Publications by authors named "Alexander Rakin"

40 Publications

Cyclohexane, naphthalene, and diesel fuel increase oxidative stress, CYP153, sodA, and recA gene expression in Rhodococcus erythropolis.

Microbiologyopen 2019 09 22;8(9):e00855. Epub 2019 May 22.

Southern Federal University, Rostov-on-Don, Russian Federation.

In this study, we compared the expression of CYP153, sodA, sodC, and recA genes and ROS generation in hydrocarbon-degrading Rhodococcus erythropolis in the presence of cyclohexane, naphthalene, and diesel fuel. The expression of cytochrome P450, sodA (encoding Fe/Mn superoxide dismutase), recA, and superoxide anion radical generation rate increased after the addition of all studied hydrocarbons. The peak of CYP153, sodA, and recA gene expression was registered in the presence of naphthalene. The same substrate upregulated the Cu/Zn superoxide dismutase gene, sodC. Cyclohexane generated the highest level of superoxide anion radical production. Hydrogen peroxide accumulated in the medium enriched with diesel fuel. Taken together, hydrocarbon biotransformation leads to oxidative stress and upregulation of antioxidant enzymes and CYP153 genes, and increases DNA reparation levels in R. erythropolis cells.
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http://dx.doi.org/10.1002/mbo3.855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816061PMC
September 2019

Development of a flow cytometry based assay to determine the invasion of enteropathogenic Yersiniae into C2BBe1 cells.

J Microbiol Methods 2018 06 24;149:29-35. Epub 2018 Apr 24.

Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute for Bacterial Infections and Zoonoses, Naumburger Str. 96a, D-07743 Jena, Germany. Electronic address:

A rapid method was developed to determine the invasion frequency of enteropathogenic Yersinia into intestinal C2BBe1 cells by means of flow cytometry. Bacteria are labelled with a thiol-cleavable amine-reactive biotin and subsequently incubated with the fluorochrome-labelled biotin-ligand neutravidin. After infection of the intestinal cells with the labelled bacteria, the neutravidin-coupled fluorochrome is detached by breaking up the linker through reduction of the disulphide. Despite reduced adhesion and invasion frequencies of the labelled bacteria into C2BBe1 cells this procedure offers the basis for the development of a fast single-step staining protocol for the recovery of invading bacteria in in a host-pathogen system for further transcriptome or proteome analysis.
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http://dx.doi.org/10.1016/j.mimet.2018.04.017DOI Listing
June 2018

Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC).

Evol Bioinform Online 2016 21;12:185-91. Epub 2016 Aug 21.

Far Eastern Federal University, Vladivostok, Russia.; Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia.

The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system.
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http://dx.doi.org/10.4137/EBO.S40346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993215PMC
August 2016

Adaptive responses of outer membrane porin balance of Yersinia ruckeri under different incubation temperature, osmolarity, and oxygen availability.

Microbiologyopen 2016 08 1;5(4):597-603. Epub 2016 Apr 1.

G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, 690022 Prospect 100-let Vladivostoku 159, Vladivostok, Russia.

The capability of Yersinia ruckeri to survive in the aquatic systems reflects its adaptation (most importantly through the alteration of membrane permeability) to the unfavorable environments. The nonspecific porins are a key factor contributing to the permeability. Here we studied the influence of the stimuli, such as temperature, osmolarity, and oxygen availability on regulation of Y. ruckeri porins. Using qRT-PCR and SDS-PAGE methods we found that major porins are tightly controlled by temperature. Hyperosmosis did not repress OmpF production. The limitation of oxygen availability led to decreased expression of both major porins and increased transcription of the minor porin OmpY. Regulation of the porin balance in Y. ruckeri, in spite of some similarities, diverges from that system in Escherichia coli. The changes in porin regulation can be adapted in Y. ruckeri in a species-specific manner determined by its aquatic habitats.
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http://dx.doi.org/10.1002/mbo3.354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985593PMC
August 2016

A molecular scheme for Yersinia enterocolitica patho-serotyping derived from genome-wide analysis.

