Deciphering the Role of Colicins during Colonization of the Mammalian Gut by Commensal E. coli

Colicins are specific and potent toxins produced by Enterobacteriaceae that result in the rapid elimination of sensitive cells. Colicin production is commonly found throughout microbial populations, suggesting its potential importance for bacterial survival in complex microbial environments. Nonetheless, as colicin biology has been predominately studied using synthetic models, it remains unclear how colicin production contributes to survival and fitness of a colicin-producing commensal strain in a natural environment. To address this gap, we took advantage of MP1, an E. coli strain that harbors a colicinogenic plasmid and is a natural colonizer of the murine gut. Using this model, we validated that MP1 is competent for colicin production and then directly interrogated the importance of colicin production and immunity for MP1 survival in the murine gut. We showed that colicin production is dispensable for sustained colonization in the unperturbed gut. A strain lacking colicin production or immunity shows minimal fitness defects and can resist displacement by colicin producers. This report extends our understanding of the role that colicin production may play for E. coli during gut colonization and suggests that colicin production is not essential for a commensal to persist in its physiologic niche in the absence of exogenous challenges.

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Absolute requirement for polyamines for growth of Escherichia coli mutants (mnmE/G) defective in modification of the wobble anticodon of transfer-RNA

FEMS Microbiology Letters ◽

10.1093/femsle/fnz110 ◽

2019 ◽

Vol 366 (10) ◽

Cited By ~ 2

Author(s):

Christopher Keller ◽

Manas Chattopadhyay ◽

Herbert Tabor

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Protein Biosynthesis ◽

Transfer Rna ◽

Coli Strain ◽

E Coli ◽

Wobble Position ◽

Inhibition Of Growth ◽

Absolute Requirement

Abstract The genes mnmE and mnmG are responsible for the modification of uridine 34, ‘the wobble position’ of many aminoacyl-tRNAs. Deletion of these genes affects the strength of the codon-anticodon interactions of the aminoacyl-tRNAs with the mRNAs and the ribosomes. However, deletion of these genes does not usually have a significant effect on the growth rate of the standard Escherichia coli strains. In contrast, we have found that if the host E. coli strain is deficient in the synthesis of polyamines, deletion of the mnmE or mnmG gene results in complete inhibition of growth unless the medium contains polyamines. The finding of an absolute requirement for polyamines in our current work will be significant in studies on polyamine function, in studies on the function of the mnmE/G genes, and in studies on the role of aminoacyl-tRNAs in protein biosynthesis.

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#48: Perinatal Transmission of Multi-Drug Resistant Enterobacteriaceae

Journal of the Pediatric Infectious Diseases Society ◽

10.1093/jpids/piab031.032 ◽

2021 ◽

Vol 10 (Supplement_2) ◽

pp. S14-S15

Author(s):

Leena B Mithal ◽

Aspen Kremer ◽

Sebastian Otero ◽

Sotirios N Markuly ◽

Randy A McCool ◽

...

Keyword(s):

Mouse Model ◽

Perinatal Transmission ◽

Coli Strain ◽

International Travel ◽

Transmission Model ◽

Clinical Samples ◽

Invasive Infection ◽

Drug Resistant ◽

E Coli ◽

Gut Colonization

Abstract Background There is a global rise in multi-drug resistant (MDR) Gram-negative Enterobacteriaceae. Both maternal and neonatal gut colonization with pathogenic E. coli is associated with risk of invasive infection in infancy. Infections caused by MDR strains have delayed effective therapy and higher morbidity. Additionally, extended spectrum beta-lactamase producing (ESBL) E. coli are persistent gut colonizers. Perinatal transmission of ESBL E. coli could therefore have profound impact on the infant microbiota and health. Our aims were to 1) Determine the burden of ESBL Enterobacteriaceae (ESBL-E) colonization and rate of perinatal transmission among healthy mother-infant dyads in the Chicago area and 2) Compare rates of perinatal transmission among commensal, antibiotic-susceptible human E. coli strains versus ESBL E. coli strains in a mouse model. Methods This is an ongoing prospective study of healthy mothers and term infants born vaginally between 7/2020-11/2020. Maternal demographic and medical history data were collected, including age, race/ethnicity, international travel, and pregnancy/delivery information. Maternal rectal and vaginal swabs (n=62) were collected during labor. Clinical samples were initially grown in media supplemented with ampicillin and vancomycin to suppress growth of most susceptible commensal organisms. The sample was then plated on MacConkey agar (+/-ceftriaxone). We also developed a mouse model of perinatal transmission to determine the rate of transmission among ESBL E. coli compared to the commensal E. coli strain MG1655 and the pan-sensitive uropathogenic E. coli strain UTI89. Pups were sacrificed at 24–48 hours of life or 7 days of life, and presence of antibiotic resistant E. coli was noted. Results Human: Median maternal age was 32 years (IQR 30–35). Race was primarily white (84%), 11% Asian, and 10% were of Latina ethnicity. There were 30 (48%) female infants, and 42 (68%) infants received exclusive breastmilk. About half of the mothers reported international travel in the past 2 years (28/62, 45%), and 17/62 (27%) were born outside the USA. Ampicillin resistant (Amp-R) Enterobacteriaceae were recovered from rectal samples of 59/62 (95%) and vaginal samples of 16/62 (26%) mothers. Ten of 62 (16%) mothers were colonized with ESBL-E. Over half of infant stool samples (27/49, 55%) grew Amp-R Enterobacteriaceae. Of 6 mother-infant dyads with maternal ESBL-E colonization and infant stool available, 2 (33%) infants grew out ESBL-E. Murine We tested 4 ESBL E. coli strains and 2 control strains (MG1655 and UTI89) in our perinatal transmission model. We noted that while all strains adequately colonized the gut of the dams, only 2 (ST132 and ST511) of the 4 ESBL E. coli strains and none of the control strains were transmitted efficiently to the mouse pups (Figure 1). Conclusions There is notable community colonization of resistant Enterobacteriaceae among healthy mothers in Chicago with significant transmission to infants. Murine model data shows that human ESBL E. coli strains can colonize pregnant dams, and some ESBL E. coli strains appear to be perinatally transmitted more efficiently than non-drug resistant and commensal E. coli strains. Persistence of colonization, genomic factors related to transmission, and impact on the developing infant microbiome are future directions of study.

