Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications.

A computer analysis of 2328 protein sequences comprising about 60% of the Escherichia coli gene products was performed using methods for database screening with individual sequences and alignment blocks. A high fraction of E. coli proteins--86%--shows significant sequence similarity to other proteins in current databases; about 70% show conservation at least at the level of distantly related bacteria, and about 40% contain ancient conserved regions (ACRs) shared with eukaryotic or Archaeal proteins. For > 90% of the E. coli proteins, either functional information or sequence similarity, or both, are available. Forty-six percent of the E. coli proteins belong to 299 clusters of paralogs (intraspecies homologs) defined on the basis of pairwise similarity. Another 10% could be included in 70 superclusters using motif detection methods. The majority of the clusters contain only two to four members. In contrast, nearly 25% of all E. coli proteins belong to the four largest superclusters--namely, permeases, ATPases and GTPases with the conserved "Walker-type" motif, helix-turn-helix regulatory proteins, and NAD(FAD)-binding proteins. We conclude that bacterial protein sequences generally are highly conserved in evolution, with about 50% of all ACR-containing protein families represented among the E. coli gene products. With the current sequence databases and methods of their screening, computer analysis yields useful information on the functions and evolutionary relationships of the vast majority of genes in a bacterial genome. Sequence similarity with E. coli proteins allows the prediction of functions for a number of important eukaryotic genes, including several whose products are implicated in human diseases.

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament.

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.

Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil

OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC) strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk -- types B and C -- of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1%) were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2%). CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication.

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge.

Needle-free procedures are very attractive ways to deliver vaccines because they diminish the risk of contamination and may reduce local reactions, pain or pain fear especially in young children with a consequence of increasing the vaccination coverage for the whole population. For this purpose, the possible development of a mucosal malaria vaccine was investigated. Intranasal immunization was performed in BALB/c mice using a well-studied Plasmodium berghei model antigen derived from the circumsporozoite protein with the modified heat-labile toxin of Escherichia coli (LTK63), which is devoid of any enzymatic activity compared to the wild type form. Here, we show that intranasal administration of the two compounds activates the T and B cell immune response locally and systemically. In addition, a total protection of mice is obtained upon a challenge with live sporozoites.

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of holliday junction resolution.

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.

The homologous regulators ANR of Pseudomonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters.

The anaerobic transcriptional regulator ANR induces the arginine deiminase and denitrification pathways in Pseudomonas aeruginosa during oxygen limitation. The homologous activator FNR of Escherichia coli, when introduced into an anr mutant of P. aeruginosa, could functionally replace ANR for anaerobic growth on nitrate but not for anaerobic induction of arginine deiminase. In an FNR-positive E. coli strain, the ANR-dependent promoter of the arcDABC operon, which encodes the enzymes of the arginine deiminase pathway, was not expressed. To analyse systematically these distinct induction patterns, a lacZ promoter-probe, broad-host-range plasmid containing various -40 regions (the ANR/FNR recognition sequences) and -10 promoter sequences was constructed. These constructs were tested in P. aeruginosa and in E. coli expressing either ANR or FNR. In conjunction with the consensus -10 hexamer of E. coli sigma 70 RNA polymerase (TATAAT), the consensus FNR site (TTGAT ..... ATCAA) was recognized efficiently by ANR and FNR in both hosts. By contrast, when promoters contained the Arc box (TTGAC .... ATCAG), which is found in the arcDABC promoter, or a symmetrical mutant FNR site (CTGAT .... ATCAG), ANR was a more effective activator than was FNR. Conversely, an extended 22 bp, fully symmetrical FNR site allowed better activation with FNR than with ANR. Combination of the arc promoter -10 sequence (CCTAAT) with the Arc box or the consensus FNR site resulted in good ANR-dependent expression in P. aeruginosa but gave practically no expression in E. coli, suggesting that RNA polymerase of P. aeruginosa differs from the E. coli enzyme in -10 recognition specificity. In conclusion, ANR and FNR are able to activate the RNA polymerases of P. aeruginosa and E. coli when the -40 and -10 promoter elements ae identical or close to the E. coli consensus sequences.

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

Characterization of the 4.5S RNA molecule in Escherichia coli

The 4.5S RNA molecule of Escherichia coli is essential to cell viability. It has been shown that depletion of this molecule inhibits protein synthesis, induces the heat shock response, and generally slows cell growth. The molecule has also been implicated in protein secretion, as in cells depleted of 4.5S RNA, an unsecreted precursor to ?-lactamase accumulates (pre-?-lactamase). A role in protein secretion is further supported by structural similarities with the 7S RNA molecule of eukaryotic SRP, specific binding to SRP54, and its homolog in E. coli, P48, and the ability of 7S RNA from certain archaebacteria to suppress 4.5S RNA depletion. In this study I have utilized strains with mutant forms of the 4.5S RNA genes in order to study the effect of altered 4.5S RNA on cell physiology. These strains have their mutant 4.55 RNA under the control of the tryptophan synthetic operon. Decreased growth rates, inhibited cell division, and altered protein synthesis all result from these mutations.

The C-terminal domain of MinC inhibits assembly of the Z ring in Escherichia coli.

In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC(122-231)) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC(122-231), the C terminus of full-length MinC, or the C terminus of MinC(122-231) perturbed MinC function, which may explain why cell division inhibition by MinC(122-231) was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea among children, human immunodeficiency virus-infected patients, and travelers to developing regions of the world. The pathogenesis of EAEC strains involves the production of biofilm. In this study, we determined the association between presence of putative EAEC virulence genes and biofilm formation in 57 EAEC isolates (as defined by HEp-2 adherence) from travelers with diarrhea and in 18 EAEC isolates from travelers without diarrhea. Twelve nondiarrheagenic E. coli isolates from healthy travelers were used as controls. Biofilm formation was measured by using a microtiter plate assay with the crystal violet staining method, and the presence of the putative EAEC virulence genes aap, aatA, aggR, astA, irp2, pet, set1A, and shf was determined by PCR. EAEC isolates were more likely to produce biofilm than nondiarrheagenic E. coli isolates (P = 0.027), and the production of biofilm was associated with the virulence genes aggR, set1A, aatA, and irp2, which were found in 16 (40%), 17 (43%), 10 (25%), and 27 (68%) of the biofilm producers versus only 4 (11%), 6 (6%), 2 (6%), and 15 (43%) in non-biofilm producers (P = 0.008 for aggR, P = 0.0004 for set1A, P = 0.029 for aatA, and P = 0.04 for irp2). Although the proportion of EAEC isolates producing biofilm in patients with diarrhea (51%) was similar to that in patients without diarrhea (61%), biofilm production was related to the carriage of aggR (P = 0.015), set1A (P = 0.001), and aatA (P = 0.025). Since aggR is a master regulator of EAEC, the presence of aap (P = 0.004), astA (P = 0.001), irp2 (P = 0.0006), pet (P = 0.002), and set1A (P = 0.014) in an aggR versus an aggR-lacking background was investigated and was also found to be associated with biofilm production. This study suggests that biofilm formation is a common phenomenon among EAEC isolates derived from travelers with or without diarrhea and that multiple genes associated with biofilm formation are regulated by aggR.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Influence of host interleukin-10 polymorphisms on development of traveler's diarrhea due to heat-labile enterotoxin-producing Escherichia coli in travelers from the United States who are visiting Mexico.

Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position -1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position -819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position -592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD.