Arginine Dihydrolase Research Articles

An Atypical Arginine Dihydrolase Involved in the Biosynthesis of Cyclic Hexapeptide Longicatenamides

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A-D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.

On etiology of <italic>Pyropia yezoensis</italic> filament macular disease

The experiment carried out an pathogenic analysis of <italic>Pyropia yezoensis</italic> filaments from a laver nursery in the Lanshan alage cultivation cooperative in Rizhao City of Shandong Province. We collected <italic>P</italic>.<italic>yezoensis</italic> filaments with typical symptoms of macular disease,cultured on three mediums of 2216E,TSB and TCBS.The dominant strain PZ201809121102 was isolated and purified from the focus of <italic>P</italic>.<italic>yezoensis</italic> filaments body.The strain PZ201809121102 was identified and obtained.Through Gram staining, electron microscope scanning, negative staining observation,16S rDNA identification, physiological and biochemical analysis and drug sensitivity test, we identified and analyzed the strains and combined with regression infection experiments to confirm the pathogenicity of the strain PZ201809121102. Results showed that: 1) The colony shape of the predominant strain PZ201809121102 after separation and purification on 2216E medium was small and round, with intermediate impurities, smooth and bright yellow surface,growing in creeping status, belonging to Gram-negative bacteria,short rot-shape and with the cell size of (0.5-1.0)μm × (1.5-5.0)μm. Under transmission electron microscope, it was observed that there was a single flagellum outside the spore. 2) 16S rDNA sequencing analysis showed that the bacterium clustered with <italic>Pseudomonas</italic> in the 16S rDNA phylogenetic tree, and had the highest homology with <italic>Pseudomonas luteola</italic>, which was 100%. 3) Physiological and biochemical results showed that the strain PZ201809121102 could grow in 6% NaCl tryptone water and 10% NaCl tryptone water. Oxidase reaction was positive. Ornithine decarboxylase,lysine decarboxylase,arginine dihydrolase reaction was positive. 3% NaCl ONPG reaction was positive. It could assimilate and utilize glucose, but not polysaccharides as sucrose, lactose, denbiose and sorbitol. 4) In regression infection experiment,10³-10⁸ CFU·mL^-1 of PZ201809121102 bacterial suspension was used to soak and infect healthy <italic>P</italic>.<italic>yezoensis</italic> filaments. After 13 days, 10⁸ CFU·mL^-1 <italic>P</italic>.<italic>yezoensis</italic> filaments appeared in different sizes in the experimental group. The yellow spots were consistent with the symptoms of naturally-occurring filaments macular disease, and the bacteria was isolated again to obtain the same domain isolate,which proved that PZ201809121102 was a pathogen of <italic>P</italic>.<italic>yezoensis</italic> filaments macular disease. However in treatments of <italic>P</italic>.<italic>yezoensis</italic> suspension concentrations lower than 10⁸ CFU·mL^-1, there was no obvious symptom of macular disease. 5) Results of drug sensitivity experiments showed that PZ201809121102 was highly sensitive to two drugs, romefloxacin and fleroxacin ,but not sensitive to cefazolin, cephalexin, and cefraditin. In this study, a species of <italic>P</italic>. <italic>yezoensis</italic> filamentous macular disease caused by <italic>P</italic>. <italic>luteola</italic> in seedling stage was discovered, providing a useful reference for the prevention and control of <italic>Pyropia yezoensis</italic> disease.

Grimontia sedimenti sp. nov., isolated from benthic sediments near coral reefs south of Kuwait.

A Gram-stain-negative, rod and rod-curved shaped motile bacterium designated strain S25T was obtained from benthic sediment collected near the Kubbar Island coral reefs south of Kuwait. Phenotypic analysis revealed that strain S25T was slightly halophilic, mesophilic and facultative anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose, fructose, gentiobiose, cellobiose, melibiose, trehalose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. It contained C16 : 1 ω7c/C16 : 1 ω6c (summed feature 3), C18 : 1 ω7c (summed feature 8) and C16 : 0 as the major fatty acids. Strain S25T grew optimally at 30 °C and pH 8 in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA sequences revealed that strain S25T is related to species of the genus Grimontia, having 99.15 % similarity to ‘Grimontia indica’ AK16T, 99.08 % to Grimontia celer 96-237T and 98.66 % to Grimontia marina IMCC 5001T. The DNA G+C content was 48.8 mol% and the full genome analysis for the strain S25T showed that the bacterium has a genome size of 5 158 621 bp and contains 4730 predicted protein-encoding genes. The average nucleotide identity values between the S25T genome and the genomes of its nearest matches ranged between 81.39 and 94.16 %. The strain was distinguishable from the phylogenetically related genera through differences in several phenotypic properties. On the basis of the phenotypic, phylogenetic and genetic data, strain S25T represents a novel species in the genus Grimontia, for which the name Grimontia sedimenti sp. nov. is proposed. The type strain of Grimontia sedimenti is S25T (=DSM 28878T=LMG 28315T).

First Report of Bacterial Leaf Spot of Cucurbita pepo Caused by Erwinia persicina in China.

