Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.     

Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups

The genetic relatedness among 96 invasive Escherichia coli belonging to several serogroups and 13 non-invasive of several serotypes that share the same O antigen was investigated by multilocus enzyme electrophoresis analysis. The invasive strains were isolated in different parts of the world and most of them recovered from dysentery. Twenty-nine electrophoretic types were distinguished and the most invasive strains were found to belong to two major lineages. These results suggested that the invasive ability in these strains has evolved in divergent chromosomal backgrounds, presumably through the horizontal spread of plasmid-borne invasion genes. The maintenance of invasive phenotypes in separate lineages suggests that this ability confers a selective advantage to invasive strains.     

Congo red binding test in enteroinvasive and nonpathogenic Escherichia coli strains.

Out of 6 variants the appropriate media to perform Congo red binding test for enteroinvasive E. coli strains were established (trypto-soy agar Eiken, T.S.A.--Cantacuzino Institute and B.T.S.D.). 12 E. coli strains belonging to enteroinvasive O-serogroups formed on Congo red agar red-coloured, non-coloured colonies or both; cultures from 59 red colonies and 61 white colonies were inoculated in guinea pig eyes. The correlation between positive Congo red binding test and positive Sereny test was 91% (out of 59 red colonies, 47 evoked keratoconjunctivitis in both infected eyes and 7 in only one eye). The negative Congo red binding test corresponds (98.4%) to the failure to induce illness in the guinea pigs' eye (only one out of 61 Crb = colonies was Sereny positive, evoking keratoconjunctivitis in only one of the two infected eyes of a guinea pig). Comparing in vivo lack of pathogenicity in 44 E. coli strains isolated from human normal intestinal flora and negative Congo red binding test, a correlation of 72.73% on B.T.S.D. and 65.91% on T.S.A. medium was found. Developing an appropriate method based on Crb test about 70% of the nonpathogenic E. coli colonies could be eliminated from the laborious agglutination with enteroinvasive O-serogroups E. coli antisera.     

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.     

Ecovariant differences of Escherichia coli strains with respect to colicinogenicity and colicin resistance

The analysis of 173 Escherichia coli strains, isolated from different sources, for colicinogenicity and colicin resistance revealed that frequency of these signs increased in the following order: water in open reservoirs, intestine, extraintestinal localizations. In most cases resistance to 5 or more bacterial colicins was due to the absence of the corresponding receptors to colicins. Colicin resistance and colicinogenicity render E. coli selective advantages under the conditions of intestinal microbiocenosis.     

Exchange of colicin receptor capacity between strains of Escherichia coli sensitive or resistant to colicin K-K235

Phage and colicin-resistant mutants were derived from Escherichia coli K-12P678. Two classes of phage T6 and colicin K-resistant mutants (genotype tsx) were isolated. Tsx-2 mutants, which demonstrated mucoid growth and increased sensitivities to many antibiotics, became sensitive to colicin K when pretreated with ethylenediaminetetraacetate (EDTA), whereas Tsx-1 mutants did not. Reassociation of EDTA-released material partially restored resistance to colicin K for Tsx-2 mutants. When EDTA-released material from strain P678 was associated with either class of K-resistant mutant, an increase in colicin K sensitivity resulted. Observations suggest that colicin K can act on its target site once it penetrates the cell surface. In addition, results suggest that functional colicin K receptors can be transferred from sensitive to resistant strains, thus conferring colicin sensitivity.Non-standard Abbreviations SDS sodium dodecyl sulfate     

The role of colicin receptors in the uptake of ferrienterochelin by Escherichia coli K-12.

The amino terminal sequences of the 4 caseins synthesized by translation of ovine mammary mRNAs in a wheat germ cell-free system have been investigated by automated Edman degradation. The 3 “calcium-sensitive” caseins (α_s1, α_s2 and β) and κ-casein were synthesized as precaseins with amino terminal hydrophobic extensions of 15 and 21 amino acid residues respectively, resembling “signal peptides” of other secretory proteins. The extra pieces of the 4 caseins, which start with a methionyl residue, end with an alanyl residue which may be one of the signals recognized by the mammary membrane-bound enzyme responsible for the specific cleavage of precaseins. The amino terminal extensions of α_s1, α_s2 and β-caseins show a high degree of homology suggesting that they have derived from a common ancestor.     

