Malonate Utilization Research Articles

Malonate is relevant to the lung environment and induces genome-wide stress responses in Pseudomonas aeruginosa.

Versatility in carbon source utilization is a major contributor to niche adaptation in Pseudomonas aeruginosa. Malonate is among the abundant carbon sources in the lung airways, yet it is understudied. Recently, we characterized how malonate impacts quorum sensing regulation, antibiotic resistance, and virulence factor production in P. aeruginosa. Herein, we show that malonate as a carbon source supports more robust growth in comparison to glycerol in several cystic fibrosis isolates of P. aeruginosa. Furthermore, we show phenotypic responses to malonate were conserved among clinical strains, i.e., formation of biomineralized biofilm-like aggregates, increased tolerance to kanamycin, and increased susceptibility to norfloxacin. Moreover, we explored transcriptional adaptations of P. aeruginosa UCBPP-PA14 (PA14) in response to malonate versus glycerol as a sole carbon source using transcriptomics. Malonate utilization activated glyoxylate and methylcitrate cycles and induced several stress responses, including oxidative, anaerobic, and metal stress responses associated with increases in intracellular aluminum and strontium. We identified several genes that were required for optimal growth of P. aeruginosa in malonate. Our findings reveal important remodeling of P. aeruginosa gene expression during its growth on malonate as a sole carbon source that is accompanied by several important phenotypic changes. These findings add to the accumulating literature highlighting the role of different carbon sources in the physiology of P. aeruginosa and its niche adaptation.

Plant growth promoting, biochemical and antifungal properties of microbes isolated from cow dung

Cow dung is a rich source of microbial diversity and being used since long time in India for seed treatment, plastering cut ends of vegetative propagated plant parts and sprinkling of diluted solution on crops. In present study, microbes from fresh cow dung of indigenous cow were isolated and biochemically characterized. Cow dung enriched with diversity of microorganisms in different medium i.e 38 x 106cfu g-1 (Nutrient agar), 26.0 x 106 cfu g-1 (Actinomycetes isolation agar), 36 x 106 cfu g-1 (Kings B agar), 16 x 106 cfu g-1(CV agar),13 x 106 cfu g-1 (Methyl red), 15 x 106 cfu g-1 (Pikovskyas agar), 30 x 106 cfu g-1 (CRYEMA), Azospirillum 70 x 106 cfu g-1 (N-free maleate) and 30 x 106 cfu g-1(Jenson agar medium). Based on microbial enumeration, total 18 bacterial isolates were selected for biochemical, PGPR characterization and biocontrol activity. All isolates were tested positive for phosphate and Zn solubilisation. Isolate CD2, CD5, CD13, CD14 and CD19 were tested highly positive for zinc, phosphate, K-solubilisation, IAA and amylase production. Test isolates CD2, CD5, CD6, CD7, CD10 and CD13 were tested highly positive for β-galactosidase activity, ornithine utilization, nitrate reduction, citrate utilization, malonate utilization, esculin hydrolysis, trehalose, glucose and lactose utilization. All isolated microbes tested for biocontrol activity (antifungal property) against standard pathogen procured from microbial type culture collection (MTCC), i.e. C. gloeosporioides (MTCC 2190), F. oxysporum (MTCC 10247), C. fimbriota (MTCC 2281) and P. aphanidermatum (MTCC 284). CD2, CD34 and CD20 isolates significantly inhibited the growth of C. gloeosporioides and P. aphanidermatum. Result of the study advocates that cow dung and cow dung based formulations are cheap source for soil, plant health and management of Colletotrichum gloeosporioides, P. aphanidermatum and Fusarium oxysporum in various crops.

Directed evolution of tripartite ATP-independent periplasmic transporter for 3-Hydroxypropionate biosynthesis.

