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Control of Bacterial Phenotype and Chromosomal Gene Expression by Single Plasmids of Lactococcus lactis IL594
Get PDFPlasmid-free Lactococcus lactis IL1403 is one of the best-characterized representatives of lactic acid bacteria (LAB), intensively used in broad microbiology worldwide. Its parent strain, L. lactis IL594, contains seven plasmids (pIL1–pIL7) with resolved DNA sequences and an indicated role for overall plasmid load in enhancing host-adaptive potential. To determine how individual plasmids manipulate the expression of phenotypes and chromosomal genes, we conducted global comparative phenotypic analyses combined with transcriptomic studies in plasmid-free L. lactis IL1403, multiplasmid L. lactis IL594, and its single-plasmid derivatives. The presence of pIL2, pIL4, and pIL5 led to the most pronounced phenotypic differences in the metabolism of several carbon sources, including some β-glycosides and organic acids. The pIL5 plasmid also contributed to increased tolerance to some antimicrobial compounds and heavy metal ions, especially those in the toxic cation group. Comparative transcriptomics showed significant variation in the expression levels of up to 189 chromosomal genes due to the presence of single plasmids and 435 unique chromosomal genes that were resultant of the activity of all plasmids, which may suggest that the observed phenotypic changes are not only the result of a direct action of their own genes but also originate from indirect actions through crosstalk between plasmids and the chromosome. The data obtained here indicate that plasmid maintenance leads to the development of important mechanisms of global gene regulation that provide changes in the central metabolic pathways and adaptive properties of L. lactis and suggest the possibility of a similar phenomenon among other groups of bacteria
Genetic engineering of low‑temperature polyhydroxyalkanoate production by Acidovorax sp. A1169, a psychrophile isolated from a subglacial outflow
Get PDFIn recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates
(PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria
have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax
sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning
PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature
of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at
17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L
hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the
polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight
into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation,
production of PHA and also on the methodology for genetic engineering of psychrophiles
Biological aspects of phage therapy versus antibiotics against Salmonella enterica serovar Typhimurium infection of chickens
Get PDFPhage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed
Deciphering the Regulatory Circuits of RA3 Replication Module - Mechanisms of the Copy Number Control
Get PDFThe RA3 plasmid, the archetype of IncU incompatibility group, represents a mosaic-
modular genome of 45.9 kb. The replication module encompasses repA and repB (initiator) surrounded
by two long repetitive sequences DR1 and DR2 of unknown function. Here, we mapped the origin
of replication oriV to the 3′ end of repB and showed that oriV was activated by the transcription
coming from orf02revp in the adjacent stability module. Using various in vivo and in vitro methods
we demonstrated that the repB expression proceeded either from repBp located in the intergenic repA-
repB region or from the upstream strong repAp that was autoregulated by RepA. Additionally, the
repBp activity was modulated by the transcription from the overlapping, divergently oriented repXp.
Both repXmRNA (antisense for repAmRNA) and its small polypeptide product, RepX, were strong
incompatibility determinants. Hence, we showed that the sophisticated RA3 copy number control
combined the multivalent regulation of repB expression, RepB titration by DR1, and transcriptional
activation of oriV, dependent on the RA3 global regulatory network. Similarly organized replicons
have been found in diverse bacterial species confirming the significance of these mechanisms in
establishing the IncU plasmids in a broad spectrum of host
Yeast as a Model to Find New Drugs and Drug Targets for VPS13-Dependent Neurodegenerative Diseases
Get PDFMutations in human VPS13A-D genes result in rare neurological diseases, including chorea-acanthocytosis. The pathogenesis of these diseases is poorly understood, and no effective treatment is available. As VPS13 genes are evolutionarily conserved, the effects of the pathogenic mutations could be studied in model organisms, including yeast, where one VPS13 gene is present. In this review, we summarize advancements obtained using yeast. In recent studies, vps13Δ and vps13-I2749 yeast mutants, which are models of chorea-acanthocytosis, were used to screen for multicopy and chemical suppressors. Two of the suppressors, a fragment of the MYO3 and RCN2 genes, act by downregulating calcineurin activity. In addition, vps13Δ suppression was achieved by using calcineurin inhibitors. The other group of multicopy suppressors were genes: FET4, encoding iron transporter, and CTR1, CTR3 and CCC2, encoding copper transporters. Mechanisms of their suppression rely on causing an increase in the intracellular iron content. Moreover, among the identified chemical suppressors were copper ionophores, which require a functional iron uptake system for activity, and flavonoids, which bind iron. These findings point at areas for further investigation in a higher eukaryotic model of VPS13-related diseases and to new therapeutic targets: calcium signalling and copper and iron homeostasis. Furthermore, the identified drugs are interesting candidates for drug repurposing for these diseases
Trimethylamine, a gut bacteria metabolite and air pollutant, increases blood pressure and markers of kidney damage including proteinuria and KIM-1 in rats
