scholarly journals Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis

2020 ◽  
Vol 8 (9) ◽  
pp. 1269
Author(s):  
Gro Herredsvela Rørvik ◽  
Krystyna Anna Liskiewicz ◽  
Fedor Kryuchkov ◽  
Ali-Oddin Naemi ◽  
Hans-Christian Aasheim ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Adenosine Monophosphate ◽  
Opportunistic Pathogen ◽  
Deletion Mutants ◽  
Bacterial Cells ◽  
Bacterial Signaling ◽  
Intermediary Metabolite ◽  
Genes Encoding ◽  
Biochemical Analyses ◽  
Reduced Rate

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as an important bacterial signaling molecule that functions both as an intracellular second messenger in bacterial cells and an extracellular ligand involved in bacteria-host cross-talk. In this study, we identify and characterize proteins involved in controlling the c-di-AMP concentration in the oral commensal and opportunistic pathogen Streptococcusmitis (S. mitis). We identified three known types of c-di-AMP turnover proteins in the genome of S. mitis CCUG31611: a CdaA-type diadenylate cyclase as well as GdpP-, and DhhP-type phosphodiesterases. Biochemical analyses of purified proteins demonstrated that CdaA synthesizes c-di-AMP from ATP whereas both phosphodiesterases can utilize c-di-AMP as well as the intermediary metabolite of c-di-AMP hydrolysis 5′-phosphadenylyl-adenosine (pApA) as substrate to generate AMP, albeit at different catalytic efficiency. Using deletion mutants of each of the genes encoding c-di-AMP turnover proteins, we show by high resolution MS/MS that the intracellular concentration of c-di-AMP is increased in deletion mutants of the phosphodiesterases and non-detectable in the cdaA-mutant. We also detected pApA in mutants of the DhhP-type phosphodiesterase. Low and high levels of c-di-AMP were associated with longer and shorter chains of S. mitis, respectively indicating a role in regulation of cell division. The deletion mutant of the DhhP-type phosphodiesterase displayed slow growth and reduced rate of glucose metabolism.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

2022 ◽  
Author(s):  
Baopeng Yang ◽  
Yujun Jiang ◽  
Yongxin Jin ◽  
Fang Bai ◽  
Zhihui Cheng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Defense Mechanism ◽  
Extracellular Polysaccharide ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Guanosine Monophosphate ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Bacterial Survival

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

scholarly journals Draft Genome Sequences of Two Enterobacter cloacae subsp. cloacae Strains Isolated from Australian Hematology Patients with Bacteremia

2017 ◽  
Vol 5 (33) ◽  
Author(s):  
Lex E. X. Leong ◽  
David Shaw ◽  
Lito Papanicolas ◽  
Diana Lagana ◽  
Ivan Bastian ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Draft Genome ◽  
Enterobacter Cloacae ◽  
Opportunistic Pathogen ◽  
Healthy Individuals ◽  
Genome Sequences ◽  
Content Type ◽  
Extended Spectrum ◽  
Genes Encoding

ABSTRACT Enterobacter cloacae is a common member of the gut microbiota in healthy individuals. However, it is also an opportunistic pathogen, capable of causing bacteremia. We report the draft genomes of two Enterobacter cloacae subspecies cloacae strains isolated from hematology patients with bacteremia. Both isolates carry genes encoding extended-spectrum β-lactamases.

scholarly journals Identification and Characterization of Quorum-Quenching Activity of N-Acylhomoserine Lactonase from Coagulase-Negative Staphylococci

Antibiotics ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 483
Author(s):  
Tomohiro Morohoshi ◽  
Yaoki Kamimura ◽  
Nobutaka Someya
Keyword(s):  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Quorum Quenching ◽  
Coagulase Negative Staphylococci ◽  
Sensing Systems ◽  
Genes Encoding ◽  
Acyl Chains ◽  
Gene Homologue ◽  
Identification And Characterization

