A novel thermophilic ß-carotene 15,15'-monooxygenase with broad substrate specificity from the marine bacterium Candidatus Pelagibacter sp. HTCC7211.

To characterize a novel thermophilic ß-carotene 15,15'-monooxygenase BCMO7211 isolated from the marine bacterium Candidatus Pelagibacter sp. HTCC7211. BCMO7211 was functionally overexpressed in Escherichia coli and purified to homogeneity by Ni-NTA affinity chromatography and Superdex-200 gel filtration chromatography. Labeling experiments with H218O demonstrated that the oxygen atom in the terminal aldehyde group of the produced retinal molecules was provided from both molecular oxygen and water, indicating that BCMO7211 is the first characterized bacterial ß-carotene 15,15'-monooxygenase. BCMO7211 exhibited broad carotenoid substrate specificity toward α-carotene, ß-cryptoxanthin, ß-carotene, zeaxanthin, and lutein. The optimum temperature, pH, and concentrations of the substrate and enzyme for retinal production were 60 °C, 9.0, 500 mg ß-carotene/L, and 2.5 U/ml, respectively. Under optimum conditions, 888.3 mg/L retinal was produced in 60 min with a conversion rate of 89.0% (w/w). BCMO7211 is a potential candidate for the enzymatic synthesis of retinal in biotechnological applications.

Biodegradation of flonicamid by Ensifer adhaerens CGMCC 6315 and enzymatic characterization of the nitrile hydratases involved.

BACKGROUND: Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinamide insecticide that regulates insect growth. Because of its wide application in agricultural production and high solubility in water, it poses potential risks to aquatic environments and food chain. RESULTS: In the present study, Ensifer adhaerens CGMCC 6315 was shown to efficiently transform FLO into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) via a hydration pathway mediated by two nitrile hydratases, PnhA and CnhA. In pure culture, resting cells of E. adhaerens CGMCC 6315 degraded 92% of 0.87 mmol/L FLO within 24 h at 30 °C (half-life 7.4 h). Both free and immobilized (by gel beads, using calcium alginate as a carrier) E. adhaerens CGMCC 6315 cells effectively degraded FLO in surface water. PnhA has, to our knowledge, the highest reported degradation activity toward FLO, Vmax = 88.7 U/mg (Km = 2.96 mmol/L). Addition of copper ions could increase the enzyme activity of CnhA toward FLO by 4.2-fold. Structural homology modeling indicated that residue ß-Glu56 may be important for the observed significant difference in enzyme activity between PnhA and CnhA. CONCLUSIONS: Application of E. adhaerens may be a good strategy for bioremediation of FLO in surface water. This work furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and provides effective transformation strategies for microbial remediation of FLO contamination.

Computational characterizations of GDP-mannose 4,6-dehydratase (NoeL) Rhizobial proteins.

A growing body of evidence suggests that Nod Factors molecules are the critical structural components in nitrogen fixation. These molecules have been implicated in plant-microbe signaling. Many enzymes involved in Nod factors biosynthesis; however, the enzymes that decorate (modify) nod factor main structure play a vital role. Here, the computational analysis of GDP-mannose 4,6-dehydratase (NoeL) proteins with great impact in modification of nod factor structure in four genomes of agriculturally important rhizobia (Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium) presented. The NoeL number of amino acids was in the range of 147 (M5AMF5) to 372 (A0A023XWX0, Q89TZ1). The molecular weights were around 41 KDa. The results showed that the strain-specific purification strategy should apply as the pI of the sequences varied significantly (in the range of 5.59 to 9.12). The enzyme sequences and eight 3-dimensional structures predicted with homology modeling and machine learning representing the phylogenetic tree revealed the stability of enzymes in different conditions (Instability and Aliphatic index); however, this stability is also strain-specific. Disulphide bonds were observed in some species; however, the pattern was not detected in all members of the same species. Alpha helix was the dominant secondary structure predicted in all cytoplasmic NoeL. All models were homo-tetramer with acceptable sequence identity, GMEAN and coverage (60, - 1.80, 88, respectively). Additionally, Ramachandran maps showed that more than 94% of residues are in favored regions. We also highlight several key characterizations of NoeL from four rhizobia genomes annotation. These findings provide novel insights into the complexity and diversity of NoeL enzymes among important rhizobia and suggest considering a broader framework of biofilm for future research.

