Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Regulating Pathways of Bacillus pumilus Adamalysin-like Metalloendopeptidase Expression.

The minor secreted proteinase of B. pumilus 3-19 MprBp classified as the unique bacillary adamalysin-like enzyme of the metzincin clan. The functional role of this metalloproteinase in the bacilli cells is not clear. Analysis of the regulatory region of the mprBp gene showed the presence of potential binding sites to the transcription regulatory factors Spo0A (sporulation) and DegU (biodegradation). The study of mprBp activity in mutant strains of B. subtilis defective in regulatory proteins of the Spo- and Deg-systems showed that the mprBp gene is partially controlled by the Deg-system of signal transduction and independent from the Spo-system.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Genomic and metabolomic profiling of endolithic Rhodococcus fascians strain S11 isolated from an arid serpentine environment.

Genomic and metabolomic studies of endolithic bacteria are essential for understanding their adaptations to extreme conditions of the rock environment and their contributions to mineralization and weathering processes. The endoliths of arid serpentine rocks are exposed to different environmental stresses, including desiccation and re-hydration, temperature fluctuations, oligotrophy, and high concentrations of heavy metals. Bacteria of the genus Rhodococcus commonly inhabit endolithic environments. Here, we describe genomic and metabolomic analyses of the non-pathogenic wild-type Rhodococcus fascians strain S11, isolated from weathered serpentine rock at the arid Khalilovsky massif, Russia. We found that strain S11 lacks the virulence plasmid that functions in the phytopathogenecity of some R. fascians strains. Phenotypic profiling revealed a high pH tolerance, phytase activity and siderophore production. A widely untargeted metabolome analysis performed using an Orbitrap LC-MS/MS method demonstrated the presence of chrysobactin-type siderophores in the culture medium of strain S11. The natural variation of secondary metabolites produced by strain S11 might provide a practical basis for revealing antibacterial, fungicide or insecticidal activities. Finally, plant infection and plant growth stimulation studies showed no observable effect of exposure strain S11 bacteria on the aerial and root parts of Arabidopsis thaliana plants. Based on our findings, R. fascians strain S11 might be promising tool for investigations of organo-mineral interactions, heavy metal bioremediation, and mechanisms of bacterial mediated weathering of plant-free serpentine rock to soil.

Diversity in the swimming motility and flagellar regulon structure of uropathogenic Morganella morganii strains.

In current times, the opportunistic pathogen Morganella morganii is increasingly becoming a cause of urinary tract infections. The condition has been further complicated by the multiple drug resistance of most isolates. Swimming motility plays an important role in the development of urinary tract infections, allowing bacteria to colonize the upper urinary tract. We determined the differences between the growth, swimming motility, and biofilm formation of two M. morganii strains MM 1 and MM 190 isolated from the urine of patients who had community-acquired urinary tract infections. MM 190 showed a lower growth rate but better-formed biofilms in comparison to MM 1. In addition, MM 190 possessed autoaggregation abilities. It was found that a high temperature (37 °C) inhibits the flagellation of strains and makes MM 190 less motile. At the same time, the MM 1 strain maintained its rate of motility at this temperature. We demonstrated that urea at a concentration of 1.5% suppresses the growth and swimming motility of both strains. Genome analysis showed that MM 1 has a 17.7-kb-long insertion in flagellar regulon between fliE and glycosyltransferase genes, which was not identified in corresponding loci of MM 190 and 9 other M. morganii strains with whole genomes. Both strains carry two genes encoding flagellin, which may indicate flagellar antigen phase variation. However, the fliC2 genes have only 91% identity to each other and exhibit some variability in the regulatory region. We assume that all these differences influence the swimming motility of the strains.

Genomic and phenotypic analysis of siderophore-producing Rhodococcus qingshengii strain S10 isolated from an arid weathered serpentine rock environment.

The success of members of the genus Rhodococcus in colonizing arid rocky environments is owed in part to desiccation tolerance and an ability to extract iron through the secretion and uptake of siderophores. Here, we report a comprehensive genomic and taxonomic analysis of Rhodococcus qingshengii strain S10 isolated from eathered serpentine rock at the arid Khalilovsky massif, Russia. Sequence comparisons of whole genomes and of selected marker genes clearly showed strain S10 to belong to the R. qingshengii species. Four prophage sequences within the R. qingshengii S10 genome were identified, one of which encodes for a putative siderophore-interacting protein. Among the ten non-ribosomal peptides synthase (NRPS) clusters identified in the strain S10 genome, two show high homology to those responsible for siderophore synthesis. Phenotypic analyses demonstrated that R. qingshengii S10 secretes siderophores and possesses adaptive features (tolerance of up to 8% NaCl and pH 9) that should enable survival in its native habitat within dry serpentine rock.

Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria.

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

Structure and variation of root-associated microbiomes of potato grown in alfisol.

