Significance of Herbaspirillum sp. in biodegradation and biodetoxification of herbicides, pesticides, hydrocarbons and heavy metals - A review.

In today's industrialized world, contamination of soil and water with various substances has emerged as a pressing concern. Bioremediation, with its advantages of degradation or detoxification, non-polluting nature, and cost-effectiveness, has become a promising method due to technological advancements. Among the bioremediation agents, bacteria have been highly explored and documented as a productive organism. Recently, few studies have reported on the significance of Herbaspirillum sp., a Gram-negative bacterium, in bioremediating herbicides, pesticides, polycyclic aromatic hydrocarbons, metalloids, and heavy metals, as well as its role in augmenting phytoremediation efforts. Herbaspirillum sp. GW103 leached 66% of Cu from ore materials and significantly enhanced the phytoaccumulation of Pb and Zn in plumule and radical tissues of Zea mays L. plants. Additionally, Herbaspirillum sp. WT00C reduced Se6+ into Se0, resulting in an increased Se0 content in tea plants. Also, Herbaspirillum sp. proved effective in degrading 0.6 mM of 4-chlorophenol, 92.8% of pyrene, 77.4% of fluoranthene, and 16.4% of trifluralin from aqueous solution and soil-water system. Considering these findings, this review underscores the need for further exploration into the pathways of pollutant degradation, the enzymes pivotal in the degradation or detoxification processes, the influence of abiotic factors and pollutants on crucial gene expression, and the potential toxicity of intermediate products generated during the degradation process. This perspective reframes the numerical data to underscore the underutilized potential of Herbaspirillum sp. within the broader context of addressing a significant research gap. This shift in emphasis aligns more closely with the problem-necessity for solution-existing unexplored solution framework.

MAW point mutation impairs H. Seropedicae RecA ATP hydrolysis and DNA repair without inducing large conformational changes in its structure.

RecA is a multifunctional protein that plays a central role in DNA repair in bacteria. The structural Make ATP Work motif (MAW) is proposed to control the ATPase activity of RecA. In the present work, we report the biochemical activity and structural effects of the L53Q mutation at the MAW motif of the RecA protein from H. seropedicae (HsRecA L53Q). In vitro studies showed that HsRecA L53Q can bind ADP, ATP, and ssDNA, as does wild-type RecA. However, the ATPase and DNA-strand exchange activities were completely lost. In vivo studies showed that the expression of HsRecA L53Q in E. coli recA1 does not change its phenotype when cells were challenged with MMS and UV. Molecular dynamics simulations showed the L53Q point mutation did not cause large conformational changes in the HsRecA structure. However, there is a difference on dynamical cross-correlation movements of the residues involved in contacts within the ATP binding site and regions that hold the DNA binding sites. Additionally, a new hydrogen bond, formed between Q53 and T49, was hypothesized to allow an independent motion of the MAW motif from the hydrophobic core, what could explain the observed loss of activity of HsRecA L53Q.

Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate production by a prpC mutant of Herbaspirillum seropedicae Z69.

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters produced by a wide range of bacteria as carbon and energy reserves. PHA accumulation is typically increased under unbalanced growth conditions and with carbon source in excess. Although polyhydroxybutyrate (PHB) could be used for specific applications, it is brittle and not a useful alternative for plastics like polypropylene. Far more useful polypropylene-like PHAs, are copolymers composed of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Propionic acid is one of the carbon sources that can be used to generate 3HV. A mutant derived from Herbaspirillum seropedicae Z69, a strain previously described as capable of producing P(3HB-co-3HV) from propionic acid, was constructed to increase 3HV biosynthetic efficiency. The strategy involved elimination of a catabolic route for propionyl-CoA by deficiency marker exchange of a selected gene. The mutant (Z69Prp) was constructed by elimination of the 2-methylcitrate synthase (PrpC) gene of the 2-methylcitrate cycle for propionate catabolism. Strain Z69Prp was unable to grow on sodium propionate, but in cultures with glucose-propionate accumulated 50% of its dry weight as copolymer. Z69Prp had 14.1 mol% 3HV; greater than that of strain Z69 (2.89 mol%). The 3HV yield from propionic acid (Y3HV/prop) was 0.80 g g-1, and below the maximum theoretical value (1.35 g g-1).

