Arp Genes Research Articles

Molecular Detection and Typing of Treponema pallidum in Non-Ocular Samples from Patients with Ocular Syphilis

ABSTRACT Introduction Ocular syphilis is a rare but potentially sight-threatening manifestation of infection with the spirochete Treponema pallidum subspecies pallidum. Molecular strain typing of clinical specimens obtained from patients with syphilis can provide useful epidemiological and clinical information. In this study, we assess the utility of non-ocular clinical samples in strain typing for patients with diagnosed ocular syphilis. Methods We collected samples of excess blood, serum, and cerebrospinal fluid (CSF) from 6 patients with ocular syphilis treated in 2013–2016. DNA was extracted, purified, and then analyzed using an enhanced molecular typing method including sequence analysis of tp0548, number of repeats in the arp gene, and restriction fragment length polymorphism of the tpr gene. Results Molecular strain typing based on tp0548 gene sequence analysis revealed two cases of type F and two cases of type G in 3 of 6 (50%) cases with CSF samples, 1 of which was obtained after starting antibiotics. In a patient with 2 distinct episodes, the same tp0548 type (type G) was identified in both episodes using different sample types (CSF, whole blood). Serum samples were available in 6 cases, but none were successfully typed with any of the methods. Amplification of the tpr and arp genes was unsuccessful in all cases. Overall, strain types were identified in 4 of the 7 episodes. Conclusion Treponema pallidum strain types F and G were detected in CSF or whole blood in 4 of 7 episodes in this series. We demonstrate moderate sensitivity of strain typing in ocular syphilis using non-ocular clinical specimens.

High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny.

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Epidemiology, molecular strain types, and macrolide resistance of Treponema pallidum in Japan, 2017–2018

BackgroundJapan has seen a substantial increase in syphilis cases since 2013 and Tokyo and Osaka prefectures accounted for about 40% of all cases in Japan. Therefore, focusing on these 2 prefectures, we assessed syphilis cases detected during 2017–2018, combining epidemiological information with molecular typing data. MethodsUsing data from surveillance reports, we described syphilis cases by gender, age, transmission route, and stage of syphilis. Clinical specimens were collected from syphilis patients in Tokyo and Osaka prefectures. Molecular typing was performed by analyzing Treponema pallidum arp, tpr, and tp0548 genes, with partial sequencing of the 23S rRNA genes for macrolide resistance. ResultsBetween 2017 and 2018, the number of syphilis cases increased from 3934 to 4588 among males and 1895 to 2414 among females, with similar age and gender distributions during the period. The predominant strain type was 14d/f (71%, 73/103), found more frequently in women who have sex with men (86%, 25/29) and men who have sex with women (83%, 39/47) than in men who have sex with men (MSM) (33%, 9/27). The majority of the strains from heterosexuals (97%, 76/78) were macrolide-resistant, considerably higher than those from MSM (59%, 20/34). The molecular profiles in each sexual-transmission group remained similar during the 2 years. ConclusionsThe epidemiological and molecular features of syphilis remained similar throughout the period, with consistent differences in strain type and macrolide resistance distributions between MSM and heterosexual cases. These findings suggest a predominantly heterosexual epidemic where the dynamics of syphilis transmission remained unchanged during 2017–2018.

Molecular typing of T. pallidum clinical isolates and their resistance to macrolides in the Russian Federation during 2018–2019

