The RNA subunit of telomerase is an essential component whose primary sequence and length are poorly conserved among eukaryotic organisms. The phytopathogen Ustilago maydis is a dimorphic fungus of ...the order Ustilaginales. We analyzed several species of Ustilaginales to computationally identify the TElomere RNA (TER) gene ter1. To confirm the identity of the TER gene, we disrupted the gene and characterized telomerase-negative mutants. Similar to catalytic TERT mutants, ter1Δ mutants exhibit phenotypes of growth delay, telomere shortening and low replicative potential. ter1-disrupted mutants were unable to infect maize seedlings in heterozygous crosses and showed defects such as cell cycle arrest and segregation failure. We concluded that ter1, which encodes the TER subunit of the telomerase of U. maydis, have similar and perhaps more extensive functions than trt1.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Actinobacillus seminis is the causal agent of epididymitis and has other effects on the reproductive tracts of small ruminants and bovines. This bacterium causes infection when luteinizing ...(LH) or follicle-stimulating hormones increase, and hosts reach sexual maturity. LH induces female ovulation and male testosterone production, suggesting that these hormones affect A. seminis pathogenicity. In the present study, we evaluated the effect of testosterone (1–5 ng/ml) or estradiol (5–25 pg/ml) added to culture medium on the in vitro growth, biofilm production, and adhesin expression of A. seminis. Estradiol does not promote the growth of this bacterium, whereas testosterone increased A. seminis planktonic growth 2-fold. Both hormones induced the expression of the elongation factor thermo unstable (EF-Tu) and phosphoglycerate mutase (PGM), proteins that A. seminis uses as adhesins. Estradiol (5 or 10 pg/ml) decreased biofilm formation by 32%, whereas testosterone, even at 5 ng/ml, showed no effect. Both hormones modified the concentrations of carbohydrates and eDNA in biofilms by 50%. Amyloid proteins are characterized by their capacity to bind Congo red (CR) dye. Actinobacillus seminis binds CR dye, and this binding increases in the presence of 5–20 pg/ml estradiol or 4 ng/ml testosterone. The A. seminis EF-Tu protein was identified as amyloid-like protein (ALP). The effect of sexual hormones on the growth and expression of virulence factors of A. seminis seems to be relevant for its colonization and permanence in the host.
Sex hormones could alter the Actinobacillus seminis growth and virulence factors expression, and in consequence, could permit to A. seminis to colonize target tissues easier.
causes gallibacteriosis in birds. These bacteria produce biofilms and secrete several fimbrial appendages as tools to cause disease in animals.
strains contain up to three types of fimbriae.
is the ...strategy currently used to determine variations in the gene content of
, although today only the completely circularized genome of
UMN179 is available.
The appearance of growth of various strains of
in liquid culture medium was studied. Biofilm production and how the amount of biofilm was affected by DNase, Proteinase K, and Pronase E enzymes were analyzed. Fimbrial gene expression was performed by protein analysis and qRT-PCR. In an avian model, the pathogenesis generated by the strains
ESV200 and 12656-12 was investigated. Using bioinformatic tools, the complete genome of G. anatis ESV200 was comparatively studied to search for virulence factors that would help explain the pathogenic behavior of this strain.
ESV200 strain differs from the 12656-12 strain because it produces a biofilm at 20%.
ESV200 strain express fimbrial genes and produces biofilm but with a different structure than that observed for strain 12656-12. ESV200 and 12656-12 strains are pathogenic for chickens, although the latter is the most virulent. Here, we show that the complete genome of the ESV200 strain is similar to that of the UNM179 strain. However, these strains have evolved with many structural rearrangements; the most striking chromosomal arrangement is a Maverick-like element present in the ESV200 strain.
We analyzed the global expression patterns of telomerase-negative mutants from haploid cells of Ustilago maydis to identify the gene network required for cell survival in the absence of telomerase. ...Mutations in either of the telomerase core subunits (trt1 and ter1) of the dimorphic fungus U. maydis cause deficiencies in teliospore formation. We report the global transcriptome analysis of two ter1Δ survivor strains of U. maydis, revealing the deregulation of telomerase-deleted responses (TDR) genes, such as DNA-damage response, stress response, cell cycle, subtelomeric, and proximal telomere genes. Other differentially expressed genes (DEGs) found in the ter1Δ survivor strains were related to pathogenic lifestyle factors, plant–pathogen crosstalk, iron uptake, meiosis, and melanin synthesis. The two ter1Δ survivors were phenotypically comparable, yet DEGs were identified when comparing these strains. Our findings suggest that teliospore formation in U. maydis is controlled by key pathogenic lifestyle and meiosis genes.
Actinobacillus seminis
is an autochthonous gram-negative bacterium that affects reproductive organs, causing epididymitis, low fertility, and occasional abortions in ovine and goats. The virulence ...factors and the pathogenicity mechanisms of
A. seminis
have not been clearly elucidated yet. In this work, biofilm production by
A. seminis
in in vitro assay
s
is described and characterized. After 48-h incubation at 37 °C in trypticase soy broth,
A. seminis
formed biofilms containing an extracellular matrix comprised mainly of fibrillar material. Microaerophilia or the presence of calcium diminished biofilm formation in approximately 50% and 70%, respectively, but low iron concentrations increased it 40%. Through enzymatic digestion, it was found that proteins were the main component of these biofilms. Structural observations through scanning electron microscopy indicated the presence of a high amount of fibrillar material in which bacteria were immersed. Antibodies against different bacterial surface proteins, such as anti-biofilm matrix and anti-adhesin, diminished biofilm formation in 70% and 25%, respectively; whereas furanone C-30 and LED-209, compounds described as quorum-sensing inhibitors, completely inhibited biofilm formation. In conclusion, environmental conditions can influence strongly biofilm formation in
A. seminis
, and this could be an advantageous strategy that allows bacteria to persist inside a host.