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).

Noviherbaspirillum agri sp. nov., isolated from reclaimed grassland soil, and reclassification of Herbaspirillum massiliense (Lagier et al., 2014) as Noviherbaspirillum massiliense comb. nov.

A straw-coloured, Gram-staining-negative, aerobic, motile and rod-shaped bacterium, designated strain K-1-15T, was isolated from reclaimed grassland soil from Biratnagar, Morang, Nepal. This strain was non-spore-forming, catalase-negative and oxidase-positive. It was able to grow at 10-45 °C, pH 6.5-9.5 and 0-1.5 % (w/v) NaCl concentration. This strain was taxonomically characterized by a polyphasic approach. Based on the results of 16S rRNA gene sequence analysis, K-1-15T formed a distinct lineage within the family Oxalobacteraceae and was most closely related to members of the genera Herbaspirillum(96.99-95.34 % sequence similarity), Noviherbaspirillum(96.72-95.45 % sequence similarity) and Paraherbaspirillum (95.85 % sequence similarity). The only respiratory quinone was ubiquinone-8. The polar lipid profile revealed the presence of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major fatty acids of K-1-15T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16  :   0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C10 : 0 3-OH, and iso-C16 : 0. The genomic DNA G+C content of this novel strain was 65.2 mol %. The DNA-DNA relatedness between K-1-15T and Herbaspirillum massiliense DSM 25712T and Noviherbaspirillum soli LMG 26149T were 18.3 and 13.7 % repectively. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, K-1-15T represents a novel species of the genus Noviherbaspirillum in the family Oxalobacteraceae, for which the name Noviherbaspirillum agri sp. nov. is proposed. The type strain is K-1-15T (=KEMB 9005-422T=KACC 18909T=JCM 31463T). Based on new data obtained in this study, we also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov. (type strain JC206T=CSUR P159T=DSM 25712T).

Serobactins-mediated iron acquisition systems optimize competitive fitness of Herbaspirillum seropedicae inside rice plants.

Herbaspirillum seropedicae Z67 is a diazotrophic endophyte able to colonize the interior of many economically relevant crops such as rice, wheat, corn and sorghum. Under iron-deficient conditions, this organism secretes serobactins, a suite of lipopetide siderophores. The role of siderophores in the interaction between endophytes and their plant hosts are not well understood. In this work, we aimed to determine the importance of serobactins-mediated iron acquisition systems in the interaction of H. seropedicae with rice plants. First we provide evidence, by using a combination of genome analysis, proteomic and genetic studies, that the Hsero_2345 gene encodes a TonB-dependent receptor involved in iron-serobactin complex internalization when iron bioavailability is low. Our results show that survival of the Hsero_2345 mutant inside rice plants was not significantly different from that of the wild-type strain. However, when plants were co-inoculated at equal ratios with the wild-type strain and with a double mutant defective in serobactins synthesis and internalization, recovery of mutant was significantly impaired after 8 days post-inoculation. These results demonstrate that serobactins-mediated iron acquisition contributes to competitive fitness of H. seropedicae inside host plants.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

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.

Description of Noviherbaspirillum malthae gen. nov., sp. nov., isolated from an oil-contaminated soil, and proposal to reclassify Herbaspirillum soli, Herbaspirillum aurantiacum, Herbaspirillum canariense and Herbaspirillum psychrotolerans as Noviherbaspirillum soli comb. nov., Noviherbaspirillum aurantiacum comb. nov., Noviherbaspirillum canariense comb. nov. and Noviherbaspirillum psychrotolerans comb. nov. based on polyphasic analysis.

