scholarly journals Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence

2014 ◽  
Vol 82 (7) ◽  
pp. 2728-2735 ◽  
Author(s):  
Swarnava Chaudhuri ◽  
Siddharth Pratap ◽  
Victor Paromov ◽  
Zhijun Li ◽  
Chinmay K. Mantri ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Second Messenger ◽  
Major Protein ◽  
Protein Subunit ◽  
Content Type ◽  
Diguanylate Cyclases ◽  
Obligate Anaerobic ◽  
Genes Encoding ◽  
Ggdef Domain

ABSTRACTPorphyromonas gingivalisis a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3′-5′) cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated inP. gingivalis, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in thepgn_1932gene had a significant effect on the intracellular c-di-GMP level inP. gingivalis. Genetic analysis showed that expression of thefimAandrgpAgenes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in thepgn_1932mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in thepgn_1932gene also had a significant impact on the adhesive and invasive capabilities ofP. gingivalis, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion byP. gingivalisby controlling the expression and biosynthesis of FimA.

scholarly journals Enzymatically Active and Inactive Phosphodiesterases and Diguanylate Cyclases Are Involved in Regulation of Motility or Sessility in Escherichia coli CFT073

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Rachel R. Spurbeck ◽  
Rebecca J. Tarrien ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Biofilm Formation ◽  
Second Messenger ◽  
Content Type ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
K 12 ◽  
Messenger Molecule

ABSTRACTIntracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. UropathogenicEscherichia coliregulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n= 11), DGC (n= 13), PilZ (n= 2), and both PDE and DGC (n= 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolateE. coliCFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenicE. coli.IMPORTANCEUropathogenicEscherichia coli(UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in anE. coliK-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain,E. coliCFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged fromE. coliK-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

scholarly journals Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

2016 ◽  
Vol 113 (7) ◽  
pp. 1790-1795 ◽  
Author(s):  
Zachary F. Hallberg ◽  
Xin C. Wang ◽  
Todd A. Wright ◽  
Beiyan Nan ◽  
Omer Ad ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Predictive Power ◽  
Geobacter Sulfurreducens ◽  
Bacterial Genomes ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Cyclic Dinucleotides ◽  
Ggdef Domain ◽  
Cyclic Dinucleotide

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

scholarly journals Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

2015 ◽  
Vol 198 (3) ◽  
pp. 521-535 ◽  
Author(s):  
Simon Schäper ◽  
Elizaveta Krol ◽  
Dorota Skotnicka ◽  
Volkhard Kaever ◽  
Rolf Hilker ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Sinorhizobium Meliloti ◽  
Acid Stress ◽  
Second Messenger ◽  
Lifestyle Changes ◽  
Single Domain ◽  
Receptor Protein ◽  
Leguminous Plant ◽  
Content Type

ABSTRACTSinorhizobium melilotiundergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG inS. melilotiRm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation ofN-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of theS. melilotigenespleD,SMb20523,SMb20447,SMc01464, andSMc03178encoding putative DGCs and ofSMb21517encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, anS. melilotistrain that did not produce detectable levels of cdG (cdG0) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. TheSMc01790-SMc01796locus, homologous to theAgrobacterium tumefaciensuppABCDEFcluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility.IMPORTANCEWe present the first systematic genome-wide investigation of the role of 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of anS. melilotistrain unable to produce cdG (cdG0) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility ofS. melilotiand uncovered a single-domain PilZ protein as regulator of motility.

scholarly journals Characterization of Epidemic IncI1-Iγ Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella enterica from Animals and Humans

2015 ◽  
Vol 59 (9) ◽  
pp. 5357-5365 ◽  
Author(s):  
Hilde Smith ◽  
Alex Bossers ◽  
Frank Harders ◽  
Guanghui Wu ◽  
Neil Woodford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Phylogenetic Trees ◽  
Functional Study ◽  
Content Type ◽  
Gene Associations ◽  
Genes Encoding ◽  
Marked Resistance

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained fromEscherichia coliandSalmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation intraYandexcAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

scholarly journals Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

2015 ◽  
Vol 59 (8) ◽  
pp. 4817-4825 ◽  
Author(s):  
Xinlong He ◽  
Feng Lu ◽  
Fenglai Yuan ◽  
Donglin Jiang ◽  
Peng Zhao ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Gene Transcription ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Content Type ◽  
Potential Association ◽  
Genes Encoding ◽  
Clinical Environments

ABSTRACTChronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency ofAcinetobacter baumanniiand the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolatedA. baumanniistrains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designatedA. baumanniiABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI,adeB,adeG,adeJ,carO, andompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation ofadeGcorrelated with biofilm induction. The consistent upregulation ofadeGandabaIwas detected in A-III-typeA. baumanniiin response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused byA. baumannii. This study provides useful information for the development of antibiofilm strategies.

scholarly journals A Role for the EAL-Like Protein STM1344 in Regulation of CsgD Expression and Motility in Salmonella enterica Serovar Typhimurium

2009 ◽  
Vol 191 (12) ◽  
pp. 3928-3937 ◽  
Author(s):  
Roger Simm ◽  
Uwe Remminghorst ◽  
Irfan Ahmad ◽  
Katherina Zakikhany ◽  
Ute Römling
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Second Messenger ◽  
Direct Role ◽  
Diguanylate Cyclases ◽  
Serovar Typhimurium ◽  
Ggdef Domain ◽  
Major Target

ABSTRACT The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively. STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.

scholarly journals Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Rudolph E. Sloup ◽  
Ashley E. Konal ◽  
Geoffrey B. Severin ◽  
Michelle L. Korir ◽  
Mira M. Bagdasarian ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Protein Complexes ◽  
Second Messenger ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type ◽  
Severe Diarrhea ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae. Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface. IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.

scholarly journals Conserved Regulators of Mating Are Essential for Aspergillus fumigatus Cleistothecium Formation

2010 ◽  
Vol 9 (5) ◽  
pp. 774-783 ◽  
Author(s):  
Edyta Szewczyk ◽  
Sven Krappmann
Keyword(s):  
Aspergillus Fumigatus ◽  
Genetic Recombination ◽  
Content Type ◽  
Hyphal Fusion ◽  
Expression Of Genes ◽  
Body Development ◽  
Fruiting Body Development ◽  
Genes Encoding ◽  
Long Time

ABSTRACT Sexual reproduction of the human pathogen Aspergillus fumigatus (teleomorph: Neosartorya fumigata) was assumed to be absent or cryptic until recently, when fertile crosses among geographically restricted environmental isolates were described. Here, we provide evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated, clinical isolates of A. fumigatus, and this evidence demonstrates the generality and reproducibility of this long-time-undisclosed phase in the life cycle of this heterothallic fungus. Successful mating requires the presence of both mating-type idiomorphs MAT1-1 and MAT1-2, as does expression of genes encoding factors presumably involved in this process. Moreover, analysis of an A. fumigatus mutant deleted for the nsdD gene suggests a role of this conserved regulator of cleistothecium development in hyphal fusion and hence heterokaryon formation.

scholarly journals Phosphoserine Phosphatase Is Required for Serine and One-Carbon Unit Synthesis in Hydrogenobacter thermophilus

2017 ◽  
Vol 199 (21) ◽  
Author(s):  
Keugtae Kim ◽  
Yoko Chiba ◽  
Azusa Kobayashi ◽  
Hiroyuki Arai ◽  
Masaharu Ishii
Keyword(s):  
Physiological Role ◽  
Tca Cycle ◽  
Content Type ◽  
Phosphoserine Phosphatase ◽  
Genes Encoding ◽  
Hydrogenobacter Thermophilus ◽  
Glycine Cleavage ◽  
Major Donor ◽  
Serine Biosynthesis

ABSTRACT Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA, the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH2-THF, in H. thermophilus. Determining the mechanism of 5,10-CH2-THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH2-THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilus. IMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH2-THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus. In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH2-THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus.

scholarly journals Expression of Type IV Pili by Moraxella catarrhalis Is Essential for Natural Competence and Is Affected by Iron Limitation

2004 ◽  
Vol 72 (11) ◽  
pp. 6262-6270 ◽  
Author(s):  
Nicole R. Luke ◽  
Amy J. Howlett ◽  
Jianqiang Shao ◽  
Anthony A. Campagnari
Keyword(s):  
Moraxella Catarrhalis ◽  
Pathogenic Bacteria ◽  
Type Iv Pili ◽  
Protein Subunit ◽  
Electron Microscopic ◽  
Type Iv ◽  
Wide Range ◽  
Genes Encoding ◽  
Microscopic Studies

ABSTRACT Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.

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