Multiple exposures to high concentrations of selenate significantly improve selenate tolerability, red elemental selenium (Se0) and selenoprotein biosynthesis in Herbaspirillum camelliae WT00C.

Herbaspirillum camelliae WT00C is a gram-negative endophyte isolated from the tea plant. It has an intact selenate metabolism pathway but poor selenate tolerability. In this study, microbiological properties of the strain WT00C were examined and compared with other three strains CT00C, NCT00C and NT00C, which were obtained respectively from four, six and eight rounds of 24-h exposures to 200 mM selenate. The selenate tolerability and the ability to generate red elemental selenium (Se0) and selenoproteins in H. camelliae WT00C has significantly improved by the forced evolution via 4-6 rounds of multiple exposures a high concentration of selenate. The original strain WT00C grew in 200 mM selenate with the lag phase of 12 h and 400 mM selenate with the lag phase of 60 h, whereas the strains CT00C and NCT00C grew in 800 mM selenate and showed a relatively short lag phase when they grew in 50-400 mM selenate. Besides selenate tolerance, the strains CT00C and NCT00C significantly improved the biosynthesis of red elemental selenium (Se0) and selenoproteins. Two strains exhibited more than 30% selenium conversion efficiency and 40% selenoprotein biosynthesis, compared to the original strain WT00C. These characteristics of the strains CT00C and NCT00C make them applicable in pharmaceuticals and feed industries. The strain NT00C obtained from eight rounds of 24-h exposures to 200 mM selenate was unable to grow in ≥ 400 mM selenate. Its selenium conversion efficiency and selenoprotein biosynthesis were similar to the strain WT00C, indicating that too many exposures may cause gene inactivation of some critical enzymes involving selenate metabolism and antioxidative stress. In addition, bacterial cells underwent obviously physiological and morphological changes, including gene activity, cell enlargement and surface-roughness alterations during the process of multiple exposures to high concentrations of selenate.

Herbaspirillum camelliae sp. nov., a novel endophytic bacterium isolated from Camellia sinensis L.

Bacterial strain WT00CT is an endophytic bacterium that was isolated from the tea plant (Camellia sinensis L.). The phylogenetic analysis of 16S rRNA genes demonstrated that strain WT00CT was a member of the genus Herbaspirillum. This strain is microaerobic, gram-negative and non-pigmented, and its cells are rod shaped, with a polar flagellum. It grew optimally at 34-37 °C, pH 5.0-8.0 and 0-1.5% NaCl (w/v). The G + C content of its genomic DNA was 62.36 mol%. C16:0, iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 were major fatty acids. The strain WT00CT contained six polar lipids, namely DPG (diphosphatidylglycerol), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PC (phosphatidylcholine), GL (glycolipid) and APL (aminophospholipids), and its respiratory quinone was Q8. The strain WT00CT had a genome size of 6.08 Mb with a total ORF of 5,537, in which one gene cluster (36 genes) encoding a type IV secretion system was absent in other members of the Herbaspirillum genus. ANI values of genomic comparison between the strain WT00CT and other Herbaspirillum species were 75-96%. Based on the phylogenetic, chemotaxonomic and phenotypic data presented here, the strain WT00CT represents a novel species in the Herbaspirillum genus, for which the name Herbaspirillum camelliae sp. nov. is proposed. The type strain of H. camelliae sp. nov. is WT00CT (AB 2018017 T and KCTC 62527 T).

Herbaspirillum robiniae sp. nov., isolated from root nodules of Robinia pseudoacacia in a lead-zinc mine.

A novel endophytic bacterium, designated strain HZ10T, was isolated from root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. The bacterium was Gram-stain-negative, aerobic, motile, slightly curved- and rod-shaped, methyl red-negative, catalase-positive, and did not produce H2S. Strain HZ10T grew at 4-45 °C (optimum, 25-30 °C), pH 5-9 (optimum, pH 7-8) and 0-1 % (w/v) NaCl. The major fatty acids were identified as C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), and the quinone type was Q-8. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of the genomic DNA was 64.9 mol% based on the whole genome sequence. According to the 16S rRNA gene sequence analysis, the closest phylogenetic relative to strain HZ10T is Herbaspirillum chlorophenolicum CPW301T (98.72 % sequence identity). Genome relatedness of the type strains H. chlorophenolicum CPW301T, Herbaspirillum seropedicae Z67T and Herbaspirillum aquaticum IEH 4430T, was quantified by using the average nucleotide identity (86.9-88.0 %) and a genome-to-genome distance analysis (26.6 %-29.3 %), with both strongly supporting the notion that strain HZ10T belongs to the genus Herbaspirillum as a novel species. Based on the results from phylogenetic, chemotaxonomic and physiological analyses, strain HZ10T represents a novel Herbaspirillum species, for which the name Herbaspirillum robiniae sp. nov. is proposed. The type strain is HZ10T (=JCM 31754T=CCTCC AB 2014352T).

