A reconsideration of the evidence for Escherichia coli STa (heat stable) enterotoxin-driven fluid secretion: a new view of STa action and a new paradigm for fluid absorption

A review of the evidence for Escherichia coli STa causing fluid secretion in vito leads to the conclusion that the concept of STa acting through enhanced chloride secretion in order to derange intestinal function is unproven. However, a consistent effect of STa in the small intestine is on Na+/H+ exchange, leading to interruption of luminal acidification. A model for the action of STa, involving inhibition of Na+/H+ exchange, is proposed which explains the ability of STa to reduce absorption in vito but its inability to cause secretion in vito in contrast to its apparent secretory effect in vitro. The apparent ability to demonstrate secretion in vitro is shown to derive from methodologies which do not involve measurement of mass transport of water but instead, infer it from in vitro and in vivo proxy measurements. The in vitro demonstration of notional secretion after STa exposure can be reconciled with the proposed new model for fluid absorption in that cell swelling is argued to arise as a transient consequence of STa challenge followed by regulatory volume decrease. Evidence for this derangement model is presented in the form of observations derived from acute in vivo physiological studies and clinical studies on patients without the exchanger. This process of appraisal of the evidence for the mechanism of action of STa has led to a new model for fluid absorption. This is based on the formation of hypotonicity at the brush border luminal surface rather than hypertonicity within the lateral spaces as required by the present standing gradient model of fluid absorption. Evidence from the literature is presented for this new paradigm of water absorption, which may only be relevant for small intestine and other tissues that have Na+/H+ exchangers in contact with HCO-3-containing solutions but which may also be generalizable to all mammalian absorbing epithelial membranes.     

Lack of evidence in vivo for nitrergic inhibition by Escherichia coli (STa) enterotoxin of fluid absorption from rat proximal jejunum

Fluid absorption from the proximal jejunum of the anaesthetised rat was measured in vivo by fluid recovery. As expected, heat stable (STa) enterotoxin from E. coli reduced fluid absorption. Neither intraperitoneal L-NAME, thought to inhibit a putative neurally mediated action of STa, nor similar doses of D-NAME, ameliorated the inhibitory effect on jejunal fluid absorption of STa. Luminally perfused 10 mM sodium nitroprusside (SNP) had no effect on fluid absorption when expressed per gram dry weight per hour but reduced fluid absorption when expressed per cm length per hour. Similarly, 80 but not 40 mg/Kg of L-NAME reduced fluid absorption when expressed per cm length per hour, while the same dose of D-NAME did not. L-NAME and SNP significantly increased the wet weight to dry weight and the length to dry weight ratio of perfused loops. We conjecture that smooth muscle relaxation caused by these compounds increases interstitial fluid volumes that can be misconstrued as changes in absorption when this is expressed per cm length or per tissue wet weight. When fluid absorption is expressed per gram dry weight of tissue, there is no evidence for a role of nitric oxide in normal or STa inhibited fluid absorption.     

A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.

The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.     

A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies

A recombinant fusion protein consisting of native Escherichia coli heat-stable enterotoxin (STa) and a dimer of a synthetic IgG-binding fragment (ZZ), derived from Staphylococcus aureus protein A was produced in E. coli. The fusion protein (ZZSTa) was secreted in large quantities into the growth medium and recovered by affinity chromatography on IgG-Sepharose. Rabbits immunized with the fusion protein responded by producing high serum levels of anti-STa antibodies that also effectively neutralized STa toxicity in infant mice. The fusion peptide ZZSTa had a substantially decreased toxicity as compared with native STa. A polymeric form of ZZSTa separated by size fractionation was about 100 times less toxic than the monomeric fusion protein, yet both forms had the same capacity to induce neutralizing antibodies. This suggests that modified non-toxic forms of ZZSTa with retained immunogenicity may be produced and tested for their usefulness as functional components in a vaccine against diarrhoea caused by enterotoxigenic E. coli.     

In vivo expression of the heat stable (estA) and heat labile (eltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.     

Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco-2)

Escherichia coli heat stable enterotoxin (ST_a) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared ST_a binding and affinity crosslinking of ST_a receptors in human small intestine to those in the Caco-2 human colon carcinoma cell line. ST_a had similar kinetics of binding in human intestinal and Caco-2 brush border membranes. In both human intestine and Caco-2 brush border membranes, multiple specifically radiolabeled bands, including a 140–165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned ST_a receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco-2 cells could be explained by the existence of several members of a family of ST_a receptors and/or the existence of smaller ST_a binding proteins generated by the protease cleavage of a larger complete ST_a receptor. © 1993 Wiley-Liss, Inc.     

The gene for a small stable RNA (10Sa RNA) of Escherichia coli

A gene that codes for a small stable RNA (362 nucleotides) has been sequenced. It is a monocistronic gene, with its own promoter and terminator. It produces a precursor that is about 100 nucleotides longer than the mature RNA with all the extra nucleotides at the 3' end. The gene contains an open reading frame that corresponds to a small protein 25 amino acids long.     

Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: extended X-ray absorption fine structure evidence for a metal-binding domain

Formamidopyrimidine-DNA glycosylase (Fpg) is a 30.2 kDa protein that plays an important role in the base excision repair of oxidatively damaged DNA in Escherichia coli. Sequence analysis and genetic evidence suggest that zinc is associated with a C4-type motif, C(244)-X(2)-C(247)-X(16)-C(264)-X(2)-C(267), located at the C-terminus of the protein. The zinc-associated motif has been shown to be essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc-associated protein (ZnFpg) in the lyophilized state and in 10% frozen aqueous glycerol solution show directly that the metal is coordinated to the sulfur atom of four cysteine residues. The average Zn-S bond length is 2.33 +/- 0.01 and 2.34 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. Fpg was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily cobalt-associated (CoFpg). The profiles of the circular dichroism spectra for CoFpg and ZnFpg are identical, suggesting that the substitution of Co(2+) for Zn(2+) does not alter the structure of Fpg. A similar conclusion is reached upon the analysis of two-dimensional (15)N/(1)H HSQC spectra of uniformly (15)N-labeled samples of ZnFpg and CoFpg; the spectra are similar and display features characteristic of a structured protein. Biochemical assays with a 54 nt DNA oligomer containing 7, 8-dihydro-8-oxoguanine at a specific location show that CoFpg and ZnFpg are equally active at cleaving the DNA at the site of the oxidized guanine. EXAFS spectra of CoFpg indicate that the cobalt is coordinated to the sulfur atom of four cysteine residues with an average Co-S bond length of 2.28 +/- 0.01 and 2.29 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. The structural similarity between CoFpg and ZnFpg suggests that it is biologically relevant to use the paramagnetic properties of Co(2+) as a structural probe.     

Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli

Bacteroides fragilis TMP10, which is clindamycin-erythromycin resistant (Clnr) and tetracycline resistant (Tetr), contains several plasmids and is capable of transferring drug resistance markers to suitable recipients. We were able to separate a 14.6-kilobase self-transmissible Clnr plasmid, pBFTM10, from the other plasmids of TMP10 in a tetracycline-sensitive recipient strain, B. fragilis TM4000. All Clnr transconjugants acquired an unaltered pBFTM10 and became plasmid donor strains. Transfer is proposed to occur by conjugation since it required to cell-to-cell contact of filter matings and was insensitive to DNase, but sensitive to chloroform treatment of donor cells. The efficiency of transfer of pBFTM10 in a Tets background (TM4003) was not affected by pretreatment of donor cells with clindamycin. A spontaneously occurring Clns derivative, pBFTM10 delta 1, suffered a deletion of DNA, which included a 4.4-kilobase EcoRI fragment. A complex interaction between the autonomous plasmid pBFTM10 and a tetracycline transfer element also present in strain TMP10 was observed since pretreatment of this donor with tetracycline or clindamycin resulted in a marked increase in transfer of both tetracycline and clindamycin resistance.     

