Structural Analysis of Spermidine Synthase from Kluyveromyces lactis.

Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5'-deoxy-5'-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.

The Y430F mutant of Salmonella d-ornithine/d-lysine decarboxylase has altered stereospecificity and a putrescine allosteric activation site.

Tyrosine-430 of d-ornithine/d-lysine decarboxylase (DOKDC) is located in the active site, and was suggested to be responsible for the D-stereospecificity of the enzyme. We have prepared the Y430F mutant form of Salmonella enterica serovar typhimurium DOKDC and evaluated its catalytic activity with D- and l-lysine and ornithine. The kinetic results show that the Y430F mutant has measurable decarboxylase activity with both D- and l-lysine and ornithine, which wild type DOKDC does not. Spectroscopic experiments show that these amino acids bind to form external aldimine complexes with the pyridoxal-5'-phosphate with λmax = 425 nm. In addition, we have obtained crystal structures of Y430F DOKDC bound to HEPES, putrescine, d-ornithine, d-lysine, and d-arginine. The d-amino acids bind in the crystals to form equilibrium mixtures of gem-diamine and external aldimine complexes. Furthermore, the crystal structures reveal an unexpected allosteric product activator site for putrescine located on the 2-fold axis between the two active sites. Putrescine binds by donating hydrogen bonds from the ammonium groups to Asp-361 and Gln-358 in the specificity helix of both chains. Addition of 0.1-1 mM putrescine eliminates the lag in steady state kinetics and abolishes the sigmoid kinetics. The catalytic loop was modeled with AlphaFold2, and the model shows that Glu-181 can form additional hydrogen bonds with the bound putrescine, likely stabilizing the catalytic closed conformation.

The effect of putrescine on the lysozyme activity and structure: Spectroscopic approaches and molecular dynamic simulation.

The present research addressed the influence of polyamine (putrescine) on the compound as well as function of lysozyme; accordingly, UV- Visible, fluorescence spectroscopy and simulation method were applied to fulfill this goal. Lysozyme's structural variability was examined at various putrescine |concentrations; also, the putrescine binding to lysozyme was addressed using spectrofluorescence, circular dichroism (CD) and UV-Vis measurements. The obtained results indicated that with raising the putrescine concentration, the intrinsic quenching fluorescence of lysozyme was decreased based on the static mechanism. Analysis of thermodynamic parameters also indicated that van der Waals as well as hydrogen bond forces served a fundamental role in determining the resulting stability; this was in agreement with modeling studies. Measurement of UV absorption spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy also demonstrated that lysozyme's second and tertiary structures were altered in a putrescine concentration-dependent manner. Putrescine inhibited lysozyme's enzymatic activity, displaying its affinity with the lysozyme's active site. Further, molecular simulation conducted revealed that putrescine could have spontaneous binding to lysozyme, changing its structure, thus further emphasizing the experimental results.

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers.

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min-1. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 µM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 µM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 µg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 µg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.

Crystal structure of Z-DNA in complex with the polyamine putrescine and potassium cations at ultra-high resolution.

The X-ray crystal structure of the d(CGCGCG)2/putrescine(2+)/K+ complex has been determined at 0.60 Šresolution. Stereochemical restraints were used only for the putrescinium dication, and 23 bonds and 18 angles of the Z-DNA nucleotides with dual conformation. The N atoms of the putrescine(2+) dication form three direct hydrogen bonds with the N7_G atoms of three different Z-DNA molecules, plus three water-mediated hydrogen bonds with cytosine, guanine and phosphate acceptors. A unique potassium cation was also unambiguously identified in the structure, albeit at a ∼0.5 occupation site shared with a water molecule, providing the first example of such a complex with Z-DNA. The K+ cation has coordination number of eight and an irregular coordination sphere, formed by four water molecules and four O atoms from two phosphate groups of the Z-DNA, including ligands present at fractional occupancy. The structural disorder of the Z-DNA duplex is manifested by the presence of alternate conformations along the DNA backbone. Comparison of the position and interactions of putrescine(2+) in the present structure with other ultra-high-resolution structures of Z-DNA in complexes with Mn2+ and Zn2+ ions shows that the dicationic putrescinium moiety can effectively substitute these metal ions for stabilization of Z-type DNA duplexes. Furthermore, this comparison also suggests that the spermine(4+) tetracation has a higher affinity for Z-DNA than K+.