Based on arp, tprII and tp0548 genes variability molecular typing of Treponema pallidum is a worldwide method for syphilis epidemiology analysis, including the ability to monitor antimicrobial-resistant variants of the causative agents of the disease. The aim of the study is to characterize the molecular subtypes of T. pallidum isolated from syphilis patients’ clinical material in 2018–2019, to assess the known antibiotic resistance determinants presence, and to compare the results with known Russian and foreign data. Materials and methods . 64 T. pallidum clinical isolates obtained from 10 regions of the Russian Federation: Astrakhan, Irkutsk, Kaluga, Moscow, Novosibirsk and Omsk regions, the Stavropol Territory, the Republic of Sakha (Yakutia), Tuva and Chuvashia were studied. The arp gene variability was estimated by the number of internal tandem repeats from 60 base pairs analyzed by PCR. The variability of tprII genes was based on the restriction fragment length polymorphism. The nucleotide sequence of the tp0548 gene with a length of 84 base pairs (positions 131–215) variable region, as well as the genetic determinants of macrolide resistance in the 23S rRNA gene, were studied by capillary sequencing. Results. 14 d/f molecular subtype of T. pallidum was absolutely prevalent in 9 of 10 examined subjects of the Russian Federation, taking 93.75 % of the total number of clinical isolates, and it was consistent with data on its stable dominance over 2011–2017. The most common in Western Europe molecular subtype of T. pallidum 14 d/g was found in the Kaluga and Omsk regions, taking 4.69 % of the analyzed samples. The molecular subtype of T. pallidum 14 b/f , sporadically but stably occurring over the many years of monitoring, was represented by a single clinical isolate (1.56 %) from the Republic of Tuva. A2058G 23S rRNA mutation causing resistance to macrolides was detected both in all (3 of 3) representatives molecular subtype 14 d/g and in 2 of 60 clinical isolates molecular subtype 14 d/f (from Moscow and Omsk region). In fact, the resistance to macrolides was found in 7.81 % T. pallidum Russian clinical isolates found during 2018–2019, that exceeds the WHO threshold level allowing to recommend a drug for the STI treatment. Conclusion . The contemporary molecular epidemiology of syphilis in the Russian Federation is characterized by the dominance of the molecular subtype T. pallidum 14 d/f , and significantly distinguishes the local population from those in the neighboring states of Europe and Asia. The continued spread of the determinants of resistance to macrolides makes us cautious about the use of this group of drugs for the treatment of syphilis and is an argument in favor of an appropriate review of current clinical recommendations.

Interactive Effects of Salinity and ZnO Nanoparticles on Physiological and Molecular Parameters of Rapeseed (Brassica napus L.)

ABSTRACTSalinity is considered as the main factor limiting the yield of crops in arid and semi-arid regions. There are still many uncertainties about the nanotechnology and its potential applications, as well as doubts about its efficacy and safety in the long term. The aim of this study was to examine the alleviative effects of ZnO NPs (nanoparticles) (0, 20 and 80 mgL−1) on toxicity damage caused by NaCl (0, 50 and 100 mM) at physiological and molecular parameters in Rapeseed. Rapeseed plants were treated at the rosette stage by different levels of salinity and ZnO NPs based on a completely randomized design (CRD) with three replications. At the physiological level, salinity stress significantly increased root ion leakage and decreased relative water content (RWC), stomata density and Hill reaction while application of ZnO NPs improved Hill reaction, and reduced ion leakage. At the molecular level, salinity stress significantly reduced the expression of ARP, MYC and SKRD2 genes compared to non-stressed plants while MPK4 gene expression increased under a high level of NaCl imposition. Foliar spraying of ZnO NPs considerably decreased the expression of MYC, MPK4, and SKRD2 genes and increased the expression of the ARP gene. It can be concluded that ZnO NPs had the ability to reduce the toxicity created under salinity stress at the optimal concentration (20 mgL−1) in rapeseed and could play an important role in increasing the resistance of rapeseed plants to salinity stress.

Genetic stability of the typing loci used in the multilocus sequence typing and enhanced CDC typing of syphilis treponemes