In February 2020, the common symptoms of water-soaked spots on Cucurbita pepo L. cotyledon were observed in Guangrao county in Shandong province, China. Field investigation showed that 40% of the Cucurbita pepo cotyledons in an area of approximately 0.8 ha were infected. The disease resulted in a severe loss in seedling production. Samples of C. pepo with water-soaked leaf spots were collected and prepared for pathogen analysis. Symptomatic cotyledon tissue was surface disinfested in 75% ethanol for 30 sec, then rinsed three times in sterilized water. Bacteria were released in sterile water in Petri dish for 2 min by cutting symptomatic tissue into small sections and stirring the plant tissue mixture fully. The diffusate was streaked onto plates containing nutrient agar (NA) and plates were incubated at 28℃ for 2 days. Three representative isolates were purified eventually from each of the plates. Colonies on NA were small, round and with smooth margins. All bacterial isolates characterized as gram-negative, white to cream color, and pink pigment was formed on the plates over long-term culture. The isolates were positive for catalase, Voges-Proskauer, potato rot, methyl red, acetoin production, nitrate utilization and citrate utilization, and acid production from maltose, glucose, melezitose, sucrose, D-arabinose, D-trehalose, cellobiose, lactose, raffinose, mannitol, D-sorbitol, melibiose and xylitol. KOH production was demonstrated according to strand formation within the potassium hydroxide test (Suslow et al. 1982). Isolates were negative for oxidase, arginine dihydrolase, phenylalanine deaminase, gelatinase, esculine, indole production and H2S production. Total genomic DNA was extracted from isolate XHL2002230201 with TIANamp Bacteria DNA Kit (TIANGEN). Universal primers 27F and 1492R (Monciardini et al. 2002) were used in PCR to amplify a 1,307-bp DNA fragment of the 16S rRNA region for molecular identification. Furthermore, four additional housekeeping genes (gyrB, atpD, rho, and rpoS) were selected and amplified using specially designed primers. The amplification products of 16S rRNA were sequenced and submitted to GenBank under accession number (MT568607.1). Sequence analysis showed 99% similarity to Erwinia persicina strains B57 (LM651373.1) and B64 (CI789_17875) by BLAST search in GenBank database (Gálvez et al. 2015; Cho et al. 2019). A phylogenetic tree was constructed, and the taxonomic position of strain XHL2002230201 was determined from the multilocus sequence analysis (MLSA) on 16S rRNA and other four housekeeping genes with E. persicina and not with other closely related Erwinia species. Pathogenicity tests and re-isolation and re-identification of the bacteria were performed to confirm the isolate and fulfill the Koch' postulates. The strain XHL2002230201 suspensions (108 CFU ml-1) were spray inoculated onto fifteen Cucurbita pepo seedlings with two true leaves, and the same number of control plants were inoculated with water. Experiments were repeated three times. All inoculated plants were kept in a moist chamber placed in a greenhouse at 28℃. Initial symptoms were observed on leaves of inoculated plants at 5 days post-inoculation, whereas no symptoms appeared on the plants inoculated with sterile distilled water. Based on morphological and biochemical characteristics, phylogenetic analysis, and Koch's postulates, the bacterial isolates were identified as E. persicina. To our knowledge, this is the first report of E. persicina causing leaf spot disease on Cucurbita pepo in China.

