Stationary Growth Phase Research Articles

Azotobacter vinelandii N 2 fixation increases in co-culture with the PGPR Bacillus subtilis in a nitrogen concentration-dependent manner

ABSTRACT Biological nitrogen fixation (BNF) is an essential source of new nitrogen (N) for terrestrial ecosystems. The abiotic factors regulating BNF have been extensively studied in various ecosystems and laboratory settings. Despite this, our understanding of the impact of neighboring bacteria on N 2 fixer activity remains limited. Here, we explored this question using a co-culture of the two model species: the free-living diazotroph Azotobacter vinelandii and the non-fixing plant growth-promoting rhizobacteria Bacillus subtilis . We observed that the interaction between the two bacteria was modulated by N availability. Under N-replete conditions, B. subtilis outcompeted A. vinelandii in the co-culture. Under N-limiting conditions, BNF activity by A. vinelandii was enhanced in the presence of B. subtilis . Reciprocally, the presence of A. vinelandii repressed sporulation by B. subtilis and supported its growth likely through N transfer. N inputs by A. vinelandii were doubled in the presence of B. subtilis compared to the monoculture, primarily due to the retention of a robust N 2 fixation activity in the stationary phase. A proteomic analysis revealed that A. vinelandii N metabolism, particularly the molybdenum nitrogenase isoform protein levels (NifK and NifD), was upregulated during the stationary growth phase in the presence of B. subtilis . This study revealed that N stress drives bacterial interactions and activity in a two-species community, especially in the stationary phase. IMPORTANCE Reducing inputs of chemical N fertilizers is essential to develop a more sustainable agriculture. The stimulation of biological nitrogen fixation by N2 fixers in multispecies cultures, here the plant growth-promoting rhizobacteria Azotobacter vinelandii and Bacillus subtilis , opens opportunities for the formulation of biofertilizers consortia. While most research on N2 fixation historically focussed on the exponential growth phase of microorganisms, we observed that Bacillus subtilis stimulated Azotobacter vinelandii N2 fixation mostly during the stationary phase. This result highlights that more research on the factors controlling N2 fixation repression during the stationary growth phase, especially bacteria-bacteria interactions, is eagerly needed.

Biological evaluation of CH3OH and C2H5OH of Berberis vulgaris for in vivo antileishmanial potential against Leishmania tropica in murine models

Abstract Leishmaniasis is one of the global health issues and is still being handled with costly and sometimes unsuccessful compounds having serious side effects, highlighting the importance of seeking new potent antileishmanial compounds. Herbal medicines have been considered as the main source of prevention and treatment for a wide variety of diseases as well as other photogenetic diseases over the last few centuries. The current study was designed to investigate the in vitro and in vivo antileishmanial efficacy of ethanolic and methanolic extracts of Berberis vulgaris root collected from Kurez region of District Orakzai, Khyber Pakhtunkhwa, Pakistan. Stationary growth-phase metacyclic promastigotes form of Leishmania tropica was incubated in vitro in methanolic and ethanolic extracts using amphotericin B as a positive control. The antileishmanial activity of B. vulgaris extracts was measured after the incubation period using methoxynitrosulfophenyl-tetrazolium carboxanilide assay. For the in vivo study, BALB/c mice were infected with metacyclic L. tropica promastigotes, and lesions appeared after 28 days of inoculum. The results of study research showed that B. vulgaris extract had a powerful antileishmanial activity on promastigotes of L. tropica at different concentrations (5, 10, 25, 50, and 75 μg/mL). The inhibition concentration 50 values for ethanolic and methanolic extracts of B. vulgaris were determined to be 15.37 and 17.55 μg/mL, respectively. In the in vivo activity, it was observed that B. vulgaris ethanolic extract at the concentration of 1.5 mg/kg decreased the lesion diameter in BALB/c infected mice. The ethanolic extract topically and orally reduced the lesion size 0.51 ± 0.023 and 0.56 ± 0.008 mm, while methanolic extract both topically and orally decrease the lesion diameter of 0.63 ± 0.008 and 0.68 ± 0.009 mm relatively, in comparison with negative control (1.45 ± 0.016 mm). Hematological parameters of mice including blood red blood cells, hematocrit, and hemoglobin in mice groups (infected non-treated) were found to be decreased, while the mice group treated with extract demonstrated nearly similar results to non-infected group. It is concluded from the current study that antileishmanial activity of B. vulgaris from Pakistan against L. tropica in both in vitro and in vivo showed a promising antileishmanial activity. This study might be helpful in the control strategies of cutaneous leishmaniasis caused by L. tropica.

