scholarly journals A native conjugative plasmid confers potential selective advantages to plant growth-promoting Bacillus velezensis strain GH1-13

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yunhee Choi ◽  
Ha Pham ◽  
Mai Phuong Nguyen ◽  
Le Viet Ha Tran ◽  
Jueun Kim ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Marker Gene ◽  
Selective Advantage ◽  
Conjugative Plasmid ◽  
Pcr Analysis ◽  
Wild Type ◽  
Bacillus Velezensis ◽  
Reverse Complement ◽  
Extracellular Matrix Components ◽  
Plant Growth Promoting

AbstractThe conjugative plasmid (pBV71) possibly confers a selective advantage to Bacillus velezensis strain GH1-13, although a selective marker gene is yet to be identified. Here we show that few non-mucoid wild-type GH1-13 cells are spontaneously converted to mucoid variants with or without the loss of pBV71. Mucoid phenotypes, which contain or lack the plasmid, become sensitive to bacitracin, gramicidin, selenite, and tellurite. Using the differences in antibiotic resistance and phenotype, we isolated a reverse complement (COM) and a transconjugant of strain FZB42 with the native pBV71. Transformed COM and FZB42p cells were similar to the wild-type strain GH1-13 with high antibiotic resistance and slow growth rates on lactose compared to those of mucoid phenotypes. RT-PCR analysis revealed that the expression of plasmid-encoded orphan aspartate phosphatase (pRapD) was coordinated with a new quorum-sensing (QS) cassette of RapF2–PhrF2 present in the chromosome of strain GH1-13, but not in strain FZB42. Multi-omics analysis on wild-type and plasmid-cured cells of strain GH1-13 suggested that the conjugative plasmid expression has a crucial role in induction of early envelope stress response that promotes cell morphogenesis, biofilm formation, catabolite repression, and biosynthesis of extracellular-matrix components and antibiotics for protection of host cell during exponential phase.

scholarly journals Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

2000 ◽  
Vol 66 (11) ◽  
pp. 4854-4862 ◽  
Author(s):  
Kornelia Smalla ◽  
Holger Heuer ◽  
Antje Götz ◽  
Dagmar Niemeyer ◽  
Ellen Krögerrecklenfort ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Ralstonia Eutropha ◽  
Blot Hybridization ◽  
Restriction Pattern ◽  
Pcr Analysis ◽  
Dot Blot ◽  
High Prevalence ◽  
Resistance Plasmids ◽  
Incq Plasmids ◽  
K 12

ABSTRACT Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors inEscherichia coli CV601 and Pseudomonas putidaUWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in severalP. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriTprobes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobactersp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals ε2-Phages Are Naturally Bred and Have a Vastly Improved Host Range in Staphylococcus aureus over Wild Type Phages

2021 ◽  
Vol 14 (4) ◽  
pp. 325
Author(s):  
David Sáez Moreno ◽  
Zehra Visram ◽  
Michele Mutti ◽  
Marcela Restrepo-Córdoba ◽  
Susana Hartmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Detection Limit ◽  
Host Range ◽  
Alternative Treatment ◽  
Standard Of Care ◽  
Wild Type ◽  
Rapid Spread ◽  
Pharmaceutical Properties ◽  
Human Infections

Due to the rapid spread of antibiotic resistance, and the difficulties of treating biofilm-associated infections, alternative treatments for S. aureus infections are urgently needed. We tested the lytic activity of several wild type phages against a panel of 110 S. aureus strains (MRSA/MSSA) composed to reflect the prevalence of S. aureus clonal complexes in human infections. The plaquing host ranges (PHR) of the wild type phages were in the range of 51% to 60%. We also measured what we called the kinetic host range (KHR), i.e., the percentage of strains for which growth in suspension was suppressed for 24 h. The KHR of the wild type phages ranged from 2% to 49%, substantially lower than the PHRs. To improve the KHR and other key pharmaceutical properties, we bred the phages by mixing and propagating cocktails on a subset of S. aureus strains. These bred phages, which we termed evolution-squared (ε2) phages, have broader KHRs up to 64% and increased virulence compared to the ancestors. The ε2-phages with the broadest KHR have genomes intercrossed from up to three different ancestors. We composed a cocktail of three ε2-phages with an overall KHR of 92% and PHR of 96% on 110 S. aureus strains and called it PM-399. PM-399 has a lower propensity to resistance formation than the standard of care antibiotics vancomycin, rifampicin, or their combination, and no resistance was observed in laboratory settings (detection limit: 1 cell in 1011). In summary, ε2-phages and, in particular PM-399, are promising candidates for an alternative treatment of S. aureus infections.

scholarly journals Genomic characterization and computational phenotyping of nitrogen-fixing bacteria isolated from Colombian sugarcane fields

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Luz K. Medina-Cordoba ◽  
Aroon T. Chande ◽  
Lavanya Rishishwar ◽  
Leonard W. Mayer ◽  
Lina C. Valderrama-Aguirre ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Nitrogen Fixation ◽  
Plant Growth ◽  
Phosphate Solubilization ◽  
Nitrogen Fixing ◽  
Nitrogen Fixing Bacteria ◽  
Promising Tool ◽  
A Genome ◽  
Plant Growth Promoting ◽  
Growth Promoting

AbstractPrevious studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.

scholarly journals Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana

2021 ◽  
Vol 22 (10) ◽  
pp. 5064
Author(s):  
Qinghua Chen ◽  
Linghui Guo ◽  
Yanwen Yuan ◽  
Shuangling Hu ◽  
Fei Guo ◽  
...  
Keyword(s):  
Drought Stress ◽  
Transmembrane Domain ◽  
Tea Plant ◽  
Microtubule Assembly ◽  
Pcr Analysis ◽  
Drought Response ◽  
Stomatal Development ◽  
Wild Type ◽  
Over Expression ◽  
Histone H3k4