Оccurrence of bacterial spot caused by Xanthomonas arboricola pv. pruni on peach and apricot in Serbia.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of leaves and fruits, defoliation, fruit dropping and twigs, branches or trunk cankers in most cultivated and ornamental Prunus species. The bacterium is listed as an EPPO (European and Mediterranean Plant Protection Organisation) A2 quarantine pathogen. Xap was first detected in 2019 on peach [Prunus persica L., unknown cultivar (cv.)] leaves in a 13-year-old orchard located in Irig (GPS: 45°6'10.538'' N, 19°54'8.04'' E), with a disease incidence of 10-20%. Thereafter, Xap was detected in 2020 on apricot (Prunus armeniaca L., cvs. NS4, NS Rodna and Roxana) leaves and fruits in a 5-year-old orchard located in Bešenovo (GPS: 45°04'59.0'' N, 19°41'23.0'' E), with disease incidence of 30-50%. Symptoms on leaves appeared along leaf midribs or margins in form of brown to black spots, with a pale green to yellow halo, evident on both leaf surfaces. The diseased area on leaves dropped out giving a shot-hole appearance, leaves turn yellow and drop prematurely. Bacterial spots of apricot fruits appeared in form of water-soaked or dark brown sunken lesions. Primarly detection of Xap in collected samples was obtained using polymerase chain reaction (PCR) with Xap - specific primers XapY17-F/XapY17-R, after amplifying target fragments of 943-bp (1). Xap reference strain NCPPB 3156 served as positive control. Isolations performed on yeast extract-dextrose-calcium carbonate agar (YDC) resulted in pale yellow, circular, raised and mucoid colonies after 3 days at 26 °C. A total of 20 representative isolates were aerobic, gram negative, catalase positive, oxidase and arginine dihydrolase negative; they induced hypersensitivity reaction, reduced nitrates, hydrolyzed aesculin and gelatine, but not starch, produced H2S, not produced indole (2). NCBI BLASTn search of the ftsX (ABC transporter ATP-binding protein) sequences (1) indicated 100% identity of Serbian isolates with Xap strains XAP HU2 (P. persica, Acc. no. MG049921) and XAP HU1 (P. armeniaca, Acc. no. KY039173) from Hungary. The nucleotide sequences of one isolate from peach (Xp219) and one from apricot (Xp320) were deposited in GenBank under Acc. nos. MT890969 and MT890970, respectively. Pathogenicity was performed on detached peach and apricot leaves for all 20 isolates and on leaves and shoots of potted 1-year-old plants of peach (cv. Vineyard peach) and apricot (cv. NS Rodna) for three isolates from each host (3, 4). Sterile distilled water and reference strain NCPPB 3156 were used as negative and positive controls. On detached leaves all isolates caused typical water-soaked spots 3 days after inoculation (DAI), while 10 DAI spots became brown and necrotic. Same symptoms were appeared on the leaves of potted plants. On peach and apricot shoots water-soaked, slightly reddish lesions emerged on inoculation sites 7-10 DAI, while 20 DAI lesions become dark, circular to elliptical 3.5-4 and 2.5-3 cm in size for peach and apricot, respectively. Negative controls were symptomless. Reisolated bacteria were confirmed to be the same as the original using PCR (1), fulfilling Koch's postulates. This is the first report of Xap in Serbia, which has occurred with a limited distribution in the Fruška Gora region (Vojvodina). Only two orchards in Serbia have been deteched with Xap so far. In the diseased peach orchard Xap was eradicated by uprooting trees. The apricot orchard is still under official control to limit disease spread. Appropriate cultivation practices, national inspection and surveillance is in place to prevent further pathogen spread and establishment to new hosts and regions in Serbia.

Data on proteome of Mycoplasma hominis cultivated with arginine or thymidine as a carbon source.

Mycoplasma hominis is an opportunistic bacterium that can cause acute and chronic infections of the urogenital tract. This bacterium, like all other Mycoplasma species, is characterized by the reduced genome size, and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway. However, several bacteria can use nucleosides as the sole energy source. Biochemical studies using Salmonella typhimurium have shown that three enzymes (thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase) are involved in the thymidine catabolic pathway. All these enzymes are present in M. hominis. For understanding changes in the energy metabolism of M. hominis we performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon source. LC-MS analysis was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Data are available via ProteomeXchange with identifier PXD018714 (https://www.ebi.ac.uk/pride/archive/projects/PXD018714).

Streptococcus downii sp. nov., isolated from the oral cavity of a teenager with Down syndrome

A new α-haemolytic streptococcal strain has been isolated from the dental plaque of a teenager with Down syndrome. Genetic and taxonomic analyses place this Streptococcus within the oralis group. It is a Gram-stain-positive, non-motile, non-spore-forming spherical alpha-haemolytic coccus arranged in chains, and it ferments a large number of monosaccharides and disaccharides, as well as polymeric carbohydrates. It differs biochemically from closely related species of Streptococcus due to its production of α-galactosidase, β-galactosidase and N-acetyl-β-d-glucosaminidase and by the absence of arginine dihydrolase deiminase and IgA1-protease. It grows in a temperature range of 25 to 40 °C (optimal growth temperature at 37 °C) and in a pH range of 4.5 to 8 (optimal pH at 7.0). A phylogenetic analysis based on its 16S and 23S rRNA gene sequences placed it close to Streptococcus dentisani CECT 7747T. The ANIb and ANIm values were 93.19 and 93.61 %, respectively, both below the accepted threshold to designate it as a new species of bacteria. A phylogenetic tree based on its core genome placed it close to Streptococcus oralis subsp. dentisani strain CECT 7747T with a distance in the expanded core phylogeny of 0.1298. The in silico DNA-DNA hybridization value was 52.2 % with respect to the closest species, S. oralis subsp. dentisani CECT 7747T. Based on these data, a new species of bacteria within the genus Streptococcus, family Streptococcaceae and order Lactobacillales is described, for which the name of Streptococcus downii sp. nov. is proposed (type strain CECT 9732T=CCUG 73139T).

Pseudomonas fildesensis sp. nov., a psychrotolerant bacterium isolated from Antarctic soil of King George Island, South Shetland Islands.