To the Editor, We read, with interest, the recently published article “Increased discrimination of Treponema pallidum strains by subtyping with a 4-component system incorporating a mononucleotide tandem repeat in rspA” in Sexually Transmitted Diseases by Pillay et al.1 The authors have combined determination of the guanine mononucleotide repeat length in the rspA gene with enhanced Centers for Disease Control and Prevention (CDC) typing for molecular typing of syphilis treponemes. The authors dedicated a significant part of their article to issues concerning genetic stability of mutations causing resistance to macrolides analyzed during the newly developed multilocus sequence typing.2–4 However, the examination of the 23S rDNA locus carrying the potential resistance markers was proposed only as an additional, clinically relevant analysis and is not a part of the typing scheme.2 In actuality, the recently proposed multilocus sequence typing is based on the sequence analyses of the TP0136, TP0548, and TP0705 loci. To assess the stability of these typing loci, we have analyzed 11 serial passages of the treponemal reference strain in experimental rabbits, and all passages revealed the same allelic profile suggesting stability of these typing loci for over a hundred treponemal generations. Moreover, the recent studies revealed a low mutation rate in both yaws and syphilis treponemes consistent with the stability of treponemal genome sequences for a decade or a longer period.5,6 However, the above described genetic stability of treponemal genome sequences does not apply to the length stability of the homopolymeric regions and other repetitive or paralogous sequences,7 including the homopolymeric region present in the rspA gene (TP0279) used in the recent work by Pillay et al.1 This locus was recently characterized as a locus showing genetic intrastrain heterogeneity among recent clinical isolates, and this variability likely drives phase variation in T. pallidum during human infection.8 In addition, the restriction fragment length polymorphism analyses of tpr genes and the determination of repeats in the arp gene were previously shown to be variable in multiple samples from the same patient, a phenomenon that was not detected in sequence-based typing.9 The enhanced CDC typing served as an adequate tool for mapping the epidemiology of syphilis for about 20 years with the first report appearing in the same year as the first treponemal genome sequence. Since then, a significant expansion of our knowledge regarding T. pallidum genetics and genomics has occurred and revealed a limited genetic stability of tpr, arp, and rspA genes in comparison to other treponemal genomic loci.7,8 Linda Grillová, PhD Biology of Spirochetes Unit Institut Pasteur, Paris, France [email protected]David Šmajs, MD, PhD Department of Biology Faculty of Medicine Masaryk University, Brno Czech Republic

New Insights into the Biosynthesis Pathway of Polyketide Alkaloid Argimycins P in Streptomyces argillaceus.

Argimycins P are a recently identified family of polyketide alkaloids encoded by the cryptic gene cluster arp of Streptomyces argillaceus. These compounds contain either a piperideine ring, or a piperidine ring which may be fused to a five membered ring, and a polyene side chain, which is bound in some cases to an N-acetylcysteine moiety. The arp cluster consists of 11 genes coding for structural proteins, two for regulatory proteins and one for a hypothetical protein. Herein, we have characterized the post-piperideine ring biosynthesis steps of argimycins P through the generation of mutants in arp genes, the identification and characterization of compounds accumulated by those mutants, and cross-feeding experiments between mutants. Based in these results, a biosynthesis pathway is proposed assigning roles to every arp gene product. The regulation of the arp cluster is also addressed by inactivating/overexpressing the positive SARP-like arpRI and the negative TetR-like arpRII transcriptional regulators and determining the effect on argimycins P production, and through gene expression analyses (reverse transcription PCR and quantitative real-time PCR) of arp genes in regulatory mutants in comparison to the wild type strain. These findings will contribute to deepen the knowledge on the biosynthesis of piperidine-containing polyketides and provide tools that can be used to generate new analogs by genetic engineering and/or biocatalysis.

Genetic antimicrobial resistance determinants and their prevalence in molecular subtypes of Treponema pallidum subsp. pallidum

