scholarly journals The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium

2021 ◽  
Author(s):  
German Pozdeev ◽  
Michael C Beckett ◽  
Aalap Mogre ◽  
Nicholas Thomson ◽  
Charles J Dorman
Keyword(s):  
Mass Spectrometry ◽  
Similar Rate ◽  
Integration Host Factor ◽  
Host Factor ◽  
Wild Type ◽  
Secretion Systems ◽  
Protein Coding ◽  
Bacterial Physiology ◽  
Serovar Typhimurium ◽  
The Right

Integration host factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes encode the subunits and are located 350 kilobase pairs apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1:1 stoichiometry, mass spectrometry revealed that IhfB is produced in 2-fold excess over IhfA. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the other's gene. Mass spectrometry showed that in this 'rewired' strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild type, had identical cell morphology, and was similar in competitive fitness. However, compared to the wild type, it was less motile, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes.

scholarly journals The Escherichia coli K-12 NarL and NarP Proteins Insulate the nrf Promoter from the Effects of Integration Host Factor

2006 ◽  
Vol 188 (21) ◽  
pp. 7449-7456 ◽  
Author(s):  
Douglas F. Browning ◽  
David J. Lee ◽  
Alan J. Wolfe ◽  
Jeffrey A. Cole ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Regulatory Region ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Host Factor ◽  
Receptor Protein ◽  
E Coli ◽  
Serovar Typhimurium ◽  
K 12

ABSTRACT The Escherichia coli K-12 nrf operon promoter can be activated fully by the FNR protein (regulator of fumarate and nitrate reduction) binding to a site centered at position −41.5. FNR-dependent transcription is suppressed by integration host factor (IHF) binding at position −54, and this suppression is counteracted by binding of the NarL or NarP response regulator at position −74.5. The E. coli acs gene is transcribed from a divergent promoter upstream from the nrf operon promoter. Transcription from the major acsP2 promoter is dependent on the cyclic AMP receptor protein and is modulated by IHF and Fis binding at multiple sites. We show that IHF binding to one of these sites, located at position −127 with respect to the nrf promoter, has a positive effect on nrf promoter activity. This activation is dependent on the face of the DNA helix, independent of IHF binding at other locations, and found only when NarL/NarP are not bound at position −74.5. Binding of NarL/NarP appears to insulate the nrf promoter from the effects of IHF. The acs-nrf regulatory region is conserved in other pathogenic E. coli strains and related enteric bacteria but differs in Salmonella enterica serovar Typhimurium.

scholarly journals Differential Regulation of Multiple Proteins of Escherichia coli and Salmonella enterica Serovar Typhimurium by the Transcriptional Regulator SlyA

2002 ◽  
Vol 184 (13) ◽  
pp. 3549-3559 ◽  
Author(s):  
Andrea Spory ◽  
Armin Bosserhoff ◽  
Christine von Rhein ◽  
Werner Goebel ◽  
Albrecht Ludwig
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Transcriptional Regulator ◽  
Host Cells ◽  
Wild Type ◽  
E Coli ◽  
Serovar Typhimurium

ABSTRACT SlyA is a transcriptional regulator of Escherichia coli, Salmonella enterica, and other bacteria belonging to the Enterobacteriaceae. The SlyA protein has been shown to be involved in the virulence of S. enterica serovar Typhimurium, but its role in E. coli is unclear. In this study, we employed the proteome technology to analyze the SlyA regulons of enteroinvasive E. coli (EIEC) and Salmonella serovar Typhimurium. In both cases, comparative analysis of the two-dimensional protein maps of a wild-type strain, a SlyA-overproducing derivative, and a corresponding slyA mutant revealed numerous proteins whose expression appeared to be either positively or negatively controlled by SlyA. Twenty of the putative SlyA-induced proteins and 13 of the putative SlyA-repressed proteins of the tested EIEC strain were identified by mass spectrometry. The former proteins included several molecular chaperones (GroEL, GroES, DnaK, GrpE, and CbpA), proteins involved in acid resistance (HdeA, HdeB, and GadA), the “starvation lipoprotein” (Slp), cytolysin ClyA (HlyE or SheA), and several enzymes involved in metabolic pathways, whereas most of the latter proteins proved to be biosynthetic enzymes. Consistently, the resistance of the EIEC slyA mutant to heat and acid stress was impaired compared to that of the wild-type strain. Furthermore, the implication of SlyA in the regulation of several of the identified E. coli proteins was confirmed at the level of transcription with lacZ fusions. Twenty-three of the Salmonella serovar Typhimurium proteins found to be affected by SlyA were also identified by mass spectrometry. With the exception of GroEL these differed from those identified in the EIEC strain and included proteins involved in various processes. The data suggest that gene regulation by SlyA might be crucial for intracellular survival and/or replication of both EIEC and Salmonella serovar Typhimurium in phagocytic host cells.

scholarly journals Synthesis and Localization of the Salmonella SPI-1 Type III Secretion Needle Complex Proteins PrgI and PrgJ

2003 ◽  
Vol 185 (11) ◽  
pp. 3480-3483 ◽  
Author(s):  
Anand Sukhan ◽  
Tomoko Kubori ◽  
Jorge E. Galán
Keyword(s):  
Mutant Strain ◽  
Type Iii Secretion ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Mutant Strains ◽  
Serovar Typhimurium ◽  
Needle Protein ◽  
Protein Instability