The strain KG01T was isolated from a soil sample from King George Island, Antarctica. Cells of KG01T are rod-shaped and motile by means of multiple polar flagella. The absence of arginine dihydrolase activity could be a key feature to readily distinguish KG01T from its closest phylogenetic relative species. The main fatty acids of the strain include summed feature 3 (C16 : 1 ω7c and/or C15 : 0 iso 2-OH), C16 : 0 and C18 : 1 ω7c. Phylogenetic analysis based on the 16S rRNA gene sequence and on a multilocus sequence analysis (MLSA) using housekeeping genes (16S rRNA, rpoB, rpoD, gyrB) were carried out. These analyses allowed us to include the strain within the Pseudomonas fluorescens group, presenting the highest similarity of multilocus sequence with Pseudomonas veronii LMG 17761T (96.67 %). The genome of KG01T was sequenced and in silico compared with genomes of the most closely related species of the P. fluorescens group. The average nucleotide identity (ANIb) and average amino acid identity (AAI) values of the species phylogenetically closest to KG01T were less than 95-96 %, threshold currently accepted to define strain as belonging to a bacterial species, the highest scores being those to Pseudomonas veronii LMG 17761T (87.98 %) and Pseudomonas marginalis ICMP 3553T (91.90 %). Therefore, the phenotypic and genotypic analyses results, allow us to propose that KG01T represents a member of a novel species of the genus Pseudomonas, for which the name Pseudomonas fildesensis is proposed, and KG01T (=CECT 9084T;=DSM 102036T) is established as the type strain .

Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine-ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure-function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase-ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a “bond rotation” catalytic mechanism

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn71, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.

Occurrence of Xanthomonas arboricola pv. pruni Causing Bacterial Leaf Spot and Shot-Hole on Peach in Montenegro

During May and June 2019, bacterial leaf spot was observed on peach (Prunus persica L. Batsch) in a young plantation (2017, 2018) on four different cultivars (Sugar Time, Britney Lane, Royal Bell, and Royal Time) in the Podgorica region of Montenegro. Diseased trees appeared sporadically, and incidence of symptomatic leaves per infected tree was 10 to 15%. Angular water-soaked spots surrounded by chlorotic tissue were observed along leaf midribs or margins, turning light brown in later stages. Necrotic lesions sometimes dropped out, giving the leaves a “shot-hole” appearance. When spots coalesced, large areas of necrotic foliar tissue formed. Eventually leaves turned yellow, resulting in premature defoliation. Margins taken from healthy tissue and necrotic lesions were homogenized in sterile distilled water (SDW) and spread onto YDC agar (Schaad et al. 2001), on which mostly pale yellow, translucent, circular, raised, and mucoid Xanthomonas-like colonies were observed after 3 days incubation at 26°C. Twelve representative isolates coded as Xp1 to 12 (Sugar Time, Xp1 to 3; Britney Lane, Xp4 to 6; Royal Bell, Xp7 to 9; and Royal Time, Xp10 to 12) were aerobic, gram negative, catalase positive, and oxidase and arginine dihydrolase negative; hydrolyzed esculin and gelatin but not starch; produced H₂S but not indole; and did not reduce nitrate (Schaad et al. 2001). All reactions corresponded with the reactions of the reference strain NCPPB 3156 of Xanthomonas arboricola pv. pruni (Xap), causal agent of leaf spot, shot-hole, and canker of stone fruit. The identity of the isolates was confirmed by PCR using the Xap-specific primer pair XapY17-F/XapY17-R for the ABC transporter ATP-binding protein (Pothier et al. 2011). A single unique target band of 943 bp was amplified for all isolates tested. Furthermore, sequencing of the housekeeping genes fyuA, rpoD, and gyrB (Young et al. 2008) showed 100% identity of tested isolates with genomic sequences of Xap strains deposited in the NCBI database (CFBP 411, 3921, 5580, 6653, 7099, and 7100) based on BLAST analysis and a neighbor-joining phylogenetic tree. The sequences of four isolates representing each cultivar were deposited in GenBank under accession numbers MN520623 to 26 for fyuA, MN520627 to 30 for rpoD, and MN520631 to 34 for gyrB gene for Xp1, Xp4, Xp7, and Xp10, respectively. Pathogenicity was confirmed on (i) young, fully expanded, healthy, detached peach leaves using a hypodermic syringe without a needle, pressed against the abaxial side of the leaf (bacterial suspension at 10⁷ CFU/ml from a 72-h YDC culture) and (ii) immature fruits stabbed and pressing the bacterial suspension (10⁷ CFU/ml) until overflow. Xap reference strain NCPPB 3156 served as a positive and SDW as a negative control. Inoculated leaves were kept under controlled conditions at 25°C, 16/8-h day/night photoperiod, and 60 to 80% relative humidity. All tested bacterial isolates and the reference strain developed water-soaked spots on leaves 3 days and on fruits 4 days after inoculation. Ten days after inoculation the spots became dark brown and necrotic. SDW-treated controls were negative. Xap was successfully reisolated and confirmed to be Xap using PCR (XapY17-F/XapY17-R). This study demonstrates Xap on peach as a new host in Montenegro. Xap was previously reported from other (15 of 44) European countries (https://gd.eppo.int/taxon/XANTPR/distribution), mainly on peach and plum, and from Montenegro on almond (Panic et al. 1998). As the causal agent of a major disease of stone fruits, Xap is a quarantine organism in Europe (EPPO A2 List of Pests recommended for regulation for EU). Therefore, it is necessary to implement containment and/or eradication measures to prevent further spread to new areas and/or new hosts. Furthermore, a nation-wide survey on the (re)occurrence of Xap in other hosts such as almond (reported by Panic et al. 1998), apricot, and plum advised.

