scholarly journals Improvement in K+-Limited Growth Rate Associated with Expression of the N-Terminal Fragment of One Subunit (KdpA) of the Multisubunit Kdp Transporter in Escherichia coli

2001 ◽  
Vol 183 (11) ◽  
pp. 3515-3520 ◽  
Author(s):  
Abhijit A. Sardesai ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Membrane Potential ◽  
Cytoplasmic Membrane ◽  
Inducible Promoter ◽  
Limited Growth ◽  
Atpase Subunit ◽  
Terminal Fragment ◽  
P Type

ABSTRACT Mutations in any one of three genes, kdpA, -B, or -C, in Escherichia coli abolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA′), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA′ was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA′ was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA′ provides an incipient pathway for K+ translocation which can serve to transport K+ into the cells in response to the cytoplasmic membrane potential.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

scholarly journals Rapid Growth of UropathogenicEscherichia coliduring Human Urinary Tract Infection

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Valerie S. Forsyth ◽  
Chelsie E. Armbruster ◽  
Sara N. Smith ◽  
Ali Pirani ◽  
A. Cody Springman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Urinary Tract ◽  
Growth Rates ◽  
High Growth ◽  
Replication Forks ◽  
Chromosomal Replication ◽  
E Coli ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenicE. coli(ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than otherE. coliisolates and survive in that niche. To date, there has not been a reliable method available to measure their growth ratein vivo. Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robustin vivo, matching or exceedingin vitrogrowth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth ratesin vivoat 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR,E. coliin urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth ratesin vivoand resistance to the innate immune response appear to be critical phenotypes of UPEC strains.IMPORTANCEUropathogenicEscherichia coli(UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized byE. colito colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measurein vivogrowth rates of other bacterial pathogens during host colonization.

scholarly journals In Vivo Synthesis of the Periplasmic Domain of TonB Inhibits Transport through the FecA and FhuA Iron Siderophore Transporters of Escherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 5885-5895 ◽  
Author(s):  
S. Peter Howard ◽  
Christina Herrmann ◽  
Chad W. Stratilo ◽  
V. Braun
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Proton Motive Force ◽  
Motive Force ◽  
In Vivo Studies ◽  
Siderophore Transport

ABSTRACT The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage φ80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuAΔ5-160 was also inhibited by TonB(33-239), although FhuAΔ5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.

scholarly journals The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

2016 ◽  
Vol 198 (23) ◽  
pp. 3186-3199 ◽  
Author(s):  
Amit Pathania ◽  
Arvind Kumar Gupta ◽  
Swati Dubey ◽  
Balasubramanian Gopal ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cytoplasmic Membrane ◽  
Basic Amino Acid ◽  
Intramolecular Interactions ◽  
Membrane Topology ◽  
Content Type ◽  
E Coli ◽  
Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

scholarly journals Additional In Vitro and In Vivo Evidence for SecA Functioning as Dimers in the Membrane: Dissociation into Monomers Is Not Essential for Protein Translocation in Escherichia coli

2007 ◽  
Vol 190 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Hongyun Wang ◽  
Bing Na ◽  
Hsiuchin Yang ◽  
Phang C. Tai
Keyword(s):  
Escherichia Coli ◽  
Lipid Bilayers ◽  
Cytoplasmic Membrane ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Oligomeric State ◽  
Dependent Protein ◽  
Ts Mutant

ABSTRACT SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.

scholarly journals Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP, LacY, ProW, AqpZ, MscS, and MscL

2009 ◽  
Vol 192 (4) ◽  
pp. 912-924 ◽  
Author(s):  
Tatyana Romantsov ◽  
Andrew R. Battle ◽  
Jenifer L. Hendel ◽  
Boris Martinac ◽  
Janet M. Wood
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Cytoplasmic Membrane ◽  
Protein Localization ◽  
General Characteristic ◽  
Mechanosensitive Channel ◽  
E Coli ◽  
Polar Localization ◽  
Cytoplasmic Membrane Proteins

ABSTRACT Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).

scholarly journals Characterization of Escherichia coli ExbD protein modification in vivo

2020 ◽  
Author(s):  
Aruna Kumar ◽  
Kathleen Postle
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Active Transport ◽  
Protein Modification ◽  
Cytoplasmic Membrane ◽  
Physical Contact ◽  
Protonmotive Force ◽  
Iron Starvation ◽  
Tonb System

ABSTRACTThe TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

EVOLUTION OF TRANSPOSONS: NATURAL SELECTION FOR Tn5 IN ESCHERICHIA COLI K12

Genetics ◽  
1983 ◽  
Vol 103 (4) ◽  
pp. 581-592
Author(s):  
Susan Wurster Biel ◽  
Daniel L Hartl
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rate Effect ◽  
Coding Region ◽  
Bacterial Populations ◽  
The Right ◽  
Derivatives Of ◽  
Intact Element ◽  
Prior Adaptation

ABSTRACT A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.

scholarly journals Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli

Open Biology ◽  
2017 ◽  
Vol 7 (8) ◽  
pp. 170091 ◽  
Author(s):  
Johann Habersetzer ◽  
Kristoffer Moore ◽  
Jon Cherry ◽  
Grant Buchanan ◽  
Phillip J. Stansfeld ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Protein Transport ◽  
Cytoplasmic Membrane ◽  
Polar Cluster ◽  
Additional Sequence ◽  
Tat System ◽  
Folded Proteins ◽  
Related Proteins

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

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