ABSTRACT An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.

scholarly journals Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor

1999 ◽  
Vol 181 (14) ◽  
pp. 4245-4249 ◽  
Author(s):  
Eun Hee Cho ◽  
Chan-Eun Nam ◽  
Renato Alcaraz ◽  
Jeffrey F. Gardner
Keyword(s):  
Integration Host Factor ◽  
Host Factor ◽  
Maximum Efficiency ◽  
Wild Type ◽  
Bacteriophage P22 ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Mutant Cells ◽  
Formation Of Complexes

ABSTRACT Site-specific recombination by phages λ and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates λ site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting λ site-specific recombination than wild-type cells. Although the attPregion of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.

scholarly journals Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Is Necessary for Complete Virulence in a Mouse Model of Infectious Enterocolitis

2005 ◽  
Vol 73 (6) ◽  
pp. 3219-3227 ◽  
Author(s):  
Bryan Coburn ◽  
Yuling Li ◽  
David Owen ◽  
Bruce A. Vallance ◽  
B. Brett Finlay
Keyword(s):  
Salmonella Enterica ◽  
Intestinal Inflammation ◽  
Systemic Disease ◽  
Intestinal Disease ◽  
Intercellular Adhesion Molecule ◽  
Wild Type ◽  
Secretion Systems ◽  
Wide Range ◽  
Serovar Typhimurium ◽  
Infectious Enterocolitis

ABSTRACT Salmonella species cause a wide range of disease in multiple hosts. Salmonella enterica serovar Typhimurium causes self-limited intestinal disease in humans and systemic typhoid-like illness in susceptible mice. The prevailing dogma in murine S. enterica serovar Typhimurium pathogenesis is that distinct virulence mechanisms—Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2)—perform distinct roles in pathogenesis, the former being important for invasion and intestinal disease and the latter important for intracellular survival and systemic persistence and disease. Although evidence from bovine infections has suggested that SPI2 has a role in ileal disease, there is no evidence that SPI2 is important for inflammation in a disease that more closely recapitulates human colitis. Using S. enterica serovar Typhimurium strains that lack functional type III secretion systems, we demonstrate that SPI2 is essential for complete virulence in murine infectious enterocolitis. Using a recently characterized murine model (M. Barthel,S. Hapfelmeier, L. Quintanilla-Martinez, M. Kremer, M. Rohde, M. Hogardt, K. Pfeffer, H. Russmann, and W. D. Hardt, Infect. Immun. 71:2839-2858, 2003), we demonstrate that SPI1 mutants are unable to cause intestinal disease 48 h after infection and that SPI2-deficient bacteria also cause significantly attenuated typhlitis. We show that at the peak of inflammation in the cecum, SPI2 mutants induce diminished intercellular adhesion molecule 1 expression and neutrophil recruitment but that wild-type and mutant Salmonella are similarly distributed in the lumen of the infected organ. Finally, we demonstrate that attenuation of intestinal inflammation is accompanied by resolution of typhlitis in the mutant, but not wild-type, infections. Collectively, these results indicate that SPI2 is needed for enterocolitis, as well as for systemic disease.

scholarly journals Expression of the Transposase Gene tnpAof Tn4652 Is Positively Affected by Integration Host Factor

1998 ◽  
Vol 180 (11) ◽  
pp. 2822-2829 ◽  
Author(s):  
Rita Hõrak ◽  
Maia Kivisaar
Keyword(s):  
Promoter Activity ◽  
Integration Host Factor ◽  
Host Factor ◽  
Specific Factor ◽  
Positive Role ◽  
Transposase Gene ◽  
Negative Effect ◽  
A Cell ◽  
The Right

ABSTRACT Tn4652 is a derivative of the toluene degradation transposon Tn4651 that belongs to the Tn3family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet. 210:270–276, 1987). We have sequenced the transposase genetnpA of transposon Tn4652 and mapped its promoter to the right end of the element. The deduced amino acid sequence of tnpA revealed 96.2% identity with the putative transposase of Tn5041. Homology with other Tn3family transposases was only moderate (about 20 to 24% identity), suggesting that Tn4652 and Tn5041 are distantly related members of the Tn3 family. Functional analysis of the tnpA promoter revealed that it is active inPseudomonas putida but silent in Escherichia coli, indicating that some P. putida-specific factor is required for the transcription from this promoter. Additionally,tnpA promoter activity was shown to be modulated by integration host factor (IHF). The presence of an IHF-binding site upstream of the tnpA promoter enhanced the promoter activity. The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the P. putida ihfA-deficient strain A8759. Moreover, the Tn4652 terminal sequences had a negative effect on transcription from the tnpA promoter in theihfA-defective strain. This finding suggests that IHF not only enhances transcription from the tnpA promoter but also alleviates the negative effect of terminal sequences of Tn4652 on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn4652 bind IHF from a cell lysate of E. coli.

scholarly journals Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila

2021 ◽  
pp. 074873042199811
Author(s):  
Franziska Ruf ◽  
Oliver Mitesser ◽  
Simon Tii Mungwa ◽  
Melanie Horn ◽  
Dirk Rieger ◽  
...  
Keyword(s):  
Molecular Clock ◽  
Time Window ◽  
Fruit Fly ◽  
Diel Activity ◽  
Wild Type ◽  
Statistical Measures ◽  
Clock Mutant ◽  
Full Complement ◽  
The Right

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling ( Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling

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