Comparison of iPSC-derived human intestinal epithelial cells with Caco-2 cells and human in vivo data after exposure to Lactiplantibacillus plantarum WCFS1

To investigate intestinal health and its potential disruptors in vitro, representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However, the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently, the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge, IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model, while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately, comparison to in vivo transcriptomics data showed limited similarities, which could be explained by essential differences in the study setups. Altogether, hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.

Suppression of a transcriptional regulator, HexA, is essential for triggering the bacterial virulence of the entomopathogen, Xenorhabdus hominickii

A nematode-symbiotic bacterium, Xenorhabdus hominickii, exhibits two distinct lifestyles. Upon infection of its host nematode into a target insect, X. hominickii is released into the insect hemocoel and becomes pathogenic. This study examines the critical transformation in bacterial life forms concerning the activity of a transcriptional regulator, HexA. When X. hominickii was cultured in tryptic soy broth, HexA was expressed during the stationary phase of bacterial growth. Conversely, HexA was expressed in the early growth stage within the insect host, Spodoptera exigua, when infected with X. hominickii. The transient expression of HexA was succeeded by the expression of another transcriptional regulator, Lrp, which led to the production of bacterial virulent factors. Expression of HexA was manipulated by replacing its promoter with an inducible promoter controlled by the inducer, l-arabinose. In the absence of the inducer, the mutant bacteria expressed HexA at a low level, resulting in a bacterial culture broth that was more effective at suppressing insect immune responses than the wild type. When the inducer was added, HexA was expressed at high levels, rendering the culture broth ineffective in immunosuppression. Interestingly, expression of HexA inhibited the expression of another transcriptional regulator, Lrp, which in turn induced the expression of a non-ribosomal peptide synthetase, gxpS, leading to the production of an immunosuppressive metabolite, GXP. Suppression of HexA expression in mutant bacteria augmented GXP levels in secondary metabolites. This indicates that infection of X. hominickii into the insect host represses HexA expression and upregulates Lrp expression, leading to GXP production. The GXP metabolites inhibit insect immunity, thus protecting the bacteria-nematode complex. Therefore, the suppression of HexA expression in the insect hemocoel is crucial for the bacteria’s transition from a symbiotic to a pathogenic life form.

Effects of flavonoids on yeast ABC transporters activity

Flavonoids are known to be effective biomodulators of various processes in eukaryotic cells. As these compounds are present in wine and beer raw materials, they can influence the qualitative characteristics of the ethanol content in wine-making and brewing products, including directly through the mechanisms of regulation of gene expression and the activity of ATP-binding cassette (ABC) proteins. The main function of ABC transporters in yeast cells is to transport various substrates, including ethanol. This process ensures the survival of yeast cells under conditions of ethanol stress. It has been found that flavonoids, as well as their Schiff base derivatives, are effective stimulators and inhibitors of mRNA expression and activity of ABC proteins both in logarithmic and stationary phases of growth, which has a direct impact on bioethanol production by yeast.

The sigma factor σB is required for the development of the growth phase- and temperature-dependent increases in thermoresistance and membrane rigidity in Staphylococcus aureus

The aim of this study is to determine the role that σB factor plays in the development of growth phase- and temperature-dependent thermoresistance in Staphylococcus aureus cells and its possible relationship with membrane fluidity. Cells of S. aureus strain Newman and its isogenic ΔsigB mutant (strain IK184) were grown at different temperatures to exponential and stationary growth phases, and the relationship between σB activity (estimated by RT-qPCR), heat resistance, and membrane fluidity (fatty acid profiles and DPH fluorescence anisotropy) was assessed. σB was required for the development of the growth phase- and temperature-dependent increases in thermoresistance and membrane rigidity (up to 37 °C), although a direct correlation between σB activity and heat resistance or membrane fluidity in S. aureus could not be established. Results obtained also revealed the existence of σB-independent growth phase and temperature (>37 °C) heat adaptation mechanisms. Moreover, it was demonstrated that σB does not control the fatty acid synthesis in S. aureus. Results obtained in this work contribute to a deeper knowledge of S. aureus physiology and to the development and design of more effective processes used by the food industry for microbial inactivation.

