Hin Genes Research Articles

Development and application of a multiplex PCR method to differentiate Salmonella enterica serovar Typhimurium from its monophasic variants in pig farms

Salmonella enterica serovar Typhimurium monophasic variants (Salmonella 4,[5],12:i:-) has increased dramatically, causing human salmonellosis and colonization in pigs. With a difference to S. Typhimurium, the monophasic variants of S. Typhimurium lose the gene cassettes encoding the second phase flagellin. To establish a rapid method to detect and differentiate the two serotypes, we analyzed the published 679 genomes of S. Typhimurium and its monophasic variants and found that no Salmonella 4,[5],12:i:- strains carry both fljB and hin genes. Therefore, we established a novel multiplex PCR method using the fljB-hin region and mdh gene as target sequences to detect and differentiate both serotypes. This method can be used to specifically detect both serotypes with a detection limit for DNA concentration at 10 pg/μL. In addition, the PCR assay successfully differentiated 36 S. Typhimurium isolates from 62 isolates of monophasic variants preserved in our laboratory from 2009 to 2017, which corresponds to the whole-genome-based serotyping results. Application of the multiplex PCR method to 60 fecal samples from a pig farm identified 11.7% (7/60) of S. Typhimurium monophasic variants, which is consistent with the whole-genome-based serotyping results. The multiplex PCR assay is a rapid and precise method for the detection of S. Typhimurium monophasic variants from samples across food production chains.

Genetic variability of Hin genes in selected barley germplasm and potential implications for grain texture

Grain hardness (texture) in barley is a commercially important trait, associated with water-uptake during the steeping phase of malting, quantified by measuring endosperm hardness. Hordoindolines are seed proteins of barley, related to the wheat puroindoline proteins. The Hordoindoline genes Hina, Hinb-1 and Hinb-2 are orthologs of wheat Pin genes. The information on contributions of Hin genes to barley grain hardness is limited and somewhat ambiguous. Hence this study aimed to investigate the genetic diversity of Hin genes in 12 Australian barley cultivars and 14 landraces originating from geographic centers of diversity. The grain hardness was determined by the Single Kernel Characterisation System (SKCS) and ranged between 24.2–78.5 SKCS units for cultivars and 48.7–94.4 SKCS units for landraces. Hin gene amplifications and DNA sequencing indicated notable genetic diversity, with seven Hina, six Hinb-1 alleles and eight Hinb-2 alleles detected. Some of the SNPs led to substitutions at the potentially functionally important tryptophan rich domain and certain basic and hydrophobic residues in the putative proteins. A novel Hinb-1 allele with an insertion at nucleotide position 210, leading to a frame-shift mutation, was discovered in landrace L400211 from Ethiopia that had a high SKCS hardness value, indicating harder grain. The study suggests that Hin gene variations may contribute to barley grain hardness, and selection of certain alleles may be useful for breeding for varying barley grain textures.

Salmonella Typhimurium in Iran: Contribution of molecular and IS200 PCR methods in variants detection.

Salmonella Typhimurium, a zoonotic pathogen, is regarded as a major health and economic concern worldwide. Recently, monophasic variants of this serovar have been significantly associated with human gastroenteritis outbreaks globally, making its accurate identification essential for epidemiological and control purposes. We have identified and analyzed 150 S. Typhimurium from 884 Salmonella genus isolated from humans, domestic animals, poultry, food items and abattoirs origins. The Salmonella isolates were obtained from Iranian National Veterinary Reference Laboratories of 9 provinces during 2007–2016, and from five hospitals in Tehran in 2015. The isolates were evaluated biochemically, serologically, and by PCR amplification of invA, mdh, STM4492, fliC, fljA, fljB, hin genes, IS200 and DT104. invA and mdh genes were used to confirm the S. Typhimurium serotype, fliC and fljB genes for determination of monophasic variants and amplification of IS200 to discriminate the monophasic variants from the closely related serotypes. We identified 78.6% (118/150) as classical S. Typhimurium (fliC, fljB and IS200 positive), 12.6% (19/150) were IS200 negative from all isolates. DT104 is another marker for S.Typhimurium serovar typing. Contrary to EFSA guidelines 20.6% (19/29) of human isolates that lacked IS200 insertion sequence, were confirmed as S.Typhimurium. Compared to the North American/European isolates the low prevalence of fljB negative 6% (9/150) and the high abundance of fliC negative 23.3% (35/150) isolates also were indicative of a different regional atypical population. Studies have shown that the prevalence of monophasic (fljB-) S. Typhimurium worldwide is promoted by the Swine industry. Thus, one reason for this high number of different atypical strains could be inhibition of swine breeding system (house hold and industry) in Iran. These results demonstrate a need for a modified identifying protocol to overcome the regional differences.