Our previous study's introduction of the malonic acid assimilation pathway into Escherichia coli enabled biosynthesis of 3-Hydroxypropionate (3-HP) from malonate. However, the relatively low uptake activity of tripartite ATP-independent periplasmic (TRAP) malonic acid transporter (MatPQM) is considered rate-limiting in malonate utilization. Here, to improve the transport performance of this importer, MatP variants were obtained via directed evolution and a novel developed enzyme-inhibition-based high throughput screening approach. This plate chromogenic screening method is based on the fact that malonic acid inhibits both of succinate dehydrogenase activity and further the capability of the reduction of methylene-blue to methylene-white. The best mutant E103G/S194G/Y218H/L235P/N272S showed twofold increased transport efficiency compared to the wild-type. ITC assay and structural analysis revealed that increased binding affinity of the mutant to the ligand was the reason for improved uptake activity of MatPQM. Finally, the engineered strain harboring the evolved mutant produced 20.08g/L 3-HP with the yield of 0.87mol/mol malonate in a bioreactor. Therefore, the well-established directed evolution strategy can be regarded as the reference work for other TRAP-type transporters engineering. And, this transporter mutant with enhanced malonic acid uptake activity has broad applications in the microbial biosynthesis of malonyl-CoA-derived valuable compounds in bacteria. KEY POINTS: • We reported directed evolution of a TRAP-type malonic acid transporter. • We found the enhanced malonate uptake activity of mutant lies in improved affinity. •We enhanced 3-HP bioproduction with high yield by employing the best mutant.

Molecular Characterization of Plant Growth Promoting Pseudomonas from Rhizosphere of Euphorbia caducifolia Haines

A study was conducted during kharif 2020 at the Department of Microbiology, Chimanbhai Patel College of Agriculture, S. D. Agricultural University for molecular characterization of plant growth promoting Pseudomonas isolates from rhizosphere of Euphorbia caducifolia. Twenty Pseudomonas isolates (designated as PsEc1 to PsEc20) were axenically isolated from rhizospheric soil sample of Euphorbia caducifolia using Pseudomonas Isolation Agar Base (a selective medium). These isolates were tested gram negative, rod shaped, and oxidase positive. All 20 isolates were positive for utilization of maltose, esculin, citrate and malonate whereas these isolates showed varied results for utilization of xylose, fructose, galactose, sucrose and sorbitol. 16Sr DNA sequencing data revealed similarity of the isolates to different species of Pseudomonas, i.e., P. fluroscens (11), P. aeruginosa (3), P. putida (3), P. stutzeri (2) and P. syringe (1). These nucleotide sequences were submitted to the NCBI GenBank database with accession numbers respectively MT775484 to MT775503. The 16S rDNA sequences were aligned and used to reconstruct a phylogenetic tree with bootstrap values using software MEGA 6.06. The phylogenetic tree comprised of three clades. Clade I comprised of sixteen isolates, clade II four isolates and clade III only one isolate. The members of clade I belonged to P. fluorescens, P. putida and P. stutzeri whereas members of clade II comprised of P. aeruginosa. However, clade III consisted of P. syringae. In vitro test showed PsEc17 (P. fluorescens, MT775500) as most efficient in solubilizing tricalcium phosphate (78.53 µg ml-1) and IAA production (78.53 µg ml-1). PsEc17 also proved most efficient and showed 69.5% inhibition of Fusarium oxysporum f. sp. cumini.

Providencia heimbachae Associated with Post-weaning Diarrhea in Piglets: Identification, Phenotype, and Pathogenesis.

Despites Providencia heimbachae has been isolated from human, penguin, and bovine fetus, relatively little information is available regarding the pathogenicity and biologic characteristics of P. heimbachae. Here, we report that investigation of post-weaning diarrhea yielded bacterial isolates identified as P. heimbachae based on the biochemical tests and 16S ribosomal DNA sequence analysis. The two isolates were positive for utilization of Malonate, no gas production from glucose, and non-fermentation of D-mannitol, D-Galactose, and L-Rhamnose that were different from those of the type strain, and both of them have the ability of adhesion and invasion to IPEC-J2 cells, and were resistant to 21 out of the 41 antibiotics tested. In addition, the isolate 99101 was highly pathogenic to mice and piglets. Histopathology studies on nerve tissue of piglets that developed hindlimb paralysis showed microglia cell infiltration and neuron damage in the spinal cord. Notably, the strains could grow under low temperature (4°C), which raise attention of a new risk factor for food safety. To the best of our knowledge, this is the first report of P. heimbachae strain caused post-weaning diarrhea in piglets in both natural and experimental conditions. These findings extended the knowledge of P. heimbachae as an important zoonotic agent, which should be given more attention during surveillance and diagnostics.

