Enzyme activity and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesis

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found

Detection of Yersinia enterocolitica in Food by Biomolecular Techniques

In Europe yersiniosis related to Yersinia enterocolitica is the third most numerously reported zoonoses. In Italy, notification of yersiniosis is not compulsory; thus, no true incidence rates are available from this country. Yersinia species are ubiquitous and they are also isolated from a wide variety of foods.\ud The objectives of this work were to study a multiplex PCR that could be applicable for a screening of food samples for pathogenic Y. enterocolitica, to characterize strains isolated from human patients, swine carcasses, meat, vegetables, burrata cheese and wild boars collected in Umbria and Marche Regions during 2004-2011, to investigate the detection of genotypic virulence markers ail,\ud ystA, ystB, myfA and hreP and to characterize the recovered isolates by PFGE and MLVA.\ud Among 120 swine carcasses investigated Y. enterocolitica were isolated from 25,8% of the samples and 2 of them were identified as Y. enterocolitica biotype 4 showing all the virulence factors tested and identical PFGE and MLVA profiles. Ten pathogenic Y. enterocolitica strains, recovered from stools of patients, exhibited the genotype ystA+ yadA+ myfA+ hreP+, 6 showed the genotype ystA+ yadA- myfA+ hreP+ and 2 were biotype 1A ystB+. Most of the colonies isolated from the other sources was nonpathogenic and harboured the ystB gene.\ud The multiplex PCR method was effective, fast and simple, capable of detecting pathogenic Yersinia enterocolitica in 48-72 hours. It offers significant advantages over the microbiological methods of isolation, allowing shorter response times and early detection of strains carrying the pathogenicity factor

Evolutionary dynamics of the Yersinia enterocolitica complex

The genus Yersinia consists of a heterogeneous collection of organisms, comprising the highly pathogenic species Yersinia pestis, enteropathogens Yersinia pseudotuberculosis and Yersinia enterocolitica as well as environmental species. The evolutionary history of Y. pestis has been well documented, but information on the evolutionary relationship between the other Yersiniae is less characterized. Y. enterocolitica is a diverse species classed into six different biotypes (BT), but only a single genome sequence for high-pathogenic BT 1B was available at the start of the project

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications.

Recombinant enzyme expression in Escherichia coli is one of the most popular methods to produce bulk concentrations of protein product. However, this method is often limited by the inadvertent formation of inclusion bodies. Our analysis systematically reviews literature from 2010 to 2021 and details the methods and strategies researchers have utilized for expression of difficult to express (DtE), industrially relevant recombinant enzymes in E. coli expression strains. Our review identifies an absence of a coherent strategy with disparate practices being used to promote solubility. We discuss the potential to approach recombinant expression systematically, with the aid of modern bioinformatics, modelling, and 'omics' based systems-level analysis techniques to provide a structured, holistic approach. Our analysis also identifies potential gaps in the methods used to report metadata in publications and the impact on the reproducibility and growth of the research in this field.Non

Defining the mechanism of Yersinia entomophaga MH96 exoprotein release : a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

There is an increasing need in New Zealand for the development of new solutions and improvement of currently available biological control agents of pasture pests. In the last two decades, the entomopathogen Yersinia entomophaga was isolated, characterised, and defined as an economically viable agent for use in biopesticides against pasture pests, such as the New Zealand grass grub Costelytra giveni, the black beetle Hetreronychus arator, and other insects from the orders Coleoptera and Lepidoptera. Y. entomophaga produces an ABC toxin complex, called the Yen-TC, which is its main orally active virulence factor. Although previous studies have revealed how the toxin is affecting the host, the mechanism for Yen-TC production and cellular release have yet to be fully elucidated. Furthermore, the roles of other virulence factors of Y. entomophaga and their effects and mechanisms of cellular release are yet to be determined. In silico analysis of the draft genome sequence revealed several gene clusters encoding for virulence factors such as Rhs with predicted function in the type 6 secretion system, YenT as a possible heat-stable enterotoxin, and PirAB, a haemocoelic-active toxin. This study aimed to identify regulators of exoproteome release in Y. entomophaga MH96. The Yersinia entomophaga region of exoproteome release (YeRER) was identified by an exoproteome screening assay developed in this study. The YeRER is comprised of the transcriptional regulator RoeA, a regulatory ncRNA, and the Yersinia lysis cassette (YLC). Transcriptomics, mutagenesis, and trans complementation experiments were used to elucidates the importance of YeRER in Y. entomophaga protein release and changes in cell morphology. The global regulator, RoeA, strictly down-regulates the YLC expression which is involved in protein release and vesicle formation. Expression levels of the encoded secretion systems in MH96, T1SS, T3SS, T3SS2, and T6SS are not under the control of YeRER or quorum sensing (QS), which are involved in protein secretion. The T2SS expression is increased in roeA and QS mutants, which in turn showed reduced global exoproteome concentration. It is unlikely that the MH96 secretion systems are involved in MH96 exoproteome production in vitro. While underlying mechanisms have yet to be investigated, this study strongly suggests that exoproteins such as the Yen-TC are secreted by membrane vesicles which are induced by activation of a holin-endolysin complex

Comparative phenotypic, proteomic and genomic approaches to assess lipopolysaccharide and outer membrane protein diversity among isolates of Yersinia ruckeri recovered from Atlantic salmon and rainbow trout