PSLBII-27 Investigating the emergence of antimicrobial resistance in Mycoplasma bovis from feedlot cattle

Abstract Bovine respiratory disease (BRD) is the most significant disease affecting feedlot cattle in North America. Mycoplasma bovis is among the main BRD pathogenic bacteria. Infection control of M. bovis is hampered by a lack of effective vaccines and increasing antimicrobial resistance (AMR). Integrative and conjugative elements (ICEs) have been documented as a mechanism of horizontal gene transfer (HGT) in mycoplasmas. Mycoplasma ICEs (MICE) do not carry AMR genes but have been associated with the HGT of genes that confer AMR through point mutations. Hence, there is a need to gain a better understanding of the role of MICE in M. bovis AMR. Randomly selected M. bovis isolates (n = 54) collected from Western Canadian feedlot cattle (AB and SK) underwent whole-genome sequencing by short-reads (Illumina), with a subset (n = 6) selected based on AMR and ICE profiles for Oxford Nanopore long-read sequencing. Sequences were screened for the presence of MICE. MICE circularization (cMICE) is the first step of conjugation, and an in-house collection of 472 M. bovis field isolates (feedlot cattle just from AB) was screened by PCR for the presence of cMICE as a proxy for conjugation capabilities. Environmental conditions that could impact cMICE quantity, including growth phase, temperature, cell density, pH, starvation, UV light, CO2 concentration, and sub-inhibitory concentrations of mitomycin C were investigated by qPCR, followed by growth of M. bovis isolates under the most favorable conditions identified for cMICE-detection and screening by a high throughput qPCR method that eliminated the need for DNA extraction. Nineteen different mating pairs were carried out and 4 different environmental factors were tested for their possible impact on conjugation occurrence (incubation time, incubation volume, conjugation parent proportions, atmosphere). All isolates carried MICEs with 23/54 having a structure/sequence comparable to previously described functional MICE in mycoplasma, 43/54 possessing non-functional ICE, and 23 having >1 ICE. The presence of more than one MICE/genome suggests a lack of MICE (entry) exclusion systems in M. bovis, a trait that may promote the flow of genetic information among M. bovis isolates including AMR. Even though some factors including growth phase (early stationary phase) and pH (9.0) generated greater levels (≥1.5 fold) of cMICE, none of the examined parameters were statistically significant. However, as early stationary growth phase generated the greatest cMICE quantity (2.3 fold), M. bovis isolates were cMICE-screened at this growth stage for the expeditious identification of M. bovis conjugation candidates. A total of 25.7% M. bovis were cMICE-positive. Of 19 mating pairs tested, transconjugants were recovered from only 2 different mating pairs, possibly reflective of the need for further optimization of conjugation conditions.

Untargeted Liquid Chromatography-High-Resolution Mass Spectrometry Metabolomic Investigation Reveals Altered Lipid Content in Leishmania infantum Lacking Lipid Droplet Protein Kinase.

Leishmaniasis is a complex disease caused by different species of Leishmania. To date, no vaccine for humans or ideal therapy has been developed owing to the limited efficacy and toxicity of available drugs, as well as the emergence of resistant strains. Therefore, it is necessary to identify novel therapeutic targets and discover therapeutic options for leishmaniasis. In this study, we evaluated the impact of deleting the lipid droplet protein kinase (LDK) enzyme in Leishmania infantum using an untargeted metabolomics approach performed using liquid chromatography and high-resolution mass spectrometry. LDK is involved in lipid droplet biogenesis in trypanosomatids. Thirty-nine lipid metabolites altered in the stationary and logarithmic growth phases were noted and classified into five classes: (1) sterols, (2) fatty and conjugated acids, (3) ceramides, (4) glycerophosphocholine and its derivatives, and (5) glycerophosphoethanolamine and its derivatives. Our data demonstrated that glycerophosphocholine and its derivatives were the most affected after LDK deletion, suggesting that the absence of this enzyme promotes the remodeling of lipid composition in L. infantum, thus contributing to a better understanding of the function of LDK in this parasite.

Biochemical and Antioxidant Characteristics of Chlorococcum oleofaciens (Chlorophyceae, Chlorophyta) under Various Cultivation Conditions.