Circulation and etiological characterization of Salmonella enterica serotype in human in Guangdong province, 2007-2016

Objective: To understand the circulation, drug resistance and molecular characteristics of Salmonella1, 4, [5], 12: i:- in human in Guangdong province. Methods:Salmonella1, 4, [5], 12: i:- isolated from diarrhea patients in Guangdong during 2007-2016 were detected for drug resistance, genes and PFGE characteristics. Results: A total of 2 960 strains Salmonella1, 4, [5], 12: i: - were isolated from human diarrhea cases during this period. The positive rates of the isolation increased year by year. The male to female ratio of the infection cases was 1.58∶1, and the infection mainly occurred in infants and young children. Except imipenem, Salmonella1, 4, [5], 12: i: - was resistant to other 17 antibiotics to some extent. The drug resistant rates to ceftazidime, cefotaxime and ciprofloxacin increased from 2011 to 2016. Multi-drug resistance was serious, for example, the multi-drug resistant strains with ASSuT accounted for 70.62% (435/616) and the multi-drug resistant strains with ACSuGSTTm accounted for 27.11% (167/616). The lack of fljA, fljB and hin genes, as well as the retaining of iroB, STM2740, STM2757 genes, resulted in the unable expression of FljBenx gene with 8 different defection profiles. There were 934 different PFGE patterns observed in 2 347 strains, which displayed a relatively large fingerprint polymorphism. The major PFGE pattern was JPXX01. GD0226, which was found in 97 strains, accounting for 4.13% (97/2 347). The PFGE patterns in 168 Salmonella1, 4, [5], 12: i: - strains were consistent with that of Salmonella typhimurium. Conclusions:Salmonella1,4,[5], 12: i: - strains has become the major serotype of Salmonella that cause diarrhea in human in Guangdong. The multi-drug resistance of Salmonella1,4, [5], 12: i: - was serious, and since the defection of fljA, fljB and hin genes, the expression of FljBenx protein failed. The PFGE results were diverse, which displayed polymorphism in inheritance.

Molecular Characterization of “Inconsistent” Variants ofSalmonellaTyphimurium Isolated in Italy

Salmonella 4,[5],12:i:- is a variant of Salmonella Typhimurium, which lacks the expression of phase-2 flagellar antigen, generally associated with the deletion of the fljB gene. Additional mechanisms involving the fljAB operon ( fljA, fljB, and hin genes) lead to the lack of expression of phase-2 flagellar antigens also in Salmonella strains harboring the fljB gene. For 20 S. 4,[5],12:i:- strains, defined as "inconsistent" Salmonella Typhimurium variants since they had phenotypically behaved as monophasic, even though the fljB gene was conserved, the fljAB operon was characterized in order to explain the ineffective expression of the phase-2 flagellar antigen. The monophasic phenotype for a first group of strains (9) was likely due to the absence of the hin gene, leading to the inhibited switch between the expression of phase-1 and phase-2 flagellar genes. For a second group of strains (5), the monophasic phenotype could be attributed to nonconservative point mutations identified in fljA and hin genes, which could hamper the proper expression of invertase gene and the fljA, acting as repressor of the phase-1 flagellar gene. Finally, for a last group of inconsistent strains (6), a plausible reason for their monophasic phenotype was not found, since the genes involved in the expression of phase-2 flagellar antigen were fully conserved. Moreover, the collection of inconsistent Salmonella Typhimurium isolates investigated were characterized by distinct molecular profiles, as demonstrated by multiple-locus variable-number tandem repeat analysis, and phenotype variability, as demonstrated by phage-typing. This study highlights the usefulness of investigating the entire fljAB operon when a definitive identification of the monophasic or biphasic status of Salmonella Typhimurium strains is needed (for instance, in the context of epidemiological investigations aimed to identify the relatedness among strains).