An aerobic, Gram-negative, rod-shaped bacterium with polar flagella, strain CC-AFH3(T), was isolated from an oil-contaminated site located in Kaohsiung county, Taiwan. Strain CC-AFH3(T) grew at 20-40 °C, pH 5.0-10.0 and <2 % (w/v) NaCl. 16S rRNA gene sequence analysis indicated that strain CC-AFH3(T) showed the greatest degree of similarity to Herbaspirillum soli SUEMI10(T) (96.5 %), H. aurantiacum SUEMI08(T) (96.3 %), H. canariense SUEMI03(T) (96.0 %), H. psychrotolerans PB1(T) (95.4 %) and members of other Herbaspirillum species (94.1-95.2 %), and lower similarity to members of other genera (<94 %). Phylogenetic analyses also positioned the novel strain in the genus Herbaspirillum as an independent lineage. The major fatty acids in strain CC-AFH3(T) were C10 : 0 3-OH, C12 : 0, C14 : 0 2-OH, C16 : 0, iso-C15 : 0 3-OH, C17 : 0 cyclo, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The major polar lipids of strain CC-AFH3(T) were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The predominant quinone was ubiquinone 8 (Q-8) and the DNA G+C content was 63.4 mol%. On the basis of 16S rRNA gene sequence analysis in combination with physiological and chemotaxonomic data, strain CC-AFH3(T) represents a novel species in a new genus, for which we propose the name Noviherbaspirillum malthae gen. nov., sp. nov.; the type strain of Noviherbaspirillum malthae is CC-AFH3(T) ( = BCRC 80516(T) = JCM 18414(T)). We also propose the reclassification of Herbaspirillum soli, Herbaspirillum aurantiacum, Herbaspirillum canariense and 'Herbaspirillum psychrotolerans' as Noviherbaspirillum soli comb. nov. (type strain SUEMI10(T) = LMG 26149(T) = CECT 7840(T)), Noviherbaspirillum aurantiacum comb. nov. (type strain SUEMI08(T) = LMG 26150(T) = CECT 7839(T)), Noviherbaspirillum canariense comb. nov. (type strain SUEMI03(T) = LMG 26151(T) = CECT 7838(T)) and Noviherbaspirillum psychrotolerans comb. nov. (type strain PB1(T) = DSM 26001(T) = LMG 27282(T)), respectively. An emended description of Herbaspirillum seropedicae is also presented.

Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.

Herbaspirillum species: a potential pathogenic bacteria isolated from acute lymphoblastic leukemia patient.

Herbaspirillum species, colonized the plant rhizosphere, also called rhizobacteria, are plant growth-promoting bacteria. Recently we isolated Herbaspirillum from blood cultures of acute lymphoblastic leukemia (ALL) and identified by PCR and gene sequencing. Herbaspirillum may be a potential pathogenic bacteria. Although the exact role that these species play in ALL patients is unknown, their differentiation from other species has serious implications for clinical care and patient well-being.

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Associative diazotrophic bacteria in grass roots and soils from heavy metal contaminated sites

This work aimed to evaluate density of associative diazotrophic bacteria populations in soil and grass root samples from heavy metal contaminated sites, and to characterize isolates from these populations, both, phenotypically (Zinc, Cadmium and NaCl tolerance in vitro, and protein profiles) and genotypically (16S rDNA sequencing), as compared to type strains of known diazotrophic species. Densities were evaluated by using NFb, Fam and JNFb media, commonly used for enrichment cultures of diazotrophic bacteria. Bacterial densities found in soil and grass root samples from contaminated sites were similar to those reported for agricultural soils. Azospirillum spp. isolates from contaminated sites and type strains from non-contaminated sites varied substantially in their in vitro tolerance to Zn+2 and Cd+2, being Cd+2 more toxic than Zn+2. Among the most tolerant isolates (UFLA 1S, 1R, S181, S34 and S22), some (1R, S34 and S22) were more tolerant to heavy metals than rhizobia from tropical and temperate soils. The majority of the isolates tolerant to heavy metals were also tolerant to salt stress as indicated by their ability to grow in solid medium supplemented with 30 g L-1 NaCl. Five isolates exhibited high dissimilarity in protein profiles, and the 16S rDNA sequence analysis of two of them revealed new sequences for Azospirillum.