Root-associated fungi and bacteria play a pivotal role in the plant-soil ecosystem by influencing both plant growth and immunity. The aim of this study was to unravel the biodiversity of the bacterial and fungal rhizosphere (RS) and rhizoplane (RP) microbiota of Zhukovskij rannij potato (Solanum tuberosum L.) cultivar growing in the Alfisol of Tatarstan, Russia. To assess the structure and diversity of microbial communities, we employed the 16S rRNA and internal transcribed spacer gene library technique. Overall, sequence analysis showed the presence of 3982 bacterial and 188 fungal operational taxonomic units (OTUs) in the RP, and 6018 bacterial and 320 fungal OTUs for in the RS. Comparison between microbial community structures in the RS and RP showed significant differences between these compartments. Biodiversity was higher in the RS than in the RP. Although members of Proteobacteria (RS-59.1 ± 4.9%; RP-54.5 ± 9.2%), Bacteroidetes (RS-23.19 ± 10.2%; RP-34.52 ± 10.4%) and Actinobacteria (RS-11.55 ± 4.9%; RP-7.7 ± 5.1%) were the three most dominant phyla, accounting for 94-98% of all bacterial taxa in both compartments, notable variations were observed in the primary dominance of classes and genera in RS and RP samples. In addition, our results demonstrated that the potato rhizoplane was significantly enriched with the genera Flavobacterium, Pseudomonas, Acinetobacter and other potentially beneficial bacteria. The fungal community was predominantly inhabited by members of the Ascomycota phylum (RS-81.4 ± 8.1%; RP-81.7 ± 5.7%), among which the genera Fusarium (RS-10.34 ± 3.41%; RP-9.96 ± 4.79%), Monographella (RS-7.66 ± 4.43%; RP-9.91 ± 5.87%), Verticillium (RS-4.6 ± 1.43%; RP-8.27 ± 3.63%) and Chaetomium (RS-4.95 ± 2.07%; RP-8.33 ± 4.93%) were particularly abundant. Interestingly, potato rhizoplane was significantly enriched with potentially useful fungal genera, such as Mortierella and Metacordiceps. A comparative analysis revealed that the abundance of Fusarium (a cosmopolitan plant pathogen) varied significantly depending on rotation variants, indicating a possible control of phytopathogenic fungi via management-induced shifts through crop rotational methods. Analysis of the core microbiome of bacterial and fungal community structure showed that the formation of bacterial microbiota in the rhizosphere and rhizoplane is dependent on the host plant.

Regulatory Characteristics of Bacillus pumilus Protease Promoters.

Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5' regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3-19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445 bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250 bp in length, respectively. Promoter analysis of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of P aprBp -gfpmu3 and P gseBp -gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis.

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1.

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.

Telomere Length Variation in Model Bryophytes.

The ends of linear chromosomes of most eukaryotes consist of protein-bound DNA arrays called telomeres, which play essential roles in protecting genome integrity. Despite general evolutionary conservation in function, telomeric DNA is known to drastically vary in length and sequence between different eukaryotic lineages. Bryophytes are a group of early diverging land plants that include mosses, liverworts, and hornworts. This group of ancient land plants recently emerged as a new model for important discoveries in genomics and evolutionary biology, as well as for understanding plant adaptations to a terrestrial lifestyle. We measured telomere length in different ecotypes of model bryophyte species, including Physcomitrium patens, Marchantia polymorpha, Ceratodon purpureus, and in Sphagnum isolates. Our data indicate that all analyzed moss and liverwort genotypes have relatively short telomeres. Furthermore, all analyzed ecotypes and isolates of model mosses and liverworts display evidence of substantial natural variation in telomere length. Interestingly, telomere length also differs between male and female strains of the dioecious liverwort M. polymorpha and dioecious moss C. purpureus. Given that bryophytes are extraordinarily well adapted to different ecological niches from polar to tropical environments, our data will contribute to understanding the impact of natural telomere length variation on evolutionary adaptations in this ancient land plant lineage.

Genomic insights and anti-phytopathogenic potential of siderophore metabolome of endolithic Nocardia mangyaensis NH1.