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signals in Gram-negative bacteria. Many genes encoding AHL-degrading enzymes have been cloned and characterized in various microorganisms. Coagulase-negative staphylococci (CNS) are present on the skin of animals and are considered low-virulent species. The AHL-lactonase gene homologue, ahlS, was present in the genomes of the CNS strains Staphylococcus carnosus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus sciuri. We cloned the candidate ahlS homologue from six CNS strains into the pBBR1MCS5 vector. AhlS from the CNS strains showed a higher degrading activity against AHLs with short acyl chains compared to those with long acyl chains. AhlS from S. sciuri was expressed and purified as a maltose-binding protein (MBP) fusion. Pseudomonas aeruginosa is an opportunistic pathogen that regulates several virulence factors such as elastase and pyocyanin by quorum-sensing systems. When MBP-AhlS was added to the culture of P. aeruginosa PAO1, pyocyanin production and elastase activity were substantially reduced compared to those in untreated PAO1. These results demonstrate that the AHL-degrading activity of AhlS from the CNS strains can inhibit quorum sensing in P. aeruginosa PAO1.

scholarly journals Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA

1999 ◽  
Vol 65 (2) ◽  
pp. 514-522 ◽  
Author(s):  
Silvia G. Acinas ◽  
Josefa Antón ◽  
Francisco Rodríguez-Valera
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Western Mediterranean ◽  
Bacterial Cells ◽  
Free Living ◽  
Attached Bacteria ◽  
Genes Encoding ◽  
Community Fingerprinting ◽  
Close Relatives ◽  
Alteromonas Macleodii

ABSTRACT In a previous study (S. G. Acinas, F. Rodrı́guez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria, Alteromonas macleodii, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated γProteobacteria predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the α Proteobacteria of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the A. macleodii cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.

scholarly journals Isolation, Screening and Identification of Cellulolytic Bacteria from Soil

2020 ◽  
pp. 1-8 ◽  
Author(s):  
Pratibha Maravi ◽  
Anil Kumar
Keyword(s):  
Plant Cell Wall ◽  
Paper Mill ◽  
Soil Samples ◽  
Bacterial Cells ◽  
Cellulolytic Bacteria ◽  
Nutrient Media ◽  
Phylogenetic Tree Analysis ◽  
Parasite Relationship ◽  
Screening And Identification ◽  
Biochemical Analyses

Background: Cellulose is the most abundant carbohydrate on earth and is considered as a good candidate for production of second generation biofuel (ethanol) and many other products of routine use. For degradation, cellulases are used which are mostly secreted by microbes such as fungi. Cellulases also play an important role in senescence of plants and in host-parasite relationship for invading the plant cell wall. However, comparatively lesser studies have been carried out on cellulase producing bacteria. Therefore, present study was aimed to isolate cellulase (Endo-β-1,4-D-glucanase; EC. 3.2.1.4.) from bacterial sources. Methodology: To isolate thermophilic/ mesophilic cellulase producing bacteria, soil samples were collected from wood furnishing area and agricultural farm around Indore. Besides, soil sample was also collected from the vicinity of Amlai Paper Mill in Budhar district, Madhya Pradesh. These soil samples after suitable dilutions were streaked on different nutrients agar petri-dishes having carboxymethyl cellulose (CMC) as an inducer. After screening, four colonies were isolated capable of producing good amount of cellulase. Screening was done using Congo red staining and confirmation was done after growth of the bacteria in liquid nutrient medium having CMC. These colonies individually were grown in suitable nutrient media having CMC as an inducer and enzyme activity was determined in the nutrient media after harvesting bacterial cells by centrifugation. Results: The highest enzyme producing bacteria were identified as Bacillus lichenoformis and Ochrobactrum anthropi after biochemical analyses, 16S rRNA sequencing and subsequently phylogenetic tree analysis.

scholarly journals F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Senjuti Saha ◽  
Chidozie D. Ojobor ◽  
Erik Mackinnon ◽  
Olesia I. North ◽  
Joseph Bondy-Denomy ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bactericidal Activity ◽  
Pathogenic Bacteria ◽  
Therapeutic Potential ◽  
Experimental Studies ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Antibiotic Resistant ◽  
Genes Encoding ◽  
Intraspecies Competition

ABSTRACTMost Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3’-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

scholarly journals GuUGT, a glycosyltransferase from Glycyrrhiza uralensis , exhibits glycyrrhetinic acid 3- and 30-O-glycosylation

2019 ◽  
Vol 6 (10) ◽  
pp. 191121 ◽  
Author(s):  
Ying Huang ◽  
Da Li ◽  
Jinhe Wang ◽  
Yi Cai ◽  
Zhubo Dai ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Glycyrrhetinic Acid ◽  
Glycyrrhiza Uralensis ◽  
Transcriptome Data ◽  
Bioactive Components ◽  
Triterpenoid Saponins ◽  
Genes Encoding ◽  
Engineered Yeast ◽  
Biochemical Analyses