In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic ...TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•A. seminis phosphoglycerate mutase and elongation factor-Tu function as adhesins.•These proteins interact with sheep fibrinogen and fibronectin.•Immune sera against them inhibit the A. seminis ...adherence to epithelial cells.
Actinobacillus seminis is an autochthon of ovine reproductive organs. However, it is also considered to be a pathogen of these animals and is responsible for causing epididymitis and low fertility, among other pathological diseases. In this study, we describe the identification of two proteins functioning as adhesins in A. seminis. After obtaining surface proteins of this bacterium, two proteins of approximately 25 and 40 kDa were partially purified by ion interchange chromatography. Both proteins interacted with bovine fibronectin and fibrinogen, and they cross-reacted with a polyclonal serum from a sheep infected with A. seminis. Both proteins were identified by mass spectrometry analysis as phosphoglycerate mutase and elongation factor-Tu, respectively. A. seminis was able to adhere to cultured human bladder cells, and pre-incubation of A. seminis with the polyclonal sera against these proteins significantly inhibited their ability to adhere to cells but adherence was not affected by pre-immune serum, indicating their activity as adhesins. The identification of proteins involved in the adhesion of A. seminis to tissues and that serve as virulence factors will help furtherour understanding of the pathogenic mechanisms of this bacterium.
The pOV plasmid isolated from the Pasteurella multocida strain PMOV is a new plasmid, and its molecular characterization is important for determining its gene content and its replicative properties ...in Pasteurellaceae family bacteria.
Antimicrobial resistance mediated by the pOV plasmid was tested in bacteria. Purified pOV plasmid DNA was used to transform E. coli DH5α and Gallibacterium anatis 12656-12, including the pBluescript II KS(−) plasmid DNA as a control for genetic transformation. The pOV plasmid was digested with EcoRI for cloning fragments into the pBluescript II KS(−) vector to obtain constructs and to determine the full DNA sequence of pOV.
The pOV plasmid is 13.5 kb in size; confers sulfonamide, streptomycin and ampicillin resistance to P. multocida PMOV; and can transform E. coli DH5α and G. anatis 12656-12. The pOV plasmid was digested for the preparation of chimeric constructs and used to transform E. coli DH5α, conferring resistance to streptomycin (plasmid pSEP3), ampicillin (pSEP4) and sulfonamide (pSEP5) on the bacteria; however, similar to pBluescript II KS(−), the chimeric plasmids did not transform G. anatis 12656-12. A 1.4 kb fragment of the streptomycin cassette from pSEP3 was amplified by PCR and used to construct pSEP7, which in turn was used to interrupt a chromosomal DNA locus of G. anatis by double homologous recombination, introducing strA-strB into the G. anatis chromosome.
The pOV plasmid is a wide-range, low-copy-number plasmid that is able to replicate in some gamma-proteobacteria. Part of this plasmid was integrated into the G. anatis 12656-12 chromosome. This construct may prove to be a useful tool for genetic studies of G. anatis.
•The plasmid pOV can replicate independently from chromosomal DNA in several Pasteurellaceae bacterial species and in E. coli.•The plasmid belongs to IncX incompatibility group.•pOV sequence analysis revealed a novel integron arrangement.•A chimeric plasmid can to transform G. anatis and to recombine within the chromosome.
Pasteurellaceae
family members obtain iron directly from host proteins or through siderophore-dependent mechanisms. Although
Gallibacterum anatis
expresses different virulence factors, its response ...to growth under iron restriction is unknown.
G. anatis
cultured in the presence of 2,2′-dipyridyl, up-expressed an approximately 65 kDa protein and repressed the expression of a 70 kDa protein. MALDI-TOF analysis of those proteins indicated homology with CirA (65 kDa), a protein involved in iron-siderophore acquisition in
Mannheimia succinoproducens
and a TonB-dependent receptor (70 kDa protein), a protein that binds chicken hemoglobin; however,
G. anatis
siderophore production was not detected by chromo azurol S (CAS)-BHI agar determination. This putative
G. anatis
siderophore receptor is under Fur control, but not the hemoglobin binding protein, as observed in
G. anatis
12656-12
fur
mutant (Ω
fur
126.13) grown in the presence or not of 2,2′-dipyridyl. The addition of FeCl
3
to the culture medium diminished the growth and biofilm production in approximately 30% and 35%, respectively, in the wild-type strain, but the growth of Ω
fur
126.13 strain was not affected and biofilm production increased in 35%.
G. anatis
Ω
fur
126.13 presented lower virulence when it was inoculated to 35-day-old chickens in comparison to the wild-type strain. The induction of more than one iron uptake mechanism could benefit pathogenic microorganisms such as
Gallibacterium
.
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