Discovery of antibacterial biogenic magnetosome nanoparticles from Providencia sp. MTBPRB-1: Screening, purification and characterization.

Bacterial species referred to as magnetotactic bacteria (MTB) biomineralize iron oxides and iron sulphides inside the cell. Bacteria can arrange themselves passively along geomagnetic field lines with the aid of these iron components known as magnetosomes. In this study, magnetosome nanoparticles, which were obtained from the taxonomically identified MTB isolate Providencia sp. PRB-1, were characterized and their antibacterial activity was evaluated. An in vitro test showed that magnetosome nanoparticles significantly inhibited the growth of Staphylococcus sp., Pseudomonas aeruginosa, and Klebsiella pneumoniae. Magnetosomes were found to contain cuboidal iron crystals with an average size of 42 nm measured by particle size analysis and scanning electron microscope analysis. The energy dispersive X-ray examination revealed that Fe and O were present in the extracted magnetosomes. The extracted magnetosome nanoparticles displayed maximum absorption at 260 nm in the UV-Vis spectrum. The distinct magnetite peak in the Fourier transform infrared (FTIR) spectroscopy spectra was observed at 574.75 cm-1. More research is needed into the intriguing prospect of biogenic magnetosome nanoparticles for antibacterial applications.

Structural and phylogenetic analyses of resistance to next-generation aminoglycosides conferred by AAC(2') enzymes.

Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2')-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2')-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2')-Ia-acetylated plazomicin complex, these structures outline AAC(2')-Ia's specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2')-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.

Poly-lysine supported cross-linked enzyme aggregates of penicillin G acylase and its application in synthesis of ß-lactam antibiotics.

Penicillin G acylase (PGA) from Providencia rettgeri PX04 (PrPGA) was utilized to synthesize ß-lactam antibiotics. Poly-lysine supported cross-linked enzyme aggregates (PL-CLEAs) were prepared using PGA. Addition of poly-lysine significantly increased retention of PGA activity in CLEAs, with a decrease in the synthesis/hydrolysis (S/H) ratio. PL-CLEAs with 0.56 mg/mL poly-lysine retained 83% of free PGA activity, and displayed a higher S/H ratio than that of the free enzyme. Both PL-CLEAs and CLEAs exhibited high pH and thermal stabilities. PL-CLEAs possessed the best stability profile, and the lowest α value [(kcat/Km)Ps/(kcat/Km)AD], and was most effective at amoxicillin synthesis. A >94% yield of amoxicillin was achieved using a D-HPGME/6-APA ratio of 1.2:1 (240 mM, 200 mM), with fed-batch addition of D-HPGME. PL-CLEAs displayed excellent operational stability during amoxicillin synthesis. Over 97% of initial conversion was retained after twenty rounds of catalysis. PL-CLEAs exhibited greater potency than CLEAs in practical catalysis, permitting a higher concentration of reactants.

Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities.

The gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact, before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, we hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii, Omp-Pst1 and Omp-Pst2. Our crystal structures reveal that these porins can self-associate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication.

In vitro metal catalyzed oxidative stress in DAH7PS: Methionine modification leads to structure destabilization and induce amorphous aggregation.

The first committed step of the shikimate pathway is catalyzed by a metalloenzyme 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), which exhibits vulnerability to the oxidative stress. DAH7PS undergoes inactivation in multiple ways in the presence of redox metal, H2O2, and superoxide. The molecular mechanism and susceptibility of its inactivation might differ in different organisms and are presently unclear. In the present work, we have cloned, expressed and purified a DAH7PS from Providencia alcalifaciens (PaDAH7PS). The oligomeric state and effect of redox metal treatment on its stability were analyzed through the size exclusion chromatography. The FTIR, MALDI-TOF/TOF-MS studies revealed that methionine residues were modified to methionine sulfoxide in PaDAH7PS. During oxidation, PaDAH7PS is altered into partially folded protein and unfolded states as determined by CD and Fluorescence studies. A significant loss in enzymatic activity of PaDAH7PS was determined and the formation of amorphous aggregates was visualized using AFM imaging and also confirmed by ThT binding based assay. This is the first report where we have shown a hexameric DAH7PS and the methionine residues of PaDAH7PS get oxidize in the presence of oxidative stress. The partially folded and unfolded oligomeric states with high ß-content of PaDAH7PS might be the critical precursors for aggregation.