Biomolecular characterization and efficacy evaluation of pathogen Erwinia tracheiphila against plant oils

Aim: To investigate patho-dyanamics of Erwinia tracheiphila on Cucurbita pepo with biochemical, molecular characterization and eco-friendly management approaches. Methodology: Variability study of Erwinia tracheiphila was done by collecting the isolates from different places in Odisha. Disease severity was calculated and analyzed from 2016-18 to predict trend of emergence and disease progress. Molecular detection of pathogen was done through 16S rRNA sequencing. Biochemical characterization and identification of pathogenic nature of Erwinia tracheiphila was performed through Ortho-Nitrophenyl β-Galactoside, Esculin hydrolysis, Citrate and Malonate utilization in the presence of different carbohydrate molecules. Efficacy of eight different plant oils were tested by in-vitro inhibition zone (IVIZ) assay against Erwinia tracheiphila as eco-friendly management approaches. Results: Analysis of disease severity data from 2016-18 ranged between 29% to 33%. Molecular detection of causal pathogen was further confirmed by 16S rRNA region sequencing with 99% identity of NCBI-GenBank published Erwinia tracheiphila (MK356446 and MK356441) database and phylogenetic analysis. The results of IVIZT revealed that the clove oil treatment showed maximum inhibitory effect on the growth of Erwinia tracheiphila followed by mustard oil and neem oil. Sesamum oil showed the lowest efficacy against Erwinia tracheiphila. Optimum temperature range between 25-28°C, 80-85% relative humidity and 150-180 mm average rainfall triggered the disease progress and turned to be a severe one under experimental agro-climatic situation. Interpretation: Biochemical characterization and molecular detection of causal isolate confirmed the pathogen as Erwinia tracheiphila, causing wilt disease of Cucurbita pepo. This study also revealed the potential efficacy of clove oil and mustard oil against Erwinia tracheiphila as compared to other selected plant oils.

Malonate utilization by Pseudomonas aeruginosa affects quorum-sensing and virulence and leads to formation of mineralized biofilm-like structures.

Pseudomonas aeruginosa is an opportunistic pathogen that uses malonate among its many carbon sources. We recently reported that, when grown in blood from trauma patients, P. aeruginosa expression of malonate utilization genes was upregulated. In this study, we explored the role of malonate utilization and its contribution to P. aeruginosa virulence. We grew P. aeruginosa strain PA14 in M9 minimal medium containing malonate (MM9) or glycerol (GM9) as a sole carbon source and assessed the effect of the growth on quorum sensing, virulence factors, and antibiotic resistance. Growth of PA14 in MM9, compared to GM9, reduced the production of elastases, rhamnolipids, and pyoverdine; enhanced the production of pyocyanin and catalase; and increased its sensitivity to norfloxacin. Growth in MM9 decreased extracellular levels of N-acylhomoserine lactone autoinducers, an effect likely associated with increased pH of the culture medium; but had little effect on extracellular levels of PQS. At 18hr of growth in MM9, PA14 formed biofilm-like structures or aggregates that were associated with biomineralization, which was related to increased pH of the culture medium. These results suggest that malonate significantly impacts P. aeruginosa pathogenesis by influencing the quorum sensing systems, the production of virulence factors, biofilm formation, and antibiotic resistance.

Isolation and identification of two Serratia marcescens strains from silkworm, Bombyx mori.

