Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates.

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Understanding the role of bacterial cellular adsorption, accumulation and bioavailability regulation by biosurfactant in affecting biodegradation efficacy of polybrominated diphenyl ethers.

Microbiological degradation is often considered as an important strategy to reduce the risks of polybrominated diphenyl ethers (PBDEs), which are environmentally widespread and harmful to human health and wildlife. With the well-identified aerobic bacteria, i.e. B. xenovorans LB400, the biodegradation of 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) occurred efficiently in conformity to the first-order kinetics and showed the strong dependence on initial concentration of pollutant and bioavailability regulation by biosurfactant. The mild increase of initial concentration of BDE-47 would enhance biodegradation whereas the excessive increase failed due to the oxidative stress or cytotoxicity to bacteria. Rather than the bacterial extracellular adsorption that was bioactively-mediated in thermodynamics, the intracellular accumulations at different time gradients showed the negative correlation with biodegradation efficiency of BDE-47. The spontaneous biodegradation of pollutant should be sourced from the gradual reduction of intracellular accumulation. Though the improved bioavailability of BDE-47 by sucrose fatty acid ester (SFAE) hardly altered the extracellular adsorption, the bacterial intracellular accumulation was indicated to increase continuously with used amount of biosurfactant and then decrease for the cellular morphological damage, and interestingly it appeared to be temporary reservoir for prompt delivery to biodegradation in light of the opposite variation tendency with time.

Identification of Holrhizins E-Q Reveals the Diversity of Nonribosomal Lipopeptides in Paraburkholderia rhizoxinica.

The products of a nonribosomal peptide synthetase gene, holA, from Paraburkholderia rhizoxinica were investigated using our recently established recombineering technique. Fifteen products, including 13 new linear lipopeptides, holrhizins E-Q (2-8, 10-15), together with the two known holrhizins A and B (1, 9), were detected in the activated mutant, and their structures were identified using HRESIMS, NMR spectroscopy, Marfey's analysis, and feeding experiments with labeled amino acids. The lipohexapeptides 1-3 and 7-14 differ in three amino acid residues and the N-terminal fatty acid chains. The diversity of the holrhizins originates from the substrate flexibility of the A4, A5, and A6 domains as well as the starter C domain in the biosynthetic pathway.

Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry.

In recent years, use of supercritical-fluid chromatography (SFC) with CO2 as the mobile phase has been expanding in the research laboratory and industry since it is considered to be a green analytical method. This technique offers numerous advantages, such as good separation and sensitive detection, short analysis times, and stability of analytes. In this study, a method for quantification of N-acyl homoserine lactones (AHLs), signaling molecules responsible for cell-to-cell communication initially discovered in bacteria, by SFC coupled with high-resolution mass spectrometry (HRMS) was developed. The SFC conditions and MS ionization settings were optimized to obtain the best separation and greatest sensitivity. The optimal analysis conditions allowed quantification of up to 30 AHLs in a single run within 16 min with excellent linearity (R2 > 0.998) and sensitivity (picogram level). This method was then applied to study AHL production by one Gram-negative endophytic bacterium, Paraburkholderia sp. BSNB-0670. Nineteen known AHLs were detected, and nine abundant HSLs were quantified. To further investigate the production of uncommon AHLs, a molecular networking approach was applied on the basis of the SFC-HRMS/MS data. This led to additional identification of four unknown AHLs annotated as N-3-hydroxydodecanoylol homoserine lactone, N-3-hydroxydodecadienoyl homoserine lactone, and N-3-oxododecenoyl homoserine lactones (two isomers).

Genome Mining and Heterologous Expression Reveal Two Distinct Families of Lasso Peptides Highly Conserved in Endofungal Bacteria.

Genome mining identified the fungal-bacterial endosymbiosis Rhizopus microsporus-Mycetohabitans (previously Burkholderia) rhizoxinica as a rich source of novel natural products. However, most of the predicted compounds have remained cryptic. In this study, we employed heterologous expression to isolate and characterize three ribosomally synthesized and post-translationally modified peptides with lariat topology (lasso peptides) from the endosymbiont M. rhizoxinica: burhizin-23, mycetohabin-16, and mycetohabin-15. Through coexpression experiments, it was shown that an orphan gene product results in mature mycetohabin-15, albeit encoded remotely from the core biosynthetic gene cluster. Comparative genomics revealed that mycetohabins are highly conserved among M. rhizoxinica and related endosymbiotic bacteria. Gene knockout and reinfection experiments indicated that the lasso peptides are not crucial for establishing symbiosis; instead, the peptides are exported into the environment during endosymbiosis. This is the first report on lasso peptides from endosymbiotic bacteria.