The functional state of enrichment cultures of the Chlorophycean strain Chlorococcum oleofaciens CAMU MZ-Ch4 under various cultivation conditions was studied. Experiments with different aeration conditions, cultivation durations, and nitrogen and phosphorus concentrations in the medium were carried out to evaluate the growth dynamics of the strain and its biochemical characteristics. The contents of chlorophylls, carotenoids, proteins, lipids, retinol, α-tocopherol, ascorbic acid, phenolic compounds, lipid peroxidation products, antioxidant enzymes (glutathione peroxidase, catalase, superoxide dismutase), and succinate dehydrogenase activity were measured. The lipid content on the fully supplemented Bold's basal medium increased to 381.03 mg g-1 dry weight at the late stationary growth phase. This value is 1.3-2.8 times higher than in other experiments. The use of aeration was associated with an increased content of proteins at 283.56 mg g-1 and of carotenoids at 2.12 mg g-1. Also, cultures at the early stationary growth phase with aeration showed the ability to accumulate phenolic compounds and ascorbic acid in amounts up to 0.32 mg g-1 and 0.19 mg g-1. The 74-day-old cultures had the highest contents of retinol (0.16 mg g-1) and α-tocopherol (0.68 mg g-1). Growth in nitrogen- and phosphorus-depleted media increased catalase and superoxide dismutase activity. A comprehensive analysis of all data showed that the antioxidant defence system is stress-resistant and flexible under varying aeration conditions and nitrogen and phosphorus availabilities. Thus, the strain CAMU MZ-Ch4 can be considered a potential producer of lipids, pigments, proteins, and vitamins under various culturing conditions.

Effect of O-Polysaccharide Modifications on Successful Plant Colonization by Bacteria

O-polysaccharides of gram-negative bacteria are a highly variable component of the lipopolysaccharide molecules located at the cell wall surface and involved in microbial interaction with plant and animal cells. Activity of prophage genes often results in various non-stoichiometric modifications (methylation, acetylation, etc.) of glycans at bacterial cell surface. The share of modified O-polysaccharides increases during the stationary growth phase and results in increased hydrophobicity of microbial surface. Bacterial cells with different hydrophobicity showed difference in attachment to plant roots. Increased cell hydrophobicity index was found to result in a significant increase in the number of adsorbed microorganisms per unit root length. Thus, acetyl transferase and methyl transferase genes of viral origin may be indirectly involved in successful colonization of plant roots by rhizosphere bacteria.

Unveiling the role of PA0730.1 sRNA in Pseudomonas aeruginosa virulence and biofilm formation: Exploring rpoS and mucA regulation

Small RNA (sRNA) in bacteria serve as the key messengers in regulating genes associated with quorum sensing controlled bacterial virulence. This study was aimed to unveil the regulatory role of sRNA PA0730.1 on the expression of various traits of Pseudomonas aeruginosa linked to pathogenicity, with special emphasis on the growth, colony morphology, cell motility, biofilm formation, and the expression of diverse virulence factors. PA0730.1 sRNA was found to be upregulated both during planktonic stationary growth phase and at biofilm state of P. aeruginosa PAO1. PA0730.1 deleted strain showed significant growth retardation with increased doubling time. Overexpression of PA0730.1 led to enhanced motility and biofilm formation, while the ∆PA0730.1 strain displayed significant inhibition in motility and biofilm formation. Furthermore, PA0730.1 was found to regulate the synthesis of selected virulence factors of P. aeruginosa. These observations in PA0730.1+ and ∆PA0730.1 were found to be correlated with the PA0730.1-mediated repression of transcription regulators, mucA and rpoS, both at transcriptional and translational levels. The results suggest that PA0730.1 sRNA might be a promising target for developing new drug to counter P. aeruginosa pathogenesis, and could also help in RNA oligonucleotide based therapeutic research for formulating a novel therapeutant.

Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens.