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

Bioconversion of indole-3-acetonitrile by the N2-fixing bacterium Ensifer meliloti CGMCC 7333 and its Escherichia coli-expressed nitrile hydratase.

An N2-fixing bacterium, Ensifer meliloti CGMCC 7333, has been reported to degrade the cyano-containing neonicotinoid insecticides acetamiprid and thiacloprid using a nitrile hydratase (NHase). Here, the bioconversion of indole-3-acetonitrile (IAN) by E. meliloti, Escherichia coli overexpressing the NHase, and purified recombinant NHase was studied. E. meliloti converted IAN to the product indole-3-acetamide (IAM), and no nitrilase or amidase activities, or indole-3-acetic acid formation, were detected. Whole cells of E. meliloti converted IAN from the initial content of 6.41 to 0.06 mmol/L in 48 h. Meanwhile, forming 5.99 mmol/L IAM, the molar conversion of 94.4%. E. coli Rosetta overexpressing the NHase from E. meliloti produced 4.46 mmol/L IAM in 5 min, with a conversion rate of 91.1%. The purified NHase had a Vmax for IAN conversion of 294.28 U/mg. Adding 2% and 10% (v/v) dichloromethane to 50 mmol/L sodium phosphate buffer containing 200 mg/L IAN increased the NHase activity by 26.8% and 11.5% respectively, while the addition of 20% hexane had no inhibitory effect on IAN bioconversion. E. meliloti shows high NHase activity without forming a byproduct carboxylic acid, and its tolerance of dichloromethane and hexane increases its potential for application in the green biosynthesis of high-value amide compounds.

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus.

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.

Proteolytic Activity in Meadow Soil after the Application of Phytohormones.

Phytohormones, similar to soil enzymes, are synthesized and secreted into the soil environment by fungi and microorganisms. Phytohormones are involved in regulating microbial community activity in the rhizosphere. This paper examines how auxins, cytokinins, ethephon and chlorocholine chloride affect the activity of native soil proteases in the organo-mineral horizon of an alpine meadow. In the meadow habitat, native soil proteases were inhibited by auxins whereas the effect of cytokinins on these enzymes was not statistically significant. A similar inhibitory effect on the activity of proteases was shown for ethephon and chlorocholine chloride, both of which also inhibited the activity of native soil proteases in the alpine meadow soil. Overall, the inhibitory effect of phytohormones on the activity of native protease activity may affect plant nutrition by retarding the nitrogen cycle in the soil. This work contributes to our understanding of the influence of substances produced by the rhizosphere that can actively participate in the activity of soil microorganisms and consequently influence the soil nitrogen cycle.

Structure and product relationship analysis of acyl homoserine lactone synthases among Ensifer adhaerens reveals distinct chromosome and plasmid origins.

Despite the conservative DNA sequences among LuxI (Acyl Homoserine Lactones synthase gene) homologs, structure-product relationship of AHL synthase remains to be elucidated. In this study, through degenerate primers and in vitro expression methods, we collected the information of the gene sequences and AHL profiles from nine LuxIs among Ensifer adhaerens strains. The chromosome-encoded LuxI (C-LuxI) distinguished themselves from the plasmid-encoded ones (P-LuxI) not only in the DNA sequences, but also in AHL profiles. The C-LuxIs produced only C14-HSL, while the P-LuxIs produced predominantly C8-HSL and 3-oxo-C8-HSL. Sequence-product relationship analysis updated our recognition of the role of T140 (EsaI) in the 3-oxo-HSL production. Computational calculation based on 3D structures of these LuxIs revealed the linear relationship between the chain length and the affinity of amides to AHL synthase in C-LuxI, which was not found in the P-LuxI. We hereby proposed the linear docking affinity as a criterion for the prediction of long-chain AHL production by an AHL synthase. This study extends our understanding on the structure-product relationship of AHL synthases and revealed the distinct chromosome and plasmid origin of this enzyme among E. adhaerens.

Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways.

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L-1.

Examining the molecular characteristics of glycoside hydrolase family 20 ß-N-acetylglucosaminidases with high activity.

ß-N-Acetylglucosaminidases (GlcNAcases) possess many important biological functions and are used for promising applications that are often hampered by low-activity enzymes. We previously demonstrated that most GlcNAcases of the glycoside hydrolase (GH) family 20 showed higher activities than those of other GH families, and we presented two novel GH 20 GlcNAcases that showed higher activities than most GlcNAcases. A highly flexible structure, which was attributed to the presence of to a high proportion of random coils and flexible amino acid residues, was presumed to be a factor in the high activity of GH 20 GlcNAcases. In this study, we further hypothesized that two special positions might play a key role in catalytic activity. The increase in GH 20 GlcNAcase activity might correspond to the increased structural flexibility and substrate affinity of the two positions due to an increase in random coils and amino acid residues, notably acidic Asp and Glu.

'Candidatus Liberibacter asiaticus' peroxiredoxin (LasBCP) suppresses oxylipin-mediated defense signaling in citrus.

The Lasbcp (CLIBASIA_RS00445) 1-Cys peroxiredoxin gene is conserved among all 13 sequenced strains of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing or "citrus greening" disease. LasBCP was previously characterized as a secreted peroxiredoxin with substrate specificity for organic peroxides, and as a potential pathogenicity effector. Agrobacterium-mediated transient expression of LasBCP in citrus leaves provided significant protection against peroxidation of free and membrane-bound lipids, thereby preserving the molecular integrity of the chlorophyll apparatus and reducing accumulation of lipid peroxidation products (oxylipins) following exposure to tert-butyl hydroperoxide (tBOOH, an organic peroxide). Oxylipins extracted from GUS-expressing citrus leaves reduced viability of L. crescens, the only Liberibacter species cultured to date. However, similar extracts obtained from LasBCP-expressing leaves were less inhibitory to L. crescens growth and viability in culture. Quantitative RT-PCR analyses showed coordinated transcriptional downregulation of oxylipin biosynthetic (CitFAD, CitLOX, CitAOS and CitAOC), and jasmonic acid (JA) (CitJAR1, CitCOI1 and CitJIN1) and salicylic acid (SA) (CitPAL, CitICS and CitPR1) signaling pathway genes in citrus leaves expressing LasBCP and treated with tBOOH. The negative response regulator of jasmonic acid CitJAZ1 was upregulated in LasBCP-expressing citrus leaves under similar conditions. These data clearly demonstrated a protective role of secreted LasBCP in favor of Las survival and colonization by alleviating ROS-induced lipid peroxidation in citrus host, preventing accumulation of antimicrobial oxylipins, and suppressing both localized and systemic immune responses in planta.

A Novel Nutrient Deprivation-Induced Neonicotinoid Insecticide Acetamiprid Degradation by Ensifer adhaerens CGMCC 6315.

Biodegradation of pesticide pollution is often restricted by environmental pressures, such as nutrient deprivation. Ensifer adhaerens CGMCC 6315 could overcome this issue and degrade neonicotinoid acetamiprid (ACE) efficiently under low nutrient stimuli. The ACE degradation rate improved by 33.1-fold when the lysogeny broth content for cell culture was decreased to 1/15-fold. Resting cells of CGMCC 6315 degraded 94.4% of 200 mg/L ACE in 12 h and quickly eliminated 87.8% of 5 mg/kg of residual soil ACE within 2 d. ACE degradation by CGMCC 6315 was via a nitrile hydratase (NHase) pathway. Genome sequencing showed that CGMCC 6315 had two NHase genes ( cnhA and pnhA). PnhA had the highest reported activity of 28.8 U/mg for ACE. QPCR and proteomic analysis showed that the improved ACE degradation ability was attributed to the up-regulated expression of PnhA. This biodegradation system of CGMCC 6315 has great potential for use in pesticide pollution remediation.

