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.

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.

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.

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.

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.

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 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.

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.

[Bacterial antigen polysaccharides. 43. Structure of O-specific polysaccharide of Providencia alcalifaciens O46].

Acid O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. Consideration of the data revealed the following structure of the hexasaccharide repeating unit of O-specific polysaccharide under methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H, (1)H-COSY, TOCSY-, ROESY-,(1)H, (13)C-HSQC-, and HMQC-TOCSY experiments: [Formula: see text].

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.

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.

Structure of the O-antigen of Providencia stuartii O20, a new polysaccharide containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid.

The O-polysaccharide chain of the lipopolysaccharide (LPS) of Providencia stuartii O20 was found to contain d-glucuronic acid, N-acetyl-d-glucosamine, and a rarely occurring higher sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). Degradation of the LPS with dilute acetic acid caused depolymerization of the polysaccharide chain by the ketosidic linkage to give a tetrasaccharide corresponding to the repeating unit of the polysaccharide. Based on sugar and methylation analyses of the tetrasaccharide and O-deacylated LPS as well as ESIMS, (1)H and (13)C NMR spectroscopy data, the structure of the O-polysaccharide of P. stuartii O20 was established.

New structure for the O-polysaccharide of Providencia alcalifaciens O27 and revised structure for the O-polysaccharide of Providencia stuartii O43.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O27 and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments. It was found that the polysaccharide is built up of linear partially O-acetylated tetrasaccharide repeating units and has the following structure: [structure: see text] where Qui4NFo stands for 4-formamido-4,6-dideoxyglucose (4-formamido-4-deoxyquinovose). The O-polysaccharide structure of Providencia stuartii O43 established earlier was revised with respect to the configuration of the constituent 4-amino-4,6-dideoxyhexose (from Rha4N to Qui4N).

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O36 containing 3-deoxy-d-manno-oct-2-ulosonic acid.

An oligosaccharide that corresponds to the repeating unit of the O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O36. Structural studies of the oligosaccharide and O-deacylated lipopolysaccharide were performed using sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC and HMBC experiments. It was found that the O-polysaccharide is built up of linear trisaccharide repeating units containing 2-acetamido-2-deoxyglucose, 6-deoxy-l-talose (l-6dTal), and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and has the following structure. [structure: see text]

Structure of a colitose-containing O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O6.

The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O6 and studied by sugar and methylation analysis, selective hydrolytic removal of 3,6-dideoxy-L-xylo-hexose (colitose, Col), (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments. The polysaccharide was found to have a branched pentasaccharide repeating unit with the following structure: [see text] Remarkably, the trisaccharide side chain of the O6-polysaccharide represents a colitose ('3-deoxy-L-fucose') analogue of the H type 1 (precursor) antigenic determinant.

Structure of the O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Providencia alcalifaciens O32 containing N-acetylisomuramic acid.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O32 and studied by sugar and methylation analyses, solvolysis with triflic acid, 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. It was found that the polysaccharide has a branched tetrasaccharide repeating unit containing 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (D-GlcNAc3Slac, N-acetylisomuramic acid) with the following structure: [ STRUCTURE: SEE TEXT]. Serological studies with O-antisera showed antigenic relationships between P. alcalifaciens O32 and O29 as well as several other Providencia and Proteus strains sharing putative epitopes on the O-polysaccharides.

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.

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O30.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O30. Studies by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, HMBC, and HMQC-TOCSY experiments, showed that the polysaccharide has a linear pentasaccharide repeating unit of the following structure:

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.

The O-polysaccharide from the lipopolysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides.

The O-polysaccharide (O-antigen) of Providencia stuartii O44:H4 (strain 3768/51) was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure: [Formula: see text].