X-ray Crystallographic Snapshots of Substrate Binding in the Active Site of Histone Deacetylase 10.

Histone deacetylase 10 (HDAC10) is a zinc-dependent polyamine deacetylase enriched in the cytosol of eukaryotic cells. The active site of HDAC10 contains catalytic residues conserved in other HDAC isozymes that function as lysine deacetylases: Y307 assists the zinc ion in polarizing the substrate carbonyl for nucleophilic attack, and the H136-H137 dyad serves general base-general acid functions. As an inducer of autophagy, HDAC10 is an attractive target for the design of selective inhibitors that may be useful in cancer chemotherapy. Because detailed structural information regarding the catalytic mechanism of HDAC10 may inform new approaches to inhibitor design, we now report X-ray crystal structures of HDAC10 in which reaction intermediates with substrates N8-acetylspermidine and N-acetylputrescine are trapped in the active site. The Y307F substitution prevents activation of the substrate carbonyl for nucleophilic attack by the zinc-bound water molecule, thereby enabling crystallographic isolation of intact enzyme-substrate complexes. The H137A substitution removes the catalytically obligatory general acid, thereby enabling crystallographic isolation of oxyanionic tetrahedral intermediates. Finally, the acetate complex with the wild-type enzyme represents a product complex after dissociation of the polyamine coproduct. Taken together, these structures provide snapshots of the reaction coordinate of acetylpolyamine hydrolysis and are consistent with a mechanism in which tandem histidine residues H136 and H137 serve as general base and general acid catalysts, respectively. The function of the histidine dyad in the HDAC10 mechanism appears to be similar to that in HDAC6, but not HDAC8 in which both functions are served by the second histidine of the tandem pair.

Noviherbaspirillum aerium sp. nov., isolated from air.

A Gram-stain-negative, strictly aerobic, non-spore-forming, rod-shaped, motile with polar flagella and pale-orange bacterium, designated strain 122213-3T, was isolated from air, collected at the foot of the Xiangshan Mountain, located in Beijing, PR China. Optimal growth occurred at 28 °C, at pH 7 and in the presence of 0-1 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that 122213-3T clustered with species of the genus Noviherbaspirillum and formed a distinct sublineage, showing highest similarities to Noviherbaspirillum malthae CC-AFH3T (96.88 %), Noviherbaspirillum massiliense JC206T (95.78 %) and Noviherbaspirillum aurantiacum SUEMI08T (95.78 %). The predominant cellular fatty acids were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The predominant quinone was ubiquinone 8 (Q-8). The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, unidentified phospholipid and two unidentified polar lipids. The polyamine pattern showed the presence of putrescine as the major polyamine, with minor amounts of 2-hydroxyputrescine. The DNA G+C content was 60.1 mol%. The phylogenetic analysis and physiological and biochemical data showed that strain 122213-3T should be classified as representing a novel species in the genus Noviherbaspirillum, for which the name Noviherbaspirillum aerium sp. nov. is proposed. The type strain of N. aerium is 122213-3T (=CFCC 14286T=LMG 30131T).

Removal of biogenic amines from wines by chemisorption on functionalized silica and effects on other wine components.

The effectiveness of several functionalized silica materials (cation-exchange materials) for the removal of biogenic amines from wines, and the effects on other wine components and organoleptic characteristics were evaluated. Results have shown that mesoporous silica material bi-functionalized with phosphonic and sulfonic acids allowed the removal of histamine, putrescine, cadaverine, spermine and spermidine from wines, although the dose must be adapted for each wine according to the removal requirements and wine characteristics. A plus of the adsorbent developed is that it can be recovered and re-used for at least 3 treatments. Immediately following the treatments, a decrease in the levels of linear ethyl esters (ethyl hexanoate, ethyl octanoate and ethyl decanoate) was observed, although these levels were re-equilibrated after several days reducing this undesired side effect. A slight, but perceptible, effect on wine color was observed, probably due to the slight decrease in the pH of the wine produced by the treatments. On the basis of the sensory analysis that focused only on the aroma of the wines, the proposed technique would be more adequate for wines aged in barrels than for young wines.

Pharmacokinetic Properties of 68Ga-labelled Folic Acid Conjugates: Improvement Using HEHE Tag.

