scholarly journals A ToxR Homolog from Vibrio anguillarum Serotype O1 Regulates Its Own Production, Bile Resistance, and Biofilm Formation

2002 ◽  
Vol 184 (6) ◽  
pp. 1630-1639 ◽  
Author(s):  
Su-Yan Wang ◽  
Johan Lauritz ◽  
Jana Jass ◽  
Debra L. Milton
Keyword(s):  
Outer Membrane ◽  
Regulatory Protein ◽  
Vibrio Anguillarum ◽  
Wild Type ◽  
Environmental Signals ◽  
Environmental Stimuli ◽  
Outer Membrane Porin ◽  
Bile Resistance ◽  
Functional Analyses ◽  
Mutant Forms

ABSTRACT ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.

scholarly journals The Virulence Regulatory Protein ToxR Mediates Enhanced Bile Resistance in Vibrio cholerae and Other PathogenicVibrio Species

2000 ◽  
Vol 68 (3) ◽  
pp. 1491-1497 ◽  
Author(s):  
Daniele Provenzano ◽  
Darren A. Schuhmacher ◽  
Justin L. Barker ◽  
Karl E. Klose
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Proteins ◽  
Regulatory Protein ◽  
Wild Type ◽  
Outer Membrane Porins ◽  
Mutant Strains ◽  
Bile Resistance ◽  
El Tor

ABSTRACT The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogenVibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes. However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively. The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, andompT genes are apparently in the ancestralVibrio chromosome. The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown. We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner. In both classical and El Tor biotypes of V. cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxTmutant strains. Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS. Growth of either V. cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot. Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile. ThetoxR mutant strains of three additional human intestinal pathogenic Vibrio species, V. mimicus, V. fluvialis, and V. parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains. Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibriospecies and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile. We speculate that ToxR-mediated bile resistance was an early step in the evolution ofV. cholerae as an intestinal pathogen.

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild-type and specific knock-out mutant strains

PROTEOMICS ◽  
2009 ◽  
Vol 9 (3) ◽  
pp. 648-656 ◽  
Author(s):  
Juan Marzoa ◽  
Ana Abel ◽  
Sandra Sánchez ◽  
Hannah Chan ◽  
Ian Feavers ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Wild Type ◽  
Knock Out ◽  
Outer Membrane Porin ◽  
Mutant Strains

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

2020 ◽  
Vol 22 (1) ◽  
pp. 152
Author(s):  
Dorota Dabrowska ◽  
Justyna Mozejko-Ciesielska ◽  
Tomasz Pokój ◽  
Slawomir Ciesielski
Keyword(s):  
Chain Length ◽  
Nitrogen Limitation ◽  
Stringent Response ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain ◽  
Environmental Stimuli ◽  
Medium Chain Length ◽  
In The Wild

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

scholarly journals Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Erin N Asleson ◽  
Dennis M Livingston
Keyword(s):  
Half Life ◽  
Translational Regulation ◽  
Cellular Level ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Proteins ◽  
Gal1 Promoter ◽  
Rad52 Protein ◽  
The Stability ◽  
Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

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