Caballeronia ginsengisoli sp. nov., isolated from ginseng cultivating soil.

A Gram-stain-negative, strictly aerobic, non-motile, ivory colored and rod-shaped bacterium (designated Gsoil 652T) isolated from ginseng cultivating soil, was characterized using a polyphasic approach to clarify its taxonomic position. Strain Gsoil 652T was observed to grow optimally at 30 °C and at pH 7.0 on R2A agar medium. Phylogenetic analysis, based on 16S rRNA gene sequences similarities, indicated that Gsoil 652T belongs to the genus Caballeronia of the family Burkholderiaceae and was most closely related to Caballeronia choica LMG 22940T (98.9%), Caballeronia udeis LMG 27134T (98.9%), Caballeronia sordidicola LMG 22029T (98.2%) and Caballeronia humi LMG 22934T (98.1%). The DNA G+C content was 62.1 mol% and Q-8 was the major isoprenoid quinone. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, unidentified aminophospholipid, and unidentified phospholipid. The predominant fatty acids were C16:0, C17:0 cyclo and C19:0 cyclo ω8c. The DNA-DNA relatedness value between strain Gsoil 652T and closely related type strains of Caballeronia species were less than 36.0%. Moreover, strain Gsoil 652T could be distinguished phenotypically from the recognized species of the genus Caballeronia. The novel isolate, therefore, represents a novel species, for which the name Caballeronia ginsengisoli sp. nov. is proposed, with the type strain Gsoil 652T (= KACC 19441T = LMG 30326T).

Gramibactin is a bacterial siderophore with a diazeniumdiolate ligand system.

Genome mining and chemical analyses revealed that rhizosphere bacteria (Paraburkholderia graminis) produce a new type of siderophore, gramibactin, a lipodepsipeptide that efficiently binds iron with a logß value of 27.6. Complexation-induced proton NMR chemical shifts show that the unusual N-nitrosohydroxylamine (diazeniumdiolate) moieties participate in metal binding. Gramibactin biosynthesis genes are conserved in numerous plant-associated bacteria associated with rice, wheat, and maize, which may utilize iron from the complex.

Crystal structure analysis, covalent docking, and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase.

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3-hydroxybutyrate) depolymerase were identified in two high-resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281-295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3-hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281-295 in comparison to the apo (substrate-free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281-295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351-1361. © 2017 Wiley Periodicals, Inc.

The therapeutic potential of microbial proteasome inhibitors.

The proteasome influences cellular homeostasis through the degradation of regulatory proteins, many of which are also involved in disease pathogenesis. In particular, numerous regulatory proteins associated with tumor growth, such as cyclins, cyclin-dependent kinase inhibitors, tumor suppressors, and NF-κB inhibitors are degraded by the proteasome. Proteasome inhibitors can stabilize these regulatory proteins, resulting in the suppression of tumor development and the regulation of immune responses. Thus, proteasome inhibitors are promising candidate antitumor agents and immune-regulatory agents. Bortezomib is the first-in-class proteasome inhibitor approved for the treatment of multiple myeloma. Despite its high efficiency, however, a large proportion of patients do not attain sufficient clinical response due to toxicity and drug resistance. Therefore, the development of new proteasome inhibitors with improved pharmacological properties is needed. Natural products produced by microorganisms are a promising source of such compounds. This review provides an overview of proteasome inhibitors produced by microorganisms, with special focus on inhibitors isolated from actinomycetes.

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases.

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4'-dihydroxybiphenyl, 4-hydroxy-4'-chlorobiphenyl, 3-hydroxy-4,4'-dichlorobiphenyl, and 3,3'-dihydroxy-4,4'-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4'-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4'-chlorobiphenyl and 3,4-dihydroxy-4'-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4'-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4'-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3'-trihydroxy-4'-chlorobiphenyl with concomitant dechlorination, and 2,3,3'-trihydroxy-4'-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3'-Dihydroxy-4,4'-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3'-trihydroxy-4,4'-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.

Degradation pathway and kinetic analysis for p-xylene removal by a novel Pandoraea sp. strain WL1 and its application in a biotrickling filter.