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.

scholarly journals FlrA, a σ54-Dependent Transcriptional Activator in Vibrio fischeri, Is Required for Motility and Symbiotic Light-Organ Colonization

2003 ◽  
Vol 185 (12) ◽  
pp. 3547-3557 ◽  
Author(s):  
Deborah S. Millikan ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Transcriptional Activator ◽  
Pcr Analysis ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Flagellar Regulon ◽  
Colonization Factors ◽  
Recognition Motifs ◽  
Arbitrarily Primed Pcr ◽  
Organ Colonization

ABSTRACT Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative σ54-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent σ54 recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

scholarly journals Antagonism between Aminoglycosides and β-Lactams in a Methicillin-Resistant Staphylococcus aureus Isolate Involves Induction of an Aminoglycoside-Modifying Enzyme

2002 ◽  
Vol 46 (5) ◽  
pp. 1516-1521 ◽  
Author(s):  
Takashi Ida ◽  
Ryoichi Okamoto ◽  
Masato Nonoyama ◽  
Kazuhiko Irinoda ◽  
Mizuyo Kurazono ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Antimicrobial Activities ◽  
Hybrid Structure ◽  
Conjugative Plasmid ◽  
Pcr Analysis ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Methicillin Resistant ◽  
Aureus Isolate

ABSTRACT We encountered three clinical isolates of methicillin-resistant Staphylococcus aureus which were susceptible to netilmicin and arbekacin in the absence of β-lactam antibiotics but which were resistant to them in the presence of β-lactam antibiotics. One of these strains, KU5801, was used to further investigate the antagonism between aminoglycosides and β-lactam antibiotics. β-Lactam antibiotics induced bacterial synthesis of aminoglycoside-6′-N-acetyltransferase and 2"-O-phosphotransferase [AAC(6′)-APH(2")] in association with decreased antimicrobial activities of aminoglycosides. A 14.4-kb EcoRI fragment that included the genes that control for β-lactam-inducible aminoglycoside resistance was cloned from a 31-kb conjugative plasmid present in KU5801. Restriction fragment mapping and PCR analysis suggested that a Tn4001-like element containing a gene encoding AAC(6′)-APH(2") was located downstream from a truncated blaZ gene. The DNA sequence between blaR1 and a Tn4001-like element was determined. The Tn4001-IS257 hybrid structure was cointegrated into the blaZ gene, and the typical sequences for the termination of transcription were not found between these regions. We deduced that antagonism of aminoglycosides by β-lactam antibiotics in isolate KU5801 involved transcription of the aac(6′)-Ie-aph(2")-Ia gene under the influence of the system regulating penicillinase production.

scholarly journals Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

2006 ◽  
Vol 188 (6) ◽  
pp. 2063-2072 ◽  
Author(s):  
Preeti M. Tendolkar ◽  
Arto S. Baghdayan ◽  
Nathan Shankar
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Insertional Mutagenesis ◽  
Surface Proteins ◽  
Conjugative Plasmid ◽  
Sequence Information ◽  
Wild Type ◽  
Abiotic Surfaces ◽  
Mutant Bank

ABSTRACT Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

scholarly journals Bacillus velezensisWall Teichoic Acids Are Required for Biofilm Formation and Root Colonization

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Zhihui Xu ◽  
Huihui Zhang ◽  
Xinli Sun ◽  
Yan Liu ◽  
Wuxia Yan ◽  
...  
Keyword(s):  
Plant Growth ◽  
Biofilm Formation ◽  
Root Colonization ◽  
Molecular Networks ◽  
Model Organisms ◽  
Bacillus Velezensis ◽  
Content Type ◽  
Plant Growth Promoting ◽  
Colonization Process ◽  
Growth Promoting

ABSTRACTRhizosphere colonization by plant growth-promoting rhizobacteria (PGPR) along plant roots facilitates the ability of PGPR to promote plant growth and health. Thus, an understanding of the molecular mechanisms of the root colonization process by plant-beneficialBacillusstrains is essential for the use of these strains in agriculture. Here, we observed that ansfpgene mutant of the plant growth-promoting rhizobacteriumBacillus velezensisSQR9 was unable to form normal biofilm architecture, and differential protein expression was observed by proteomic analysis. A minor wall teichoic acid (WTA) biosynthetic protein, GgaA, was decreased over 4-fold in the Δsfpmutant, and impairment of theggaAgene postponed biofilm formation and decreased cucumber root colonization capabilities. In addition, we provide evidence that the major WTA biosynthetic enzyme GtaB is involved in both biofilm formation and root colonization. The deficiency in biofilm formation of the ΔgtaBmutant may be due to an absence of UDP-glucose, which is necessary for the synthesis of biofilm matrix exopolysaccharides (EPS). These observations provide insights into the root colonization process by a plant-beneficialBacillusstrain, which will help improve its application as a biofertilizer.IMPORTANCEBacillus velezensisis a Gram-positive plant-beneficial bacterium which is widely used in agriculture. Additionally,Bacillusspp. are some of the model organisms used in the study of biofilms, and as such, the molecular networks and regulation systems of biofilm formation are well characterized. However, the molecular processes involved in root colonization by plant-beneficialBacillusstrains remain largely unknown. Here, we showed that WTAs play important roles in the plant root colonization process. The loss of thegtaBgene affects the ability ofB. velezensisSQR9 to sense plant polysaccharides, which are important environmental cues that trigger biofilm formation and colonization in the rhizosphere. This knowledge provides new insights into theBacillusroot colonization process and can help improve our understanding of plant-rhizobacterium interactions.

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