Bacterial septicemia commonly occurs and usually cause huge losses in sericulture industry. Here, two pathogenic bacterial strains were isolated from dead silkworm and named as ZJ-1 and ZJ-2. Phenotypic and genotypic analysis results revealed that both of these two strains are closely related to Serratia marcescens (S. marcescens). The morphological as well as physiological and biochemical characteristics of ZJ-1 were accordant with S. marcescens mentioned in Bergey's manual of determinative bacteriology, whereas ZJ-2 showed some discrepancies such as the utilization of malonate and starch, fermentation of maltose and sucrose, and tests of urease, etc. Surprisingly, ZJ-2 could produce red pigment at high temperature (37°) but ZJ-1 could not. Besides, by analyzing the lethal concentration 50 (LC50) of ZJ-1 and ZJ-2, it was found that the virulence of ZJ-2 was lower than that of ZJ-1. These results revealed that ZJ-1 and ZJ-2 were two different strains of S. marcescens and that ZJ-2 was expected to be a safe (low-toxicity) and efficient strain for the production of red pigment. Nonetheless, further research in molecular level is needed to understand the regulation mechanism of pigment production and infection of ZJ-2.

Plant growth promoting, biochemical and antifungal properties of microbes isolated from cow dung

Cow dung is a rich source of microbial diversity and being used since long time in India for seed treatment, plastering cut ends of vegetative propagated plant parts and sprinkling of diluted solution on crops. In present study, microbial isolation from fresh cow (indigenous) dung observed in different generalized and selective medium. Cow dung enriched with diversity of microorganisms as it contained various microbial population as observed in different mediumi.e38 x 106cfu g−1 (in nutrient agar), 26.0 x 106 cfu g−1 (actinomycetes isolation agar), 36 x 106 cfu g−1 (Kings B agar), 16 x 106 cfu g−1 (CV agar),13 x 106 cfu g−1 (Methyl red), 15 x 106 cfu g−1 (Pikovskyas agar), 30 x 106 cfu g−1 (CRYEMA), Azospirillum 70 x 106 cfu g−1 (N-free maleate) and 30 x 106 cfu g−1 (Jenson agar medium). Based on microbial enumeration, total 18 bacterial isolates were selected for biochemical, PGPR characterization and biocontrol activity. All isolates were tested positive for phosphate and Zn solubilisation. Isolate CD2, CD5, CD13, CD14 and CD19 were tested highly positive for zinc, phosphate, K-solubilisation, IAA and amylase production. Test isolates CD2, CD5, CD6, CD7, CD10 and CD13 were tested highly positive for β-galactosidase activity, ornithine utilization, nitrate reduction, citrate utilization, malonate utilization, esculin hydrolysis, trehalose, glucose and lactose utilization. All isolated microbes tested for biocontrol activity (antifungal property) against standard pathogen procured from microbial type culture collection (MTCC), i.e. C. gloeosporioides (MTCC 2190), F. oxysporum (MTCC 10247), C. fimbriota (MTCC 2281) and P. aphanidermatum (MTCC 284). CD2, CD34 and CD20 isolates significantly inhibited the growth of C. gloeosporioides and P. aphanidermatum. Result of the study advocates that cow dung and cow dung based formulations are cheap source for soil, plant health and management of Colletotrichum gloeosporioides, P. aphanidermatum and Fusarium oxysporum in various crops.

Genomic characterization of malonate positive Cronobacter sakazakii serotype O:2, sequence type 64 strains, isolated from clinical, food, and environment samples

BackgroundMalonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains.ResultsIn this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene.ConclusionsUntil this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.

Crystal structure of a Pseudomonas malonate decarboxylase holoenzyme hetero-tetramer

Pseudomonas species and other aerobic bacteria have a biotin-independent malonate decarboxylase that is crucial for their utilization of malonate as the sole carbon and energy source. The malonate decarboxylase holoenzyme contains four subunits, having an acyl-carrier protein (MdcC subunit) with a distinct prosthetic group, as well as decarboxylase (MdcD–MdcE) and acyl-carrier protein transferase (MdcA) catalytic activities. Here we report the crystal structure of a Pseudomonas malonate decarboxylase hetero-tetramer, as well as biochemical and functional studies based on the structural information. We observe a malonate molecule in the active site of MdcA and we also determine the structure of malonate decarboxylase with CoA in the active site of MdcD–MdcE. Both structures provide molecular insights into malonate decarboxylase catalysis. Mutations in the hetero-tetramer interface can abolish holoenzyme formation. Mutations in the hetero-tetramer interface and the active sites can abolish Pseudomonas aeruginosa growth in a defined medium with malonate as the sole carbon source.

