scholarly journals Speed fluctuations of bacterial replisomes

2021 ◽  
Author(s):  
Deepak Bhat ◽  
Samuel Hauf ◽  
Charles Plessy ◽  
Yohei Yokobayashi ◽  
Simone Pigolotti
Keyword(s):  
Bacterial Population ◽  
Protein Complexes ◽  
Bacterial Species ◽  
Abundance Distribution ◽  
E Coli ◽  
Speed Error ◽  
Different Temperatures ◽  
Speed Fluctuations ◽  
Speed And Accuracy

Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, the dynamics of replisomes along the genome is poorly characterised, especially in vivo. In this paper, we link the replisome dynamics with the DNA abundance distribution measured in an exponentially growing bacterial population. Our approach permits to accurately infer the replisome dynamics along the genome from deep sequencing measurements. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures. We find that the average replisome speed increases nearly five-fold between 17°C and 37°C. Further, we observe wave-like variations of the replisome speed along the genome. These variations are correlated with previously observed variations of the mutation rate along the genome. We interpret this correlation as a speed--error trade-off and discuss its possible dynamical origin. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.

scholarly journals Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

2010 ◽  
Vol 76 (14) ◽  
pp. 4655-4663 ◽  
Author(s):  
Sean M. Lee ◽  
Aaron Wyse ◽  
Aaron Lesher ◽  
Mary Lou Everett ◽  
Linda Lou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Bacterial Species ◽  
Intestinal Flora ◽  
Average Density ◽  
Host Bacterium ◽  
E Coli ◽  
K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

scholarly journals FtsW exhibits distinct processive motions driven by either septal cell wall synthesis or FtsZ treadmilling in E. coli

2019 ◽  
Author(s):  
Xinxing Yang ◽  
Ryan McQuillen ◽  
Zhixin Lyu ◽  
Polly Phillips-Mason ◽  
Ana De La Cruz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Single Molecule ◽  
Bacterial Cell ◽  
Spatial Information ◽  
Bacterial Species ◽  
E Coli ◽  
Slow Moving

AbstractDuring bacterial cell division, synthesis of new septal peptidoglycan (sPG) is crucial for successful cytokinesis and cell pole morphogenesis. FtsW, a SEDS (Shape, Elongation, Division and Sporulation) family protein and an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a new monofunctional peptidoglycan glycosyltransferase (PGTase). FtsW and its cognate monofunctional transpeptidase (TPase) class B penicillin binding protein (PBP3 or FtsI in E. coli) may constitute the essential, bifunctional sPG synthase specific for new sPG synthesis. Despite its importance, the septal PGTase activity of FtsW has not been documented in vivo. How its activity is spatiotemporally regulated in vivo has also remained unknown. Here we investigated the septal PGTase activity and dynamics of FtsW in E. coli cells using a combination of single-molecule imaging and genetic manipulations. We show that FtsW exhibits robust activity to incorporate an N-acetylmuramic acid analog at septa in the absence of other known PGTases, confirming FtsW as the essential septum-specific PGTase in vivo. Notably, we identified two populations of processive moving FtsW molecules at septa. A fast-moving population is driven by the treadmilling dynamics of FtsZ and independent of sPG synthesis. A slow-moving population is driven by active sPG synthesis and independent of FtsZ’s treadmilling dynamics. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving, sPG synthesis-dependent population. Our results support a two-track model, in which inactive sPG synthase molecules follow the fast treadmilling “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model explains how the spatial information is integrated into the regulation of sPG synthesis activity and suggests a new mechanistic framework for the spatiotemporal coordination of bacterial cell wall constriction.

scholarly journals Regulation of ribosomal protein synthesis in mycobacteria: autogenous control of rpsO

2021 ◽  
Author(s):  
Leonid V Aseev ◽  
Ludmila S Koledinskaya ◽  
Oksana S Bychenko ◽  
Irina V Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Bacterial Species ◽  
Shuttle Vector ◽  
Gc Content ◽  
E Coli ◽  
Autogenous Control ◽  
Autogenous Regulation

ABSTRACTAutogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. Our main goal was to develop techniques for investigating the r-protein synthesis regulation in mycobacteria, Gram-positive organisms with a high GC-content, which has never been addressed. We started with the rpsO gene known to be autoregulated by its product, r-protein S15, in a broad range of bacterial species. To study the in vivo regulation of rpsO from Mycobacterium smegmatis (Msm), we first applied an approach based on chromosomally integrated Msm rpsO’-’lacZ reporters by using E. coli as a surrogate host. The β-galactosidase assay has shown that mycobacterial rpsO expression is feedback regulated at the translation level in the presence of Msm S15 in trans, like in E. coli. Next, to overcome difficulties caused by the inefficiency of mycobacterial gene expression in E. coli, we created a fluorescent reporter system based on M. smegmatis. To this end, the integrative shuttle plasmid pMV306 was modified to provide insertion of the Msm or Mtb (M. tuberculosis) rpsO-egfp reporters into the Msm chromosome, and a novel E. coli-mycobacteria replicative shuttle vector, pAMYC, a derivative of pACYC184, was built. Analysis of the eGFP expression in the presence of the pAMYC derivative expressing Msm rpsO vs an empty vector confirms the autogenous regulation of the rpsO gene in mycobacteria. Additionally, we have revealed that the mycobacterial rpsO core promoters are rather weak and require upstream activating elements to enhance their strength.IMPORTANCEBacterial ribosomes are targets for a majority of as-yet reported antibiotics, hence ribosome biogenesis and its regulation are central for development of new antimicrobials. One of the key mechanisms regulating ribosome biogenesis in bacteria is the autogenous control of r-protein synthesis, which has been so far explored for E. coli and Bacillus spp. but not yet for mycobacteria. Here, we describe experimental approaches for in vivo analysis of mechanisms regulating r-protein synthesis in mycobacteria, including M. tuberculosis, and show, for the first time, that the autogenous control at the translation level is really functioning in these microorganisms. The developed system paves the way for studying various regulatory circuits involving proteins or sRNAs as mRNA- targeting trans-regulators in mycobacteria as well as in other actinobacterial species.

scholarly journals 2134. Differential Changes in Breath Volatile Metabolites to Identify Carbapenem-Resistant Enterobacteriaceae (CRE) in a Murine Pneumonia Model

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S722-S723
Author(s):  
Nour Ismail ◽  
Hazem Albashash ◽  
Mahesh J Thalavitiya Acharige ◽  
Mohamad Hejazi ◽  
Carmen Leon Astudillo ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Rapid Identification ◽  
Metabolic Responses ◽  
P Value ◽  
Species Pair ◽  
E Coli ◽  
Volatile Metabolites ◽  
Carbapenem Resistant ◽  
Carbapenem Resistant Enterobacteriaceae

Abstract Background CRE infections cause significant mortality, in large part because rapid identification of these infections is challenging. We tested the hypothesis that CRE and their isogenic carbapenem-susceptible counterparts have differential metabolic responses to carbapenem therapy. Methods We generated isogenic pairs of E. coli, E. cloacae, and K. pneumoniae by inserting a blaNDM-1-containing plasmid into carbapenem-susceptible E. coli, E. cloacae, and K. pneumoniae. We confirmed phenotypic meropenem (MPM) resistance per CLSI breakpoints for Enterobacteriaceae (MIC ≥4) in the NDM-1+ member and susceptibility (MIC≤1) in the NDM-1- member of each pair. We administered 2 × 108 CFU of each isolate intranasally to 23–28 g male C57BL/6J mice, infecting 6 mice with the NDM-1+ member and 6 with the NDM-1− member of each species pair (12 mice per bacterial species). 24 hours after infection, we treated 3 mice in each NDM-1+ and NDM-1− bacterial species cohort with MPM over 4 hours, and the other 3 mice in each cohort with saline over 4 hours as controls, confirming adequate infection (a target of 106 CFU/g of lung tissue) in quantitative lung homogenate cultures. We then collected breath samples from each mouse via tracheostomy using a murine ventilator, identifying all volatile metabolites in each sample using thermal desorption-gas chromatography/tandem mass spectrometry. We used Wilcoxon tests to examine differences in metabolite abundance between MPM and saline-treated control mice in the NDM-1+ and NDM-1− a member of each species pair, with a two-sided P-value threshold of < 0.1. Results Several breath volatile metabolites changed differentially within each NDM-1+/NDM-1- pair, outlined in Table 1 (E. coli), Table 2 (E. cloacae), and Table 3 (K. pneumoniae). Each listed metabolite that changed with MPM did not change with MPM in mice infected with each isogenic counterpart Conclusion There are differential in vivo metabolic responses with effective vs. ineffective treatment of mice with pneumonia caused by E. coli, E. cloacae, and K. pneumoniae pairs that are genetically identical other than blaNDM-1; this differential treatment response can potentially be used to identify these infections. Disclosures All authors: No reported disclosures.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals Effective Control of Salmonella Infections by Employing Combinations of Recombinant Antimicrobial Human β-Defensins hBD-1 and hBD-2

2014 ◽  
Vol 58 (11) ◽  
pp. 6896-6903 ◽  
Author(s):  
Soumitra Maiti ◽  
Sunita Patro ◽  
Sukumar Purohit ◽  
Sumeet Jain ◽  
Shantibhusan Senapati ◽  
...  
Keyword(s):  
Bacterial Population ◽  
Antibacterial Activities ◽  
Effective Control ◽  
Content Type ◽  
E Coli ◽  
Salmonella Infections ◽  
Functional Peptides ◽  
Lethal Doses