Int J Med Microbiol 2014 May 24;304(3-4):275-83. Epub 2013 Oct 24.

Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians-University, Munich, Germany. Electronic address:

Yersinia enterocolitica is a food-borne, gastro-intestinal pathogen with world-wide distribution. Only 11 serotypes have been isolated from patients, with O:3, O:9, O:8 and O:5,27 being the serotypes most commonly associated with human yersiniosis. Serotype is an important characteristic of Y. enterocolitica strains, allowing differentiation for epidemiology, diagnosis and phylogeny studies. Conventional serotyping, performed by slide agglutination, is a tedious and laborious procedure whose interpretation tends to be subjective, leading to poor reproducibility. Here we present a PCR-based typing scheme for molecular identification and patho-serotyping of Y. enterocolitica. Genome-wide comparison of Y. enterocolitica sequences allowed analysis of the O-antigen gene clusters of different serotypes, uncovering their formerly unknown genomic locations, and selection of targets for serotype-specific amplification. Two multiplex PCRs and one additional PCR were designed and tested on various reference strains and isolates from different origins. Our genotypic assay proved to be highly specific for identification of Y. enterocolitica species, discrimination between virulent and non-virulent strains, distinguishing the main human-related serotypes, and typing of conventionally untypeable strains. This genotyping scheme could be applied in microbiology laboratories as an alternative or complementary method to the traditional phenotypic assays, providing data for epidemiological studies.
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http://dx.doi.org/10.1016/j.ijmm.2013.10.007DOI Listing
May 2014

Genome Sequences of Four Yersinia enterocolitica Bioserotype 4/O:3 Isolates from Mammals.

Genome Announc 2013 Jul 11;1(4). Epub 2013 Jul 11.

Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians-University, Munich, Germany.

We report here the complete genome sequences of four European Yersinia enterocolitica mammalian isolates of bioserotype 4/O:3. The genomes have an average size of 4.50 Mb, a G+C content of 47%, and between 4,231 and 4,330 coding sequences (CDSs). No relevant differences were detected by genome comparison between mammalian and human isolates.
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http://dx.doi.org/10.1128/genomeA.00466-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709148PMC
July 2013

Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis.

Proc Natl Acad Sci U S A 2013 Jan 27;110(2):577-82. Epub 2012 Dec 27.

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

The genetic diversity of Yersinia pestis, the etiologic agent of plague, is extremely limited because of its recent origin coupled with a slow clock rate. Here we identified 2,326 SNPs from 133 genomes of Y. pestis strains that were isolated in China and elsewhere. These SNPs define the genealogy of Y. pestis since its most recent common ancestor. All but 28 of these SNPs represented mutations that happened only once within the genealogy, and they were distributed essentially at random among individual genes. Only seven genes contained a significant excess of nonsynonymous SNP, suggesting that the fixation of SNPs mainly arises via neutral processes, such as genetic drift, rather than Darwinian selection. However, the rate of fixation varies dramatically over the genealogy: the number of SNPs accumulated by different lineages was highly variable and the genealogy contains multiple polytomies, one of which resulted in four branches near the time of the Black Death. We suggest that demographic changes can affect the speed of evolution in epidemic pathogens even in the absence of natural selection, and hypothesize that neutral SNPs are fixed rapidly during intermittent epidemics and outbreaks.
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http://dx.doi.org/10.1073/pnas.1205750110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545753PMC
January 2013

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

Front Cell Infect Microbiol 2012 30;2:151. Epub 2012 Nov 30.

Phylogenomics of the Enteropathogenic Yersinia, Max von Pettenkofer-Institute, LMU Munich, Germany.

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.
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http://dx.doi.org/10.3389/fcimb.2012.00151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510459PMC
December 2013

Tracing genomic variations in two highly virulent Yersinia enterocolitica strains with unequal ability to compete for host colonization.

BMC Genomics 2012 Sep 11;13:467. Epub 2012 Sep 11.

Max von Pettenkofer-Institute, LMU, Munich, Germany.

Background: Yersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081.

Results: Regions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity <50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed.