In this study, a novel Pandoraea sp. strain WL1 capable of mineralizing p-xylene as sole carbon and energy source was isolated from the activated sludge of a pharmaceutical wastewater treatment plant. A nearly complete degradation of 16.6∼99.4 mg L(-1)p-xylene in the liquid-phase was achieved within 6∼18 h accompanied by 15.9∼56.3 mg dry cell weight (DCW)L(-1) for bacterial growth. A complete pathway for p-xylene degradation by strain WL1 was presented through identification of a major intermediate (p-toluic acid) and final products (2.193 gCO2 gp-xylene(-1) of CO2 production and 0.215 g DCW gp-xylene(-1) of bacterial yield). Kinetics of bacterial growth and p-xylene degradation were evaluated using Haldane-Andrews model and pseudo first-order model, respectively. Furthermore, a biotrickling filter (BTF) was employed to evaluate the application of strain WL1 on the removal of gas-phase p-xylene under gas flow rates of 0.41∼1.98 m(3)h(-1) for inlet loading rates of 5∼248 gm(-3)h(-1). The BTF inoculated with strain WL1 proved to be robust against fluctuations of gas flow rates and inlet p-xylene concentrations. All the results obtained highlight the potential of strain WL1 for the treatment of p-xylene.

Structural characterization of Pandoraea pnomenusa B-356 biphenyl dioxygenase reveals features of potent polychlorinated biphenyl-degrading enzymes.

The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a heterohexameric (αß)(3) Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO(B356) with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO(B356) and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2'-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.

Clinical and microbiological features of a cystic fibrosis patient chronically colonized with Pandoraea sputorum identified by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Clonal isolates identified as various nonfermentative Gram-negative bacilli over a 5-year period from sputum cultures of a 30-year-old cystic fibrosis patient were successfully reidentified as Pandoraea sputorum by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Decreased lung function improved after 1 year of azithromycin and inhaled 7%-hypertonic saline treatment.

Polynucleobacter difficilis sp. nov., a planktonic freshwater bacterium affiliated with subcluster B1 of the genus Polynucleobacter.

Strain AM-8B5(T), isolated from Lake Sevan in Armenia, was characterized phenotypically, chemotaxonomically and phylogenetically. This chemo-organoheterotrophic, aerobic, facultatively anaerobic, catalase- and oxidase-positive, non-motile strain grew on NSY medium at NaCl concentrations of 0.0-0.2 % (w/v) and at 4-30 °C. Whole-cell fatty acids were dominated by summed feature 3 (including C(16 : 1)ω7c and iso-C(15 : 0) 2-OH), C(16 : 0) and C(18 : 1)ω7c. C(12 : 0) 2-OH and C(16 : 1) 2-OH were the only hydroxylated fatty acids detected. Phylogenetic analysis as well as phenotypic and chemotaxonomic similarities indicated that the novel isolate was affiliated with the genus Polynucleobacter. 16S rRNA gene similarity values with the four previously described Polynucleobacter species ranged from 96.2 to 98.7 %. DNA-DNA hybridization experiments showed that the isolate did not belong to any of the previously described Polynucleobacter species. The isolate could be distinguished from all previously established Polynucleobacter species based on chemotaxonomic and phenotypic traits. The bacterium possessed a free-living lifestyle and represents a group of bacteria inhabiting the water column of many freshwater lakes. Based on the revealed phylogeny, and chemotaxonomic and phenotypic differences to previously described Polynucleobacter species, it is proposed that the isolate represents a novel species, Polynucleobacter difficilis sp. nov.; the type strain is AM-8B5(T) ( = DSM 22349(T) = CIP 110078(T)).

Insight into the metabolism of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by biphenyl dioxygenases.

In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356)) to metabolize DDT. Data show BphAE(LB400) is unable to metabolize this substrate but BphAE(B356) metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE(LB400) and BphAE(B356) identified residue Phe336 of BphAE(LB400) as critical to prevent productive binding of DDT to BphAE(LB400). Furthermore, the fact that residue Gly319 of BphAE(B356) is less constrained than Gly321 of BphAE(LB400) most likely contributes to the ability of BphAE(B356) to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE(LB400) were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE(B356). Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247-260 are likely involved in stereospecificity.

The structure of PhaZ7 at atomic (1.2 A) resolution reveals details of the active site and suggests a substrate-binding mode.

