scholarly journals Phenylacetic Acid Catabolism and Its Transcriptional Regulation in Corynebacterium glutamicum

2012 ◽  
Vol 78 (16) ◽  
pp. 5796-5804 ◽  
Author(s):  
Xi Chen ◽  
Thomas A. Kohl ◽  
Christian Rückert ◽  
Dmitry A. Rodionov ◽  
Ling-Hao Li ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Phenylacetic Acid ◽  
Specific Binding ◽  
Genomic Analysis ◽  
Negative Regulator ◽  
Comparative Genomic ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type ◽  
Transcription Regulator

ABSTRACTThe industrially important organismCorynebacterium glutamicumhas been characterized in recent years for its robust ability to assimilate aromatic compounds. In this study,C. glutamicumstrain AS 1.542 was investigated for its ability to catabolize phenylacetic acid (PAA). Thepaagenes were identified; they are organized as a continuouspaagene cluster. The type strain ofC. glutamicum, ATCC 13032, is not able to catabolize PAA, but the recombinant strain ATCC 13032/pEC-K18mob2::paagained the ability to grow on PAA. ThepaaRgene, encoding a TetR family transcription regulator, was studied in detail. Disruption ofpaaRin strain AS 1.542 resulted in transcriptional increases of allpaagenes. Transcription start sites and putative promoter regions were determined. An imperfect palindromic motif (5′-ACTNACCGNNCGNNCGGTNAGT-3′; 22 bp) was identified in the upstream regions ofpaagenes. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of PaaR to this motif, and phenylacetyl coenzyme A (PA-CoA) blocked binding. It was concluded that PaaR is the negative regulator of PAA degradation and that PA-CoA is the PaaR effector. In addition, GlxR binding sites were found, and binding to GlxR was confirmed. Therefore, PAA catabolism inC. glutamicumis regulated by the pathway-specific repressor PaaR, and also likely by the global transcription regulator GlxR. By comparative genomic analysis, we reconstructed orthologous PaaR regulons in 57 species, including species ofActinobacteria,Proteobacteria, andFlavobacteria, that carry PAA utilization genes and operate by conserved binding motifs, suggesting that PaaR-like regulation might commonly exist in these bacteria.

scholarly journals MgrA Negatively Regulates Biofilm Formation and Detachment by Repressing the Expression of psm Operons in Staphylococcus aureus

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Qiu Jiang ◽  
Zeyu Jin ◽  
Baolin Sun
Keyword(s):  
Staphylococcus Aureus ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Negative Regulator ◽  
Regulatory Mechanisms ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type ◽  
Multiple Functions ◽  
Biofilm Development

ABSTRACT Phenol-soluble modulins (PSMs) are amphipathic peptides that are produced by staphylococci and play important roles in Staphylococcus aureus biofilm formation and dissemination. Although the multiple functions of PSMs have been recognized, the regulatory mechanisms controlling the expression of psm operons remain largely unknown. In this study, we identified MgrA in a DNA pulldown assay and further demonstrated, by electrophoretic mobility shift assays and DNase I footprinting assays, that MgrA could bind specifically to the promoter regions of psm operons. We then constructed an isogenic mgrA deletion strain and compared biofilm formation and detachment in the wild-type and isogenic mgrA deletion strains. Our results indicated that biofilm formation and detachment were significantly increased in the mgrA mutant strain. Real-time quantitative reverse transcription-PCR data indicated that MgrA repressed the transcription of psm operons in cultures and biofilms, suggesting that MgrA is a negative regulator of psm expression. Furthermore, we analyzed biofilm formation by the psm mutant strains, and we found that PSMs promoted biofilm structuring and development in the mgrA mutant strain. These findings reveal that MgrA negatively regulates biofilm formation and detachment by repressing the expression of psm operons through direct binding to the psm promoter regions.IMPORTANCE Staphylococcus aureus is a human and animal pathogen that can cause biofilm-associated infections. PSMs have multiple functions in biofilm development and virulence in staphylococcal pathogenesis. This study has revealed that MgrA can negatively regulate psm expression by binding directly to the promoter regions of psm operons. Furthermore, our results show that MgrA can modulate biofilm structuring and development by repressing the production of PSMs in S. aureus. Our findings provide novel insights into the regulatory mechanisms of S. aureus psm gene expression, biofilm development, and pathogenesis.