Microbial remediation of fluoride-contaminated water via a novel bacterium Providencia vermicola (KX926492).

The present study emphasizes on the isolation, identification and characterization of a fluoride-resistant bacteria from contaminated groundwater of a severely affected rural area. The isolate was investigated for its possible role towards bioremediation of fluoride. Bacterial growth was determined by various carbon and nitrogen sources. Influence of parameters like initial fluoride concentration (5-25 mg L-1), pH (3-9) and temperature (15-42 °C) on fluoride removal by Providencia sp. KX926492 were also examined. SEM, EDX and FTIR were performed to analyse the surface texture, elemental composition and functional groups of the bacterium involved in the uptake of fluoride ions. 16S rRNA sequencing was performed to identify the isolate. Plackett-Burman design was employed to optimize the various parametric conditions of fluoride removal. Maximum removal of 82% was achieved when the initial fluoride concentration was 20 mgL-1 at pH 7 and 37 °C temperature with dextrose and nitrogen concentrations of 5 and 4 g per 50 mL respectively. Results suggested that Providencia vermicola (KX926492) could be a potential bacterium in removal of fluoride from contaminated water.

Porin flexibility in Providencia stuartii: cell-surface-exposed loops L5 and L7 are markers of Providencia porin OmpPst1.

Epidemiologically unrelated Providencia stuartii strains isolated in hospitals in the south of France were investigated for their porin sequences and profiles. Noticeable resistance to ß-lactams was found to be associated with production of extended spectrum ß-lactamases or AmpC overproduction, but not metallo-ß-lactamases. At the same time, the expression level of outer membrane porins was unmodified in these isolates. The identity of the amino acid sequences of the major porin OmpPst1 was less than 90% in the tested clinical strains, whereas sequences of the second major porin OmpPst2 were found to be identical in all isolates. Sequence diversity identified in the OmpPst1 porins was mainly located in two cell-surface-exposed loops (L5 and L7): these loops were found to be responsible for 80% of the main movements of the protein. Parallel tempering MD simulations indicated possible coordinated movement of these loops that might affect the electrostatic interaction of the porin with membrane components (e.g. LPS) or with external molecules/surfaces. This suggests that such flexibility of surface-exposed domains of OmpPst1 may participate in bacterial adaptation to the environment.

Structure of the O-polysaccharide of Providencia alcalifaciens O2 containing ascarylose and N-(L-alanyl)-D-glucosamine.

The O-polysaccharide was obtained by degradation of the lipopolysaccharide of Providencia alcalifaciens O2 under mild acidic conditions followed by GPC. The polysaccharide was found to contain two unusual components: 3,6-dideoxy-L-arabino-hexose (ascarylose, Asc) and 2-(L-alanyl)amino-2-deoxy-D-glucose (GlcNAla). Ascarylose was partially split off during lipopolysaccharide degradation and could be eliminated completely by selective acid hydrolysis, which also partially cleaved the ß-GAlNAc-(1 → 6) linkage. The following structure of the branched pentasaccharide repeating unit was established by (1)H and (13)C NMR spectroscopy of the O-polysaccharide and O-deacetylated polysaccharide, as well as products of partial acid hydrolysis: α-Ascp-(1 → 4)-α-D-GlcpA-(1 → 4) → 6)-ß-D-GlcpNAla-(1 → 4)-ß-D-GlpA-(1 → 3)-ß-D-GalpNAc-(1 → ~60% OAc--3).

Structure and gene cluster organization of the O-antigen of Providencia alcalifaciens O45:H25.

O-Polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O45:H25 and studied by sugar analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeat of the O-polysaccharide was established: [structure: see text]. The O-antigen gene cluster of P. alcalifaciens O45 was sequenced and found to be in full agreement with the O-polysaccharide structure established.

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures.

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the 'water retention site', suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.

Detection of tetrodotoxin-producing Providencia rettgeri T892 in Lagocephalus pufferfish.