PM 7/60 (2) Pantoea stewartii subsp. stewartii

Specific scopeThis Standard describes a diagnostic protocol for Pantoea stewartii subsp. stewartii.Specific approval and amendmentApproved in 2005‐09.Revised in 2016‐04.

Isolation and characterization of heavy metal resistant cellulosimicrobium sp. From paper mill polluted soil

A Gram-positive, rod-shaped, yellow-pigmented bacterium was isolated from the polluted soil of paper mill contaminated with various heavy metals. The strain was tested for its resistance to different heavy metals (Ni, Cu, Zn and Cd) by the growth in nutrient broth tubes containing various concentrations of (0.1, 0.5, 2.0, 4.0 mM). The relative growths (%) at 2mM concentration were observed as Ni (23.52%)>Zn (20.58%)>Cu (19.20%) > Cd (15.81 %). The heavy metal resistant in the isolated strain was found to be Ni>Zn>Cu>Cd at higher concentrations. The strain showed positive activity towards urease, nitrate, citrate utilization, methyl red, Malonate utilization, starch amylase and showed negative activity against ONPG, H 2 S production, phynylalanine, Lysine utilization, Voges Proskauer’s (VP) test, catalase and oxidase activity. The strain was found susceptible to various antibiotics Vancomycin, Streptomycin, Rifamycin Amikacin and Ciprofloxacin. In silico study was conducted to understand the major evolutionary relationship among the different strains of Cellulosimicrobium species of nucleotide sequence of 16s ribosomal RNA with the isolated strain (KC602297) of Cellulosimicrobium sp. obtained from Gene Bank.

FIRST REPORT OF PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM CAUSING SOFT ROT ON WATERMELON IN IRAN

Iran is the 4th largest producer of watermelon (Citrullus lanatus) in the world. In 2012 a disease characterized by water-soaked lesions and soft rot was observed on mature and immature fruits of watermelon cv. Crimson sweet. Fruit samples with transpicuous symptoms were transferred to laboratory and bacterial colonies were isolated from these on nutrient agar. Hypersensitivity reaction (HR) assays (Bauer et al., 1994) were successfully done using 108 CFU/ml bacterial suspension into tobacco leaf epidermis. Bacterial isolates were Gram-negative, facultative anaerobes, able to soften potato slices and growing at 37°C. They were negative for oxidase, urease and sensitivity to erythromycin, positive for catalase, gelatinase and utilization of malonate and citrate. Isolates produced acid from lactose, cellobiose, raffinose and trehalose (Schaad et al., 2001). The pathogenicity of bacterial isolates was confirmed by injecting cell suspension (calibrated at 107 CFU/ml) in watermelon fruits. Symptoms developed on fruits 3 to 4 days post inoculation looking the same as those shown by naturally infected fruits. Control samples injected with sterilized distilled water remained healthy. A 16S ribosomal RNA fragment of 1100 bp was amplified from bacterial isolates and the partial 16S rRNA gene sequence was deposited in GenBank under the accession No. KF956742. Based on phenotypic characteristics and the 99.7% homology of 16S rRNA sequence to Pectobacterium carotovorum subsp. carotovorum (Pcc), the bacterium that causes water-soaked and soft rot of watermelon fruit was identified as Pcc. To our knowledge, this is the first report of soft rot caused by Pcc on watermelon from Iran.

First Report of Bacterial Soft Rot of Horn Lian (Typhonium giganteum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China.