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PBP4 Is Likely Involved in Cell Division of the Longitudinally Dividing Bacterium Candidatus Thiosymbion Oneisti

Antibiotics ◽

10.3390/antibiotics10030274 ◽

2021 ◽

Vol 10 (3) ◽

pp. 274

Author(s):

Jinglan Wang ◽

Laura Alvarez ◽

Silvia Bulgheresi ◽

Felipe Cava ◽

Tanneke den Blaauwen

Keyword(s):

Cell Division ◽

Cell Shape ◽

Cellular Localization ◽

Average Length ◽

Bacterial Survival ◽

E Coli ◽

Polar Localization ◽

Cross Linkage ◽

Longitudinal Cell

Peptidoglycan (PG) is essential for bacterial survival and maintaining cell shape. The rod-shaped model bacterium Escherichia coli has a set of seven endopeptidases that remodel the PG during cell growth. The gamma proteobacterium Candidatus Thiosymbion oneisti is also rod-shaped and attaches to the cuticle of its nematode host by one pole. It widens and divides by longitudinal fission using the canonical proteins MreB and FtsZ. The PG layer of Ca. T. oneisti has an unusually high peptide cross-linkage of 67% but relatively short glycan chains with an average length of 12 disaccharides. Curiously, it has only two predicted endopeptidases, MepA and PBP4. Cellular localization of symbiont PBP4 by fluorescently labeled antibodies reveals its polar localization and its accumulation at the constriction sites, suggesting that PBP4 is involved in PG biosynthesis during septum formation. Isolated symbiont PBP4 protein shows a different selectivity for β-lactams compared to its homologue from E. coli. Bocillin-FL binding by PBP4 is activated by some β-lactams, suggesting the presence of an allosteric binding site. Overall, our data point to a role of PBP4 in PG cleavage during the longitudinal cell division and to a PG that might have been adapted to the symbiotic lifestyle.

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Characterization of the flexible genome complement of the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31)

Microbiology ◽

10.1099/mic.0.27469-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 385-398 ◽

Cited By ~ 33

Author(s):

Jana Hejnova ◽

Ulrich Dobrindt ◽

Radka Nemcova ◽

Christophe Rusniok ◽

Alojz Bomba ◽

...