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat (CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

The intracellular accumulation of iron coincides with enhanced biohydrogen production by Thermoanaerobacterium thermosaccharolyticum

The aim of this study was to investigate the effects of different doses (10–200 mg/L) of magnetite (FeO·Fe2O3), Fe0, Ni0, and FeCl3 on the efficiency of dark fermentative H2 production from cheese whey using Thermoanaerobacterium thermosaccharolyticum SP-H2. Energy conversion efficiency and hydrogen yield increased significantly by 22.7 % and 34.8 %, respectively, when 10 mg/L of magnetite was added, and hydrogenase activity increased by 23.6 % when 100 mg/L of Fe0 was added compared to the control (p < 0.001). Using scanning electron microscopy and energy dispersive X-ray spectroscopy, it was found that during the early exponential phase of hydrogen evolution, T. thermosaccharolyticum cells accumulated up to 11.2 times more Fe (up to 2.8 wt%) in the presence of optimal concentrations of Fe additives compared to the control. Intracellular iron accumulation and hydrogen yield were positively correlated with acetate concentration (p < 0.001), suggesting an efficient acetate pathway for dark fermentation. During the stationary growth phase, with almost no H2 production, the intracellular iron content decreased by up to 67.9 %. The phenomenon of changes in the amount of metal in cells, depending on the phase of hydrogen production, was demonstrated for the first time. The findings suggest that intracellular metals can be used in cell metabolism to produce H2, and the mechanisms behind this process need further investigation. Overall, this study provides valuable insights into the optimal forms and doses of metals for enhancing biohydrogen production, as well as possible research directions for the potential use of metals by microorganisms during additive-assisted dark fermentation of carbohydrate-rich wastes.

Structural basis for the distinct core-antenna assembly of cryptophyte photosystem II

Photosystem II (PSII) catalyzes the light-driven charge separation and water oxidation reactions of photosynthesis. Eukaryotic PSII core is usually associated with membrane-embedded light-harvesting antennae, which greatly increase the absorbance cross-section of the core. The peripheral antennae in different phototrophs vary considerably in protein composition and arrangement. Photosynthetic cryptophytes possess chlorophyll a/c binding proteins (CACs) that serve as their antennae. How these CACs assemble with the PSII core remains unclear. Here, we report the 2.57-Å resolution structure of cryptophyte PSII-CAC purified from cells at nitrogen-limited stationary growth phase. We show that each monomer of the PSII homodimer contains a core complex, six chlorophyll a/c binding proteins (CACs) and a previously unseen chlorophyll-binding protein (termed CAL-II). Six CACs are arranged as a double-layered arc-shaped non-parallel belt, and two such belts attach to the dimeric core from opposite sides. The CAL-II simultaneously interacts with a number of core subunits and five CACs. The distinct organization of CACs and the presence of CAL-II may play a critical role in stabilizing the dimeric PSII-CAC complex under stress conditions. Our study provides mechanistic insights into the assembly and function of the PSII-CAC complex as well as the possible adaptation of cryptophytes in response to environmental stresses.

Salinity Stress Acclimation Strategies in Chlamydomonas sp. Revealed by Physiological, Morphological and Transcriptomic Approaches.

Climate changes may include variations in salinity concentrations at sea by changing ocean dynamics. These variations may be especially challenging for marine photosynthetic organisms, affecting their growth and distribution. Chlamydomonas spp. are ubiquitous and are often found in extreme salinity conditions. For this reason, they are considered good model species to study salinity adaptation strategies. In the current study, we used an integrated approach to study the Chlamydomonas sp. CCMP225 response to salinities of 20‱ and 70‱, by combining physiological, morphological, and transcriptomic analyses, and comparing differentially expressed genes in the exponential and stationary growth phases under the two salinity conditions. The results showed that the strain is able to grow under all tested salinity conditions and maintains a surprisingly high photosynthetic efficiency even under high salinities. However, at the highest salinity condition, the cells lose their flagella. The transcriptomic analysis highlighted the up- or down-regulation of specific gene categories, helping to identify key genes responding to salinity stress. Overall, the findings may be of interest to the marine biology, ecology, and biotechnology communities, to better understand species adaptation mechanisms under possible global change scenarios and the potential activation of enzymes involved in the synthesis of bioactive molecules.