Detection of virulent Shigella and enteroinvasive Escherichia coli by induction of the 43 kDa invasion plasmid antigen, ipaC

The invasion plasmid antigen, ipaC (43 kDa) of Shigella spp. and enteroinvasive Escherichia coli (EIEC) could be induced in vitro by growing them in the presence of Congo red. An enzyme-linked immunosorbent assay (ELISA) using antibodies to the 43 kDa protein of Shigella has been developed for specific detection of virulent Shigella spp and EIEC. The test is independent of initial isolation of individual colonies. As few as 10² CFU/ml of virulent Shigella present in mixed cultures could be detected and concurrently their susceptibility to antibiotics could be analysed after an initial growth of 8–16 h in Congo red-containing medium. The test may prove useful in the diagnosis and treatment of bacillary dysentery caused either by Shigella or EIEC through their rapid identification and proper antimicrobial therapy.     

Analysis of threonine uptake in Escherichia coli threonine production strains

Threonine uptake in Escherichia coli wild-type and in threonine-producing strains decreased throughout threonine production. In contrast to previously published results, the SstT uptake system is not the sole serine/threonine permease in E. coli, since a novel transport system was detected in an sstT deletion strain.     

Invasion of tissue culture cells by diarrhoeagenic strains of Escherichia coli which lack the enteroinvasive inv gene

Abstract Invasive Escherichia coli strains of certain serotypes invade by the same mechanism as the Shigella sp. It has been proposed that invasion of epithelial cells by EPEC strains may also occur; this is a previously overlooked property. In the present study E. coli strains isolated from patients with diarrhoea or ulcerative colitis, lacking the inv plasmid mediating classical invasion, but hybridizing with probes for different adhesins, were analyzed for their ability to invade HeLa and Caco-2 cells. The majority of strains invaded Caco-2 cells to a higher extent than HeLa cells. Adhesion to Caco-2 cells was a prerequisite for subsequent invasion of the cells but EAF, eae , EAgg and other known virulence factors were not sufficient to mediate invasion. In 8/9 E. coli strains invasion was enhanced after growth under iron restriction. Growth during anaerobic conditions did not influence subsequent invasion by E. coli strains whereas 6/9 strains had their invasive ability significantly decreased after growth in the presence of 1% glucose. The invasive process was inhibited by mannose but not by lactose, fucose or galactose. Our data indicate that strains of E. coli may invade Caco-2 cells by novel mechanisms which require adhesion to the cells but which differ from those of Salmonella sp., Yersinia sp., Shigella sp. and classical enteroinvasive E. coli .     

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.     

Fused lacZ genes code for di-, tri- and tetra-beta-galactosidase in Escherichia coli.

Plasmids were constructed which carry two, three or four active lacZ genes of Escherichia coli fused head-to-tail in phase. The products of these oligomeric lacZ genes are shown to be polypeptides with expected subunit mol. wts. of 230 kd (di-beta-galactosidase), 350 kd (tri-beta-galactosidase) and 460 kd (tetra-beta-galactosidase). Di-beta-galactosidase has the same enzymatic activity as the wild-type enzyme. It subunits are practically not degraded proteolytically in vivo. It aggregates predominantly to a dimer which has the same sedimentation constant as the wild-type tetrameric enzyme. Furthermore, it is more heat stable than the wild-type enzyme. Tri- and tetra-beta-galactosidase have strongly reduced enzymatic activities and are largely degraded. Our experiments lead us to propose that covalent joining of two subunits through proper gene duplication may possibly be an intermediate in the evolution of self aggregation of homo-oligomeric proteins.     

Relation of cell growth and colicin tolerance to vitamin B12 uptake in Escherichia coli.

The uptake of vitamin B12 was measured in cells of Escherichia coli whose growth had been inhibited by any of a variety of treatments. In all cases, the secondary, energy-dependent phase of B12 uptake was depressed in proportion to the decrease in growth rate, but uptake was constant in cells growing logarithmically at different rates. The depression of B12 uptake activity was independent of the site of cell metabolism affected by the inhibitor or by its effect on cell viability, and was both more rapid and of greater degree than the effects on the uptake of any of the six amino acids tested. The decline was not affected by inhibitors of either cell division or proteolysis and was manifested without any apparent decrease in the surface B12 binding activity. Transport activity was rapidly regained upon reversal of the inhibition of protein synthesis. Prompted by this response, the uptake of B12 was contrasted to the apparent uptake of the E colicins, which share the same outer membrane receptor. Sensitivity to colicin E1, measured by its inhibition of proline uptake, was not affected by growth inhibition by antibiotic treatment. Finally, there was no specific depression of B12 uptake in cells rendered colicin tolerant either by mutation or as a consequence of phage f1 infection.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Prevalence of ColE1-like plasmids and colicin K production among uropathogenic Escherichia coli strains and quantification of inhibitory activity of colicin K

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.