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Gene Clusters ◽

Coli Strain ◽

Bac Clone ◽

Sequence Comparisons ◽

Multiple Sequence ◽

E Coli ◽

Gut Colonization ◽

K 12

Colonization by the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31) has proved to be safe and efficient in the prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants in Czech paediatric clinics over the past three decades. In searching for traits contributing to this beneficial effect related to the gut colonization capacity of the strain, the authors have analysed its genome by DNA–DNA hybridization to E. coli K-12 (MG1655) genomic DNA arrays and to ‘Pathoarrays’, as well as by multiplex PCR, bacterial artificial chromosome (BAC) library cloning and shotgun sequencing. Four hundred and ten E. coli K-12 ORFs were absent from A0 34/86, while 72 out of 456 genes associated with pathogenicity islands of E. coli and Shigella were also detected in E. coli A0 34/86. Furthermore, extraintestinal pathogenic E. coli-related genes involved in iron uptake and adhesion were detected by multiplex PCR, and genes encoding the HlyA and cytotoxic necrotizing factor toxins, together with 21 genes of the uropathogenic E. coli 536 pathogenicity island II, were identified by analysis of 2304 shotgun and 1344 BAC clone sequences of A0 34/86 DNA. Multiple sequence comparisons identified 31 kb of DNA specific for E. coli A0 34/86; some of the genes carried by this DNA may prove to be implicated in the colonization capacity of the strain, enabling it to outcompete pathogens. Among 100 examined BAC clones roughly covering the A0 34/86 genome, one reproducibly conferred on the laboratory strain DH10B an enhanced capacity to persist in the intestine of newborn piglets. Sequencing revealed that this BAC clone carried gene clusters encoding gluconate and mannonate metabolism, adhesion (fim), invasion (ibe) and restriction/modification functions. Hence, the genome of this clinically safe and highly efficient colonizer strain appears to harbour many ‘virulence-associated’ genes. These results highlight the thin line between bacterial ‘virulence’ and ‘fitness' or ‘colonization’ factors, and question the definition of enterobacterial virulence factors.

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A stutter in the coiled coil domain of E.coli co-chaperone GrpE connects structure with function

10.1101/2020.07.03.186056 ◽

2020 ◽

Author(s):

Tulsi Upadhyay ◽

Vaibhav V Karekar ◽

Ishu Saraogi

Keyword(s):

Heat Stress ◽

Coiled Coil ◽

Bacterial Survival ◽

Nucleotide Exchange ◽

E Coli ◽

Coiled Coil Domain ◽

Nucleotide Exchange Factor ◽

First Time

AbstractIn bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a long non-ideal coiled-coil. During heat stress, this region partially unfolds and prevents DnaK nucleotide exchange, ultimately ceasing the chaperone cycle. In this study, we elucidate the role of thermal unfolding of the coiled-coil domain of E. coli GrpE in regulating its co-chaperonic activity. The presence of a stutter disrupts the regular heptad arrangement typically found in an ideal coiled coil resulting in structural distortion. Introduction of hydrophobic residues at the stutter altered the structural stability of the coiled-coil. Using an in vitro FRET assay, we show for the first time that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, the mutants were defective in in vitro functional assays, and were unable to support bacterial growth at heat shock temperature in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in the GrpE coiled-coil, and its role in regulating the DnaK-chaperone cycle for bacterial survival during heat stress. More generally, our findings illustrate how a sequence specific stutter in a coiled-coil domain regulates the structure function trade-off in proteins.

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ShiF acts as an auxiliary factor of aerobactin secretion in meningitis Escherichia coli strain S88

BMC Microbiology ◽

10.1186/s12866-019-1677-2 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Mathieu Genuini ◽

Philippe Bidet ◽

Jean-François Benoist ◽

Dimitri Schlemmer ◽

Chloé Lemaitre ◽

...

Keyword(s):

Wild Type Strain ◽

Maximum Growth ◽

Coli Strain ◽

Transcriptional Analysis ◽

Neonatal Meningitis ◽

Liquid Chromatography Mass Spectrometry ◽

Wild Type ◽

E Coli ◽

Auxiliary Factor

Abstract Background The neonatal meningitis E. coli (NMEC) strain S88 carries a ColV plasmid named pS88 which is involved in meningeal virulence. Transcriptional analysis of pS88 in human serum revealed a strong upregulation of an ORF of unknown function: shiF, which is adjacent to the operon encoding the siderophore aerobactin. The aim of this work is to investigate the role of shiF in aerobactin production in strain S88. Results Study of the prevalence of shiF and aerobactin operon in a collection of 100 extra-intestinal pathogenic E. coli strains (ExPEC) and 50 whole genome-sequenced E. coli strains revealed the colocalization of these two genes for 98% of the aerobactin positive strains. We used Datsenko and Wanner’s method to delete shiF in two S88 mutants. A cross-feeding assay showed that these mutants were able to excrete aerobactin meaning that shiF is dispensable for aerobactin excretion. Our growth assays revealed that the shiF-deleted mutants grew significantly slower than the wild-type strain S88 in iron-depleted medium with a decrease of maximum growth rates of 23 and 28% (p < 0.05). Using Liquid Chromatography-Mass Spectrometry, we identified and quantified siderophores in the supernatants of S88 and its shiF deleted mutants after growth in iron-depleted medium and found that these mutants secreted significantly less aerobactin than S88 (− 52% and - 49%, p < 0.001). Conclusions ShiF is physically and functionally linked to aerobactin. It provides an advantage to E. coli S88 under iron-limiting conditions by increasing aerobactin secretion and may thus act as an auxiliary virulence factor.