Get PDFChemerin Effect on the Endometrial Proteome of the Domestic Pig during Implantation Obtained by LC-MS/MS Analysis
Get PDFChemerin (CHEM) is a hormone mainly expressed in adipocytes involved in the regulation of energy homeostasis and inflammatory response. CHEM expression has been demonstrated in the structures of the porcine hypothalamic-pituitary-gonadal axis, as well as in the uterus, trophoblasts and conceptuses of pigs. In this study, we performed high-throughput proteomic analyses (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the influence of CHEM (400 ng/mL) on differentially regulated proteins (DRPs) in the porcine endometrial tissue explants during implantation (15 to 16 days of gestation). Among all 352 DRPs, 164 were up-regulated and 188 were down-regulated in CHEM-treated group. DRPs were assigned to 47 gene ontology (GO) terms (p-adjusted < 0.05). Validation of four DRPs (IFIT5, TGFβ1, ACO1 and PGRMC1) by Western blot analysis confirmed the veracity and accuracy of the LC-MS/MS method used in the present study. We suggest that CHEM, by modulating various protein expressions, takes part in the endometrial cell proliferation, migration and invasion at the time of implantation. It also regulates the endometrial immune response, sensitivity to P4 and the formation of new blood vessels. Additionally, CHEM appears to be an important factor involved in endothelial cell dysfunction during the pathogenesis of preeclampsia. The identification of a large number of DRPs under the influence of CHEM provides a valuable resource for understanding the molecular mechanisms of this hormone action during implantation, which is a prerequisite for better control of pig reproduction
Functional Characterization of TetR-like Transcriptional Regulator PA3973 from Pseudomonas aeruginosa
Get PDFPseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial
infections. Its ability to survive under different conditions relies on a complex regulatory network
engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use
the available resources. P. aeruginosa PA3973 encodes an uncharacterized TetR family transcriptional
regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification
of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of
PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes
in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973
binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of
PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA
dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly,
the lack of PA3973 triggered increased expression of PA3972 and PA3971. The DPA3972-71 PAO1161
strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine
or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall
our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in
the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971,
encoding proteins with a proposed role in stress response
Diet of the grey wolf Canis lupus in Roztocze and Solska Forest, south-east Poland
Get PDFThe diet composition and prey selection of grey wolves (Canis lupus) inhabiting the Roztocze and Solska Forest (south-east Poland) was studied based on an analysis of scats collected in 2001-2002 (n = 84) and 2017-2020 (n = 302). In both periods, wolves preyed mainly on wild ungulates (96.5-96.7% of consumed biomass). Roe deer (Capreolus capreolus) was the most critical wolf prey accounting for 57.8% of consumed biomass in 2001-2002 and 49.2% and 2017-2020, but wolves positively select only wild boar (Jacob's selectivity index D = 0.213 in 2001-2002 and 0.710 in 2017-2020) and fallow deer (D = 0.588 only in 2017-2020). The largest species – moose Alces alces and red deer Cervus elaphus – were consumed less than expected from their share in the ungulate community. Predation on medium-sized wild mammals and domestic animals was low, 0.8-2.2% and 1.1-2.7% of the biomass consumed, respectively. The breadth of the wolf diet was very narrow and identical in both study periods (B = 1.07), while the similarity of diet composition was high (α = 0.999). This study indicated the stability of the wolf diet over two decades and the importance of wild boar as a food source for this carnivore
Are the diets of sympatric Pygoscelid penguins more similar than previously thought?
Get PDFIn recent years, functional changes in Southern Ocean are becoming more noticeable, due to climate change and increasing human impacts, including a growing fshery that is concentrating in the Antarctic Peninsula (AP) region. Antarctic krill Euphausia superba is often the primary prey species for animals such as Pygoscelis penguins, a sentinel species for ecosystem monitoring and management. During the last two decades in the AP gentoo penguin numbers (Pygoscelis papua) have increased and their range has shifted southward, in contrast to the decline in numbers of Adélie (P. adeliae) and chinstrap (P. antarcticus) penguins. Given divergent population trends, the goal of this study was to examine diferences in their diet, and size structure of Antarctic krill recovered from penguin diet samples. The study is based on diet samples collected during the austral summers on King George Island (South Shetland Islands) where P. adeliae, P. antarcticus, and P. papua breed in mixed colonies. Results indicate that the penguins consumed krill of similar sizes during the breeding period. In contrast
to prior diet studies, we found higher proportions of krill in the gentoo diet and changes in the percentage of krill in the diet relative obtained during 1970s. The similarity in diets among all three species suggests that the availability prey items (e. g., fshes) may be changing and driving higher dietary overlap. Moreover, we also check diferences in krill length among penguin individuals and we did not fnd any statistically signifcant diferences. We also found plastic debris in penguin stomachs during both summers