysis with Biolog GN2 microplates revealed an enhanced ability of both E. coli MG1655* and the isogenic flhD mutant to oxidize a variety of carbon sources. The results show that intestine-adapted E. coli MG1655* is more fit than the wild type for intestinal colonization, because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, resulting in more efficient carbon source utilization and a higher intestinal population. Hence, mutations that enhance metabolic efficiency confer a colonization advantage.

The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence.

The MAT1-2-1 mating-type gene upregulates photo-inducible carotenoid biosynthesis in Fusarium verticillioides

Filamentous ascomycetes, including mitotic holomorphs, have constitutively transcribed MAT (mating type) genes. These genes encode transcription factors considered to be the major regulators of sexual communication. The proven targets of the MAT transcription factors are pheromone precursor and pheromone receptor genes. However, recent studies demonstrated that MAT proteins may also affect other genes not involved directly in the mating process. When grown in the light, Fusarium verticillioides produces the acidic xanthophyll neurosporoxanthin and lower amounts of nonpolar precursor carotenes, such as phytoene, torulene, β-carotene, and γ-carotene. Depending on the illumination conditions, a drastic decrease or the absence of light-inducible carotenoid accumulation was detected in three independent ΔFvMAT1-2-1 knockout mutants of F. verticillioides as compared with the parental wild-type strain. Transcript levels of the carB, carRA, and carT genes, encoding key enzymes of the carotenoid biosynthetic pathway, were also significantly reduced in the mutants. The downregulation of these genes in the ΔFvMAT1-2-1 mutant indicates that MAT genes play a role in the control of carotenogenesis in Fusarium. The finding that mating-type genes regulate important processes unrelated to sex helps to understand the presence of functional MAT genes in asexually reproducing fungus populations.