Molecular characterization of two novel alleles of Hordoindoline genes in Hordeum chilense Roem. et Schult.

Grain texture is an important trait in wheat quality, and is related to the presence or absence of puroindolines a and b. In barley, these proteins are synthesised by the Hordoindoline (Hin) genes, orthologs of the Puroindoline (Pin) genes present in bread wheat and Aegilops species. In this study, the variation of Hin genes has been characterized in two representative lines of Hordeum chilense Roem. et Schult., a wild barley species that has been used in the development of a new man-made cereal (tritordeum, ×Tritordeum Ascherson et Graebner). Two novel alleles were detected in this material, one for the Hina-Hch1 gene and another for the Hinb-Hch1 gene. These alleles demonstrated high similarity to the Pin genes of bread wheat, which may explain the soft grain texture of tritordeum. This suggests that the variation present in H. chilense could be utilized in wheat breeding through the use of tritordeum as a bridge species, to extend the range of different textures.

Molecular Characterization ofSalmonella entericaSerovar 4,[5],12:i:- DT193 ASSuT Strains from Two Outbreaks in Italy

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- DT193 is recognized as an emerging monophasic variant of Salmonella Typhimurium in many European countries. Resistance to ampicillin, streptomycin, sulphonamides, and tetracycline (R-type ASSuT) is described as one of the most common profiles of resistance within this clone. Recently, strains presenting such features were isolated from two unrelated outbreaks in Italy. Strains were characterized by pulsed-field gel electrophoresis (PFGE), performed with XbaI, BlnI, and SpeI, and multiple-locus variable-number tandem repeat analysis (MLVA). XbaI-PFGE showed strains related to the two outbreaks as indistinguishable. Conversely, both BlnI-PFGE and MLVA characterized the strains related the two outbreaks as different. XbaI-PFGE identified two profiles, differing by one band, within strains isolated from one of the two outbreaks. Also BlnI-PFGE and MLVA generated different profiles among the strains related to that outbreak. Combining the PFGE profiles obtained by XbaI and BlnI and comparing them with the MLVA profiles, the two methods grouped the same isolates based on identity. Moreover, genomic deletions of the genes included in the operon fljAB, the flanking iroB gene, and the closely located STM2757 gene were investigated. For all strains, the same profile of deletion characterized by the absence of fljA, fljB, and hin genes and the presence of STM2757 and iroB genes was identified. This profile of deletion represents a mixture between two profiles of Salmonella 4,[5],12:i:- described as the "Spanish" and the "U.S." clones. This study demonstrated that although strains of Salmonella 4,[5],12:i:- DT193 ASSuT are highly clonal, minor differences between strains may be seen during the same outbreak by using in parallel PFGE with different restriction enzymes, MLVA, and the analysis of molecular markers related to the operon fljAB. The combination of these different molecular approaches was essential to clarify the epidemiological relationship among the strains.

Virulence Function of Pectobacterium atrosepticum Secretion System Mutants on Evaluation of Some Solanum tuberosum Resistance Genes

Background: P. atrosepticum is a commercially important pathogen. It causes blackleg in the field and soft rot of tubers after the harvest. This effect is due to secretion of depolymerases and other virulence factors by several mechanisms including T3SS Objectives: The effect of bacterial T3SS on Solanum tuberosum (S. tuberosum) varieties and its relationship with S. tuberosum resistance gene expression were studied. Materials and Methods: A P. atrosepticum HrpW gene was cloned, sequenced and constructed a phylogenic tree with some phytopathogen. The virulence properties of P. atrosepticum strains were investigated and then S. tuberosum varieties were tested for their sensitivity against P. atrosepticum. PR-5 and HIN genes copy-number for infiltrated S. tuberosum tubers were assessed. Results: The results show that infiltrated tubers of P. atrosepticum T3SS mutants were significantly more macerated than the wild type ones. Conclusions: PR-5 and HIN expression amounts were depended on bacterial T3SS function.