Objetivou-se avaliar a densidade de populações de bactérias diazotróficas associativas em amostras de solos e de raízes de gramíneas oriundas de sítios contaminados com metais pesados, e caracterizar isolados destas populações através da análise fenotípica (tolerância aos metais pesados zinco e cádmio e à NaCl in vitro, perfis protéicos), e genotípica (seqüenciamento de 16S rDNA), comparados às estirpes tipo das mesmas espécies. As densidades foram avaliadas nos meios NFb, Fam e LGI, comumente utilizados para culturas de enriquecimento de populações de bactérias diazotróficas associativas. As densidades encontradas em amostras de solo e raiz de sítios contaminados foram semelhantes àquelas relatadas na literatura para solos agrícolas. Isolados de Azospirillum spp. de solos contaminados e estirpes tipo oriundas de solos não contaminados variaram substancialmente com relação à tolerância a Zn+2 e Cd+2, sendo que Cd+2 mais tóxico que Zn+2. Dentre os isolados mais tolerantes (UFLA 1S, 1R, S181, S34, e S22), alguns(1R, S34 e S22) foram mais tolerantes a metais pesados que rizóbios isolados de solos de áreas tropicais e temperadas. A maioria dos isolados mais tolerantes a metais pesados também foi tolerante ao estresse salino, o que foi indicado por seu crescimento em meio sólido suplementado com 30 g L-1 de NaCl in vitro. Cinco isolados apresentaram alta dissimilaridade em perfis protéicos e o seqüenciamento do gene 16S rDNA em dois deles revelou que apresentam novas seqüências de Azospirillum.

Herbaspirillum rhizosphaerae sp. nov., isolated from rhizosphere soil of Allium victorialis var. platyphyllum.

Two Gram-negative, milky-white-pigmented, motile, slightly curved rod-shaped bacterial isolates, UMS-37(T) and UMS-40, were isolated from rhizosphere soil of wild edible greens cultivated on Ulleung island, Korea, and their taxonomic positions were investigated by a polyphasic approach. They grew optimally at 25-30 degrees C and contained Q-8 as the predominant ubiquinone. The major cellular fatty acids (>10 % of total fatty acids) were C(16 : 0), cyclo C(17 : 0) and C(16 : 1)omega7c and/oriso-C(15 : 0) 2-OH. The DNA G+C contents of the two isolates were 59.8 and 60.0 mol%. Isolates UMS-37(T) and UMS-40 exhibited no difference in their 16S rRNA gene sequences and possessed a mean DNA-DNA relatedness level of 94 %; they exhibited 16S rRNA gene sequence similarity levels of 96.8-98.2 % to the type strains of recognized Herbaspirillum species. Phylogenetic analyses based on 16S rRNA gene sequences showed that isolates UMS-37(T) and UMS-40 formed a distinct phylogenetic lineage within the genus Herbaspirillum. DNA-DNA relatedness levels between isolates UMS-37(T) and UMS-40 and the type strains of some phylogenetically related Herbaspirillum species were in the range 3-56 %. On the basis of differences in phenotypic properties and phylogenetic distinctiveness and genomic data, isolates UMS-37(T) and UMS-40 were classified in the genus Herbaspirillum within a novel species, for which the name Herbaspirillum rhizosphaerae sp. nov. is proposed, with the type strain UMS-37(T) (=KCTC 12558(T) =CIP 108917(T)).

[Mobilization of simazine degradation genes by the plasmid pSa].

The plasmid pSa was found to mobilize the genes for simazine degradation (smz) of the rhizosphere bacterium Herbaspirillum sp.B601 by forming hybrid pSa-Smz plasmids. Independent migration of smz genes into various loci of genome during transfer and elimination of the hybrid plasmids indicated that the genes were parts of a "catabolic island" which could be unstable under certain conditions.

Iron depletion affects nitrogenase activity and expression of nifH and nifA genes in Herbaspirillum seropedicae.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.