Purification and partial characterization of LdtP, a cell envelope modifying enzyme in Liberibacter asiaticus.

BACKGROUND: The aggressive spread of Liberibacter asiaticus, a bacterium closely associated with citrus greening, has given rise to an acute crisis in the citrus industry, making it imperative to expand the scientific knowledge base regarding L. asiaticus. Despite several endeavors to culture L. asiaticus, this bacterium has yet to be maintained in axenic culture, rendering identification and analysis of potential treatment targets challenging. Accordingly, a thorough understanding of biological mechanisms involved in the citrus host-microbe relationship is critical as a means of directing the search for future treatment targets. In this study, we evaluate the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. RESULTS: This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. This work described, for the first time, an enzyme of the L,D-transpeptidase family with moonlighting enzyme activity directed to the modification of the bacterial cell wall and LPS. CONCLUSIONS: Taken together, the data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host.

Isolation and characterization of racemase from Ensifer sp. 23-3 that acts on α-aminolactams and α-amino acid amides.

Limited information is available on α-amino-ε-caprolactam (ACL) racemase (ACLR), a pyridoxal 5'-phosphate-dependent enzyme that acts on ACL and α-amino acid amides. In the present study, eight bacterial strains with the ability to racemize α-amino-ε-caprolactam were isolated and one of them was identified as Ensifer sp. strain 23-3. The gene for ACLR from Ensifer sp. 23-3 was cloned and expressed in Escherichia coli. The recombinant ACLR was then purified to homogeneity from the E. coli transformant harboring the ACLR gene from Ensifer sp. 23-3, and its properties were characterized. This enzyme acted not only on ACL but also on α-amino-δ-valerolactam, α-amino-ω-octalactam, α-aminobutyric acid amide, and alanine amide.

Concomitant Loss of the Glyoxalase System and Glycolysis Makes the Uncultured Pathogen "Candidatus Liberibacter asiaticus" an Energy Scavenger.

Methylglyoxal (MG) is a cytotoxic, nonenzymatic by-product of glycolysis that readily glycates proteins and DNA, resulting in carbonyl stress. Glyoxalase I and II (GloA and GloB) sequentially convert MG into d-lactic acid using glutathione (GSH) as a cofactor. The glyoxalase system is essential for the mitigation of MG-induced carbonyl stress, preventing subsequent cell death, and recycling GSH for maintenance of cellular redox poise. All pathogenic liberibacters identified to date are uncultured, including "Candidatus Liberibacter asiaticus," a psyllid endosymbiont and causal agent of the severely damaging citrus disease "huanglongbing." In silico analysis revealed the absence of gloA in "Ca Liberibacter asiaticus" and all other pathogenic liberibacters. Both gloA and gloB are present in Liberibacter crescens, the only liberibacter that has been cultured. L. crescens GloA was functional in a heterologous host. Marker interruption of gloA in L. crescens appeared to be lethal. Key glycolytic enzymes were either missing or significantly downregulated in "Ca Liberibacter asiaticus" compared to (cultured) L. crescens Marker interruption of sut, a sucrose transporter gene in L. crescens, decreased its ability to take up exogenously supplied sucrose in culture. "Ca Liberibacter asiaticus" lacks a homologous sugar transporter but has a functional ATP/ADP translocase, enabling it to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding MG generation via glycolysis, and (iii) directly importing ATP from the host cell. MG detoxification enzymes appear to be predictive of "Candidatus" status for many uncultured pathogenic and environmental bacteria.IMPORTANCE Discovered more than 100 years ago, the glyoxalase system is thought to be present across all domains of life and fundamental to cellular growth and viability. The glyoxalase system protects against carbonyl stress caused by methylglyoxal (MG), a highly reactive, mutagenic and cytotoxic compound that is nonenzymatically formed as a by-product of glycolysis. The uncultured alphaproteobacterium "Ca Liberibacter asiaticus" is a well-adapted endosymbiont of the Asian citrus psyllid, which transmits the severely damaging citrus disease "huanglongbing." "Ca Liberibacter asiaticus" lacks a functional glyoxalase pathway. We report here that the bacterium is able to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding (significant) MG generation via glycolysis, and (iii) directly importing ATP from the host cell. We hypothesize that failure to culture "Ca Liberibacter asiaticus" is at least partly due to its dependence on host cells for both ATP and MG detoxification.