Objective. To investigate genetic determinants of resistance to antimicrobial agents recommended for the treatment of syphilis and assess their prevalence in molecular subtypes of Treponema pallidum subsp. pallidum in the Russian Federation over the period of 2014-2017. Materials and Methods. A total of 161 clinical isolates of T. pallidum obtained from Tyva, Stavropol, Irkutsk, Kaluga, Novosibirsk and Omsk regions were included in this study. Genetic material of T. pallidum was detected by PCR with primers to polA gene. Determinants of resistance to penicillins (tromp1, tp47), tetracyclines (16S rRNA) and macrolides (23S rRNA) were determined using the gene sequence analysis. Molecular typing was performed by characterizing variable arp, tpr (E, G, J) and tp0548 genes according to the CDC protocol. Results of this study were compared to historical data on antimicrobial resistance of T. pallidum over the period of 2011-2012. Results. Analysis of tromp1 and tp47 gene sequences detected C22G and G208T substitutions, respectively. These polymorphisms were not significant for activity of the corresponding proteins, but differed the studied clinical isolates from the reference strain Nichols, therefore, linking them with epidemic genogroup T. pallidum Street Strain 14. Based on the analysis of G1058C mutation in the 16S rRNA gene, all clinical isolates obtained in 2014-2017 belonged to wild type, whereas this genetic determinant of resistance to tetracyclines was determined in 2 of 190 isolates obtained in 2011-2012. Also, A2059G/C mutation in the 23S rRNA gene was not found, whereas a significant A2058G substitution in this gene was determined in 4 isolates obtained in 2014-2017. Results of this study confirm sporadic resistance to macrolides in the Russian Federation, which was previously (2011-2012) found in 3 of 190 isolates of T. pallidum. A2058G mutation was detected predominantly in minor subtypes of T. pallidum (14 b/f, 14 b/g and 14 d/g) and was unrepresentative for molecular subtype 14 d/f which is a predominant one in the Russian Federation. Conclusions. The long-term use of penicillins for the treatment of syphilis did not result in emergence of T. pallidum resistance to this antibiotic class. An absence of genetic determinants of resistance to tetracyclines confirms them to be second-line drugs. A sporadic prevalence of determinants of resistance to macrolides requires they be used for the treatment of syphilis with caution.

Molecular epidemiology of treponema pallidum in border region of russian federa-tion (Tuva Republic)

The 111 strains of T. pallidum subsp. pallidum collected in Tuva Republic in 2013-2016 were typed by the arp, tpr (E, G, J) and tp0548 genes. The 7 subtypes were identified, in which the 14 d/f type was predominant (90.1%). The minor subtypes 14 b/f, 14 c/f, 14 d/g and 14 i/f constituted 0.9-1.8%. Single strains of 4 d/f H 9 d/f types (each 0.9%) previously described in China were detected in 2015. Both 9 and 14 arp gene variants were found in 3 clinical specimens for the first time in 2015-2016. Similarities in the molecular epidemiology of syphilis in Tuva Republic and Russia were demonstrated as well as differences from the T. pallidum subsp. pallidum population in China and Western Europe.

Research Article Differential expression of dormancy-Associated genes in fastigiata and hypogaea peanut

Seed dormancy is a temporary replacement state observed in some higher plants, which prevents germination under unfavorable conditions. In several species, dormancy process is triggered by hormonal factors, initiated by ABA and precursor enzymes. In this work, we investigated the relationship between dormancy and the expression of ARP, DMR1, and NCED genes in upright and runner peanut seeds. Eight dormancy-contrasting genotypes were previously phenotyped based on germination traits, during three storage periods. Expression of ARP, DMR1 and NCED transcripts was analyzed by qRT-PCR, from endosperm and embryo tissues. Higher expression of NCED and ARP were found in embryo tissues from runner genotypes, IAC Caiapo and LGoPE-06, confirming findings in literature as to association of these genes with seed dormancy. Higher expression of DMR1 was found only in endosperm of upright materials (L7 Bege and BR1). We suggest the NCED is a functional molecular marker to identify dormancy in seeds, contributing to selection procedures of runner genotypes in peanut breeding.

Syphilis epidemiology in 1994-2013, molecular epidemiological strain typing and determination of macrolide resistance in Treponema pallidum in 2013-2014 in Tuva Republic, Russia.