Glycyrrhiza uralensis is a well-known herbal medicine that contains triterpenoid saponins as the predominant bioactive components, and these compounds include glycyrrhetinic acid (GA)-glycoside derivatives. Although two genes encoding UDP-glycosyltransferases (UGTs) that glycosylate these derivates have been functionally characterized in G. uralensis , the mechanisms of glycosylation by other UGTs remain unknown. Based on the available transcriptome data, we isolated a UGT with expression in the roots of G. uralensis . This UGT gene possibly encodes a glucosyltransferase that glycosylates GA derivatives at the 3-OH site. Biochemical analyses revealed that the recombinant UGT enzyme could transfer a glucosyl moiety to the free 3-OH or 30-COOH groups of GA. Furthermore, engineered yeast harbouring genes involved in the biosynthetic pathway for GA-glycoside derivates produced GA-3- O -β-D-glucoside, implying that the enzyme has GA 3-O-glucosyltransferase activity in vivo . Our results could provide a frame for understand the function of the UGT gene family, and also is important for further studies of triterpenoids biosynthesis in G. uralensis .

scholarly journals Genetic Analysis of the Methanol- and Methylamine-Specific Methyltransferase 2 Genes of Methanosarcina acetivorans C2A

2008 ◽  
Vol 190 (11) ◽  
pp. 4017-4026 ◽  
Author(s):  
Arpita Bose ◽  
Matthew A. Pritchett ◽  
William W. Metcalf
Keyword(s):  
Genetic Analysis ◽  
Methane Production ◽  
Untranslated Region ◽  
Gene Fusions ◽  
Methanosarcina Acetivorans ◽  
Concerted Effort ◽  
Genes Encoding ◽  
Low Levels ◽  
Reduced Rate

ABSTRACT The entry of methanol into the methylotrophic pathway of methanogenesis is mediated by the concerted effort of two methyltransferases, namely, methyltransferase 1 (MT1) and methyltransferase 2 (MT2). The mtaA1, mtaA2, and mtbA genes of Methanosarcina acetivorans C2A encode putative methanol- or methylamine-specific MT2 enzymes. To address the in vivo roles of these genes in growth and methanogenesis from known substrates, we constructed and characterized mutants with deletions of each of these genes. The mtaA1 gene is required for growth on methanol, whereas mtaA2 was dispensable. However, the mtaA2 mutant had a reduced rate of methane production from methanol. Surprisingly, deletion of mtaA1 in combination with deletions of the genes encoding three methanol-specific MT1 isozymes led to lack of growth on acetate, suggesting that MT1 and MT2 enzymes might play an important role during growth on this substrate. The mtbA gene was required for dimethylamine and monomethylamine (MMA) utilization and was important, but not required, for trimethylamine utilization. Analysis of reporter gene fusions revealed that both mtaA1 and mtbA were expressed on all methanogenic substrates tested. However, mtaA1 expression was induced on methanol, while mtbA expression was down-regulated on MMA and acetate. mtaA2 was expressed at very low levels on all substrates. The mtaA1 transcript had a large 5′ untranslated region (UTR) (275 bp), while the 5′ UTR of the mtbA transcript was only 28 bp long.

259 PRELIMINARY INVESTIGATIONS OF THE EFFECT OF NITRIC OXIDE ON THE EXPRESSION OF GENES INVOLVED IN THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE/CYCLIC ADENOSINE MONOPHOSPHATE PATHWAY DURING MEIOSIS RESUMPTION IN BOVINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 277
Author(s):  
K. R. L. Schwarz ◽  
M. R. Chiaratti ◽  
L. G. Mesquita ◽  
C. L. V. Leal
Keyword(s):  
Nitric Oxide ◽  
Oocyte Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Temporary Increase ◽  
Expression Of Genes ◽  
Genes Encoding