Pseudomonas tructae sp. nov., novel species isolated from rainbow trout kidney.

This study describes the biochemical and phylogenetic characteristics of a Gram-negative strain, SNU WT1T, isolated from rainbow trout kidney. The 16S rRNA gene sequencing indicated that strain SNU WT1T was highly similar to Pseudomonas wadenswilerensis CCOS 864T and closely related to other Pseudomonas putida-related strains. Multilocus sequence analysis of concatenated partial gyrB, rpoB and rpoD sequences revealed that strain SNU WT1T was distinct from P. putida-related strains and formed a separate clade. The average nucleotide identity and Genome-to-Genome Distance Calculator values were 90.19 and 41.7 %with its closest relative P. wadenswilerensis CCOS 864T; however, it was phenotypically distinct from CCOS 864T with respect to arginine dihydrolase, glucose fermentation, aesculin hydrolysis and N-acetyl-glucosamine assimilation. The major polar lipid of the strain was phosphatidylethanolamine and the major quinone was Q-9. The genome of strain SNU WT1T had 5 685 196 bp with a G+C content of 61.83 mol%. We describe a novel species of genus Pseudomonas, for which the name Pseudomonastructae has been proposed, with SNU WT1T (=KCTC 72265=JCM 33436) as the type strain.

Sphingomonas populi sp. nov., isolated from bark of Populus × euramericana.

One Gram-stain-negative, aerobic, motile bacterial strain, 3-7T, was isolated from symptomatic canker bark tissue of Populus × euramericana. 16S rRNA gene sequence data revealed that the novel isolate shared highest similarity with Sphingomonas panacis DCY99T (98.8 %), and <96.5 % sequence similarity with all other species with validly published names. Growth occurred between 15 and 37 °C and at pH 6.0-9.0, and optimal growth occurred at 30 °C and pH 7.0-8.0. Nitrogen was produced from nitrates. The strain was positive for acetoin production and activity of catalase, oxidase, β-galactosidase, arginine dihydrolase and β-glucosidase. The major fatty acids were C16 : 0, C18 : 1ω7c and/or C18 : 1ω6c. The polar lipids of the novel isolate included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid, glycolipid, two uncharacterized phospholipids and two uncharacterized lipids. The respiratory quinones detected in isolate 3-7T were Q-10 (96.9 %) and Q-9 (3.1 %). The DNA G+C content was 65.1 mol%. Based on phenotypic and genotypic characteristics, the isolate represents a novel species within the genus Sphingomonas, for which the name Sphingomonas populi is proposed. The type strain is 3-7T (=CFCC 11561T=LMG 30138T).

Pseudomonas nosocomialis sp. nov., isolated from clinical specimens.

Strains A31/70T, CCUG 58779 and SD129 were Gram-stain-negative, short rods, motile by one polar flagellum and isolated from clinical specimens in Botswana, Sweden and Spain, respectively. The 16S rRNA sequence similarity values grouped them in the Pseudomonas stutzeri phylogenetic group of species. The DNA G+C content ranged from 65.5 to 65.7 mol%. The strains were characterized phenotypically by the API 20NE and Biolog GEN III tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and by their fatty acid contents. The absence of the arginine dihydrolase and the hydrolysis of gelatin differentiated these strains from the closest species, Pseudomonas azotifigens. The major fatty acid contents were summed feature 8 (38.6 %), C16 : 0 (22.6 %), summed feature 3 (20.5 %) and C12 : 0 (8.4 %). Multilocus sequence analysis with three housekeeping gene sequences (rpoD, gyrB and 16S rRNA) together with whole-genome comparisons indicated that these strains cluster together in the phylogenetic analysis and their similarity values were lower than the thresholds established for species in the genus Pseudomonas. These results permit us to conclude that strains A31/70T, CCUG 58779 and SD129 belong to a novel species in the genus Pseudomonas for which the name Pseudomonas nosocomialis sp. nov. is proposed. The type strain is A31/70T (=CECT 9981T=CCUG 73638T).

Plasmid Profiles of Pseudomonas syringae pv. savastanoi.