The incidence of syphilis in the Tuva Republic (geographical centre of Asia), Russia has been exceedingly high historically. No detailed examinations and no molecular investigations of Treponema pallidum strains transmitted in the Tuva Republic, or in general, in Russia, were published internationally. We examined the syphilis epidemiology in 1994-2013, and the molecular epidemiology and macrolide resistance in T. pallidum strains in 2013-2014 in the Tuva Republic. Among 95 mainly primary or secondary syphilis patients, the arp, tpr, tp0548 and 23S rRNA genes in 85 polA gene-positive genital ulcer specimens were characterized. The syphilis incidence in Tuva Republic peaked in 1998 (1562), however declined to 177 in 2013. Among the 70 (82%) completely genotyped specimens, six molecular strain types were found. Strain type 14d/f accounted for 91%, but also 14c/f, 14d/g, 14b/f, 14i/f, 9d/f, and 4d/f were identified. Two (2.4%) specimens contained the 23S rRNA A2058G macrolide resistance mutation. This is the first internationally published typing study regarding T. pallidum in Russia, performed in the Tuva Republic with the highest syphilis incidence in Russia. The two molecular strain types 4d/f and 9d/f have previously been described only in Eastern and Northern China and for the first time, macrolide-resistant syphilis was described in Russia.

Detection of polA gene in whole blood samples and preliminary observations with molecular subtyping of Treponema pallidum

To detect Treponema pallidum (T.pallidum) DNA with polymerase chain reaction (PCR) in whole blood samples of syphilis patients and analyze their features of sub-genotypes. The clinical data of patients were collected from July 2012 to February 2013. And polA gene of syphilis was detected by PCR. The arp and tpr genes of polA gene-positive samples were analyzed by the established genotyping system.Statistical analyses were performed to compare different clinical courses and features to examine their correlations. The common treponemal gene target (polA) of 35 samples were detected in whole blood by PCR in 181 samples. A total of 24 cases (35.8%) were positive in 67 patients with newly diagnosed non-latent syphilis untreated patients;26 cases of latent syphilis were negative;7 cases (9.7%) were positive in 72 subsequent visit patients;4 cases (25.0%) were positive in 16 patients with sero-resistant. There were 4 subtypes of 14a (n = 7), 14b (n = 10), 13c (n = 4) and 14d (n = 14). Among those positive samples, there were 4 sero-resistant samples of 3 subtypes 14d and 1 subtype 14b. The feasibility of peripheral blood is confirmed. Although the positive rate of whole blood detection of T.pallidum gene is low, the method is both simple and reliable for patients with sero-resistant syphilis.

Phenotypic characterization of the CRISPA (ARP gene) mutant of pea (Pisum sativum; Fabaceae): a reevaluation.

Leaf form and development are controlled genetically. The ARP genes encode MYB transcription factors that interact with Class 1 KNOX genes in a regulatory module that controls meristem-leaf determinations and is highly conserved in plants. ARP loss of function alleles and subsequent KNOX1 overexpression cause many unusual leaf phenotypes including loss or partial loss of the ability to produce a lamina and production of "knots" on leaf blades. CRISPA (CRI) is the ARP gene in pea, and a number of its mutant alleles are known. We made morphological and anatomical evaluations of cri-1 mutant plants while controlling for genetic background and for heteroblastic effects, and we used aldehyde fixation and resin preparations for anatomical analysis. Further, we compared gene expression in WT and cri-1 shoot tips and HOP1/PsKN1 and CRI expression in other leaf mutants. The cri-1 plants had more extensive abnormalities in the proximal than in the distal regions of the leaf, including ectopic stipules, narrow leaflets, and shortened petioles with excessive adaxial expansion. "Knots" were morphologically and anatomically variable but consisted of vascularized out-pocketing of the adaxial leaflet surface. HOP1/PsKN1 and UNI mRNA levels were higher in cri-1 shoot tips, and some auxin-regulated genes were lower. Low LE expression suggests that the GA level is high in cri-1 shoot tips. The CRISPA gene of pea suppresses KNOX1 genes and UNI and functions to (1) maintain proximal-distal regions in their appropriate positions, (2) restrict excessive adaxial cell proliferation, and (3) promote laminar expansion.