Conclusions: Our study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype.
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http://dx.doi.org/10.1186/1471-2164-13-467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469391PMC
September 2012

The molecular phylogeny of the gyrB gene: a molecular marker for systematic characterization of the genus Yersinia.

Adv Exp Med Biol 2012 ;954:53-6

Pacific Institute of Bioorganic Chemistry, FEBRAS, Prospect 100 let Vladivostoku 159, Vladivostok, 690022, Russia.

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http://dx.doi.org/10.1007/978-1-4614-3561-7_7DOI Listing
September 2012

Diversity and adaptive evolution of a major porin gene (ompF) in Yersinia pseudotuberculosis.

Adv Exp Med Biol 2012 ;954:39-43

Pacific Institute of Bioorganic Chemistry, FEBRAS, Prospekt 100-let Vladivostoku, 159, Vladivostok, 690022, Russia.

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http://dx.doi.org/10.1007/978-1-4614-3561-7_5DOI Listing
September 2012

Gains and Losses in Yersinia enterocolitica subsp. palearctica Genomes.

Adv Exp Med Biol 2012 ;954:23-9

Max von Pettenkofer Institut for Hygiene and Medical Microbiology, Ludwig Maximillians University, Pettenkoferstr. 9a, 80336 Muenchen, Munich, Germany.

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http://dx.doi.org/10.1007/978-1-4614-3561-7_3DOI Listing
September 2012

Identification of three oligo-/polysaccharide-specific ligases in Yersinia enterocolitica.

Mol Microbiol 2012 Jan 28;83(1):125-36. Epub 2011 Nov 28.

Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland.

In lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria the lipid A-core oligosaccharide (LA-core) and O-polysaccharide (O-PS) biosynthesis pathways proceed separately and converge in periplasmic space where the waaL-encoded ligase joins O-PS onto LA-core. Enterobacterial common antigen (ECA) biosynthesis follows that of O-PS except that ECA is usually ligated to phosphatidylglycerol (PG) and only rarely to LA-core. In Yersinia enterocolitica serotype O:3 LPS is composed of LA-inner core (IC) onto which a homopolymeric O-PS, a hexasaccharide called outer core (OC), and/or ECA are ligated. We found that an individual O:3 LPS molecule carries either OC or O-PS substitution but not both. Related to this, we identified three genes in Y. enterocolitica O:3 that all expressed O-PS ligase activity in the Escherichia coliΔwaaL mutant. The LPS phenotypes of Y. enterocolitica O:3 single, double and triple ligase mutants indicated that two of ligases, named as WaaL(os) and WaaL(ps) , had a preferred substrate specificity for OC and O-PS, respectively, although with some promiscuity between the ligases; the third ligase named as WaaL(xs) was not involved in LPS or ECA biosynthesis. In Y. enterocolitica O:8 the WaaL(os) homologue (Ye1727) ligated a single pentasaccharide O-unit to LA-IC suggesting that in both Y. enterocolitica O:3 and O:8 WaaL(os) is an oligosaccharide (OS)-specific ligase. Finally, Yersinia pestis and Y. pseudotuberculosis carry only the waaL(ps) gene, while either waaL(os) or waaL(xs) or both are additionally present in other Yersinia species. This is the first report on the presence of three different oligo-/polysaccharide-specific ligases in a single bacterium.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07918.xDOI Listing
January 2012

The pathogenic potential of Yersinia enterocolitica 1A.

Int J Med Microbiol 2011 Nov 28;301(7):556-61. Epub 2011 Jul 28.

Max von Pettenkofer-Institute, LMU, Munich, Germany.