scholarly journals Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum

2014 ◽  
Vol 80 (14) ◽  
pp. 4215-4225 ◽  
Author(s):  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Binding Site ◽  
Corynebacterium Glutamicum ◽  
Promoter Sequence ◽  
Negative Regulation ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Global Regulator ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type

ABSTRACTCorynebacterium glutamicumis an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription ofgenDFMandgenKHoperons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of threegenoperons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located −13 to +8 bp upstream of thegenDFMpromoter, which was involved in negative regulation ofgenDFMtranscription. The GlxR binding site R-KHx01 (located between positions −11 to +5) was upstream of thegenKHpromoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds togenRpromoter to repress its expression, was found within a footprint extending from positions −71 to −91 bp. These results reveal that GlxR represses the transcription of all threegenoperons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.

scholarly journals In VivoEmergence of Colistin Resistance in Klebsiella pneumoniae Producing KPC-Type Carbapenemases Mediated by Insertional Inactivation of the PhoQ/PhoPmgrBRegulator

2013 ◽  
Vol 57 (11) ◽  
pp. 5521-5526 ◽  
Author(s):  
Antonio Cannatelli ◽  
Marco Maria D'Andrea ◽  
Tommaso Giani ◽  
Vincenzo Di Pilato ◽  
Fabio Arena ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Negative Regulator ◽  
Resistant Isolate ◽  
Comparative Genomic ◽  
Colistin Resistance ◽  
Content Type ◽  
Insertional Inactivation ◽  
Resistant Mutants

ABSTRACTColistin is one of the few agents that retain activity against extensively drug-resistant strains ofKlebsiella pneumoniaeproducing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of themgrBgene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role ofmgrBinactivation in acquired colistin resistance was confirmed by complementation experiments with wild-typemgrB, which restored colistin susceptibility in KKBO-4, and by construction of anmgrBdeletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. InsertionalmgrBinactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of themgrBgene, upregulated transcription ofphoP,phoQ, andpmrK(which is part of thepmrHFIJKLMoperon) was detected. These findings confirmed the MgrB regulatory role inK. pneumoniaeand were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of thepmrHFIJKLMoperon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

scholarly journals Genomic Analysis of Multiresistant Staphylococcus capitis Associated with Neonatal Sepsis

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Glen P. Carter ◽  
James E. Ussher ◽  
Anders Gonçalves Da Silva ◽  
Sarah L. Baines ◽  
Helen Heffernan ◽  
...  
Keyword(s):  
Neonatal Sepsis ◽  
Neonatal Intensive Care ◽  
Genomic Analysis ◽  
Bloodstream Infections ◽  
Multidrug Resistant ◽  
Comparative Genomic ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Staphylococcus Capitis ◽  
Hospital Infection Control

ABSTRACT Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen.

scholarly journals Complete Genome Resequencing of Thermus thermophilus Strain TMY by Hybrid Assembly of Long- and Short-Read Sequencing Technologies

2021 ◽  
Vol 10 (46) ◽  
Author(s):  
Kentaro Miyazaki ◽  
Natsuko Tokito
Keyword(s):  
Complete Genome ◽  
Thermus Thermophilus ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Hybrid Assembly ◽  
Genome Resequencing ◽  
Short Read ◽  
Content Type ◽  
Sequencing Technologies ◽  
Long Read

Complete genome resequencing was conducted for Thermus thermophilus strain TMY by hybrid assembly of Oxford Nanopore Technologies long-read and MGI short-read data. Errors in the previously reported genome sequence determined by PacBio technology alone were corrected, allowing for high-quality comparative genomic analysis of closely related T. thermophilus genomes.

scholarly journals Identification of the Regulon of AphB and Its Essential Roles in LuxR and Exotoxin Asp Expression in the Pathogen Vibrio alginolyticus

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Xiating Gao ◽  
Yang Liu ◽  
Huan Liu ◽  
Zhen Yang ◽  
Qin Liu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Vibrio Alginolyticus ◽  
Physiological Adaptation ◽  
Pcr Analysis ◽  
Growth Stages ◽  
Mobility Shift ◽  
Marine Animals ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACT In Vibrio species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in Vibrio alginolyticus, a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the V. alginolyticus genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of asp and the master quorum-sensing (QS) regulator luxR. DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the luxR promoter region overlaps that of LuxR-binding site I, which mediates the positive control of luxR promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in V. alginolyticus. IMPORTANCE In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in V. alginolyticus, a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the aphB gene itself was identified to be positively autoregulated, and AphB also positively controlled asp and luxR expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression.