ABSTRACTWe successfully produced two human β-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities againstSalmonella entericaserovar Typhi,Escherichia coli, andStaphylococcus aureusemploying both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD50) of hBD-1 againstS.Typhi,E. coli, andS. aureuswere 0.36, 0.40, and 0.69 μg/μl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 μg/μl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected withS.Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging theSalmonellainfectionin vitroandin vivo. We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-derived recombinant β-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP)-based substances for the development of new therapeutics against typhoid fever.

scholarly journals Metabolic exchange and energetic coupling between nutritionally stressed bacterial species, the possible role of QS molecules

2020 ◽  
Author(s):  
David Ranava ◽  
Cassandra Backes ◽  
Ganesan Karthikeyan ◽  
Olivier Ouari ◽  
Audrey Soric ◽  
...  
Keyword(s):  
Species Interactions ◽  
Bacterial Species ◽  
Anaerobic Bacteria ◽  
Species Interaction ◽  
Desulfovibrio Vulgaris ◽  
Physical Interaction ◽  
E Coli ◽  
Metabolic Exchange

AbstractTo clarify the principles controlling inter-species interactions, we previously developed a co-culture model with two anaerobic bacteria, Clostridium acetobutylicum and Desulfovibrio vulgaris Hildenborough, in which nutritional stress for D. vulgaris induced tight cell-cell inter-species interaction. Here we show that exchange of metabolites produced by C. acetobutylicum allows D. vulgaris to duplicate its DNA, and to be energetically viable even without its substrates. Physical interaction between C. acetobutylicum and D. vulgaris (or Escherichia coli and D. vulgaris) is linked to the quorum-sensing molecule AI-2, produced by C. acetobutylicum and E. coli. With nutrients D. vulgaris produces a small molecule that inhibits in vitro the AI-2 activity, and could act as an antagonist in vivo. Sensing of AI-2 by D. vulgaris could induce formation of an intercellular structure that allows directly or indirectly metabolic exchange and energetic coupling between the two bacteria.

scholarly journals Transient protein-protein interactions perturb E.coli metabolome and cause gene dosage toxicity

2016 ◽  
Author(s):  
Sanchari Bhattacharyya ◽  
Shimon Bershtein ◽  
Jin Yan ◽  
Tijda Argun ◽  
Amy I. Gilson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Gene Dosage ◽  
Bacterial Species ◽  
Dynamic Network ◽  
Catalytic Efficiency ◽  
Evolutionary Divergence ◽  
Protein Protein Interactions ◽  
E Coli ◽  
Metabolic Imbalance

Several genes exhibit gene dosage toxicity yet its molecular underpinnings remain unknown. Here we demonstrate that overexpression of DHFR in E. coli causes toxic metabolic imbalance triggered by interactions with several enzymes involved in 1-carbon metabolism, in particular GlyA and PurH. DHFR overexpression partially inhibits activity of these enzymes, but at physiological concentrations, PurH-DHFR interaction enhances catalytic efficiency of DHFR, implying a functional interaction in vivo. Surprisingly, overexpression of orthologous DHFRs from other bacterial species caused minimal metabolic and fitness perturbations, despite pulling out more interacting partners than overexpressed endogenous DHFR. Orthologous DHFRs were less potent in inhibiting E. coli GlyA and PurH, or gaining a catalytic improvement upon interaction with PurH, indicating a partial loss of interaction specificity due to evolutionary divergence. This study shows how protein overexpression perturbs a dynamic network of weak yet potentially functional PPI with consequences for the metabolic state of cells and their fitness.

scholarly journals Analysis of the pmsCEAB Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain Pseudomonas fluorescensWCS374

2001 ◽  
Vol 183 (6) ◽  
pp. 1909-1920 ◽  
Author(s):  
Jesús Mercado-Blanco ◽  
Koen M. G. M. van der Drift ◽  
Per E. Olsson ◽  
Jane E. Thomas-Oates ◽  
Leendert C. van Loon ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Histidine Decarboxylase ◽  
Bacterial Species ◽  
Open Reading Frames ◽  
Putative Promoter Region ◽  
E Coli ◽  
Low Levels ◽  
Reading Frames ◽  
Siderophore Activity

ABSTRACT Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC andpmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that thepmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsBabolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.

scholarly journals The Escherichia coli mqsR and ygiT Genes Encode a New Toxin-Antitoxin Pair

2010 ◽  
Vol 192 (11) ◽  
pp. 2908-2919 ◽  
Author(s):  
Villu Kasari ◽  
Kristi Kurg ◽  
Tõnu Margus ◽  
Tanel Tenson ◽  
Niilo Kaldalu
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Growth Inhibition ◽  
Protein Complexes ◽  
Ectopic Expression ◽  
E Coli ◽  
New Family ◽  
Single Operon ◽  
Growing Population

ABSTRACT Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.

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