Pseudomonas syringae pv. savastanoi (Psv) is the causal agent of olive knot disease. Forty nine bacterial isolates of Psv were isolated from knots on several hosts at the western area of Libya: 31 isolates from olive Olea europaea, 17 isolates from athel Tamarix aphylla (on which the disease is documented for the first time) and one isolate from retem Retama raetam. The isolates were identified on the basis of their morphological characteristics and LOPAT profile. They produced round, white creamy colonies on selective media (PAB and KB), from which 15 isolates produced fluorescent pigments. With the exception of other LOPAT analysis, all isolates were pectinolytic activity and arginine dihydrolase negative. some isolates were levan positive (10 isolates) and oxidase positive (12 isolates), while the rest of isolates were negative for both tests. Most of the isolates induced a hypersensitive reaction on tobacco and pepper leaves. Plasmid profile analysis of Psv strains indicated high genetic variability between the isolates of the same host or different hosts. Most of the olive isolates were classified according to their plasmid profile into five groups (A, B, C, D, F), however, the athel isolates were separated into three different groups designated as G, K, N, on the other hand, group E and H contained mixed isolates from different hosts: group H included two isolates from olive (OS25w and OS42w) and one isolate from retem (Ra1); only two strains OS6w and Ta5y from olive and athel respectively were classified within the same group designated as E. The remaining seven isolates from all hosts were unique. The total number of plasmids ranged from 1-4 for the strains tested, while the DNA content varied widely ranging from 540 to 13550 bp. No plasmid were detected in 14 isolates tested. Genome analysis based on plasmid profiles indicated the great potential of this technique to discriminate between the isolates of Psv from different hosts and geographical regions.

First Report of Pseudomonas syringae pv. aptata Causing Bacterial Leaf Spot on Common Beet (Beta vulgaris) in Ohio

In July 2017, common beet leaves (cv. Red Ace) from an open field in Erie County, Ohio, showing foliar blight symptoms were received by the OSU Vegetable Pathology Laboratory. Another beet sample (cv. Boro) from Wayne County, Ohio, displaying the same symptoms was received in June 2018. Symptoms consisted of pale brown circular water-soaked necrotic lesions, 2 to 3 mm in diameter, surrounded by a red halo. Bacterial streaming was observed from the lesions when viewed microscopically. Pieces of symptomatic tissue ∼0.25 cm² were surface sterilized in 70% ethanol for 30 s, rinsed in sterile water, placed in 100 µl of sterile water, and minced with a sterile blade. After 30 s, 10 µl of the resulting suspension was streaked onto Pseudomonas F agar medium (PF) and incubated in the dark for 48 h at 28°C. White creamy colonies fluorescent on PF when exposed to UV light were selected. Isolates from both samples were gram negative, oxidase negative, arginine dihydrolase negative, levan positive, and caused a hypersensitive reaction 24 h after infiltration into tobacco (Nicotiana tabacum) leaves. None of the isolates caused soft rot when inoculated at 10⁷ CFU/ml onto sterile potato slices. Box PCR (Louws et al. 1994) profiles were similar for strain SM200-2017 (Erie County) and SM61-2018 (Wayne County), with an additional band in SM200-2017. Both strains were subjected to whole-cell DNA extraction and multilocus sequence analysis of the housekeeping genes sigma factor 70, rpoD; glyceraldehyde-3-phosphate dehydrogenase, gapA; DNA gyrase B, gyrB; and citrate synthase, gltA (Hwang et al. 2005). Sequences were compared with those of the reference pathotype strain Pseudomonas syringae pv. aptata (Psa) CFBP 1617 deposited in the PAMDB database (Almeida et al. 2010). Both SM200-2017 (MK085510) and SM61-2018 (MK085511) showed 100% identity to the rpoD gene. SM200-2017 gapA sequence (MK085516) shared 99% homology with the gapA sequence of Psa CFBP 1617, and SM61-2018 gapA sequence (MK085517) was 100% identical. Similarly, the gyrB sequences from SM200-2017 (MK085515) and SM61-2018 (MK085514) were 99 and 100% similar to Psa CFBP 1617, respectively. No match was found for SM200-2017 and SM61-2018 gltA sequences (MK085512 and MK085513, respectively) when compared with those of Psa CFBP 1617. However, the sequences were 100% identical to Psa sequence KX226440 (Arabiat et al. 2016) deposited in the NCBI database. Koch’s postulates were confirmed in a greenhouse by spray inoculating 4-week-old beet seedlings (cv. Red Ace) with 10⁶ CFU/ml water suspensions of SM200-2017 or SM61-2018, or with sterile water. Plants were maintained at 27°C with a 14-h photoperiod, in plastic bags for the first 48 h. Symptom development was monitored daily. Water-soaked necrotic lesions similar to those observed on the original samples appeared 10 days after inoculation, whereas mock-inoculated plants did not show any symptoms. Symptomatic leaves were collected and processed as described above. Colonies with the same morphology on PF and with the same biochemical characteristics previously described were isolated. Box PCR profiles matched those of the original strains. To our knowledge, this is the first report of bacterial leaf spot of beet caused by Psa in Ohio. Common beets are cultivated throughout Ohio and can be severely affected under disease-conducive environmental conditions.