Nematicidal effect of rhizobacteria on plant-parasitic nematodes associated with vineyards.

The action of metabolites and exoenzymes from rhizobacteria on different plant-parasitic nematodes has an influence on the nematicidal efficacy of the microbe. Seven rhizobacteria, divided into two bacterial groups, were evaluated in vitro for nematicidal activity on Meloidogyne ethiopica and Xiphinema index. The direct effect of their filtrates on egg hatching and juveniles of M. ethiopica as well as mobile stages of X. index was evaluated during a 72-h period. The production of four exoenzymes and two metabolites associated with nematode mortality was investigated. Molecular characterization of three isolates was performed, and the physiological profiles and lipase activity of all isolates were obtained using the BIOLOG EcoPlate system. While chitinase and collagenase were measured using the BIOLOG MT2 plate system, protease, hydrogen cyanide and hydrogen sulphide were directly determined in Petri dishes. Nematode mobile stages exposure to the bacterial filtrate revealed a nematicidal effect up to 93.7% on X. Index and up to 83.3% on M. ethiopica. The control of egg hatching varied between 35 and 85%. A positive correlation was found between the mortality of both nematode mobile stages and the concerted activities of the bacterial enzymes as well as the level of the volatile metabolites. The nematicidal effect of rhizobacteria strains varies by nematode genera and among the developmental stages evaluated.

A Shinella ß-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and molecular characteristics.

ß-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min-1 mg-1) or V max (1030.0 ± 82.1 µmol min-1 mg-1) toward p-nitrophenyl ß-N-acetylglucosaminide and N,N'-diacetylchitobiose (specificity activity of 35.4 µmol min-1 mg-1) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0-15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms.

Functional characterization of LotP from Liberibacter asiaticus.

Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host-pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N-terminus. Co-immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP-interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two-hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.

A comprehensive phylogenetic analysis of copper transporting P1B ATPases from bacteria of the Rhizobiales order uncovers multiplicity, diversity and novel taxonomic subtypes.

The ubiquitous cytoplasmic membrane copper transporting P1B-1 and P1B-3 -type ATPases pump out Cu+ and Cu2+ , respectively, to prevent cytoplasmic accumulation and avoid toxicity. The presence of five copies of Cu-ATPases in the symbiotic nitrogen-fixing bacteria Sinorhizobium meliloti is remarkable; it is the largest number of Cu+ -transporters in a bacterial genome reported to date. Since the prevalence of multiple Cu-ATPases in members of the Rhizobiales order is unknown, we performed an in silico analysis to understand the occurrence, diversity and evolution of Cu+ -ATPases in members of the Rhizobiales order. Multiple copies of Cu-ATPase coding genes (2-8) were detected in 45 of the 53 analyzed genomes. The diversity inferred from a maximum-likelihood (ML) phylogenetic analysis classified Cu-ATPases into four monophyletic groups. Each group contained additional subtypes, based on the presence of conserved motifs. This novel phylogeny redefines the current classification, where they are divided into two subtypes (P1B-1 and P1B-3 ). Horizontal gene transfer (HGT) as well as the evolutionary dynamic of plasmid-borne genes may have played an important role in the functional diversification of Cu-ATPases. Homologous cytoplasmic and periplasmic Cu+ -chaperones, CopZ, and CusF, that integrate a CopZ-CopA-CusF tripartite efflux system in gamma-proteobacteria and archeae, were found in 19 of the 53 surveyed genomes of the Rhizobiales. This result strongly suggests a high divergence of CopZ and CusF homologs, or the existence of unexplored proteins involved in cellular copper transport.