Microorganisms isolated from various traditionally fermented food products prepared in households without commercial starter cultures are designated as natural isolates. In addition, this term is ...also used for microorganisms collected from various natural habitats or products (silage, soil, manure, plant and animal material, etc.) that do not contain any commercial starters or bacterial formulations. They are characterized by unique traits that are the result of the selective pressure of environmental conditions, as well as interactions with other organisms. The synthesis of antimicrobial molecules, including bacteriocins, is an evolutionary advantage and an adaptive feature that sets them apart from other microorganisms from a common environment. This review aims to underline the knowledge of bacteriocins produced by natural isolates, with a particular emphasis on the most common location of their genes and operons, plasmids, and the importance of the relationship between the plasmidome and the adaptive potential of the isolate. Applications of bacteriocins, ranging from natural food preservatives to supplements and drugs in pharmacology and medicine, will also be addressed. The latest challenges faced by researchers in isolating new natural isolates with desired characteristics will be discussed, as well as the production of new antimicrobials, nearly one century since the first discovery of colicins in 1925.
Key points
•
Natural bacterial isolates harbor unique properties shaped by diverse interactions.
•
Horizontal gene transfer enables constant engineering of new antimicrobials.
•
Fermented food products are important source of bacteriocin-producing natural isolates.
Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally ...multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients.
We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results.
Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed.
High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates.
Purpose
Flotation tailings represent an extremely unfriendly substrate for plant colonization due to toxic metal concentrations and marked macronutrient deficiencies. The perennial grass
Miscanthus
x
...giganteus
J.M.Greef & Deuter ex Hodk. & Renvoize was successfully cultivated in this infertile substrate for two years. Our aim was to identify composition of its rhizosphere bacterial community and to analyze the effects of the selected rhizobacteria on plant growth, root development, metal and P uptake.
Methods
Using the cultivation-dependent method, 75 isolates were collected from the rhizosphere and six rhizobacterial strains were selected for further characterization based on morphological and biochemical differences. The plant rhizomes were inoculated with the consortium of rhizobacteria and cultivated in the flotation tailings substrate.
Results
Detected bacterial strains were characterized as metal-resistant and plant growth-promoting rhizobacteria (PGPR) because of their metal tolerance (NiCl
2
, Pb(C
2
H
3
O
2
)
2
, CuSO
4
, NaAsO
2
, MnCl
2
) and some or all of the plant growth-promoting (PGP) properties (indole-3-acetic acid and siderophore production, 1-aminocyclopropane-1-carboxylic acid deaminase activity and phosphate solubilization). PGPR mitigated the negative effects of high metal concentrations and macronutrient deficiency as shown by stimulated lateral roots development, increased root hair length, plant below and above ground biomass yield, higher plant P uptake and metal accumulation rate.
Conclusions
The isolated PGPR strains could be used in PGP-bacteria assisted phytoremediation of flotation tailings and metal polluted soils by
M.
x
giganteus
. Their PGP effects on various metal-tolerant target plant species in the respective substrate remain to be verified.
Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was ...constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.
Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim ...of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates.
The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates.
Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200-400 AU/ml for licheniocin 50.2 and 400-3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001).
This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.
Aggregation may play a main role in the adhesion of bacteria to the gastrointestinal epithelium and their colonization ability, as well as in probiotic effects through co-aggregation with intestinal ...pathogens and their subsequent removal. The aggregation phenomenon in lactococci is directly associated with the sex factor and lactose plasmid co-integration event or duplication of the cell wall spanning (CWS) domain of PrtP proteinase.