First Report of Pseudomonas viridiflava Causing Bacterial Blight on Globe Artichoke in Turkey

The production of globe artichoke (Cynara scolymus L.), which is a major part of Mediterranean cuisine and packs an abundance of nutritional value, has been increasing over recent decades in Turkey. In the spring of 2018, globe artichoke (cv. Sakiz) plants with blight symptoms on outer bracts, dark brown sunken lesion on inner sides, red-brown areas on margins, and small and malformed buds were found in artichoke-growing areas in the Karaburun district of Izmir, Turkey. In some areas, disease incidence was up to 5% of the plants. Several buds in harvest stage exhibiting symptoms were collected from 30-ha artichoke production fields that have heavy soil texture and humid weather conditions and processed to identify the causal agent on 15 artichoke samples. Symptomatic outer bracts showing characteristics of bacterial blight symptoms as well as spines were surface sterilized with 1% NaOCl, rinsed in sterilized distilled water (SDW), and cultured on King’s medium B (KB) for 24 h at 28°C. After incubation, a predominant bacterium was isolated from all samples. Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. Round, flat, and blue-green translucent colonies fluorescing under 360-nm UV light when grown on KB medium were observed in all samples taken. The isolates caused soft rot of potato tubers, induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: – – + – +. Acid was produced from mannitol, erythritol glycerol, D-sorbitol, D-mannose, and inositol but not from L-tyrosine, D-cellobiose, D-arabinose, and D-trehalose, all of which are typical characteristics of Pseudomonas viridiflava (Lelliott et al. 1966; Schaad et al. 2001). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using the universal bacterial primers 8F and 1492R (Galkiewicz and Kellogg 2008) and gyrase subunit B (gyrB) gene using primers gyrB-Rps/gyrB-Fps (Sawada et al. 1999). The sequences were submitted to GenBank under accession numbers MH763694 for 16S and MK546431 for gyrB. BLASTn analysis of the 1,042-bp amplicon for 16S and 545-bp amplicon for gyrB indicated that it had 99% similarity with P. viridiflava strain 4G1113 (accession no. MG972916) and 100% similarity with P. viridiflava ATCC 13223 strain (accession no. KY612187), respectively. Pathogenicity tests were conducted twice on outer bracts and main stems of artichoke cultivar Sakiz by inoculating 20 µl of bacterial suspension of a 24-h culture in KB medium (1.9 × 10⁹ CFU/ml in SDW). The inoculated plants were placed in a transparent box for 7 days and kept at 25°C and 70% relative humidity. The bract leaves inoculated with only SDW did not develop symptoms; however, the bracts treated with isolated bacterial suspension developed blight symptoms similar to those observed in the field, 7 days postinoculation. The stems inoculated with the isolate also exhibited soft rot symptoms, and the same bacterium was consistently reisolated from both bracts and stems but not from parts of negative control plants. Reisolated bacteria exhibited the same biochemical profile with our isolate, thereby fulfilling Koch’s postulates. P. viridiflava causing a bacterial blight of artichoke bracts was previously reported in Greece (Sarris et al. 2012). To our knowledge, it is the first report of this bacterium causing bacterial blight on leaves and soft rot on stems of globe artichoke in Turkey. Given the availability of climatic conditions, the potential of disease spreading can cause a serious reduction in yield and marketability of this vegetable, which has increasing popularity owing to having high nutritional value, leading to substantial economic losses.

Pseudomonas edaphica sp. nov., isolated from rhizospheric soil of Cistus ladanifer L. in Spain.

During a study on biodiversity of bacteria inhabiting rhizospheric soil of rockrose (Cistus ladanifer L.), we isolated a strain coded RD25T in a soil from Northern Spain. The 16S rRNA gene sequence showed 99.5 % identity with respect to the closest related species Pseudomonas brenneri DSM15294T, and 99.4 % with respect to P. paralactis WS4672T. The following related Pseudomonas species showed 99.3 % or less identity, and therefore RD25T was classified within genus Pseudomonas. The phylogenetic analysis of 16S rRNA and the housekeeping genes rpoB, rpoD and gyrB suggested that this strain could be a novel species. The strain RD25T has several polar-subpolar flagella. It can grow at 36 °C, at 0-6 % NaCl concentration and a range of pH 5-9. Positive for arginine dihydrolase and urease production, and negative for reduction of nitrate. The strain is catalase and oxidase positive. Major fatty acids are C16 : 1 ω7c / C16 : 1 ω6c in summed feature 3, C16 : 0, and C18 : 1 ω7c / C18 : 1 ω6c in summed feature 8. The respiratory ubiquinone is Q9. The DNA G+C content was 59.9 mol%. The digital DNA-DNA hybridisation average values (dDDH) ranged between 30-61.2 % relatedness and the ANIb values ranged between 93.9-80.5 % with respect to the type strains of the closely related species. Therefore, the genotypic, genomic, phenotypic and chemotaxonomic data support the classification of strain RD25 as a novel species of genus Pseudomonas, for which the name P. edaphica sp. nov. is proposed. The type strain is RD25T (=LMG 30152T=CECT 9373T).

Pseudomonas bijieensis sp. nov., isolated from cornfield soil.