The folate receptor (FR) is a promising cell membrane-associated target for molecular imaging and radionuclide therapy of cancer (FR-α) and potentially also inflammatory diseases (FR-ß) through use of folic acid-based radioconjugate. FR is often overexpressed by cells of epithelial tumors, including tumors of ovary, cervix, endometrium, lungs, kidneys, etc. In healthy tissues, FR can be found in small numbers by the epithelial cells, mainly in the kidneys. Extremely high undesired accumulation of the folate radioconjugates in the renal tissue is a main drawback of FR-targeting concept. In the course of this work, we aimed to reduce the undesirable accumulation of folate radioconjugates in the kidneys by introducing a histidine/glutamic acid tag into their structure. Two folic acid based compounds were synthesized: NODAGA-1,4-butanediamine-folic acid (FA-I, as control) and NODAGA-[Lys-(HE)2]-folic acid (FA-II) which contains a (His-Glu)2 fragment. In vitro studies with FR (+) cells (KB and others) showed that both compounds have specificity for FR. Introduction of (HE)2-tag does not affect FR binding ability of the conjugates. In vivo biodistribution studies with normal laboratory animals, as well as with KB tumor bearing animals, were carried out. The results showed that introduction of the (HE)2 tag into the structure of folate radioconjugates can significantly reduce the accumulation of these compounds in non-target tissues and important organs (the accumulation in the kidneys is reduced 2-4 times), leaving the accumulation in tumor at least at the same level, and even increasing it.

Change in taste-altering non-volatile components of blood and common orange fruit during cold storage.

Cold storage may cause changes in the volatile and non-volatile components of orange fruit, in association with the decrement of the characteristic fruit flavour and sensory acceptability. The aim of this work was to evaluate the changes of some non-volatile taste-altering components (total and individual sugars, acids, anthocyanins, putrescine and limonin) that may affect the organoleptic perception of cold-stored orange fruit. Three blood orange varieties ('Tarocco TDV', 'Tarocco Gallo', and 'Moro') and a common variety ('Washington navel') were stored at 6 ± 1 °C and 90-95% Relative Humidity (RH) for 60 d. Chemical and sensory assessments were performed during fruit storage at 15 d intervals. During storage, no dramatic change of the physicochemical parameters was recorded and the ascorbic acid content remained almost unchanged in all varieties. As expected, total anthocyanins significantly increased during storage. Limonin significantly decreased in all varieties. A consistent and significant increase in putrescine occurred during storage in the fruit of the pigmented varieties, not recorded in the common orange variety. Putrescine behaviour showed direct correlation with the accumulation of off-flavour in cold-stored 'Moro' and 'T. TDV' fruit, showing a clear influence of its relative concentration on the sensory perception of fruit. Finally, principal component analysis showed that the complete quality profile of the four investigated varieties represented clear differentiation without overlapping clusters. Our results suggest that the arise of a negative sensory perception in cold stored blood orange fruit might be linked to their accumulation of putrescine.

Investigation on the interaction of acid phosphatase with putrescine using docking, simulations methods and multispectroscopic techniques.

The effect of putrescine on the dynamics, conformation, and kinetics of acid phosphatase investigated via different experimental and theoretical methods. The Stern-Volmer constants (Ksv) for the acid phosphatase- putrescine compound was obtained at different temperatures. Therefore, putrescine quenched the intensity of the enzyme via the static method. Gibbs free energy displayed which binding process was spontaneous. MD simulation, docking method, and thermodynamic parameters revealed the van der Waals forces and hydrogen bonding that had the specific interaction in unfolding the compound. After putrescine binding, the Vmax amounts of acid phosphatase without changed, and the value of kcat/Km increased. The Tm the acid phosphatase-putrescine compound was decreased. Presumably because of more surface hydrophilicity and further H-bond formation underlying the putrescine modification. As confirmed by fluorescence spectra and UV-Visible spectroscopy. Circular dichroism (CD) and UV absorption investigations also demonstrated which binding of putrescine to acid phosphatase led to microenvironmental variations around the protein. Therefore, putrescine cause changes in its structure and function. The results, therefore, showed the effect of putrescine was because of its kosmotropic specifications. Molecular dynamics simulation and Molecular docking results further confirmed the results obtained by CD and spectroscopy experiments.

Engineering siderophores.

Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate siderophores by various biochemical approaches based on the example of Shewanella algae. The marine gamma-proteobacterium S. algae produces three different cyclic hydroxamate siderophores as metabolites via a single biosynthetic gene cluster and one of them is an important key player in interspecies competition blocking swarming of Vibrio alginolyticus. AvbD is the key metabolic enzyme assembling the precursors into three different core structures and hence an interesting target for metabolic and biochemical engineering. Synthetic natural and unnatural precursors can be converted in vitro with purified AvbD to generate siderophores with various ring sizes ranging from analytical to milligram scale. These engineered siderophores can be applied, for example, as swarming inhibitors against V. alginolyticus. Here, we describe the synthesis of the natural and unnatural siderophore precursors HS[X]A and provide our detailed protocols for protein expression of AvbD, conversion of HS[X]A with the enzyme to produce ring-size engineered siderophores and secondly for a biosynthetic feeding strategy that allows to extract engineered siderophores in the milligram scale.

What is the role of putrescine accumulated under potassium deficiency?

Biomarker metabolites are of increasing interest in crops since they open avenues for precision agriculture, whereby nutritional needs and stresses can be monitored optimally. Putrescine has the potential to be a useful biomarker to reveal potassium (K+ ) deficiency. In fact, although this diamine has also been observed to increase during other stresses such as drought, cold or heavy metals, respective changes are comparably low. Due to its multifaceted biochemical properties, several roles for putrescine under K+ deficiency have been suggested, such as cation balance, antioxidant, reactive oxygen species mediated signalling, osmolyte or pH regulator. However, the specific association of putrescine build-up with low K+ availability in plants remains poorly understood, and possible regulatory roles must be consistent with putrescine concentration found in plant tissues. We hypothesize that the massive increase of putrescine upon K+ starvation plays an adaptive role. A distinction of putrescine function from that of other polyamines (spermine, spermidine) may be based either on its specificity or (which is probably more relevant under K+ deficiency) on a very high attainable concentration of putrescine, which far exceeds those for spermidine and spermine. putrescine and its catabolites appear to possess a strong potential in controlling cellular K+ and Ca2+ , and mitochondria and chloroplasts bioenergetics under K+ stress.

Sphingobium fluviale sp. nov., isolated from a river.

A Gram-stain-negative, rod-shaped, non-motile, poly-ß-hydroxybutyrate-accumulating and aerobic bacterial strain, designated CHR27T, was isolated and characterized by using the polyphasic taxonomy approach. The results of phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of an up-to-date bacterial core gene set (92 protein clusters) indicated that strain CHR27T is affiliated with species in the genus Sphingobium. 16S rRNA gene sequence similarity results indicated that strain CHR27T was closely related to species of the genus Sphingobium (94.3-97.0 %), and had the highest sequence similarity to Sphingobium qiguonii X23T (97.0 %). Strain CHR27T showed 19.4-22.1 % digital DNA-DNA hybridization values and 73.2-74.8 % average nucleotide identity values with the strains of other Sphingobium species. Optimal growth occurred at 25 °C, pH 7.5 and in the absence of NaCl. The major fatty acids of strain CHR27T were C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The predominant hydroxy fatty acid was C14 : 0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, two unidentified sphingoglycolipids and an unidentified aminophospholipid. Strain CHR27T contained spermidine as the major polyamine and putrescine as a minor component. The only isoprenoid quinone was ubiquinone-10. The genomic DNA G+C content of strain CHR27Twas 61.8 mol%. On the basis of the phylogenetic inference and phenotypic data, strain CHR27T was considered a representative of a novel species within the genus Sphingobium. The name Sphingobium fluviale sp. nov. is proposed, with strain CHR27T (=BCRC 81121T=LMG 30596T=KCTC 62510T) as the type strain.

Analytical possibilities of Putrescine and Cadaverine enzymatic colorimetric determination in tuna based on diamine oxidase: A critical study of the use of ABTS.

The joint determination of putrescine (Put) and cadaverine (Cad) in the presence of other biogenic amines is studied using their enzymatic reaction with diamine oxidase (DAO). Three alternative methods are studied based on the intrinsic colorimetric properties of DAO or horseradish peroxidase (HRP), and the use of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) colorimetric reagent, respectively. In this last case an in-depth study is carried out in order to explain and solve some drawbacks usually associated with the use of this reagent (especially interferences, interaction with enzymes and instability), and to propose new analytical methodologies which this reagent allows to achieve (transient signal and the use of the violet species). Finally, the method has been applied to Put + Cad determination in a tuna sample without interference of other biogenic amines. The result has been compared with that obtained using a method based on HPLC-MS, which has allowed the new methodology to be validated.

Study of alkaline phosphatase interaction with putrescine using multi-spectroscopic and docking methods.