Yersinia ruckeri is the causative agent of enteric redmouth (ERM) disease in farmed salmonids. ERM disease is traditionally associated with rainbow trout (Oncorhynchus mykiss¸ Walbaum), but the incidence of the disease in Atlantic salmon (Salmo salar) has increased in recent years. Historically, motile (biotype 1), serotype O1 isolates of Y. ruckeri have been mostly responsible for ERM in rainbow trout worldwide but non-motile (biotype 2), serotype O1 isolates have become increasingly prevalent in this species over wide geographic areas since their first isolation in the UK in the 1980s. Yersinia ruckeri isolates responsible for infection of salmon have been less well characterised than those from rainbow trout and little is known about their diversity. The emergence of new pathogenic strains, together with vaccine breakdown in the field, has emphasised the need for greater knowledge about strain diversity which may lead to the development of improved vaccines for both species. In the present study, a unique and extensive strain collection encompassing 135 isolates of Y. ruckeri were characterised using complementary phenotypic, proteomic and genomic approaches. In the initial part of this thesis, 135 isolates recovered over a 14 year period in the UK from infected Atlantic salmon (109 isolates) and rainbow trout (26 isolates) were phenotypically characterised through biotype, serotype, and outer membrane protein (OMP) -type analysis. Atlantic salmon isolates represented a wider range of O-serotypes and associated lipopolysaccharide (LPS) types, and had more diverse OMP profiles than those from rainbow trout. Most significantly, a new O-serotype/LPS type was identified in 56 Atlantic salmon isolates; other Atlantic salmon isolates were represented by serotypes O1 (five isolates), O2 (34 isolates) and O5 (14 isolates). This new LPS type comprises a core polysaccharide region similar to that of serotype O1 but has a unique, previously unidentified O-antigen region. Atlantic salmon isolates could be assigned to one of four major OMP-types and to one of 11 OMP-sub types. Isolates recovered from rainbow trout were represented by the same non-motile clone that is responsible for the majority of ERM outbreaks in this species within the UK. This clone was not associated with any infected salmon. However, two isolates of the novel serotype O1/O5 were recovered from rainbow trout in 2010 and 2011. These data suggest that different Y. ruckeri strains are specifically adapted to cause disease in either Atlantic salmon or rainbow trout. The efficacy of current vaccine formulations against different clonal groups must be examined. Subsequently, an in-depth characterisation of the outer membrane (OM) proteome of isolates recovered from Atlantic salmon and rainbow trout was conducted. Outer membrane proteins are at the interface of host pathogen interactions, with important roles in adherence, evasion of host immune response, and transport. Using a bioinformatic prediction pipeline and four publicly available genomes, 141 proteins were confidently predicted to be OM associated. Subsequently, the OM proteomes of eight representative isolates (four from rainbow trout; four from Atlantic salmon) were analysed using a combination of gel-based and gel-free proteomic approaches. In total, 66 OMPs were identified through this combined approach, of which 28 were unique to the gel-free approach and 13 were unique to the gel-based approach. Further to this, the OM proteomes of these eight representative isolates were examined when cells were grown under conditions that aimed to mimic the in vivo and environmental conditions of Y. ruckeri. These included growing cells aerobically at 22°C and 28°C, anaerobically, under iron-depletion and in an artificial seawater medium at 22°C. In total, 76 OMPs were identified in all eight isolates under these growth conditions. Finally, a phylogenetic study was undertaken whereby the genomes of 16 representative isolates encompassing a range of biotypes, serotypes, host species (eight from rainbow trout, seven from Atlantic salmon and one from European eel), geographic locations and dates of isolation were considered. A phylogenetic species tree based on the concatenated sequences of 19 housekeeping genes revealed host specific lineages suggesting an earlier host-associated evolutionary split within Y. ruckeri. Subsequent analysis of the presence, absence and variation of the nucleotide and amino acid sequences of the 141 predicted OMPs revealed high levels of conservation (with 120 OMPs showing less than 1% nucleotide variation). One hundred and thirty proteins were identified in all 16 genomes examined. However, 11 proteins were not, and these included invasins, OmpE and proteins involved in pilus biogenesis. Further examination of the OMPs OmpA and OmpF, which were identified in the genomes of all 16 isolates, revealed variation in the surface exposed loop regions which may play a role in pathogenicity and/or host specificity. This study represents a comprehensive characterisation of Y. ruckeri isolates recovered from Atlantic salmon and rainbow trout using a range of molecular techniques, and reveals important adaptations that the bacteria may make in order to survive both inside and outside of the host. Importantly, this study provides comprehensive support for future work involving this fish pathogen

Dedicated to the 55th Anniversary of G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

The G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences (PIBOC FEB RAS) was founded in 1964 in Vladivostok in the Far East of Russia. Over many years, we have been carrying out studies on the natural products of both marine and terrestrial origin. In collaboration with many Russian and foreign scientists, we have investigated many hundreds of diverse biomolecules, including steroids and terpenoids, quinoid compounds and alkaloids, polysaccharides and lipids, enzymes and lectins, proteins, and peptides. The Institute has a collection of marine microorganisms (KMM) PIBOC, which includes more than 4000 strains of marine bacteria and more than 1000 strains of marine fungi. The biological activity of natural compounds is also being studied. This book includes the 14 manuscripts which covered almost all aspects of PIBOC research activity in the fields of bioorganic chemistry, biochemistry, organic synthesis of natural compounds, marine microbiology, and genetic engineering, and we hope it will provide interesting new information for scientists working in these fields