A new species of Kanahia (Asclepidiaceae) with a reconsideration of the genus

A new species, K. carlsbergiana , is described in what is currently considered a monotypic genus. The new species is only known from permanent streams in the Arssi and Bale regions of S Ethiopia. The delimitation and position of the genus is reconsidered in light of the additional information provided by the new species. The distinctiveness of the genus is reconfirmed whilst no new clues to possible relationships with other genera were observed. The taxonomy of the other species, K. laniflora (Forssk.) R. Br., is also briefly reconsidered.     

Copy-number control of the Escherichia coli chromosome: a plasmidologist's view

The homeostatic system that sets the copy number, and corrects over-replication and under-replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy-number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative-feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number.     

Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.     

Characterization of 10S RNA: a new stable rna molecule from Escherichia coli.

Summary When cells of Escherichia coli are labeled with ³²P_i for long periods of time and the cell content is subjected to electrophoresis in polyacrylamide gels, an RNA band appears which is about 10S in size. This band seems to contain three conformers. After treatment with formamide only a single band appears in this region of the gel, which contains 550 nucleotides as determined from its mobility. The complexity of the fingerprint of this material, after digestion with T₁-RNase, is in agreement with the size as determined by the mobility, this confirming that indeed it is a single molecule. Composition of the T₁-oligonucleotides was determined by digesting the T₁-generated oligonucleotides with pancreatic RNase and T₂-RNase. The quantitative and qualitative analysis of these digestions suggests that 10S RNA contains 609 nucleotides. The molecule contains, besides the four regular bases, one copy per molecule of the modified base pseudouridine.10S RNA cannot be processed by cell extracts to tRNA-sized molecules and does not bind significantly to ribosomes, hence it is unlikely to be a tRNA precursor or an mRNA.     

The action of beta-galactosidase (Escherichia coli) on allolactose.

The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.     

Seaweed attachment to bedrock: biophysical evidence for a new geophycology paradigm

Seaweeds are amongst the most obvious and ecologically important components of rocky shore communities worldwide but until now little has been known about the processes involved in their attachment. This multidisciplinary study investigated for the first time the interactions between marine macroalgal holdfasts and their underlying substrata, requiring the development of specialized sample preparation techniques to maintain the structural integrity of the holdfast–bedrock interface. Transmitted plane polarized light microscopy, scanning electron microscopy with energy dispersive spectroscopy and structured light illumination microscopy were used in the examination of the interface between Ascophyllum nodosum (Fucales, Heterokontophyta) and crustose red algae Lithothamnion sp. (Corallinales, Rhodophyta) on granite and limestone substrates. The new evidence presented here represents a paradigm shift in the way we view seaweed attachment because results show that the holdfasts exploit the physical characteristics of the rock-forming minerals in order to penetrate the bedrock and thus facilitate the attachment process. Mineral cleavage planes together with intercrystalline and intracrystalline boundaries and fractures provide penetration pathways for the holdfast tissue. This process causes disaggregation of rock-forming minerals to depths <10 mm and therefore assists in the bioerosion of coastal bedrock. It is concluded that seaweeds are able to cause weathering of natural rock and the term 'geophycology' is introduced to describe seaweed–bedrock interactions, including seaweed-induced weathering.     

Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome.

The gene for 10Sa RNA, which is a major small, stable RNA in Escherichia coli, is a unique gene in the E. coli chromosome. The 10Sa RNA gene (ssrA) has been located between 2,760 and 2,761 kilobases on the E. coli genome.     

Linkage of K99 production and STa activity in a plasmid of an Escherichia coli porcine isolate

An Escherichia coli strain ZP118 of porcine origin was shown to harbor a 68-megadalton (Md) plasmid coding for a colonization factor K99 and heat-stable enterotoxin (STa), and a self-transmissible 51-Md plasmid coding for drug resistance. One of the transconjugants obtained by mating between ZP118 and E. coli C was found to harbor a 90-Md plasmid coding for K99, STa and drug resistance. Restriction endonuclease analysis suggested that the 90-Md plasmid could be a recombinant between the 68-Md plasmid and the 51-Md plasmid.