Yersinia enterocolitica 1A strains are generally considered apathogenic. However, besides environmental sources, foods and animals, they are repeatedly isolated from patients with gastrointestinal symptoms typical to those evoked by Yersinia of the virulent 1B and 2-4 biotypes. Also, at least 2 gastrointestinal outbreaks associated with 1A strains have been reported. There is a general controversy concerning the pathogenic potential of 1A isolates of clinical and non-clinical origin. To address the 1A puzzle, we have determined the genome sequences of 2 1A strains, a nosocomial O:5 and environmental O:36 isolates, and compared them to each other and to O:8/1B and O:3/4 representatives of the virulent serobiotypes. 1A isolates have mosaic genomes and share genes both with serobiotypes O:8/1B and O:3/4 that implies their common descent. Besides the pYV virulence plasmid, 1A strains lack the classical virulence markers, like the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. However, they still possess genes encoding such known and suspect virulence-associated determinants like the YstB enterotoxin, the InvA invasin, the mucoid Yersinia factor MyfA, and the enterochelin utilisation fepBDGC/fepA/fes gene cluster. In contrast to previous studies, we have found that the strains of the 1A group possess the MyfA antigen although with limited similarity to the highly conserved MyfA in the virulent serobiotypes. In turn, the MyfB chaperone coevolved with the MyfA fibrillae, while the MyfC usher retains 90% identity to its MyfC counterparts in O:3/O:8 group. The only notable difference between clinical and non-clinical 1A strains was the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital O:5 isolate. Taken together, Y. enterocolitica BT 1A group represents opportunistic pathogens whose opportunity to establish infection seems to rely mainly on the state of the host defence system. However, presence of known and putative virulence-associated features shared with the pathogenic serobiotypes compels to reconsider properly the pathogenic potential of this group of emerging pathogens.
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http://dx.doi.org/10.1016/j.ijmm.2011.05.002DOI Listing
November 2011

Yersinia pestis autoagglutination factor is a component of the type six secretion system.

Int J Med Microbiol 2011 Nov 23;301(7):562-9. Epub 2011 Jul 23.

Anti-plague Research Institute, Rostov-on-Don, Russia.

Autoagglutination (AA) is a protective phenotypic trait facilitating survival of bacteria in hostile environments and in the host during infection. Autoagglutination factors (AFs) that possess self-associating ability are currently characterized in many Gram-negative bacteria, but Yersinia pestis AFs are still a matter of debate. Previously, we have shown that AF of Hms(-) strain Y. pestis EV76 is a complex of the 17,485-kDa protein and a low-molecular-weight component with siderophore activity. Here, we identified the protein moiety of AF and examined its role in AA of Hms(+) and Hms(-)Y. pestis strains. Using MALDI-TOF MS of trypsin-hydrolyzed AF, we unambiguously identified the protein as YPO0502, which belongs to a family of Hcp-proteins forming pilus-like structures of the type six secretion system (T6SS). To address the role of YPO0502 in AA, we cloned ypo0502 in E. coli, overexpressed it in Y. pestis and constructed its knock-out mutant in Y. pestis. However, all these approaches failed: YPO0502 was not secreted in E. coli, formed inclusion bodies when overexpressed in Y. pestis, and could probably be compensated by other Hcp-like proteins in Y. pestis. In contrast, downregulation of ypo0502 expression by its antisense RNA supported the contribution of YPO0502 in AA of Hms(+) and Hms(-)Y. pestis strains. The results of the present study indicate that the Hcp-like component of T6SS encoded by ypo502 is involved in Y. pestis AA and suggest that at least one (ypo0499-0516) of the 6 T6SS clusters of Y. pestis is involved in bacterial interaction.
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http://dx.doi.org/10.1016/j.ijmm.2011.03.004DOI Listing
November 2011

Unique cell adhesion and invasion properties of Yersinia enterocolitica O:3, the most frequent cause of human Yersiniosis.

PLoS Pathog 2011 Jul 7;7(7):e1002117. Epub 2011 Jul 7.

Department of Molecular Infection Biology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany.

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.
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http://dx.doi.org/10.1371/journal.ppat.1002117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131269PMC
July 2011

Yersinia enterocolitica palearctica serobiotype O:3/4--a successful group of emerging zoonotic pathogens.

BMC Genomics 2011 Jul 6;12:348. Epub 2011 Jul 6.

Max von Pettenkofer-Institute, LMU, Munich, Germany.