Actinobacteria are one of the predominant groups that successfully colonize and survive in various aquatic, terrestrial and rhizhospheric ecosystems. Among actinobacteria, Nocardia is one of the most important agricultural and industrial bacteria. Screening and isolation of Nocardia related bacteria from extreme habitats such as endolithic environments are beneficial for practical applications in agricultural and environmental biotechnology. In this work, bioinformatics analysis revealed that a novel strain Nocardia mangyaensis NH1 has the capacity to produce structurally varied bioactive compounds, which encoded by non-ribosomal peptide synthases (NRPS), polyketide synthase (PKS), and post-translationally modified peptides (RiPPs). Among NRPS, five gene clusters have a sequence homology with clusters encoding for siderophore synthesis. We also show that N. mangyaensis NH1 accumulates both catechol- and hydroxamate-type siderophores simultaneously under iron-deficient conditions. Untargeted LC-MS/MS analysis revealed a variety of metabolites, including siderophores, lipopeptides, cyclic peptides, and indole-3-acetic acid (IAA) in the culture medium of N. mangyaensis NH1 grown under iron deficiency. We demonstrate that four CAS (chrome azurol S)-positive fractions display variable affinity to metals, with a high Fe3+ chelating capability. Additionally, three of these fractions exhibit antioxidant activity. A combination of iron scavenging metabolites produced by N. mangyaensis NH1 showed antifungal activity against several plant pathogenic fungi. We have shown that the pure culture of N. mangyaensis NH1 and its metabolites have no adverse impact on Arabidopsis seedlings. The ability of N. mangyaensis NH1 to produce siderophores with antifungal, metal-chelating, and antioxidant properties, when supplemented with phytohormones, has the potential to improve the release of macro- and micronutrients, increase soil fertility, promote plant growth and development, and enable the production of biofertilizers across diverse soil systems.

Biofilm Formation by Mutant Strains of Bacilli under Different Stress Conditions.

Bacillus subtilis is traditionally classified as a PGPR that colonizes plant roots through biofilm formation. The current study focused on investigating the influence of various factors on bacilli biofilm formation. In the course of the study, the levels of biofilm formation by the model strain B. subtilis WT 168 and on its basis created regulatory mutants, as well as strains of bacilli with deleted extracellular proteases under conditions of changes in temperature, pH, salt and oxidative stress and presence of divalent metals ions. B. subtilis 168 forms halotolerant and oxidative stress-resistant biofilms at a temperature range of 22 °C-45 °C and a pH range of 6-8.5. The presence of Ca2+, Mn2+ and Mg2+ upsurges the biofilm development while an inhibition with Zn2+. Biofilm formation level was higher in protease-deficient strains. Relative to the wild-type strain, degU mutants showed a decrease in biofilm formation, abrB mutants formed biofilms more efficiently. spo0A mutants showed a plummeted film formation for the first 36 h, followed by a surge after. The effect of metal ions and NaCl on the mutant biofilms formation is described. Confocal microscopy indicated that B. subtilis mutants and protease-deficient strains differ in matrix structure. The highest content of amyloid-like proteins in mutant biofilms was registered for degU-mutants and protease-deficient strains.

Phosphate Solubilization and Plant Growth Promotion by Pantoea brenneri Soil Isolates.

Phosphate solubilizing microorganisms (PSMs) in soil have been shown to reduce mineral phosphate fertilizer supplementation and promote plant growth. Nevertheless, only several P-solubilizing microorganisms capable of solubilizing both organic and mineral sources of soil phosphorus have been identified up to now. The aim of this study was to evaluate the inorganic soil phosphate solubilizing activity of phytate-hydrolyzing Pantoea brenneri soil isolates. We showed that the strains efficiently solubilize a variety of inorganic phosphates. We optimized the media composition and culturing conditions to improve the solubilization efficiency of the strains and investigated the mechanisms of their phosphate solubilization. Through HPLC analysis, it was determined that P. brenneri produce oxalic, malic, formic, malonic, lactic, maleic, acetic, and citric acids as well as acid and alkaline phosphatases while growing on insoluble phosphate sources. Finally, we analyzed the influence of P. brenneri strains with multiple PGP-treats on plant growth in greenhouse experiments and showed their ability to promote growth of potato.

Characterization of Bacillus pumilus Strains with Targeted Gene Editing for Antimicrobial Peptides and Sporulation Factor.

Due to their capacity to produce antimicrobial peptides that can prevent the growth of diseases, many Bacillus spp. are beneficial to plants. In this study, we looked into the antagonistic activity of the B. pumilus 3-19 strain and its derivatives following targeted genome editing. Two peptide genes with antibacterial action, bacilysin (bac) and bacteriocin (bact), and the sigF gene, which encodes the sigma factor of sporulation, were specifically inactivated using the CRISPR-Cas9 system in the genome of B. pumilus 3-19. Antibacterial activity against B. cereus and Pantoea brenneri decreased as a result of the inactivation of target genes in the B. pumilus 3-19 genome, with a noticeable effect against bacilysin. The growth dynamics of the culture changed when the bac, bact, and sigF genes were inactivated, and the altered strains had less proteolytic activity. An asporogenic mutant of B. pumilus 3-19 was obtained by inactivating the sigF gene. It has been proven that bacilysin plays a unique part in the development of B. pumilus 3-19's antagonistic action against soil microorganisms.

Identification and Analysis of Antimicrobial Activities from a Model Moss Ceratodon purpureus.