Horn lian (Typhonium giganteum) is a perennial herb of the family Aracea and is commonly used for expelling phlegm and as an antispasmodic treatment. In August 2012, horn lian grown in Changchun, Jilin Province of China, exhibited soft rot disease with ~60% incidence and experienced great losses. Water-soaked and dark green lesions on leaves expanded along main veins. Semitransparent, water-soaked, and sunken lesions on stems expanded rapidly and caused the whole plant to collapse with a foul smell. Nine representative strains were isolated from infected leaves and stems on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent on King's B medium (KB), facultatively anaerobic, motile with three to six peritrichous flagella (observed by electron transmission microscope), positive for catalase and pectolytic activity test on potato slices, but negative for oxidase, urease, and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin and reduced nitrate, but did not reduce sucrose. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose and indole. Results were variable for the Voges-Proskauer test. The strains utilized sucrose, arabinose, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, maltose, raffinose, glycerol, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or dulcitol. Species identity was confirmed by molecular characterization of one of the nine strains, DJL1-2. DNA GC content indicated by high performance liquid chromatography (HPLC) was 51.7%. The 16S rDNA sequence (KC07897) of DJL1-2 showed 99% identity to that of a Pectobacterium carotovorum subsp. carotovorum (Pcc) strain (CP001657) and the sequence of the 16S-23S rDNA spacer region (KJ623257) was 93% similar to that of another known strain of Pcc (CP003776). As a result, the strains were identified as Pcc (2). Pathogenicity of the nine strains was evaluated by spraying 1 ml of bacterial cell suspension (108 CFU/ml) onto healthy leaves and injecting 0.1 ml of cell suspension into stems of 3-year-old horn lian plants with a sterile pipette tip. Three seedlings were used for each strain and sterilized water served as negative controls. Pcc SMG-2 reference strain (from milk thistle) was also inoculated into horn lian leaves and stems. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 72 h, water-soaked lesions similar to the naturally infected plants were observed on leaves and stems inoculated by the nine isolated strains and Pcc SMG-2, while negative control plants remained symptomless. Biochemical tests and 16S rDNA sequence analysis confirmed that the re-isolated bacteria were Pcc. To our knowledge, this is the first report of Pcc causing bacterial soft rot of horn lian in Changchun, Jilin Province, China.

First Report of Bacterial Soft Rot of Milk Thistle (Silybum marianum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China.

Milk thistle (Silybum marianum) is an annual or biannual plant of the Asteraceae family that produces the hepaprotectant silymarin. In 2012, almost all milk thistle grown in the medicinal herbal garden of Jilin Agricultural University (Changchun, Jilin Province, China) exhibited symptoms of a previously undetected soft rot disease. Initial symptoms on stems appeared as tan, semitransparent, and water-soaked, then became sunken. The rotted lesions expanded rapidly and inner stem tissues were rotten with a foul smell. Eventually, the whole plant became black, then collapsed and died. Economic losses were significant as the seed crop was almost completely lost. Nine bacterial strains were isolated from tissues on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies of the nine strains were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent, facultatively anaerobic, motile with two to four peritrichous flagella (observed by electron transmission microscope), positive for catalase and potato rot, but negative for oxidase and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose, and indole. Results were variable for the Voges-Proskauer test and production of H2S from cysteine. The strains utilized esculin, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or starch. Glycerol and maltose were only weakly utilized. Species identity was confirmed by molecular analysis of one of the strains, SMG-2. HPLC indicated a DNA GC content of 50.55%. The 16S rDNA sequence (KC207898) of SMG-2 showed 99% sequence identity to that of a Pectobacterium carotovorum subsp. carotovorum strain (DQ333384) and the sequence of the 16S-23S rDNA spacer region (KJ415377) was 95% similar to that of another known strain of P. carotovorum subsp. carotovorum (AF232684). Based on biochemical and physiological characteristics (2), as well as 16S rDNA gene analysis, the strains were identified as P. carotovorum subsp. carotovorum. Pathogenicity of the nine strains was evaluated by depositing a bacterial suspension (108 CFU/ml) on wounded stems (made with a disinfected razor blade) of 3-month-old milk thistle plants. Three plants were inoculated with each strain and three plants were treated with sterilized water as negative controls. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 48 h, the plants inoculated with bacteria showed similar symptoms as the naturally infected plants, while control plants remained symptomless. The symptoms observed on inoculated stems were rotten and sunken tissues. Bacteria were re-isolated from the inoculated plants and confirmed to be identical to the original strains based on 16S rDNA sequence analysis. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of milk thistle in Changchun, Jilin Province, China.