Background: High-pathogenic Y. enterocolitica ssp. enterocolitica caused several human outbreaks in Northern America. In contrast, low pathogenic Y. enterocolitica ssp. palearctica serobiotype O:3/4 is responsible for sporadic cases worldwide with asymptomatic pigs being the main source of infection. Genomes of three Y. enterocolitica ssp. palearctica serobiotype O:3/4 human isolates (including the completely sequenced Y11 German DSMZ type strain) were compared to the high-pathogenic Y. enterocolitica ssp. enterocolitica 8081 O:8/1B to address the peculiarities of the O:3/4 group.

Results: Most high-pathogenicity-associated determinants of Y. enterocolitica ssp. enterocolitica (like the High-Pathogenicity Island, yts1 type 2 and ysa type 3 secretion systems) are absent in Y. enterocolitica ssp. palearctica serobiotype O:3/4 genomes. On the other hand they possess alternative putative virulence and fitness factors, such as a different ysp type 3 secretion system, an RtxA-like and insecticidal toxins, and a N-acetyl-galactosamine (GalNAc) PTS system (aga-operon). Horizontal acquisition of two prophages and a tRNA-Asn-associated GIYep-01 genomic island might also influence the Y. enterocolitica ssp. palearctica serobiotype O:3/4 pathoadaptation. We demonstrated recombination activity of the PhiYep-3 prophage and the GIYep-01 island and the ability of the aga-operon to support the growth of the Y. enterocolitica ssp. enterocolitica O:8/1B on GalNAc.

Conclusions: Y. enterocolitica ssp. palearctica serobiotype O:3/4 experienced a shift to an alternative patchwork of virulence and fitness determinants that might play a significant role in its host pathoadaptation and successful worldwide dissemination.
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http://dx.doi.org/10.1186/1471-2164-12-348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161016PMC
July 2011

Trends of the major porin gene (ompF) evolution: insight from the genus Yersinia.

PLoS One 2011 31;6(5):e20546. Epub 2011 May 31.

Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of Russian Academy of Sciences, Vladivostok, Russian Federation.

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020546PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105102PMC
October 2011

Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3.

J Bacteriol 2011 Apr 4;193(8):2067. Epub 2011 Feb 4.

Max von Pettenkofer-Institute, LMU, Munich, Germany.

We report here the first finished and annotated genome sequence of a representative of the most epidemiologically successful Yersinia group, Y. enterocolitica subsp. palearctica strain Y11, serotype O:3, biotype 4. This strain is a certified type strain of the German DSMZ collection (DSM no. 13030; Yersinia enterocolitica subsp. palearctica) that was isolated from the stool of a human patient (H. Neubauer, S. Aleksic, A. Hensel, E. J. Finke, and H. Meyer. Int. J. Med. Microbiol. 290:61-64, 2000).
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http://dx.doi.org/10.1128/JB.01484-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133053PMC
April 2011

Structures of the arm-type binding domains of HPI and HAI7 integrases.

J Biol Chem 2009 Nov 8;284(46):31664-71. Epub 2009 Sep 8.

Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.

The structures of the N-terminal domains of two integrases of closely related but not identical asn tDNA-associated genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin biosynthesis and transport) and an Erwinia carotovora genomic island with yet unknown function, HAI7, have been resolved. Both integrases utilize a novel four-stranded beta-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded beta-sheets. Moreover, the beta-sheets in Int(HPI) and Int(HAI7) are longer than those in other integrases, and the structured helical N terminus is positioned perpendicularly to the large C-terminal helix. These differences strongly support the proposal that the integrases of the genomic islands make up a distinct evolutionary branch of the site-specific recombinases that utilize a unique DNA-binding mechanism.
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http://dx.doi.org/10.1074/jbc.M109.059261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797237PMC
November 2009

The subcutaneous inoculation of pH 6 antigen mutants of Yersinia pestis does not affect virulence and immune response in mice.

J Med Microbiol 2009 Jan;58(Pt 1):26-36

State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Serpukhov District, Moscow Region, Russia.

Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6(-) mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.
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http://dx.doi.org/10.1099/jmm.0.005678-0DOI Listing
January 2009

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli.

FEMS Microbiol Lett 2009 Jan 10;290(1):62-9. Epub 2008 Nov 10.

Robert Koch Institut, Wernigerode Branch, Wernigerode, Germany.