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Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate

Antibiotics ◽

10.3390/antibiotics10050503 ◽

2021 ◽

Vol 10 (5) ◽

pp. 503

Author(s):

Sabine Schuster ◽

Martina Vavra ◽

Ludwig Greim ◽

Winfried V. Kern

Keyword(s):

Fluorescent Dyes ◽

Efflux Pump ◽

Multidrug Resistant ◽

Coli Strain ◽

Double Knockout ◽

E Coli ◽

Knockout Mutants ◽

Antibiotic Agents ◽

Single Knockout

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

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Proteins-partners of PH domain of BCR-ABL protein: creation of DNA constructs to uncover molecular characteristics of CML development

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v20.732 ◽

1970 ◽

pp. 47-52

Author(s):

S. V. Antonenko ◽

I. V. Kravchuk ◽

D. S. Gurianov ◽

G. D. Telegeev

Keyword(s):

Protein Interactions ◽

Coli Strain ◽

Molecular Characteristics ◽

Ph Domain ◽

Protein Protein Interactions ◽

E Coli ◽

Cellular Processes ◽

Molecular Events ◽

Genetic Constructs

Aim. Impact of domains of Bcr in oncogenic effect associated with Bcr-Abl remains unclear. Investigation of protein-protein interactions can be one of the effective ways to reveal those molecular events that alter normal cellular processes and cause malignant transformation. Previous research showed that USP1, Cortactin and Hsp27 may interact with PH domain. To confirm interactions and to study their biological consequences, genetic constructs for expression and microscopy should be created. Methods. Various standard molecular cloning techniques and expression in E. coli strain Rosetta. Results. Several DNA constructs have been created (pBluescriptSKII(+)+USP1, pFastFT-N1-CTTN, pMediumFT-N1-CTTN, pSlowFT-N1- CTTN and pET42a-hsp27). Effective bacterial expression of Hsp27 has been performed. Conclusions. All DNA constructs can be effective instruments to study biological role of interactions between PH domain of Bcr and USP1, Cortactin, Hsp27.Keywords: PH domain, Bcr-Abl, USP1, Cortactin, Hsp27.

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Role of Deoxyribose Catabolism in Colonization of the Murine Intestine by Pathogenic Escherichia coli Strains

Infection and Immunity ◽

10.1128/iai.01039-08 ◽

2009 ◽

Vol 77 (4) ◽

pp. 1442-1450 ◽

Cited By ~ 15

Author(s):

Vanessa Martinez-Jéhanne ◽

Laurence du Merle ◽

Christine Bernier-Fébreau ◽

Codruta Usein ◽

Amy Gassama-Sow ◽

...

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Pathogenic Potential ◽

E Coli ◽

Intestinal Epithelia ◽

Gut Colonization ◽

Pathogenic Escherichia Coli ◽

Increased Risk ◽

Evolutionary Step

ABSTRACT We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by the deoK operon, is associated with the pathogenic potential of Escherichia coli strains. Carbohydrate metabolism is thought to provide the nutritional support required for E. coli to colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenic E. coli strains. Molecular and biochemical characterization of 1,221 E. coli clones from various collections showed this biochemical trait to be common in the E. coli species (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizing E. coli clones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenic E. coli were significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of the deoK operon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenic E. coli growth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.

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RNA Interference by Ingested Dsrna-Expressing Bacteria to Study Porphyrin Pigmentation in Crassostrea gigas

International Journal of Molecular Sciences ◽

10.3390/ijms22116120 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6120

Author(s):

Biyang Hu ◽

Qi Li ◽

Hong Yu

Keyword(s):

Rna Interference ◽

Crassostrea Gigas ◽

Coli Strain ◽

Functional Genes ◽

Unicellular Alga ◽

E Coli ◽

Genes Expression ◽

Porphyrin Synthesis

Porphyrins are a widespread group of pigments in nature which are believed to contribute to shell colors in mollusks. Previous studies have provided candidate genes for porphyrin shell coloration, however, the linkage analysis between functional genes and porphyrin pigmentation remains unclear in mollusks. RNA interference is a powerful molecular tool for analyzing the loss of functions of genes in vivo and alter gene expression. In this study, we used unicellular alga Platymonas subcordiformis and Nitzschia closterium f. minutissima as vectors to feed oysters with Escherichia coli strain HT115 engineered to express double-stranded RNAs targeting specific genes involved in porphyrin synthesis. A strain of Crassostrea gigas with orange shell was used to target key haem pathway genes expression using the aforementioned approach. We show here that feeding the oysters with E. coli, containing dsRNA targeting pigmentation genes, can cause changes in the color of the newly deposited shell. For example, the RNAi knockdown of CgALAS and CgPBGD resulted in the loss of uroporphyrin pigmentation from the shell due to the accumulation of the pigment in the oyster’s mantle. The study probed the crucial role of ALAS and PBGD genes potential functions of uroporphyrin production and shell color pigmentation in C. gigas.

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