Genome Sequence of Lactobacillus pentosus KCA1: Vaginal Isolate from a Healthy Premenopausal Woman

The vaginal microbiota, in particular Lactobacillus species, play an important role in female health through modulation of immunity, countering pathogens and maintaining a pH below 4.7. We report the isolation and genome sequence of Lactobacillus pentosus strain KCA1 (formally known as L. plantarum) from the vagina of a healthy Nigerian woman. The genome was sequenced using Illumina GA II technology. The resulting 16,920,226 paired-end reads were assembled with the Velvet tool. Contigs were annotated using the RAST server, and manually curated. A comparative analysis with the available genomes of L. pentosus IG1 and L. plantarum WCFS1 showed that over 15% of the predicted functional activities are found only in this strain. The strain has a chromosome sequence of 3,418,159 bp with a G+C content of 46.4%, and is devoid of plasmids. Novel gene clusters or variants of known genes relative to the reference genomes were found. In particular, the strain has loci encoding additional putative mannose phosphotransferase systems. Clusters of genes include those for utilization of hydantoin, isopropylmalate, malonate, rhamnosides, and genes for assimilation of polyglycans, suggesting the metabolic versatility of L. pentosus KCA1. Loci encoding putative phage defense systems were also found including clustered regularly interspaced short palindromic repeats (CRISPRs), abortive infection (Abi) systems and toxin-antitoxin systems (TA). A putative cluster of genes for biosynthesis of a cyclic bacteriocin precursor, here designated as pentocin KCA1 (penA) were identified. These findings add crucial information for understanding the genomic and geographic diversity of vaginal lactobacilli.

First Report of Enterobacter Bulb Decay of Onions Caused by Enterobacter cloacae in New York.

During the summer of 2010, onions (Allium cepa L.) of several cultivars growing in muck-land soils in Orange, Genesee, Orleans, and Oswego counties of New York exhibited leaf dieback and bulb decay consistent with disease symptoms caused by Enterobacter cloacae as described previously (1,3,4). Isolations of bacteria from symptomatic tissues and muck soil were made using onion extract medium (OEM), which contains extracts of autoclaved onions, salts, and inhibitors of fungi and gram-positive bacteria. Some presumptive strains of E. cloacae were isolated; 5 from symptomatic onions growing in Genesee County, 2 from muck-land soil, and 27 from bulbs stored for ~2.5 months in a farm storage facility in Oswego County. Tentative identification was based on colony morphology (convex, cream-color colonies, 2 to 3 mm in diameter following incubation at 28°C for 1 day on OEM), which was similar to the morphology of reference strains of E. cloacae ATCC 23355, ATCC 13047, and strain 310 (gift of H. F. Schwartz, which was derived from reference 4; personal communication). Strains were gram-negative rods, negative for oxidase and indole, positive for nitrate reductase and catalase; produced acid from glucose aerobically and anaerobically. Also, all strains produced PCR products from the 16S-23S internal transcribed spacer (ITS) DNA region of the predicted sizes using primers T5A and T3B designed for identification of E. cloacae (2). The growth of eight of the isolated strains and strains ATTC 23355 and 310 were evaluated on several carbon sources with RapiD 20E test strips (bio Mérieux, Inc, Durham, NC). All strains were positive for β-d-galactosidase, ornithine decarboxylase, utilization of citrate and malonate, and production of acetoin. Hydrolysis of esculin by β-glucosidase differed among the eight. All strains were negative for lysine decarboxylase, urease, para-phenylalanine deaminase, indole, and oxidase. All produced acid from arabinose, xylose, rhamnose, cellobiose, melibiose, saccharose, trehalose, raffinose, and glucose; no strains produced acid from adonitol. These characteristics are consistent with published data for E. cloacae. Surface-disinfested onion bulbs and sets were inoculated with 50 to 100 μl of bacterial suspensions containing ~108 CFU/ml, injected with hypodermic needles and syringes, and incubated at 37°C for 2 weeks. Bisected onions revealed dry brown discoloration in each of the four bulbs and sets that had been inoculated with each presumptive strain. Symptoms were indistinguishable from those apparent in onions inoculated with the authentic strains mentioned. Strains recovered on OEM were identified as E. cloacae based on the stated biochemical properties and analysis of the 16S rRNA gene amplified by PCR as above. The sequence of the amplicon from the isolated strains was identical to that of reference strains ATCC 23355 and 310. Amplicon sequences of the 16S rRNA gene of New York strains Ecl3, Ecl6, and Ecl7 were deposited in GenBank as JF832951, JF832952, and JF832953, respectively. The strains were accessioned as ATCC BAA-2271, ATCC BAA-2272, and ATCC BAA-2273, respectively. To our knowledge, this is the first published report of E. cloacae causing Enterobacter bulb decay of onion in New York.