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.
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http://dx.doi.org/10.1111/j.1574-6968.2008.01405.xDOI Listing
January 2009

Hypoxia-independent activation of HIF-1 by enterobacteriaceae and their siderophores.

Gastroenterology 2008 Mar 7;134(3):756-67. Epub 2007 Dec 7.

Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Tübingen, Eberhard-Karls-Universität, Tübingen, Germany.

Background & Aims: Hypoxia inducible factor-1 (HIF-1) is the key transcriptional regulator during adaptation to hypoxia. Recent studies provide evidence for HIF-1 activation during bacterial infections. However, molecular details of how bacteria activate HIF-1 remain unclear. Here, we pursued the role of bacterial siderophores in HIF-1 activation during infection with Enterobacteriaceae.

Methods: In vivo, HIF-1 activation and HIF-1-dependent gene induction in Peyer's patches were analyzed after orogastric infection with Yersinia enterocolitica. The course of an orogastric Y enterocolitica infection was determined using mice with a deletion of HIF-1alpha in the intestine. In vitro, the mechanism of HIF-1 activation was analyzed in infections with Y enterocolitica, Salmonella enterica subsp enterica, and Enterobacter aerogenes.

Results: Infection of mice with Y enterocolitica led to functional activation of HIF-1 in Peyer's patches. Because mice with deletion of HIF-1alpha in the intestinal epithelium showed a significantly higher susceptibility to orogastric Y enterocolitica infections, bacterial HIF-1 activation appears to represent a host defense mechanism. Additional studies with Y enterocolitica, S enterica subsp enterica, or E aerogenes, and, moreover, application of their siderophores (yersiniabactin, salmochelin, aerobactin) caused a robust, dose-dependent HIF-1 response in human epithelia and endothelia, independent of cellular hypoxia. HIF-1 activation occurs most likely because of inhibition of prolylhydroxylase activity and is abolished upon infection with siderophore uptake deficient bacteria.

Conclusions: Taken together, this study reveals what we believe to be a previously unrecognized role of bacterial siderophores for hypoxia-independent activation of HIF-1 during infection with human pathogenic bacteria.
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http://dx.doi.org/10.1053/j.gastro.2007.12.008DOI Listing
March 2008

Yersinia genome diversity disclosed by Yersinia pestis genome-wide DNA microarray.

Can J Microbiol 2007 Nov;53(11):1211-21

Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.

The genus Yersinia includes 11 species, 3 of which (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) are pathogenic for humans. The remaining 8 species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae) are merely opportunistic pathogens found mostly in the environment. In this work, the genomic differences among Yersinia were determined using a Y. pestis-specific DNA microarray. The results revealed 292 chromosomal genes that were shared by all Yersinia species tested, constituting the conserved gene pool of the genus Yersinia. Hierarchical clustering analysis of the microarray data revealed the genetic relationships among all 11 species in this genus. The microarray analysis in conjunction with PCR screening greatly reduced the number of chromosomal genes (32) specific for Y. pestis to 16 genes and uncovered a high level of genomic plasticity within Y. pseudotuberculosis, indicating that its different serotypes have undergone an extensively parallel loss or acquisition of genetic content, which is likely to be important for its adaptation to changes in environmental niches.
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http://dx.doi.org/10.1139/W07-087DOI Listing
November 2007

Analysis of the three Yersinia pestis CRISPR loci provides new tools for phylogenetic studies and possibly for the investigation of ancient DNA.

Adv Exp Med Biol 2007 ;603:327-38

Université Paris-Sud, Institut de Génétique et Microbiologie, Orsay, France.