Tetrodotoxin (TTX) is a potent toxin but it could be used in pharmaceutical field. Identification of TTX producing bacteria in pufferfish is necessary for TTX yield and the pufferfish conservation. In this study, Lagocephalus was collected from Cam Ranh Sea, a central part of Vietnam during spring season. The liver and intestine were incubated in 0.9 % NaCl for TTX detection in pufferfish. To be benefited from the isolation of new TTX producing bacteria, the liver and intestine were incubated in 6.5 % NaCl. The cultures were used to test the toxin and to isolate the bacterial community that could yield TTX. Surprisingly, Providencia rettgeri T892 in intestine could produce TTX identified by biochemical test and 16S rRNA sequencing. This strain was used to test the production of TTX, based on thin layer chromatography (TLC), mouse bioassay and high performance liquid chromatography (HPLC) analysis. The bacterium was optimized for TTX production in media prepared from the meat-washing water of Auxis thazard, Megalaspis cordyla and Decapterus maruadsi. Interestingly, the TTX obtained 0.106 mg/mL and 0.055 mg/mL in medium prepared from A. thazard and M. cordyla, respectively while there was no TTX production detected in medium prepared from D. maruadsi. This paper could contribute to warn to the human health care system about a possible TTX poisoning in some cases related to eating fishes.

Providencia thailandensis sp. nov., isolated from seafood processing wastewater.

The bacterial strain C1112(T) was isolated from seafood processing wastewater collected from a treatment pond of the seafood factory in Songkhla Province, Thailand. Phylogenetic analysis based on concatenated sequences from the 16S rRNA gene and five housekeeping genes, fusA, lepA, leuS, gyrB and ileS respectively showed that the strain C1112(T) belonged to the genus Providencia, and share 91.75% similarity with P. stuartii DSM 4539(T). DNA-DNA hybridization between the strain C1112(T) and P. stuartii KCTC 2568(T) was 48.1% relatedness. Moreover, some results from biochemical properties indicated that the strain C1112(T) was distinguished from the phylogenetically closest relatives. The major fatty acids of the strain C1112(T) were C16:0, iso-C15:0, C14:0 and C17:0 cyclo and the DNA G+C content was 41 mol%. Based on the genotypic and phenotypic considerations, it should be classified as a novel species of the genus Providencia for which the name Providencia thailandensis sp. nov. is proposed. The type strain is C1112(T) (= KCTC 23281(T) =NBRC 106720(T)).

Structure of the O-polysaccharide of Providencia alcalifaciens O35 containing an N-[(S)-1-carboxyethyl]-L-alanine (alanopine) derivative of 4-amino-4,6-dideoxyglucose.

The O-polysaccharide of Providencia alcalifaciens O35 was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(13)C HMBC, and NOESY experiments in D2O and, to detect correlations for NH protons, in a 9:1 H2O/D2O mixture. A unique N-(1-carboxyethyl)alanine (alanopine, Alo) derivative of 4-amino-4,6-dideoxyglucose (Qui4N) was identified as the polysaccharide component. Alanopine was isolated by solvolysis of the polysaccharide with triflic acid followed by acid hydrolysis, and its (2S,4S)-configuration was determined by the specific optical rotation. The following structure of the O-polysaccharide was established (the d configuration of Qui4N was ascribed tentatively): [structure: see text].

Structural, serological, and genetic characterization of the O-antigen of Providencia alcalifaciens O40.

The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia. In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-ß-D-Quip3NFo-(1→3)-α-D-Galp-(1→3)-ß-D-GlcpA-(1→3)-ß-D-GalpNAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico. In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-D-Qui3NFo, UDP-D-Gal, UDP-D-GlcA, and UDP-D-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb, and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called K(LPS).

Structure of the O-polysaccharide of Providencia alcalifaciens O3 containing 3,6-dideoxy-3-formamido-D-glucose and D-galacturonamide.

Mild acid degradation of the lipopolysaccharide (LPS) of Providencia alcalifaciens O3 followed by GPC on Sephadex G-50 and anion-exchange chromatography on DEAE-Trisacryl M afforded neutral and acidic polysaccharides, LPS core oligosaccharide, and an oligosaccharide composed of one repeat of the neutral polysaccharide (O-unit) linked to the LPS core. The following structure of the pentasaccharide O-unit was established by sugar and methylation analyses, 2D (1)H and (13)C NMR spectroscopy and ESI MS: [formula: see text] where Qui3NFo stands for 3,6-dideoxy-3-formamidoglucose and GalAN for galacturonamide. The LPS core is represented by the Glc(3)Gal(1)GalA(1)Hep(3)Kdo(1)Ara4N(1)P(3)EtN(2) glycoform reported earlier for P. alcalifaciens O9, O34, and O19. The acidic polysaccharide had the same peptidoglycan-like structure as a polysaccharide isolated earlier from P. alcalifaciens O24, O38, and O45, and, most likely, represents bacterial capsule material.