Clinical and Laboratory Diagnostic Characteristics and Cytotoxigenic Potential of Hafnia alvei and Hafnia paralvei Strains

A collection of 68 Hafnia strains previously identified to the species level by 16S rRNA gene sequencing were investigated for simple phenotypic properties that could aid in their recognition in the clinical laboratory. Four tests, including malonate utilization, fermentation of salicin and d-arabinose, and expression of β-glucosidase activity, correctly assigned each strain to either Hafnia alvei or H. paralvei. Antibiotic susceptibility profiles were generated for 35 H. alvei and H. paralvei isolates using Etest strips for 24 antibiotics. All strains were susceptible to aminoglycosides, quinolones, carbapenems, and monobactams. Most of the Hafnia isolates had a colistin MIC of ≥2 μg/ml. Sequencing of an internal ampC gene fragment allowed genotypic differentiation of the two Hafnia species. Approximately 70% of the hafniae tested additionally produced a cytolytic toxin active on Vero cells which may play a role in gastroenteritis.

First Report of Soft Rot of Potatoes Caused by Dickeya dadantii in Zimbabwe

A survey was carried out in the potato- (Solanum tuberosum L.) growing regions of Zimbabwe in April 2009 to assess the prevalence of bacterial soft rot. A total of 125 tubers with soft rot symptoms were collected. The disease caused severe economic losses ranging from 20 to 60% on tubers in the field and in storage. Affected tubers had symptoms that ranged from light vascular discoloration to complete seed piece decay. Infected tuber tissue was often cream colored and soft to the touch. In the field, plants showed severe wilting, often accompanied by a slimy, brown necrosis of the lower stems. Seventy-five of 125 isolations from diseased tubers yielded pectolytic bacteria on crystal violet pectate (CVP) medium and colonies were characterized after purification on King's B medium. All 75 isolates were gram-negative rods, oxidase negative, facultatively anaerobic, able to degrade pectate, and rot potato slices. They grew at 37°C, were sensitive to erythromycin, positive for phosphatase, indole production, cis-aconitate, lactose, d-arabinose, meso-tartrate, casein, d-melibiose, myo-inositol, and malonate utilization, while negative for acid production from trehalose, inuline, and α-methyl glucose. Dickeya dadantii (Erwinia chrysanthemi 3937 from the Scottish Research Institute) was included in all biochemical and pathogenicity tests. These characteristics are typical for two species, D. zeae and D. dadantii (2). Thus, the 75 isolates were further identified by PCR amplification with BOX and REP primers (3) and five isolates by gyrB sequence analysis (1). These analyses give support for the isolates being D. dadantii. Partial gyrB sequence analysis showed that the analyzed isolates had 96% sequence identity with the D. dadantii type strain Ech 586T (GenBank Accession No. CP001836.1). One-microliter suspensions (108 CFU per ml) of 20 samples were injected into the stolon end of potato tubers (S. tuberosum L.) cv. BP1. Each isolate was inoculated into three tubers, which were maintained at 25°C. Three control tubers were inoculated with sterile distilled water. Soft rot symptoms identical to those observed in the field and in storage appeared on all inoculated tubers 1 to 2 days after inoculation but not on the control tubers. A bacterium with identical characteristics to those described above was consistently reisolated from the rotted tissue of inoculated tubers. To our knowledge, this is the first report of soft rot on potato in Zimbabwe caused by D. dadantii, formerly referred to as E. chrysanthemi. This finding has implications for import and export of potato material into and out of Zimbabwe. Zimbabwe imports seed from various countries because of the current seed shortage and exports table potatoes to other African states.