Molecular typing of Treponema pallidum: a 5-year surveillance in Shanghai, China.

Previously, a small study showed that 14f was the predominant subtype of Treponema pallidum in Shanghai, China. The result was quite different from the genotype distribution in other areas of China. This study aimed to identify the strain types of Treponema pallidum in samples collected over a 5-year period in Shanghai. From 2007 to 2011, genital swabs were collected from patients with syphilis from the Shanghai Skin Disease Hospital. Positive specimens were typed by the enhanced typing method by adding a tp0548 gene to the existing arp and tpr genotype system. In total, 304 of the 372 enrolled patients yielded fully typeable DNA. Ten arp types (4, 6, 8, 9, 11, 12, 13, 14, 15, and 19), 3 tpr types (a, d, and o), and 5 tp0548 types (a, c, f, g, and i) were identified. In total, 12 subtypes were identified with a combination of the arp and tpr genes. Subtype 14d was found in 270 samples (88.8%). When the combination included the tp0548 gene, the 12 CDC subtypes identified were divided into 14 strain types. The predominant type was 14d/f (88.8%), followed by 15d/f (3.6%), 13d/f (1.3%), and 19d/c (1.3%). Two of the 44 14d/f-infected patients and both of the 19d/c-infected patients who underwent a lumbar puncture were diagnosed with neurosyphilis. This study showed that the predominant type in Shanghai was 14d/f. While this is in keeping with data from other areas in China, it is different from an earlier report showing that 14f is the most common genotype in Shanghai. Further studies are needed to better understand the association between strain types and neurosyphilis.

Sequencing-based Molecular Typing of Treponema pallidum Strains in the Czech Republic: All Identified Genotypes are Related to the Sequence of the SS14 Strain

A set of 415 clinical samples isolated from 294 patients suspected of having syphilis collected in the Czech Republic between 2004 and 2010 was tested for the presence of treponemal DNA. Standard serological tests showed that 197 patients were syphilis-seropositive and 97 patients were syphilis-seronegative. In each sample, PCR tests for polA (TP0105), tmpC (TP0319), TP0136, TP0548 and 23S rRNA genes were performed. Samples taken from 91 patients were PCR-positive. Molecular typing of treponemal DNA was based on the sequencing of TP0136, TP0548 and 23S rRNA genes. Treponemal DNA was typeable in samples taken from 64 PCR-positive patients and 9 different genotypes were found. The proportion of treponemal strains resistant to macrolide antibiotics was 37.3%. In the DNA samples taken from 39 patients, a parallel treponemal typing approved by Centers for Disease Control and Prevention was performed. The variants of arp and tpr genes appear to combine independently with sequence variants of TP0136, TP0548 and 23S rRNA genes.

Molecular typing of Treponema pallidum by analysis of three target genes

Objective To evaluate the performance of a three-gene typing system in the determination of Treponema pallidum (Tp) genotypes.Methods To determine the genotypes of Tp,three targets were assessed,including the number of 60 base-pair repeats,restriction fragment length polymorphism (RFLP) pattem of tprEGJ gene after MseI digestion and the sequence of tp0548 gene.The DNA extracted from the Nichols strain of Tp served as the positive control,and that from the moist ulcer of patients with genital herpes and negative RPR or TPPA test results served as the negative control.To validate the typing method,clinical specimens were collected from the moist skin lesions of patients with primary or secondary syphilis,and subjected to the amplification of polA gene by PCR.The enhanced molecular typing system was used to determine the genotypes of Tp in Tp DNA-positive specimens.Results The Nichols strain harbored a genotype of 14a/a.No amplification of any of the three target genes was found in the negative control.The arp gene,tprEGJ gene and tp0548 gene were amplified from 94.1％,91.2％ and 94.1％ of the 40 clinical specimens,and the genotype was successfully determined by the three-gene typing system for 91.2％ of the clinical Tp strains.The predominant type of arp,tprEGJ and tp0548 genes was 14 repeats,d and f,respectively in these clinical Tp isolates.Conclusion The enhanced molecular tying method for Tp exhibits high sensitivity,specificity and discrimination potential. Key words: Syphilis; Treponema pallidum; Molecular typing

P4-S3.04 Treponema pallidum molecular typing in China

BackgroundSyphilis is resurgent in China, but there have been limited data on molecular epidemiology. A novel Treponema Pallidum (T pallidum) typing method that uses tp0548 gene in addition to arp...