The precise nature of the pathogen having caused early plague pandemics is uncertain. Although Yersinia pestis is a likely candidate for all three plague pandemics, the very rare direct evidence that can be deduced from ancient DNA (aDNA) analysis is controversial. Moreover, which of the three biovars, Antiqua, Medievalis or Orientalis, was associated with these pandemics is still debated. There is a need for phylogenetic analysis performed on Y. pestis strains isolated from countries from which plague probably arose and is still endemic. In addition there exist technical difficulties inherent to aDNA investigations and a lack of appropriate genetic targets. The recently described CRISPRs (clustered regularly interspaced short palindromic repeats) may represent such a target. CRISPR loci consist of a succession of highly conserved regions separated by specific "spacers" usually of viral origin. To be of use, data describing the mechanisms of evolution and diversity of CRISPRs in Y. pestis, its closest neighbors, and other species which might contaminate ancient DNA, are necessary. The investigation of closely related Y. pestis isolates has revealed recent mutation events in which elements constituting CRISPRs were acquired or lost, providing essential insight on their evolution. Rules deduced represent the basis for subsequent interpretation. In the present study, the CRISPR loci from representative Y. pestis and Yersinia pseudotuberculosis strains were investigated by PCR amplification and sequence analysis. The investigation of this wider panel of strains, including other subspecies or ecotypes within Y. pestis and also Y. pseudotuberculosis strains provides a database of the existing CRISPR spacers and helps predict the expected CRISPR structure of the Y. pestis ancestor. This knowledge will open the way to the development of a spoligotyping assay, in which spacers can be amplified even from highly degraded DNA samples. The data obtained show that CRISPR analysis can provide a very powerful typing tool, adapted to the systematic, large-scale genotyping of Y. pestis isolates, and the creation of international typing databases. In addition, CRISPRs do constitute a very promising new tool and genetic target to investigate ancient DNA. The corresponding genetic targets are small (<70bp), present in multiple copies (usually more than 10), highly conserved and specific. In addition, the assay can be run in any laboratory. Interpretation of the data is not dependent on accurate sequence data.
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http://dx.doi.org/10.1007/978-0-387-72124-8_30DOI Listing
December 2007

Development of multiple-locus variable-number tandem-repeat analysis for Yersinia enterocolitica subsp. palearctica and its application to bioserogroup 4/O3 subtyping.

J Clin Microbiol 2007 Aug 6;45(8):2508-15. Epub 2007 Jun 6.

Department of Bacteriology, National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland.

Yersinia enterocolitica bioserogroup 4/O3 is the predominant causative agent of yersiniosis in Europe and North America. Multiple-locus variable-number tandem-repeat analysis (MLVA) was developed to improve the resolution power of classical genotyping methods. MLVA based on six loci was able to distinguish 76 genotypes among 91 Y. enterocolitica isolates of worldwide origin and 41 genotypes among 51 nonepidemiologically linked bioserogroup 4/O3 isolates, proving that it has a high resolution power. However, only a slight correlation of the MLVA genotypes and the geographic distribution of the isolates was observed. Although MLVA was also capable of distinguishing strains of Y. enterocolitica subsp. palearctica O9 and O5,27, there was only a minor correlation between the MLVA genotypes and serogroups. MLVA may be a helpful tool for epidemiological investigations of Y. enterocolitica subsp. palearctica outbreaks.
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http://dx.doi.org/10.1128/JCM.02252-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951228PMC
August 2007

Independent acquisition of site-specific recombination factors by asn tRNA gene-targeting genomic islands.

Int J Med Microbiol 2006 Oct 6;296(6):341-52. Epub 2006 Jun 6.

Max von Pettenkofer-Institute of Hygiene and Medical Microbiology, Pettenkofer Str. 9a, D-80336 Munich, Germany.

Two genomic islands, namely the high-pathogenicity island (HPI) and Ecoc54N target the same asn tRNA genes to integrate into the bacterial chromosome. The HPI encodes the siderophore yersiniabactin in the highly pathogenic Yersinia group (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica 1B) whilst the Ecoc54N island possibly encodes a polyketide synthase with an unknown function in the uropathogenic Escherichia coli CFT073 strain. HPI encodes the recombinase that promotes site-specific recombination (both integrative and excisive) with its corresponding attachment targets. A recombinase orthologue is also present in Ecoc54N. In addition, the HPI(Yps) of the Y. pestis/Y. pseudotuberculosis evolutionary lineage encodes the excisionase (recombination directionality factor, Xis(HPI)) that facilitates excision of the island. However, no sequence resembling the excisionase gene could be found in Ecoc54N. The rate of the HPI(Yps) excision estimated by real-time PCR was 10(-6) in Y. pseudotuberculosis. The presence of the excisionase increased the efficiency of the excisive recombination only eight fold. However, the introduction of the xis(HPI) in E. coli CFT073 did not influence the excision of Ecoc54N. The Xis(HPI) is encoded by the variable AT-rich part of the HPI(Yps) and substantially differs from its cognate recombinase in A+T content and codon usage. Also the Xis(HPI)-protected region, defined in the HPI attachment site, has suffered several nucleotide substitutions in Ecoc54N that could influence interaction with the excisionase. We propose that the pathogenicity islands (PAIs) targeting asn tRNA genes (PAIs(asn tRNA)) might have acquired recombinase and excisionase (HPI) genes independently and sequentially.
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http://dx.doi.org/10.1016/j.ijmm.2006.02.021DOI Listing
October 2006

A novel insertion sequence element, IS Yen2, as an epidemiological marker for weakly pathogenic bioserotypes of Yersinia enterocolitica.

Int J Med Microbiol 2005 Aug;295(4):213-26

Yersinia Group I, Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Pettenkofer Str. 9a, D-80336 Munich, Germany.

The structure and distribution of IS Yen2, a new Yersinia enterocolitica insertion sequence element, were investigated. ISYen2 is related to IS elements of the IS21 family and is present in two isoforms in Y. enterocolitica serotype O:3. Analysis of all copies of both isoforms, IS Yen2A and IS Yen2B, by PCR and sequencing indicated that they are not flanked by direct repeats which are typical of the IS21 family of repetitive elements. IS Yen2 is present in multiple copies in Y. enterocolitica O:3 and O:9 and in a single copy in Y. enterocolitica O:1 and O:2 serotypes. The probe for IS Yen2 efficiently detected all weakly pathogenic Y. enterocolitica bioserotypes investigated, whereas it did not hybridize with other strains. This indicates that IS Yen2 can serve as an additional tool for Y. enterocolitica differentiation and epidemiological studies. Distribution of the different groups of IS elements in two Y. enterocolitica pathotypes is in favor of the parallel evolution of American and European Y. enterocolitica strains.
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http://dx.doi.org/10.1016/j.ijmm.2005.05.007DOI Listing
August 2005

Horizontal transfer of Yersinia high-pathogenicity island by the conjugative RP4 attB target-presenting shuttle plasmid.

Mol Microbiol 2005 Aug;57(3):727-34

Max von Pettenkofer-Institut, Pettenkoferstr. 9a, 80336 Munich, Germany.

The high-pathogenicity island (HPI) encodes a highly efficient yersiniabactin system of iron acquisition responsible for mouse lethality in Yersinia. Although the HPI is widely disseminated among Enterobacteriaceae it lacks functions necessary for its replication and transmission. Therefore, the mechanism of its horizontal transfer and circulation is completely obscure. On the other hand, the HPI is a genetically active island in the bacterial cell. It encodes a functional recombinase and is able to transpose to new targets on the chromosome. Here we report on a possible mechanism of the HPI dissemination based on site-specific recombination of the excised HPI with the attB-presenting (asn tRNA gene) RP4 promiscuous conjugative shuttle plasmid. The resulting cointegrate can be transferred by conjugation to a new host, where it dissociates, and the released HPI integrates into any unoccupied asn tRNA gene target in the genome. This mechanism has been proven both with the 'mini' island carrying only the attP recognition site and genes coding for recombination enzymes and with the complete HPI labelled with an antibiotic resistance marker. After acquisition of the mobilized complete form of the HPI, the ability of the HPI-cured Yersinia enterocolitica WA-TH(-) strain to produce yersiniabactin has been restored. Such 'trapping' of pathogenicity islands and subsequent shuffling to new hosts by a conjugative replicon carrying a suitable attB site could be applied to other functional integrative elements and explain wide dissemination of PAIs.
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http://dx.doi.org/10.1111/j.1365-2958.2005.04722.xDOI Listing
August 2005