Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. METHODS: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). RESULTS: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). CONCLUSIONS: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

Complete genome sequence of the novel Escherichia coli phage phAPEC8.

Bacteriophage phAPEC8 is an Escherichia coli-infecting myovirus, isolated on an avian pathogenic Escherichia coli (APEC) strain. APEC strains cause colibacillosis in poultry, resulting in high mortality levels and important economic losses. Genomic analysis of the 147,737-bp double-stranded DNA phAPEC8 genome revealed that 53% of the 269 encoded proteins are unique to this phage. Its closest relatives include the Salmonella phage PVP-SE1 and the coliphage rv5, with 19% and 18% similar proteins, respectively. As such, phAPEC8 represents a novel, phylogenetically distinct clade within the Myoviridae, with molecular properties suitable for phage therapy applications.

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.

Human uropathogenic and bovine septicaemic Escherichia coli strains carry an identical F17-related adhesin.

Pathogenic Escherichia coli produce fimbriae which mediate binding to mucosal cells. Generally, different fimbriae are associated with different tissular tropisms and different host specificities. Genes encoding for pilin and adhesin subunits of two F17-related fimbriae were cloned and sequenced. The first, G fimbriae, are synthesized by a human uropathogenic E. coli strain, and the second, 20K fimbriae, by a bovine septicaemic E. coli strain. We showed that both fimbriae are identical and present a high homology with F17a and F17b fimbriae synthesized by bovine enterotoxigenic E. coli strains. Furthermore, data showed that the G adhesin did not mediate adhesion to human uroepithelial cells, suggesting that it is not responsible for the urinary tropism of the strain and confirming the intestinal tropism specificity of F17-related adhesins.

The trehalose operon of Pseudomonas fluorescens ATCC 17400.

The trehalose operon of Pseudomonas fluorescens ATCC 17400 consists of treP, treA and treR. The gene treP codes for a putative enzyme II subunit of the phosphotransferase system that catalyzes the phosphorylation of trehalose together with its translocation across the cell membrane and treA encodes a putative phosphotrehalase, which hydrolyzes the incoming trehalose-6-phosphate into glucose and glucose-6-phosphate. Both genes are negatively regulated by TreR, a repressor of the FadR-GntR family of transcription regulators. The operon that is induced by trehalose present in the medium shows a high similarity both in the function of genes and in the regulation with the trehalose operon of Bacillus subtilis.

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae.

Porcine Escherichia coli strains isolated from cases of postweaning diarrhea or edema disease were analysed for the presence of fedA, the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA-specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.

The pathogenesis of edema disease in pigs. A review.

Edema disease is known to cause important losses in the period shortly after weaning. Although the disease is known for many decades, intensive studies with bacterial lysates of pathogenic E. coli, followed by biotechnological research the last ten years, has led to a better understanding of its pathogenesis. Especially the impact of the toxin is clearly established. Evidence also exists that adhesion factors play a crucial role in the pathogenesis of edema disease.

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea.

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-Ilv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E. coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strains enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.

Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli.

The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.

Complete Genome Sequences of Two Prolific Biofilm-Forming Staphylococcus aureus Isolates Belonging to USA300 and EMRSA-15 Clonal Lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections that are even more difficult to treat when associated with a biofilm phenotype that facilitates evasion of the host immune system and antibiotics. As a first step toward understanding the mechanisms underlying biofilm formation, we sequenced the genomes of two prolific biofilm-forming strains belonging to the two most important globally disseminated clonal lineages, USA300 and EMRSA-15.

Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter.

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron- and pH-inducible lipoprotein gene sfbA, which is a component of a novel ABC-type transporter system required for virulence. This gene is located on a 4 kb Salmonella-specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild-type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.

In vivo random beta-glucuronidase gene fusions in Arabidopsis thaliana.

Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA. Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences. After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively. Several different patterns of tissue-specific beta-glucuronidase expression were identified. The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced. After introduction in A. thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated. These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression.

T-DNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis.

Asparagus officinalis was the first monocotyledonous plant from which hormone-independent and opine-producing crown gall tissue could be isolated. We confirm by DNA hybridization that tumor lines obtained after infection of this plant by Agrobacterium strains harboring wild-type nopaline and octopine tumor-inducing (Ti) plasmids are stably transformed and contain transferred DNA (T-DNA) segments identical to the T-DNA found in dicotyledonous plants. We have also infected Asparagus with a nononcogenic T-DNA vector that carries a chimeric aminoglycoside phosphotransferase [NOS-APH(3')II] gene and selected transformed tissues on kanamycin-containing medium. The transformed status of these tissues was then confirmed by DNA hybridization. From these calli we regenerated kanamycin-resistant shoots that were subsequently rooted. Thus we report the isolation of transgenic monocotyledonous plants engineered via the Agrobacterium vector system.

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation.

Transgenic Petunia hybrida clones harbouring the T-DNA gene 2 of Agrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In the Petunia line used, floral variegation is due to the presence of the non-autonomous transposable element dTph1 at the An1 locus. The gene 2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene 2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene 2 was no longer expressed. Southern analysis, using gene 2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene 2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene 2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene 2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene 2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene 2 in this system.

Characterization of F18 fimbrial genes fedE and fedF involved in adhesion and length of enterotoxemic Escherichia coli strain 107/86.

Infection of susceptible weaned pigs with oedema disease strains of E. coli is associated with bacterial adhesion to the small intestine. F18 fimbria (previously named F107) was the first colonisation factor described on oedema disease strains, and its genetic determinant was cloned. In the present study, genes fedE and fedF were positioned in the F18 gene cluster, downstream of the major structural subunit gene fedA. Two fedE and two fedF mutants were identified that had lost their capacity to adhere to isolated porcine villi. Moreover, these mutants produced significantly longer fimbriae. In vitro adhesion tests, electron microscopy study, transcomplementation tests, and nucleotide sequence analysis indicated that proteins FedE and FedF are F18 minor subunits essential for fimbrial adhesion and effecting fimbrial length.

The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants.

Signals necessary for in vivo expression of Ti plasmid T-DNA-encoded octopine and nopaline synthase genes were studied in crown gall tumors by constructing mutated genes carrying various lengths of sequences upstream of the 5' initiation site of their mRNAs. Deletions upstream of position -294 did not interfere with expression of the octopine synthase gene while those extending upstream of position -170 greatly reduced the gene expression. The estimated size of the octopine synthase promoter is therefore 295 bp. The maximal length of 5' upstream sequences involved in the in vivo expression of the nopaline synthase gene is 261 bp. Our results also demonstrated that Ti plasmid-derived sequences contain all signals essential for expression of opine synthase genes in plants. Expression of these genes, therefore, is independent of the direct vicinity of the plant DNA sequences and is not activated by formation of plant DNA and T-DNA border junction.