The vast application of enzymes in various industries has encouraged scientists to optimize enzymes activity and stability. Solvent engineering is a well-known method used for stabilizing enzymes. Putrescine is a polyamine that can serve as as an enzyme stabilizer. In this study, the structure and activity of bovine alkaline phosphatase (BALP) were studied in the presence of putrescine. The results confirmed that putrescine could bind to BALP and change the UV-vis spectra of the enzyme. Moreover, putrescine quenched the BALP fluorescence spectra by static quenching mechanism. Putrescine interacted with BALP spontaneously by different forces such as van der Waals and hydrogen bonding. Circular dichroism spectra studies also revealed that the BALP structure was changed in the presence of putrescine. Furthermore, the kinetic parameters showed that BALP was activated by putrescine. Moreover, docking studies confirmed that hydrogen bonds, van der Waals forces and hydrophobic interactions played the major role in the BALP-put complex formation. In general, these results showed that putrescine could modify the BALP activity and structure.

A polyamine-independent role for S-adenosylmethionine decarboxylase.

The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.

New coordination compounds of citric acid and polyamines with lanthanide ions - potential application in monitoring the treatment of cancer diseases.

Non-covalent interaction in the binary systems of polyamines (putrescine, spermidine, spermine) with citric acid and complex formation in the binary as well as ternary systems of lanthanide(III) ions, citric acid and polyamine have been investigated. The studies were performed in aqueous solution. The overall stability constants of the complexes were determined using the potentiometric method with computer analysis of the data. Only mononuclear type of complexes were found in the ternary systems and polyamines were located in the outer as well as inner coordination sphere. Non-covalent interaction between biogenic amines and citric acid in the binary and ternary systems were confirmed on the basis of the equilibrium constants analysis and spectroscopic studies.

Removal of biogenic amines from hydroalcoholic solutions by functionalized silica.

The structure and the cation-exchange functional groups of hybrid silica materials were evaluated for the effective detoxification of hydroalcoholic solutions containing eight toxic biogenic amines (BA) usually found in fermented beverages. Results show the effectiveness of the removal is related to the number of amino functions in the extracted molecule, retention by the solid being more effective in the case of multiple amino groups, since retention is stabilized through interaction with the material surface at several points. BA with one amino function (isoamylamine, tyramine, ß-phenylethylamine), in general, showed a weak retention by the solids. For BA with more than two amine groups (spermine, spermidine), the removal rate was close to 100% for all studied materials. For histamine, cadaverine and putrescine, the removal percentages were higher with a lamellar structured sulfonic acid functionalized material and with bifunctional materials (SBA-15 type and macroporous) containing sulfonic/phosphonic acid groups obtained by co-condensation sol-gel route.

Xinfangfangia humi sp. nov., isolated from soil amended with humic acid.

Two slightly beige-pigmented, Gram-stain-negative, rod-shaped bacterial strains, IMT-291T and IMT-297, were isolated from soil in a field located in Malvern, Alabama, USA. The source soil had been amended with humic acid and continuously used for the cultivation of worms used for fish bait. It is still conceivable that the source of the strains is from the humic acid amendment, although all attempts to isolate the novel phenotypes from the humic acid source have failed. The two strains were identical based on morphology, growth rate and subsequently by 16S rRNA gene sequences, but showed differences in genomic fingerprint patterns generated by rep-PCR. Phylogenetic analysis based on the 16S rRNA gene revealed a placement of the strain in a distinct cluster with Xinfangfangia soli (97.2 % 16S rRNA gene sequence similarity) and in close proximity to the genus Falsirhodobacter with highest 16S rRNA gene sequence similarity of 95.3 % to the type strain of Falsirhodobacter deserti. Sequence similarities to all other type strains were below 95.0 %. The chemotaxonomic analysis showed a clear similarity to the genus Xinfangfangia. The main cellular fatty acids of the strain were C18 : 1 ω7c, 11-methly-C18 : 1 ω7c and C16 : 0. The major quinone was ubiquinone Q-10. Phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine were predominant in the polar lipid profile. The polyamine pattern contained the major compound spermidine and moderate amounts of putrescine and cadaverine. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phylogenetic, chemotaxonomic and phenotypic analyses we propose a new species of the genus Xinfangfangia, with the name Xinfangfangiahumi sp. nov. and strain IMT-291T (=LMG 30636T=CIP 111625T=CCM 8858T) as type strain.