Effect of heavy metals on acdS gene expression in Herbaspirillium sp. GW103 isolated from rhizosphere soil.

This study aimed to understand the influence of heavy metals on 1-aminocyclopropane-1-carboxylate deaminase activity (ACCD) and acdS gene expression in Herbaspirillium sp. GW103. The GW103 strain ACCD activity decreased in cells grown in a medium supplemented with Pb and As, whereas cells grown in medium supplemented with Cu showed increase in enzyme activity. The GW103 strain produced 262.2 ± 6.17 µmol of α-ketobutyrate per milligram of protein per hour during ACC deamination at 25 °C after 24 h incubation. Using a PCR approach, an acdS coding-gene of 1.06 kbp was amplified in isolate GW103, showing 92% identity with Herbaspirillum seropedicae SmR1 acdS gene. Real time quantitative polymerase chain reaction results indicate that the acdS expression rate was increased (7.1-fold) in the presence of Cu, whereas it decreased (0.2- and 0.1-fold) in the presence of As and Pb.

Cluster and sporadic cases of herbaspirillum species infections in patients with cancer.

BACKGROUND: Herbaspirillum species are gram-negative Betaproteobacteria that inhabit the rhizosphere. We investigated a potential cluster of hospital-based Herbaspirillum species infections. METHODS: Cases were defined as Herbaspirillum species isolated from a patient in our comprehensive cancer center between 1 January 2006 and 15 October 2013. Case finding was performed by reviewing isolates initially identified as Burkholderia cepacia susceptible to all antibiotics tested, and 16S ribosomal DNA sequencing of available isolates to confirm their identity. Pulsed-field gel electrophoresis (PFGE) was performed to test genetic relatedness. Facility observations, infection prevention assessments, and environmental sampling were performed to investigate potential sources of Herbaspirillum species. RESULTS: Eight cases of Herbaspirillum species were identified. Isolates from the first 5 clustered cases were initially misidentified as B. cepacia, and available isolates from 4 of these cases were indistinguishable. The 3 subsequent cases were identified by prospective surveillance and had different PFGE patterns. All but 1 case-patient had bloodstream infections, and 6 presented with sepsis. Underlying diagnoses included solid tumors (3), leukemia (3), lymphoma (1), and aplastic anemia (1). Herbaspirillum species infections were hospital-onset in 5 patients and community-onset in 3. All symptomatic patients were treated with intravenous antibiotics, and their infections resolved. No environmental source or common mechanism of acquisition was identified. CONCLUSIONS: This is the first report of a hospital-based cluster of Herbaspirillum species infections. Herbaspirillum species are capable of causing bacteremia and sepsis in immunocompromised patients. Herbaspirillum species can be misidentified as Burkholderia cepacia by commercially available microbial identification systems.

[Microbiological properties of two endophytic bacteria isolated from tea (Camellia sinensis L.)].

OBJECTIVE: We investigated biological activities, physiological and biochemical properties of two endophytic bacteria isolated from fresh leaves of tea plants. METHOD: We did morphological observation, biological activity test, physiological and biochemical assays, 16S rDNA analysis, and compared their genotype and phenotype with those of 13 Herbaspirillum species. RESULTS: Their colonies were round, opaque, central uplift and regular edge with a milky white color. Their cells were Gram-negative, rod-shaped with the size of (0.5-0.7) mm x (1.4-1.8) mm and flagellers, but without spore. Both isolates produced indole-3-acetic acid (IAA) (18.7 mg/L for WT00C and 24.9 mg/L for WT00F), ammonia and siderophores, but no nitrogen-fixing activity. The 16S rDNA had sequences similarities of 99.7% each other and 99% with 13 Herbaspirillum species. Two isolates used carbon source as described in the genus Herbaspirillum, except for propionate salt. The neighbor-joining tree built using the 16S rDNA showed that two isolates formed an independent group, which kept certain genetic distance from the 13 Herbaspirillum species. Their physiological and biochemical characteristics and genotypes were different from those for 13 Herbaspirillum species. CONCLUSION: Two isolates WT00C and WT00F were classified as novel members in the genus Herbaspirillum.