The emergence of bacterial drug resistance is often viewed as the next great health crisis of our time. While more antimicrobial agents are urgently needed, very few new antibiotics are currently in the production pipeline. Here, we aim to identify and characterize novel antimicrobial natural products from a model dioicous moss, Ceratodon purpureus. We collected secreted moss exudate fractions from two C. purpureus strains, male R40 and female GG1. Exudates from the female C. purpureus strain GG1 did not exhibit inhibitory activity against any tested bacteria. However, exudates from the male moss strain R40 exhibited strong inhibitory properties against several species of Gram-positive bacteria, including Staphylococcus aureus and Enterococcus faecium, though they did not inhibit the growth of Gram-negative bacteria. Antibacterial activity levels in C. purpureus R40 exudates significantly increased over four weeks of moss cultivation in liquid culture. Size fractionation experiments indicated that the secreted bioactive compounds have a relatively low molecular weight of less than 1 kDa. Additionally, the R40 exudate compounds are thermostable and not sensitive to proteinase K treatment. Overall, our results suggest that the bioactive compounds present in C. purpureus R40 exudates can potentially add new options for treating infections caused by antibiotic-resistant Gram-positive bacteria.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a - deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter.

BACKGROUND: Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. RESULTS: Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the "LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. CONCLUSIONS: The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories.

Comparative Genome Analysis of Two Bacillus pumilus Strains Producing High Level of Extracellular Hydrolases.

Whole-genome sequencing of a soil isolate Bacillus pumilus, strain 7P, and its streptomycin-resistant derivative, B. pumilus 3-19, showed genome sizes of 3,609,117 bp and 3,609,444 bp, respectively. Annotation of the genome showed 3794 CDS (3204 with predicted function) and 3746 CDS (3173 with predicted function) in the genome of strains 7P and 3-19, respectively. In the genomes of both strains, the prophage regions Bp1 and Bp2 were identified. These include 52 ORF of prophage proteins in the Bp1 region and 38 prophages ORF in the Bp2 region. Interestingly, more than 50% of Bp1 prophage proteins are similar to the proteins of the phi105 in B. subtilis. The DNA region of Bp2 has 15% similarity to the DNA of the Brevibacillus Jimmer phage. Degradome analysis of the genome of both strains revealed 148 proteases of various classes. These include 60 serine proteases, 48 metalloproteases, 26 cysteine proteases, 4 aspartate proteases, 2 asparagine proteases, 3 threonine proteases, and 2 unclassified proteases. Likewise, three inhibitors of proteolytic enzymes were found. Comparative analysis of variants in the genomes of strains 7P and 3-19 showed the presence of 81 nucleotide variants in the genome 3-19. Among them, the missense mutations in the rpsL, comA, spo0F genes and in the upstream region of the srlR gene were revealed. These nucleotide polymorphisms may have affected the streptomycin resistance and overproduction of extracellular hydrolases of the 3-19 strain. Finally, a plasmid DNA was found in strain 7P, which is lost in its derivative, strain 3-19. This plasmid contains five coding DNA sequencing (CDS), two regulatory proteins and three hypothetical proteins.

Identification, characterization, and genome sequencing of Brevibacterium sediminis MG-1 isolate with growth-promoting properties.

In recent years, plant growth-promoting rhizobacteria (PGPR) have received increased attention due to their prospective use as biofertilizers for the enhancement of crop growth and yields. However, there is a growing need to identify new PGPR isolates with additional beneficial properties. In this paper, we describe the identification of a new strain of a non-sporulating Gram-positive bacterium isolated from the rhizosphere of potato plants, classified as Brevibacterium sediminis MG-1 based on whole-genome sequencing. The bacteria are aerobic; they grow in a pH range of 6.0-10.0 (optimum 6.0), and a temperature range of 20-37 °C (optimum 30 °C). At 96 h of cultivation, strain MG-1 synthesizes 28.65 µg/ml of indole-3-acetic acid (IAA) when 500 µg/ml of l-tryptophan is added. It is a producer of catechol-type siderophores and ACC deaminase (213 ± 12.34 ng/ml) and shows halotolerance. Treatment of pea, rye, and wheat seeds with a suspension of MG-1 strain cells resulted in the stimulation of stem and root biomass accumulation by 12-26% and 6-25% (P < 0.05), respectively. Treatment of seeds with bacteria in the presence of high salt concentration reduced the negative effects of salt stress on plant growth by 18-50%. The hypothetical gene lin, encoding the bacteriocin Linocin-M18, RIPP-like proteins, and polyketide synthase type III (T3PKS) loci, gene clusters responsible for iron acquisition and metabolism of siderophores, as well as gene clusters responsible for auxin biosynthesis, were identified in the B. sediminis MG-1 genome. Thus, the rhizosphere-associated strain B. sediminis MG-1 has growth-stimulating properties and can be useful for the treatment of plants grown on soils with high salinity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03392-z.