A complex case of simple leaves: indeterminate leaves co-express ARP and KNOX1 genes

The mutually exclusive relationship between ARP and KNOX1 genes in the shoot apical meristem and leaf primordia in simple leaved plants such as Arabidopsis has been well characterized. Overlapping expression domains of these genes in leaf primordia have been described for many compound leaved plants such as Solanum lycopersicum and Cardamine hirsuta and are regarded as a characteristic of compound leaved plants. Here, we present several datasets illustrating the co-expression of ARP and KNOX1 genes in the shoot apical meristem, leaf primordia, and developing leaves in plants with simple leaves and simple primordia. Streptocarpus plants produce unequal cotyledons due to the continued activity of a basal meristem and produce foliar leaves termed "phyllomorphs" from the groove meristem in the acaulescent species Streptocarpus rexii and leaves from a shoot apical meristem in the caulescent Streptocarpus glandulosissimus. We demonstrate that the simple leaves in both species possess a greatly extended basal meristematic activity that persists over most of the leaf's growth. The area of basal meristem activity coincides with the co-expression domain of ARP and KNOX1 genes. We suggest that the co-expression of ARP and KNOX1 genes is not exclusive to compound leaved plants but is associated with foci of meristematic activity in leaves.

03-P089 A simple leaf with compound gene expression: Indeterminate leaves co-express ARP and KNOX genes

03-P089 A simple leaf with compound gene expression: Indeterminate leaves co-express ARP and KNOX genes Kanae Nishii, Michael Moeller, Catherine Kidner, Alberto Spada, Raffaella Mantegazza, Hao-Chun Hsu, Toshiyuki Nagata, Chun-Neng Wang 1 National Taiwan University, Taipei, Taiwan 2 Royal Botanic Garden Edinburgh, Edinburgh, United Kingdom 3 Milan University, Milan, Italy 4 Hosei University, Tokyo, Japan

Molecular characterization and analysis of a gene encoding the acidic repeat protein (Arp) of Treponema pallidum

The acidic repeat protein (arp) genes from three subspecies of the treponeme Treponema pallidum (T. pallidum subsp. pallidum, Nichols strain; T. pallidum subsp. pertenue, CDC-1 and CDC-2 strains; and T. pallidum subsp. endemicum, Bosnia A strain) were cloned and sequenced. The predicted protein sequence contained a high percentage of glutamic acid, hence the name acidic repeat protein, or Arp. The protein had a potential membrane-spanning domain and a signal peptidase I site. The gene from the Nichols strain of T. pallidum subsp. pallidum contained a set of 14 nearly identical repeats of a 60 bp sequence, which occupied approximately 51 % of the length of the gene. Analyses of arp from laboratory strains showed that the 5' and 3' ends of the genes were conserved, but there was considerable heterogeneity in the number of repeats of this 60 bp sequence. Based on amino acid variations, the 14 sequence repeats could be classified into three types, which were named type I, type II and type III repeats. The type II repeat was the most common in the strains examined. The arp gene of the Nichols strain was subsequently cloned into the expression vector pBAD/TOPO ThioFusion. The expressed protein was detected in a Western blot assay using rabbit immune sera produced against T. pallidum, or synthetic peptides derived from the repeat sequences. Using an ELISA, rapid plasma reagin (RPR) test-positive sera reacted with synthetic peptides derived from the repeat region but not with peptides derived from N and C termini of the Arp protein. These results show that the Arp protein is immunogenic and could prove to be a useful target for serological diagnosis of T. pallidum infection.