Genetic analysis of the O-antigen of Providencia alcalifaciens O30 and biochemical characterization of a formyltransferase involved in the synthesis of a Qui4N derivative.

O-Antigen is a component of the outer membrane of Gram-negative bacteria and one of the most variable cell surface constituents, giving rise to major antigenic variability. The diversity of O-antigen is almost entirely attributed to genetic variations in O-antigen gene clusters. Bacteria of the genus Providencia are facultative pathogens, which can cause urinary tract infections, wound infections and enteric diseases. Recently, the O-antigen gene cluster of Providencia was localized between the cpxA and yibK genes in the genome. However, few genes involved in the synthesis of Providencia O-antigens have been functionally identified. In this study, the putative O-antigen gene cluster of Providencia alcalifaciens O30 was sequenced and analyzed. Almost all putative genes for the O-antigen synthesis were found, including a novel formyltransferase gene vioF that was proposed to be responsible for the conversion of dTDP-4-amino-4,6- dideoxy-D-glucose (dTDP-D-Qui4N) to dTDP-4,6-dideoxy-4-formamido-D-glucose (dTDP-D-Qui4NFo). vioF was cloned, and the enzyme product was expressed as a His-tagged fusion protein, purified and assayed for its activity. High-performance liquid chromatography was used to monitor the enzyme-substrate reaction, and the structure of the product dTDP-D-Qui4NFo was established by electrospray ionization tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Kinetic parameters of VioF were determined, and effects of temperature and cations on its activity were also examined. Together, the functional analyses support the identification of the O-antigen gene cluster of P. alcalifaciens O30.

Structure of a polysaccharide from Providencia rustigianii O11 containing a novel amide of 2-acetamido-2-deoxygalacturonic acid with L-glutamyl-L-alanine.

An acidic polysaccharide was isolated from Providencia rustigianii O11 by the phenol-water extraction. The polysaccharide was cleaved by solvolysis with triflic acid to yield disaccharides with uronic acid derivatives at the non-reducing end. The polysaccharide and the disaccharides were studied by chemical analyses, high-resolution ESI MS, and 2D (1)H and (13)C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit of the polysaccharide was established: where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcA6GluAla for N-(2-acetamido-2-deoxygalacturonoyl)-l-glutam-1-yl-l-alanine, QuiNAc4NAcyl for 2-acetamido-4-[(S)-3-hydroxybutanoylamino]-2,4,6-trideoxyglucose (∼75%) or 2,4-diacetamido-2,4,6-trideoxyglucose (∼25%); the d configuration of GalNA and QuiN4N was ascribed tentatively. To the best of our knowledge, this is for the first time that an amide of uronic acid with a dipeptide is found in bacterial polysaccharides.

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O48.

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O48 and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and (1)H,(13)C HSQC and HMBC experiments. It was found that the polysaccharide is acidic and has a linear mono-O-acetylated tetrasaccharide repeating unit with the following structure: →3)-α-D-Manp-(1→2)-α-L-Fucp-(1→2)-ß-D-GlcpA4Ac-(1→3)-α-D-GalpNAc-(1→.

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O28.

An O-polysaccharide and oligosaccharides were isolated by GPC following mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O28. The O-polysaccharide was studied by sugar and methylation analyses, (1)H and (13)C NMR spectroscopy, including 2D ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments, and the following structure of the branched pentasaccharide repeating unit was established: [see formula in text]. This structure was confirmed by ESI MS of the isolated tridecasaccharide consisting of the lipopolysaccharide core and one O-polysaccharide repeat. The ESI mass spectrum also enabled inferring the composition of the core oligosaccharide.

Structure of the O-polysaccharide of Providencia alcalifaciens O25 containing an amide of D-galacturonic acid with N(ε)-[(R)-1-carboxyethyl]-L-lysine.

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O25 followed by gel-permeation and anion-exchange chromatography. The O-polysaccharide was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional correlation (1)H,(13)C HMBC, and (1)H,(1)H ROESY experiments both in D(2)O and, to detect correlations for NH protons, in a 9 : 1 H(2)O/D(2)O mixture. An amino acid was isolated from the polysaccharide by acid hydrolysis and identified as N(ε)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine", 2S,8R-alaLys) by determination of the specific optical rotation and (13)C NMR spectroscopy, using the authentic synthetic diastereomers 2S,8R-alaLys and 2S,8S-alaLys for comparison. The structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established.