Strain L22-9T, a Gram-stain-negative and rod-shaped bacterium, motile by one polar flagellum, was isolated from cornfield soil in Bijie, Guizhou Province, PR China. Based on 16S rRNA gene sequences, it was identified as a Pseudomonas species. Multilocus sequence analysis of concatenated 16S rRNA, gyrB, rpoB and rpoD gene sequences showed that strain L22-9T formed a clearly separated branch, located in a cluster together with Pseudomonas brassicacearum LMG 21623T, Pseudomonas kilonensis DSM 13647T and Pseudomonas thivervalensis DSM 13194T. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) confirmed that strain L22-9T should be classified as a novel species. It was most closely related to P. kilonensis DSM 13647T with ANI and dDDH values of 91.87 and 46.3 %, respectively. Phenotypic features that can distinguish strain L22-9T from P. kilonensis DSM 13647T are the assimilation ability of N-acetyl-d-glucosamine, poor activity of arginine dihydrolase and failure to ferment ribose and d-fucose. The predominant cellular fatty acids of strain L22-9T are C16 : 0, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The respiratory quinones consist of Q-9 and Q-8. The polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, two unidentified phosphoglycolipids, two unidentified aminophospholipids and an unidentified glycolipid. Based on the evidence, we conclude that strain L22-9T represents a novel species, for which the name Pseudomonas bijieensis sp. nov. is proposed. The type strain is L22-9T (=CGMCC 1.18528T=LMG 31948T), with a DNA G+C content of 60.85 mol%.

First Report of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch, on Melon (Cucumis melo) in Guadeloupe (France)

HomePlant DiseaseVol. 103, No. 5First Report of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch, on Melon (Cucumis melo) in Guadeloupe (France) PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch, on Melon (Cucumis melo) in Guadeloupe (France)A. Cunty, C. Audusseau, S. Paillard, V. Olivier, C. François, C. Rivoal, and F. PoliakoffA. Cunty†Corresponding author: A. Cunty; E-mail Address: [email protected]http://orcid.org/0000-0001-8174-587XLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, C. AudusseauLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, S. PaillardLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, V. OlivierLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, C. FrançoisLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, C. RivoalLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this author, and F. PoliakoffLaboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, FranceSearch for more papers by this authorAffiliationsAuthors and Affiliations A. Cunty † C. Audusseau S. Paillard V. Olivier C. François C. Rivoal F. Poliakoff Laboratoire de la Santé des Végétaux, Agence Nationale de Sécurité Sanitaire, 49044 Angers, France Published Online:15 Mar 2019https://doi.org/10.1094/PDIS-10-18-1825-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In March 2015, symptomatic melon (Cucumis melo) plants and fruits were received from the French West Indies Island, Guadeloupe, at the Anses Plant Health Laboratory (LSV). The leaves showed brown necrotic spots surrounding by a chlorotic halo. The fruits presented small and large water-soaked lesions on the rind that did extend into the flesh, and dark brown necrotic spots into the flesh were observed under some of the lesions. The yield loss was estimated at around 50%. Symptoms were first suspected to be associated with the presence of a pathovar of Pseudomonas syringae, but the isolated strain was identified as Acidovorax citrulli and the pathogenicity was confirmed. Because leaf samples were damaged during transport (development of rot), isolations were only performed from lesions on the fruits on modified King’s medium B (mKB) supplemented with cycloheximide (50 mg/liter). A gram-negative, motile, oxidative, nonfluorescent bacterium was isolated (LSV 46.36 and then registered in the French CIRM–Plant Associated Bacteria collection as CFBP 8568). It induced a hypersensitive reaction on tobacco, had a cytochrome c oxidase activity, did not display an arginine dihydrolase activity nor hydrolyze aesculin or gelatin, did not produce levan, used l-arabinose and glucose, but did not use cellobiose and rhamnose. When plated on mKB, the colonies grew at 41°C. The strain CFBP 8568 was compared with the A. citrulli type strain CFBP 4459. Whole cell DNA, extracted by boiling bacterial suspension (1 × 106 CFU/ml) for 10 min at 100°C, was amplified by the duplex polymerase chain reaction (PCR) developed by Bahar et al. (2008). In specific, two fragments of 476 and 279 bp were obtained for both strains, indicating that the strain CFBP 8568 was A. citrulli. The real-time PCR described in appendixes 4 and 6 of the EPPO protocol PM 7/127 (EPPO 2016), which targeted complementary genes, were also applied to the DNA extract and confirmed the previous results. Application of the PCR assay developed by Zivanovic and Walcott (2017) allowed classifying the strain CFBP 8568 in group I. To fulfill all Koch’s postulates, a pathogenicity test was performed. Three melon seedlings (Charentais type) were inoculated by infiltrating a first true leaf with a cell suspension (1 × 107 CFU/ml) of the strain CFBP 8568. The type strain CFBP 4459 was used as a positive control. After 13 days, brown necrotic spots surrounded by a chlorotic halo were observed at the inoculation points for both strains. From those necrotic spots, strains with all the characteristics of A. citrulli (as previously described) were isolated. No symptoms were observed on the stem, which may be because the test was stopped just after the appearance of necrotic leaf spots. To our knowledge, this is the first report of bacterial fruit blotch (BFB) in Guadeloupe. Prophylactic measures and a strong sanitary survey of this crop allowed eradication of the outbreak. Indeed, no symptoms have been reported in 2018. However, this first report of BFB showed that it could represent a threat in Caribbean area, were it was never reported before.