scholarly journals The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT

2019 ◽  
Vol 87 (10) ◽  
Author(s):  
Tracy H. Hazen ◽  
David A. Rasko
Keyword(s):  
Escherichia Coli ◽  
Complete Genome ◽  
Genomic Analysis ◽  
Host Cells ◽  
Comparative Genomic ◽  
Content Type ◽  
E Coli ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Typical Epec

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.

scholarly journals Horizontal Gene Transfer Clarifies Taxonomic Confusion and Promotes the Genetic Diversity and Pathogenicity of Plesiomonas shigelloides

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Zhiqiu Yin ◽  
Si Zhang ◽  
Yi Wei ◽  
Meng Wang ◽  
Shuangshuang Ma ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Evolutionary Dynamics ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Content Type ◽  
Pan Genome ◽  
Long Time ◽  
Taxonomic Confusion

The taxonomic position of P. shigelloides has been the subject of debate for a long time, and until now, the evolutionary dynamics and pathogenesis of P. shigelloides were unclear. In this study, pan-genome analysis indicated extensive genetic diversity and the presence of large and variable gene repertoires. Our results revealed that horizontal gene transfer was the focal driving force for the genetic diversity of the P. shigelloides pan-genome and might have contributed to the emergence of novel properties. Vibrionaceae and Aeromonadaceae were found to be the predominant donor taxa for horizontal genes, which might have caused the taxonomic confusion historically. Comparative genomic analysis revealed the potential of P. shigelloides to cause intestinal and invasive diseases. Our results could advance the understanding of the evolution and pathogenesis of P. shigelloides, particularly in elucidating the role of horizontal gene transfer and investigating virulence-related elements.

scholarly journals Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

2014 ◽  
Vol 82 (12) ◽  
pp. 5293-5307 ◽  
Author(s):  
Marina Falaleeva ◽  
Oliwia W. Zurek ◽  
Robert L. Watkins ◽  
Robert W. Reed ◽  
Hadeel Ali ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Streptococcus Pyogenes ◽  
Regulatory Region ◽  
Regulatory System ◽  
Invasive Disease ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Putative Promoter ◽  
Transcriptional Terminator ◽  
Content Type

ABSTRACTThe important human pathogenStreptococcus pyogenes(group AStreptococcus[GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that thehasABCoperon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription ofhasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion ofhasSor of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through,hasAtranscription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

scholarly journals Biology and Genome Sequence of Streptococcus mutans Phage M102AD

2012 ◽  
Vol 78 (7) ◽  
pp. 2264-2271 ◽  
Author(s):  
Allan L. Delisle ◽  
Ming Guo ◽  
Natalia I. Chalmers ◽  
Gerard J. Barcak ◽  
Geneviève M. Rousseau ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Genome Sequence ◽  
Gc Content ◽  
Genomic Analysis ◽  
Streptococcus Thermophilus ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Content Type ◽  
Close Relationship ◽  
Virion Structure

ABSTRACTM102AD is the new designation for aStreptococcus mutansphage described in 1993 as phage M102. This change was necessitated by the genome analysis of anotherS. mutansphage named M102, which revealed differences from the genome sequence reported here. Additional host range analyses confirmed thatS. mutansphage M102AD infects only a few serotype c strains. Phage M102AD adsorbed very slowly to its host, and it cannot adsorb to serotype e and f strains ofS. mutans. M102AD adsorption was blocked by c-specific antiserum. Phage M102AD also adsorbed equally well to heat-treated and trypsin-treated cells, suggesting carbohydrate receptors. Saliva and polysaccharide production did not inhibit plaque formation. The genome of this siphophage consisted of a linear, double-stranded, 30,664-bp DNA molecule, with a GC content of 39.6%. Analysis of the genome extremities indicated the presence of a 3′-overhangcossite that was 11 nucleotides long. Bioinformatic analyses identified 40 open reading frames, all in the same orientation. No lysogeny-related genes were found, indicating that phage M102AD is strictly virulent. No obvious virulence factor gene candidates were found. Twelve proteins were identified in the virion structure by mass spectrometry. Comparative genomic analysis revealed a close relationship betweenS. mutansphages M102AD and M102 as well as withStreptococcus thermophilusphages. This study also highlights the importance of conducting research with biological materials obtained from recognized microbial collections.

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