A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

Identifying components required for OMP biogenesis as novel targets for antiinfective drugs.

The emergence of multiresistant Gram-negative bacteria requires new therapies for combating bacterial infections. Targeting the biogenesis of virulence factors could be an alternative strategy instead of killing bacteria with antibiotics. The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier. At the same time it facilitates the exchange of molecules and harbors a multitude of proteins associated with virulence. In order to insert proteins into the OM, an essential oligomeric membrane-associated protein complex, the ß-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones may serve as attractive targets to develop novel antiinfective agents. Herein, we aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, OM barrier function, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, we assume that both BamB and SurA are promising targets for the development of new antiinfective drugs in the future.

Detection, distribution and characterization of novel superoxide dismutases from Yersinia enterocolitica Biovar 1A.

BACKGROUND: Superoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism. METHODOLOGY/PRINCIPAL FINDINGS: This study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0-8.0) and temperature (4-70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA(-) SodB(-) Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB. CONCLUSIONS/SIGNIFICANCE: This is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles.

Hunger for iron: the alternative siderophore iron scavenging systems in highly virulent Yersinia.

Low molecular weight siderophores are used by many living organisms to scavenge scarcely available ferric iron. Presence of at least a single siderophore-based iron acquisition system is usually acknowledged as a virulence-associated trait and a pre-requisite to become an efficient and successful pathogen. Currently, it is assumed that yersiniabactin (Ybt) is the solely functional endogenous siderophore iron uptake system in highly virulent Yersinia (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica biotype 1B). Genes responsible for biosynthesis, transport, and regulation of the yersiniabactin (ybt) production are clustered on a mobile genetic element, the High-Pathogenicity Island (HPI) that is responsible for broad dissemination of the ybt genes in Enterobacteriaceae. However, the ybt gene cluster is absent from nearly half of Y. pseudotuberculosis O3 isolates and epidemic Y. pseudotuberculosis O1 isolates responsible for the Far East Scarlet-like Fever. Several potential siderophore-mediated iron uptake gene clusters are documented in Yersinia genomes, however, neither of them have been proven to be functional. It has been suggested that at least two siderophores alternative to Ybt may operate in the highly virulent Yersinia pestis/Y. pseudotuberculosis group, and are referred to as pseudochelin (Pch) and yersiniachelin (Ych). Furthermore, most sporadic Y. pseudotuberculosis O1 strains possess gene clusters encoding all three iron scavenging systems. Thus, the Ybt system appears not to be the sole endogenous siderophore iron uptake system in the highly virulent yersiniae and may be efficiently substituted and/or supplemented by alternative iron siderophore scavenging systems.

Inhibition of quorum sensing (QS) in Yersinia enterocolitica by an orange extract rich in glycosylated flavanones.

Flavanones, flavonoids abundant in Citrus , have been shown to interfere with quorum sensing (QS) and affect related physiological processes. We have investigated the QS-inhibitory effects of an orange extract enriched in O-glycosylated flavanones (mainly naringin, neohesperidin, and hesperidin). The QS-inhibitory capacity of this extract and its main flavanone components was first screened using the bacteriological monitoring system Chromobacterium violaceum . We next examined the ability of the orange extract and of some of the flavanones to (i) reduce the levels of the QS mediators produced by Y. enterocolitica using HPLC-MS/MS, (ii) inhibit biofilm formation, and (iii) inhibit swimming and swarming motility. Additionally, we evaluated changes in the expression of specific genes involved in the synthesis of the lactones (yenI, yenR) and in the flagellar regulon (flhDC, fleB, fliA) by RT-PCR. The results showed that the orange extract and its main flavanone components inhibited QS in C. violaceum, diminished the levels of lactones secreted by Y. enterocolitica to the media, and decreased QS-associated biofilm maturation without affecting bacterial growth. Among the tested compounds, naringin was found to inhibit swimming motility. Exposure to the orange extract and (or) to naringin was also found to be associated with induction of the transcription levels of yenR, flhDC, and fliA. This work shows the in vitro QS-inhibitory effects of an orange extract enriched in flavanones against a human enteropathogen at doses that can be achieved through the diet and suggests that consumption of these natural extracts may have a beneficial antipathogenic effect.

The active site of oligogalacturonate lyase provides unique insights into cytoplasmic oligogalacturonate beta-elimination.

Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a ß-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn(2+) atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the -1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases.

Yersinia enterocolitica type III secretion of YopR requires a structure in its mRNA.