Molecular identification in monophasic and nonmotile variants of Salmonella enterica serovar Typhimurium

Variant strains of Salmonella enterica serovar Typhimurium, lacking one or both flagellar phases have been widely reported. The monophasic S.1,4,[5],12:i:- variant has emerged worldwide in the past few years and has become one of the most frequently encountered in many countries. In contrast, monophasic S.1,4,[5],12:-:1,2 and nonmotile S.1,4,[5],12:-:- strains are rarely described. This study investigated seven molecular markers to identify and delineate monophasic S.1,4,[5],12:i:- (n = 90), S.1,4,[5],12:-:1,2 (n = 25), nonmotile S.1,4,[5],12:-:- (n = 17) strains, and some serovar Typhimurium strains (n = 124) collected through the French Salmonella network between 2001 and 2010. Three markers were commonly detected in serovar Typhimurium and in all variant strains: STM2757, mdh and fliA-B. Monophasic S.1,4,[5],12:i:- were genotypically confirmed by the absence of the fljB, fljA, and hin genes. Nevertheless, 13 (14.5%) of them were positive for these last three genes, revealing monophasic strains named “inconsistent” as previously described. All nonmotile 1,4,[5],12:-:- strains had the fliC, fljA, fljB, and hin genes and the fliC gene was detected in 88% of monophasic S.1,4,[5],12:-:1,2 strains. The combination of the seven markers detection enables to recognize eight different genotypes within the S.1,4,[5],12:i:- collection, among which the Spanish and the U.S. clones previously described could be distinguished and assigned to a genotype. Based on this molecular approach, 71% of the French S.1,4,[5],12:i:- collection belonged to the Spanish clone, whereas only 2% were assigned to the U.S. clone. This study highlights the usefulness of these molecular markers and genotypes for identifying lineages, especially among the epidemiologically important monophasic S.1,4,[5],12:i:- variant.

Distribution of Hordoindoline genes in the genus Hordeum

Hordoindoline (Hin) genes, which are known to comprise Hina, Hinb-1, and Hinb-2, are associated with grain hardness in barley. However, the interspecific variation in the Hin genes in the genus Hordeum has not been studied in detail. We examined the variation in Hin genes and used it to infer the phylogenetic relationships between the genes found in two H. vulgare subspecies (cultivated barley and H. vulgare subsp. spontaneum) and 10 wild relatives (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii). The Hina and Hinb genes of these species were amplified by PCR. We found two Hinb genes in three wild species (H. bogdanii, H. brachyantherum, and H. roshevitzii) and preliminarily named them Hinb-A and Hinb-B. Cluster analysis showed that the 17 Hinb genes present in Hordeum formed two distinct clusters (named A and B). Seven Hinb genes were included in Cluster-A, and 10 Hinb genes were included in Cluster-B. All Hinb-A genes were included in Cluster-A, while all of the Hinb-B genes were included in Cluster-B. In contrast, the Hinb-1 and Hinb-2 genes in H. vulgare were included in Cluster-B. These results suggest that the Hinb genes duplicated during the early stages of diversification in the genus Hordeum. On the other hand, the Hinb-1 and Hinb-2 genes in H. vulgare seem to have been generated by a duplication of the Hinb gene after the split of the lineages leading to H. vulgare and H. bulbosum.

Comparative Analysis of Hina Gene Sequences in Wild (Hordeum spontaneum) and Cultivated (H. vulgare) Barleys

The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley {Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgäre) lines in Europe and 23 landrace (H. vulgäre) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgäre) with unknown global distribution. C/s-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild and landrace/cultivated barleys. There were six consistent cw-acting binding sites in wild and landrace/cultivated barleys, whereas 8 to 16 inconsistent TATA-boxes were observed. In addition, three special elements (E2Fb, Spl, and boxS) were only observed in wild barley, while one (AT 1 -motif) was only found in landrace/cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace/cultivated barleys were obtained by deleting repetitive amino acid sequences, and they were clustered into two groups on the basis of Neighbor-Joining analysis. However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace/cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition.

Evidence for a DNA inversion system in Bordetella pertussis

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosamal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.

Prokaryote invertible DNA systems are highly conserved

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosamal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.