Poly-(R)-hydroxyalkanoates (PHAs) are bacterial polyesters that are degraded by a group of enzymes known as PHA depolymerases. Paucimonas lemoignei PhaZ7 depolymerase is the only extracellular depolymerase that has been described as being active towards amorphous PHAs. A previously determined crystal structure of PhaZ7 revealed an alpha/beta-hydrolase fold and a Ser-His-Asp catalytic triad. In order to address questions regarding the catalytic mechanism and substrate binding, the atomic resolution structure of PhaZ7 was determined after cocrystallization with the protease inhibitor PMSF. The reported structure has the highest resolution (1.2 A) of currently known depolymerase structures and shows a sulfur dioxide molecule covalently attached to the active-site residue Ser136. Structural comparison with the free PhaZ7 structure (1.45 A resolution) revealed no major changes in the active site, suggesting a preformed catalytic triad. The oxyanion hole was found to be formed by the amide groups of Met137 and Asn49. Nine well ordered water molecules were located in the active site. Manual docking of a substrate trimer showed that the positions of these water molecules coincide well with the substrate atoms. It is proposed that these water molecules are displaced upon binding of the substrate. Furthermore, conformational changes were identified after comparison with a previously determined PhaZ7 dimer structure in a different space group. The changes were located in surface loops involved in dimer formation, indicating some flexibility of these loops and their possible involvement in polyester binding.

Emended description of the genus Polynucleobacter and the species Polynucleobacter necessarius and proposal of two subspecies, P. necessarius subsp. necessarius subsp. nov. and P. necessarius subsp. asymbioticus subsp. nov.

Heckmann and Schmidt described the genus Polynucleobacter for bacterial endosymbionts of freshwater ciliates affiliated with the genus Euplotes, and the species Polynucleobacter necessarius for obligate endosymbionts living in the cytoplasm of Euplotes aediculatus. Pure cultures of the type strain could not be established due to the obligate nature of the symbiotic relationship between the endosymbionts and their hosts. Therefore, Polynucleobacter necessarius is one of a few bacterial species with validly published names that lack a deposited pure culture. Meanwhile, it was demonstrated that the endosymbionts used for the description of the type of Polynucleobacter necessarius are closely related to obligately free-living strains. Similarity values of the 16S rRNA gene sequences obtained from the endosymbionts in the ciliate culture and free-living isolates in the range 99.1-99.4% indicate that these organisms belong to the same species. Here, we have emended the description of Polynucleobacter necessarius by characterization of free-living strains maintained as pure cultures. The species Polynucleobacter necessarius was characterized as having low G+C contents of the DNA (44-46 mol%), small genome sizes (1.5-2.5 Mbp) and a lack of motility. Because of distinct differences in lifestyle and the genome size of Polynucleobacter necessarius strains, we propose that two novel subspecies should be established, Polynucleobacter necessarius subsp. necessarius subsp. nov. [with a type, which is a description based on endosymbionts in the culture 'stock 15' of the ciliate E. aediculatus (ATCC 30859)] and Polynucleobacter necessarius subsp. asymbioticus subsp. nov. [with the type strain QLW-P1DMWA-1T (=DSM 18221T=CIP 109841T)], for the obligate endosymbionts of E. aediculatus and Euplotes harpa and obligately free-living strains, respectively.

Involvement of cell surface structures in size-independent grazing resistance of freshwater Actinobacteria.

We compared the influences of grazing by the bacterivorous nanoflagellate Poterioochromonas sp. strain DS on ultramicrobacterial Actinobacteria affiliated with the Luna-2 cluster and ultramicrobacterial Betaproteobacteria of the species Polynucleobacter cosmopolitanus. These bacteria were almost identical in size (<0.1 microm(3)) and shape. Predation on a Polynucleobacter strain resulted in a reduction of >86% relative to the initial bacterial cell numbers within 20 days, while in comparable predation experiments with nine actinobacterial strains, no significant decrease of cell numbers by predation was observed over the period of >or=39 days. The differences in predation mortality between the actinobacterial strains and the Polynucleobacter strain clearly demonstrated size-independent grazing resistance for the investigated Actinobacteria. Importantly, this size-independent grazing resistance is shared by all nine investigated Luna-2 strains and thus represents a group-specific trait. We investigated if an S-layer, previously observed in an ultrastructure study, was responsible for the grazing resistance of these strains. Experiments aiming for removal of the S-layer or modification of cell surface proteins of one of the grazing-resistant strains by treatment with lithium chloride, EDTA, or formaldehyde resulted in 4.2- to 5.2-fold higher grazing rates in comparison to the levels for untreated cells. These results indicate the protective role of a proteinaceous cell surface structure in the size-independent grazing resistance of the actinobacterial Luna-2 strains, which can be regarded as a group-specific trait.

Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli.

An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22 degrees C but was efficiently secreted into the extracellular medium when cultivated at 37 degrees C.