Yersinia type III secretion machines transport substrate proteins into the extracellular medium or into the cytoplasm of host cells. Translational hybrids, involving genes that encode substrates as well as reporter proteins that otherwise cannot travel the type III pathway, identified signals that promote transport of effector Yops into host cells. Signals for the secretion of substrates into high calcium media were hitherto unknown. By exploiting attributes of translational hybrids between yopR, whose product is secreted, and genes that encode impassable proteins that jam the secretion machine, we isolated yopR mutations that abolish substrate recognition. Similar to effector Yops, an N-terminal or 5' signal in codons 1-11 is required to initiate YopR into the type III pathway. YopR secretion cannot be completed and translational hybrids cannot impose a block without a second signal, positioned at codons 131-149. Silent mutations in the second signal abrogate function and the phenotype of other mutations can be suppressed by secondary mutations predicted to restore base complementary in a 3' stem-loop structure of the yopR mRNA.

Correlation between Yersinia enterocolitica and type I collagen reactivity in patients with arthropathies.

We investigated the association with Yersinia infection in patients with arthropathies in our region. To assess the reactivity to articular antigens, the correlation of anti-Yersinia with anti-type I and type II collagen antibodies was studied. Sera from 124 patients with musculoskeletal symptoms, and 47 synovial fluids (SF) from patients with rheumatoid arthritis (RA), spondyloarthopathies (SpA) or osteoarthritis (OA) were examined. Immunoglobulins against Yersinia enterocolitica, type I and type II collagens were determined by enzyme-linked immunosorbent assay. Immunoglobulin (Ig) A to Yersinia lipopolysaccharide (LPS) was present in 13/124 sera (10%) and 3/47 SF (6%). By Western blot, IgA to Yersinia outer proteins (Yops) was found in 14/124 sera (11%) and 2/47 SF (4%). Yersinia DNA from SF was not amplified by polymerase chain reaction. We found a significant correlation with anti-collagen type I but not type II antibodies. These results suggest different reactivity to articular collagen in patients with Yersinia antibodies.

Growth studies of plasmid bearing and plasmid cured Yersinia enterocolitica GER O:3 in the presence of cefsulodin, irgasan and novobiocin at 25 and 37 degrees C.

AIM: In this study, the growth characteristics of Yersinia enterocolitica biotype 4, GER O:3 plasmid bearing (P+) and plasmid cured (P-) strain types were evaluated in brain heart infusion broth supplemented with cefsulodin, irgasan, and novobiocin alone or in combination. METHODS AND RESULTS: Growth curves were obtained for the two strain types in broth supplemented with selective agents at 25 or 37 degrees C for 32 h to obtain data on the lag phase durations and growth rates of the strains. Generally, the lag times and growth rates of the P+ and P- strains were similar for cultures incubated at 25 degrees C regardless of the selective agent added and where plasmid replication and expression were not under any significant burden. However, where the lag times and growth rates of the strains were examined at 37 degrees C, significant differences were observed in the lag phase durations of the plasmid bearing strain type compared the plasmid cured strain, an effect that was due to the burden of the plasmid and the influence of selective agents. Generally, when two or more agents were present, lag phase durations were longer for the plasmid bearing strain. Some exceptions noted where in the presence of irgasan or full selective agent (CIN) the opposite case was observed. When growth rates were compared, the plasmidless strain type was typically faster than the plasmid bearing strain in the presence of most selective agents at 37 degrees C and the growth rates of both strain types at 25 degrees C were similar where the temperature appeared to negate the effects of plasmid. CONCLUSIONS: The data obtained in these studies suggest that selective agents (in particular irgasan) and incubation temperature play a significant role in influencing the growth characteristics of plasmid bearing and plasmid cured strains of Y. enterocolitica. SIGNIFICANCE AND IMPACT OF THE STUDY: This data presented in this study has significant implications for enrichment methods used in the detection or recovery of plasmid bearing Y. enterocolitica strains from food, environmental or clinical samples.

Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria.

In the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.

Complete DNA sequence of Yersinia enterocolitica serotype 0:8 low-calcium-response plasmid reveals a new virulence plasmid-associated replicon.

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.

Physiological properties and production of siderophores detected in Yersinia enterocolitica strain 4-32.

A hydrophilic compound with siderophore activity was isolated from a culture of Yersinia enterocolitica 4-32 grown in an iron-deficient medium. It was found that the siderophore secreted did not belong to the catecholamide and hydroxamate type of siderophores and not yersiniabactin. Supplementation of cultures of Y. enterocolitica 4-32 with sodium chloride (300 mM) resulted in a decrease in the production of siderophores.