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We describe identification and functional characterization of ISEcll, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEcll is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEcll is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the ISllll family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter ([P.sub.juncC]) containing a--10 box in the interstitial sequence and a -35 box upstream of the right IR. A natural variant of ISEcll (ISEcllp), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEcllp forms a junction promoter ([P.sub.juncP]) which, despite carrying--10 and--35 boxes identical to those of [P.sub.juncC] exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of [P.sub.juncP] is 20-fold higher than the activity of [P.sub.juncC]. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

The virulence plasmid-carried apy (phoN2) gene of Shigella and related enteroinvasive Escherichia coli (EIEC) encodes apyrase, an ATP-diphosphohydrolase belonging to class A of the non-specific acid phosphatases (A-NSAPs). Apyrase and A-NSAPs share three domains of conserved amino acids (domains D1-D3) containing residues forming the putative active site of apyrase. In spite of their similarity, apyrase and A-NSAPs show different substrate specificity, apyrase being able to hydrolyse nucleotide tri- and diphosphates, but not monophosphates, as well as p-nitrophenyl phosphate (pNPP), while A-NSAPs are also active towards monophosphates and pNPP. In this paper, to get further insights into the structure--function relationship of apyrase, a random and site-directed mutagenesis of the apy gene of EIEC strain HN280 was conducted. Results indicate that amino acids located within the D2 and D3 conserved domains (Ser157 and Arg192, respectively) as well as residues located in the N-terminal (Ser97) and C-terminal (Glu233) domains are required for enzyme activity. Surprisingly, Ala160, located near the D2 domain and considered to be important for enzyme specificity, is required for enzyme activity, as its substitution with Thr led to the inactivation of enzyme activity. Furthermore, residue His116 is involved in apyrase specificity, since the H116L apyrase mutant shows substrate specificity resembling that of A-NSAPs.

Virulence gene expression in enteroinvasive Escherichia coli requires all three virulence regulatory genes: virB, virF and hns. The integration of the virulence plasmid pINV produces a noninvasive strain by repressing virB transcription, despite virF and hns remaining unaffected. H-NS in the pINV-strain binds more stably to virB, preventing virF activation but an increase in virF gene dosage, for autonomously replicating pINV, overcomes H-NS' negative regulatory effect on virB transcription.

Abstract Background Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains. Results All S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness. Conclusions The main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

We report a case of a vaginal infection caused by a strain of Burkholderia cenocepacia. The strain was isolated from vaginal swab specimens from a 68-year-old woman with smoldering myeloma [...]

Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the 183PAPAP187 motif of OmpA, nor the N-terminal polyproline 43PPPP46 motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented. [ABSTRACT FROM AUTHOR]

Introduction: Members of the human intestinal microbiota are key players in maintaining human health. Alterations in the composition of gut microbial community (dysbiosis) have been linked with important human diseases. Understanding the underlying processes that control community structure, including the bacterial interactions within the microbiota itself, is essential. Bdellovibrio bacteriovorus is a gram-negative bacterium that preys other gram-negative species for survival, acting as a population-balancer. It was found in terrestrial/aquatic ecosystems, and in animal intestines, postulating its presence also in the human gut. Methods: The present study was aimed to evaluate, by end-point PCR and qPCR, the presence of B. bacteriovorus in intestinal and faecal biopsy specimens from 92 paediatric healthy subjects and patients, suffering from Inflammatory Bowel Diseases (IBD), Celiac disease and Cystic fibrosis (CF). Results: i) B. bacteriovorus was present and abundant only in healthy individuals, while it was heavily reduced in patients, as in the case of IBD and Celiac, while in CF patients and relative controls we observed comparable results; ii) B. bacteriovorus seemed to be mucosa-associated, because all IBD and Celiac biopsies (and related controls) were treated with mucus-removing agents, leaving only the mucosa-attached microflora; iii) B. bacteriovorus abundance was district-dependent, with a major preponderance in duodenum, and gradually decreasing up to rectum; iv) B. bacteriovorus levels significantly dropped in disease status, in duodenum and ileum. Conclusions: Results obtained in this study could represent the first step for new therapeutic strategies aimed to restore a balance in the intestinal ecosystem, utilizing Bdellovibrio as a probiotic. [ABSTRACT FROM AUTHOR]

The relative rate constant of protein degradation for a number of enzymes as well as for rat liver fractions and for `soluble' and `insoluble' protein of rat tissues has been measured by the double-isotope technique. This technique has been evaluated and found reliable for measurements involving half-lives of about 2-8 days and therefore generally useful for liver, kidney and possibly heart but not for brain and skeletal muscle. Carbamylephosphate synthase represents the major component (≈ 20%) of the protein of rat liver mitoplasts. Particular attention was given to determine its turnover, for obviously this enzyme should be a good `marker' for comparative studies with other mitochondrial proteins. The also hitherto unknown half-lives of malate dehydrogenase and of glutamate dehydrogenase from rat liver mitochondria have been measured. The rate constants of degradation for rats fed `basal' diet were: `homogenate', kd = 0.20 day-1; 'mitochondria' kd = 0.10 day-1 `microsomes', kd = 0.28 day-1 `cytosol', kd 0.20 day-1 malate dehydrogenase, kd = 0.27 day 1; carbamyl-phosphate synthase, kd 0.09 day1; heart proteins: `insoluble', kd = 0.086 day-1; `soluble', kd = 0.175 day-1; kidney proteins: `insoluble', = 0.28 kd; `soluble', kd = 0.245 day-1 . Contrary to reports showing faster degradation of long polypeptide chains than those of smaller size, the apparent turnover rate of carbamyl-phosphate synthase is slower than those of other mitochondrial enzymes with smaller subunits, Similar measurements were carried out with tissues from rats fed `high-protein' and `protein-free' diets and after thyroidectomy. Rats fed a high-protein diet showed a general increase of the rate constant of protein degradation in all the liver fractions studied when compared with data from animals on the basal diet. Feeding a protein-free diet produced an increase of the rate constant of degradation of liver mitochondria and fractions thereof. Thyroidectomy decreased the rate constant of degradation in all the rat liver fractions and enzymes thereof. Kidney and heart insoluble and soluble proteins behaved in the same way as those from rat liver. This is the first time that a direct correlation of the often-mentioned, but hitherto not directly measured, effect on protein turnover has been determined. Small-scale purification of carbamyl-phosphate synthase, malate dehydrogenase and glutamate dehydrogenase from a single rat liver is described. The preparations showed a high degree of homogeneity by disc gel electrophoresis and by sodium dodecyl sulfate electrophoresis. Small-scale purification, when applicable, should minimize uncertainties in regard to homogeneity of enzymes used for antibody production in protein turnover studies, Moreover, it is more economical in time. [ABSTRACT FROM AUTHOR]