Nitric oxide is a chemical messenger generated by the activity of the enzyme NO synthase (NOS) and has been implicated in the control of oocyte maturation. Nitric oxide stimulates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases (PDE) that may interfere with cyclic adenosine monophosphate (cAMP) levels, a nucleotide also involved in meiosis resumption. In a previous study, we found that increasing NO levels in the presence of a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) resulted in a delayed resumption of meiosis and a lower rate of germinal vesicle breakdown after 9 h of in vitro maturation. A temporary increase in cGMP levels was also observed with the same treatment, which was reversed by inhibiting GC activity with oxadiazolo-quinoxaline-one (ODQ; unpublished data). These results suggest that NO acted via GC/cGMP and that even a temporary increase in the cGMP level led to a delay of meiosis resumption. The aim of the present study was to determine the role played by NO on the expression of genes encoding for enzymes of the NO/GC/cGMP and cAMP pathways during the first 9 h of oocyte maturation. Cumulus–oocyte complexes were in vitro matured for 9 h in a semi-defined medium (TCM-199 + 3 mg mL–1 of BSA) with 10 to 7 M SNAP associated or not associated with 100 µM ODQ, a GC inhibitor. A group of oocytes incubated in the absence of inhibitors was considered the control. Total RNA was extracted from pools of 20 denuded oocytes with TRIzol (Life Technologies, Grand Island, NY, USA) and reverse transcribed into complementary DNA using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed by real-time PCR using SYBR Green (Applied Biosystems). The genes that had their expression measured pertained to one of the following groups: 1) genes encoding for enzymes that synthesise NO (NOS2 and NOS3); 2) genes involved in the control of cGMP levels (GUCY1B3 and PDE5A) or the enzymes activated by it (PKG1 and PKG2); or 3) genes involved in the control of cAMP levels (ADCY3, ADCY6, ADCY9, PDE3A, and PDE8A) or one of the enzymes activated by it (PKA1). GAPDH and PPIA were selected as housekeeping genes using qbasePLUS version 2.3 (Biogazelle, Zwijnaarde, Belgium). Data from 5 replicates were analysed using LinRegPCR version 11.1 and SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). All genes were found to be expressed in the three experimental groups; however, a significant difference in gene expression levels was not found among groups. Results suggest that NO does not act on oocyte maturation by affecting the expression of the investigated genes in oocytes. To our knowledge, this is the first report to demonstrate the expression of the ADCY3, ADCY6, and ADCY9 genes in bovine oocytes. Further research is in progress to study the effect of the SNAP treatment on the expression of these genes in cumulus cells. Financial support from FAPESP 2010/18023-9.

scholarly journals Fast and reliable procedure developed to generate soft rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) Tn5 mutants resistant to bacteriophage infection

2020 ◽  
Vol 159 (1) ◽  
pp. 227-231
Author(s):  
Robert Czajkowski
Keyword(s):  
Growth Medium ◽  
Aminolevulinic Acid ◽  
Soft Rot ◽  
Lytic Bacteriophage ◽  
Bacterial Cells ◽  
Bacteriophage Infection ◽  
Genes Encoding ◽  
Tn5 Mutants ◽  
Bacterial Mutants ◽  
Fast Procedure

AbstractA simple and fast procedure has been developed to generate soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) Tn5 mutants in genes encoding receptors used by bacteriophages to interact with their hosts, for the follow-up studies. The procedure is inexpensive and does not require any specialized tools and/or dedicated technical support. The neomycin-resistant SRP Tn5 mutants are generated via conjugation with a transposon donor Escherichia coli ST18 strain (requiring 5-aminolevulinic acid (5-ALA) to survive) carrying pFAJ1819-mini-Tn5-neoR. The conjugation is done on solid medium supplemented with 5-ALA. After conjugation bacterial cells are collected, suspended in liquid bacterial medium, added to the suspension containing lytic bacteriophages and incubated for the additional 30 min with shaking (120 rpm). During this stage, the transposon recipients (Pectobacterium spp. and/or Dickeya spp. Tn5 mutants), susceptible to bacteriophage infection are lysed. Likewise, due to the lack of 5-ALA in the growth medium, E. coli ST18 (transposon donor) cells die at this stage. Finally, after incubation, the bacterial mutants with the Tn5 insertions, resistant to phage infection are selected on solid growth medium supplemented with neomycin. The Tn5 insertion sites are sequenced to acquire knowledge about the Tn5-distrupted genes and their putative function in phage-host interactions. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes in a short time (ca. 48 h) that can be later characterized for various other phenotypic features. In this study, as a proof-of-concept, this method has been used to generate Dickeya solani IPO2222 Tn5 mutants resistant to infection caused by the lytic bacteriophage ɸD5.

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