Effect of PCBs on Production Characteristics and Fucoxanthin / Fatty Acid Content of Cylindrotheca closterium (Ehrenberg) Reimann et Lewin Diatom Enrichment Culture

Background: Since the mid-20th century, polychlorinated biphenyls (PCBs) have emerged as one of the foremost anthropogenic organic pollutants in aquatic environments. Microphytobenthic algae of the genus Cylindrotheca have been recurrently employed in laboratory experiments to assess sediment toxicity. Recently, a novel strain of benthic diatoms belonging to the genus Cylindrotheca has been identified and characterized from PCB-contaminated sediments in the coastal region of Sevastopol Bay (Black Sea). This species of algae has high biomass productivity, the ability to synthesize fucoxanthin, and a variety of fatty acids. Cylindrotheca closterium is capable of metabolizing organic pollutants in bottom sediments. Objective: The objective of the study was to investigate the effects of PCBs on the growth patterns and physiological responses of C. closterium through a 9-day experiment, subjecting the microalgae to varying concentrations of PCBs ranging from 0.0003 to 100 mg/L. Results: The experiments revealed that C. closterium could grow in environments containing concentrations of PCBs ranging from 0.0003 to 10 mg/L, indicating its resilience to moderate levels of PCB exposure. Additionally, adaptive biochemical processes were observed in C. closterium under PCB exposure. Notably, on the sixth day of the experiment, the culture transitioned into a stationary growth phase, accompanied by significant increases in total lipid content by 1.6 times and fucoxanthin by 4.6 times compared to the control. However, a pronounced decrease in culture growth was observed at a PCB concentration of 100 mg/L, coinciding with reductions in total lipid and fucoxanthin content, suggesting a tolerance threshold of C. closterium between 10 and 100 mg/L PCB concentrations. Furthermore, alterations in the fatty acid profile of C. closterium were noted, characterized by a decrease in polyene content and an increase in monoene fatty acids, under PCB exposure. Conclusion: The study underscores the resilience of C. closterium to moderate PCB concentrations and highlights the complex physiological responses and adaptive mechanisms initiated in response to PCB exposure. The findings contribute to understanding the toxic effects of PCBs on C. closterium and provide insights into potential mechanisms underlying these effects.

Leukotoxin A Production and Release by JP2 and Non-JP2 Genotype Aggregatibacter actinomycetemcomitans in Relation to Culture Conditions.

Aggressive forms of periodontitis, especially in young patients, are often associated with an increased proportion of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans of the microbiota of the affected periodontal sites. One of the virulence factors of A. actinomycetemcomitans is a leukotoxin (LtxA) that induces a pro-inflammatory cell death process in leukocytes. A. actinomycetemcomitans exhibits a large genetic diversity and different genotypes vary in LtxA production capacity. The genotype JP2 is a heavy LtxA producer due to a 530-base pair deletion in the promoter for the toxin genes, and this trait has been associated with an increased pathogenic potential. The present study focused on the production and release of LtxA by different A. actinomycetemcomitans genotypes and serotypes under various growth conditions. Four different strains of this bacterium were cultured in two different culture broths, and the amount of LtxA bound to the bacterial surface or released into the broths was determined. The cultures were examined during the logarithmic and the early stationary phases of growth. The JP2 genotype exhibited the highest LtxA production among the strains tested, and production was not affected by the growth phase. The opposite was observed with the other strains. The composition of the culture broth had no effect on the growth pattern of the tested strains. However, the abundant release of LtxA from the bacterial surface into the culture broth was found in the presence of horse serum. Besides confirming the enhanced leucotoxicity of the JP2 genotype, the study provides new data on LtxA production in the logarithmic and stationary phases of growth and the effect of media composition on the release of the toxin from the bacterial membrane.

Isolation and characterization of novel acetogenic strains of the genera Terrisporobacter and Acetoanaerobium.

Due to their metabolic versatility in substrate utilization, acetogenic bacteria represent industrially significant production platforms for biotechnological applications such as syngas fermentation, microbial electrosynthesis or transformation of one-carbon substrates. However, acetogenic strains from the genera Terrisporobacter and Acetoanaerobium remained poorly investigated for biotechnological applications. We report the isolation and characterization of four acetogenic Terrisporobacter strains and one Acetoanaerobium strain. All Terrisporobacter isolates showed a characteristic growth pattern under a H2 + CO2 atmosphere. An initial heterotrophic growth phase was followed by a stationary growth phase, where continuous acetate production was indicative of H2-dependent acetogenesis. One of the novel Terrisporobacter isolates obtained from compost (strain COMT) additionally produced ethanol besides acetate in the stationary growth phase in H2-supplemented cultures. Genomic and physiological characterizations showed that strain COMT represented a novel Terrisporobacter species and the name Terrisporobacter vanillatitrophus is proposed (=DSM 116160T = CCOS 2104T). Phylogenomic analysis of the novel isolates and reference strains implied the reclassification of the T. petrolearius/T. hibernicus phylogenomic cluster to the species T. petrolearius and of the A. noterae/A. sticklandii phylogenomic cluster to the species A. sticklandii. Furthermore, we provide first insights into active prophages of acetogens from the genera Terrisporobacter and Acetoanaerobium.