Lactococcus lactis subsp. lactis BGKP1 was isolated from artisanal semi-hard homemade cheese and selected due to its strong auto-aggregation phenotype. Subsequently, non-aggregating derivative (Agg-) of BGKP1, designated as BGKP1-20, was isolated, too. Comparative analysis of cell surface proteins of BGKP1 and derivative BGKP1-20 revealed a protein of approximately 200 kDa only in the parental strain BGKP1. The gene involved in aggregation (aggL) was mapped on plasmid pKP1 (16.2 kb), cloned and expressed in homologous and heterologous lactococci and enterococci. This novel lactococcal aggregation protein was shown to be sufficient for cell aggregation in all tested hosts. In addition to the aggL gene, six more ORFs involved in replication (repB and repX), restriction and modification (hsdS), transposition (tnp) and possible interaction with mucin (mbpL) were also located on plasmid pKP1.
AggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci.
We examined the effect of drought-tolerant Bacillus safensis SS-2.7 and B. thuringiensis SS-29.2 strains on the response of four (133, 274, California Wonder—CalW, and Matica) sweet pepper genotypes ...to water deficiency conditions. Pepper seeds were sown in pots with (treated) and without (control) bacterial strain inoculation. After four weeks of growth under controlled conditions and regular watering, drought was imposed by completely withholding watering for seven days. Under conditions of normal watering, genotype 274 showed better seedling establishment than genotype 133 and CalW, while the slowest was genotype Matica. Antioxidant enzyme activity under drought conditions was genotype and bacterial treatment-dependent. The best response to bacterial treatment in order to cope with severe drought was found in the CalW genotype, while in genotype 133, we determined even faster plant decay during water deficiency in treated seeds. Inoculated seeds of the Matica genotype did not show different antioxidant enzyme activity under normal and drought conditions. According to the obtained results, we concluded that under drought conditions, the most susceptible was genotype 274, moderate susceptibility was detected in genotype 133, and CalW and Matica were the most tolerant genotypes. Our study demonstrates (1) that drought-tolerant Bacillus strains showed a plant growth-promoting effect on some selected pepper genotypes; (2) that there were genotype-dependent antioxidant enzyme activities under drought conditions in response to treatment with a particular bacterial strain; and (3) that we could expect a genotype-dependent response during biostimulant application, especially under stress conditions.
The diversity of plant-associated bacteria is vast and can be determined by 16S rRNA gene metabarcoding. Fewer of them have plant-beneficial properties. To harness their benefits for plants, we must ...isolate them. This study aimed to check whether 16S rRNA gene metabarcoding has predictive power in identifying the majority of known bacteria with plant-beneficial traits that can be isolated from the sugar beet (
L.) microbiome. Rhizosphere and phyllosphere samples collected during one season at different stages of plant development were analyzed. Bacteria were isolated on rich unselective media and plant-based media enriched with sugar beet leaves or rhizosphere extracts. The isolates were identified by sequencing the 16S rRNA gene and tested in vitro for their plant-beneficial properties (stimulation of germination; exopolysaccharide, siderophore, and HCN production; phosphate solubilization; and activity against sugar beet pathogens). The highest number of co-occurring beneficial traits was eight, found in isolates of five species:
,
,
,
, and
. These species were not detected by metabarcoding and have not previously been described as plant-beneficial inhabitants of sugar beets. Thus, our findings point out the necessity of a culture-dependent microbiome analysis and advocate for low-nutrient plant-based media for high-yield isolation of plant-beneficial taxa with multiple beneficial traits. A culture-dependent and -independent approach is required for community diversity assessment. Still, isolation on plant-based media is the best approach to select isolates for potential use as biofertilizers and biopesticides in sugar beet cultivation.
The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in
subsp.
BGBU1-4. Heterologous expression of pBU6 confirmed that ...production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene,
, revealed that the lactolisterin BU cluster consists of four genes: the structural gene
, the
gene encoding an ABC transporter, the
gene encoding an accessory protein and the
gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the P
promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are
constellation. Two ρ-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the
and
genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci.
•Bacillus group are well known as producers of a wide array of antimicrobials.•Most important molecules are non-ribosomally synthesized peptides and lipopeptides.•Very strong antimicrobial activity ...was confirmed through numerous studies.•Bacillus lipopeptides have strong impact through direct antibiosis on plant pathogens.
Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.
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