The efficacy of the food-grade antimicrobial xanthorrhizol against Staphylococcus aureus is associated with McsL channel expression.

The emergence and spread of multidrug-resistant Staphylococcus aureus strains demonstrates the urgent need for new antimicrobials. Xanthorrhizol, a plant-derived sesquiterpenoid compound, has a rapid killing effect on methicillin-susceptible strains and methicillin-resistant strains of S. aureus achieving the complete killing of staphylococcal cells within 2 min using 64 μg/mL xanthorrhizol. However, the mechanism of its action is not yet fully understood. The S. aureus cells treated with xanthorrhizol were studied using optical diffraction tomography. Activity of xanthorrhizol against the wild-type and mscL null mutant of S. aureus ATCC 29213 strain was evaluated in the time-kill assay. Molecular docking was conducted to predict the binding of xanthorrhizol to the SaMscL protein. Xanthorrhizol treatment of S. aureus cells revealed a decrease in cell volume, dry weight, and refractive index (RI), indicating efflux of the cell cytoplasm, which is consistent with the spontaneous activation of the mechanosensitive MscL channel. S. aureus ATCC 29213ΔmscL was significantly more resistant to xanthorrhizol than was the wild-type strain. Xanthorrhizol had an enhanced inhibitory effect on the growth and viability of exponentially growing S. aureus ATCC 29213ΔmscL cells overexpressing the SaMscL protein and led to a noticeable decrease in their viability in the stationary growth phase. The amino acid residues F5, V14, M23, A79, and V84 were predicted to be the residues of the binding pocket for xanthorrhizol. We also showed that xanthorrhizol increased the efflux of solutes such as K+ and glutamate from S. aureus ATCC 29213ΔmscL cells overexpressing SaMscL. Xanthorrhizol enhanced the antibacterial activity of the antibiotic dihydrostreptomycin, which targets the MscL protein. Our findings indicate that xanthorrhizol targets the SaMscL protein in S. aureus cells and may have important implications for the development of a safe antimicrobial agent.

Volatile organic compounds produced by thermophilic and non-thermophilic ampylobacter spp.: Influence of growth phase and nutrient composition

Bacteria produce a plethora of volatile organic compounds (VOCs) into the atmosphere as a means of interacting with their habitats, as well as for intra- and interkingdom communication, and to survive and thrive in nature and inside their hosts. Campylobacter, which is commonly found in poultry and ruminants, has demonstrated remarkable endurance in aquatic settings, making it one of the world's most hazardous foodborne pathogens that kill thousands of humans every year. In the present work, the VOCs released by both thermophilic (C. jejuni, C. coli, and C. lari) and non-thermophilic (C. fetus) Campylobacter of clinical significance, which were influenced by nutrient composition (media) and growth phase, were profiled using an active sampling approach, with active charcoal adsorbent and gas chromatography-mass spectrometry. Alcohols and ketones were detected only in the thermophilic Campylobacter strains, C. jejuni subsp. jejuni (1-heptadecanol; 1,8-nonadien-3-ol; 3,7,11-trimethyl-3-dodecanol; 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one; and isophorone), C. coli (isophorone and 3,5-bis(1,1-dimethylethyl)-phenol), and C. lari (3,7,11-trimethyl-3-dodecanol and 1s,4R,7R,11R-1,3,4,7-tetramethyltricyclo [5.3.1.0(4,11)] undec-2-en-8-one) between early stationary and stationary growth phases. The non-thermophilic C. fetus produced only a range of unidentified compounds that need to be investigated further in the future. The results from the principal component analysis showed distinctive clustering of VOC markers between the four bacteria taxa and within the strains of C. jejuni, including distinguishable groupings of VOCs throughout the growth phase of each bacterium and between different culture media. This demonstrates the influence of bacterial growth and media composition on the volatilome of the studied Campylobacter spp. Overall, the VOC profiling of these foodborne pathogens, under influencing factors of growth phase and media, paves the way for future rapid identification of Campylobacter spp. in food matrices.