Discovery of a novel symbiotic lineage associated with a hematophagous leech from the genus Haementeria.

Similarly to other strict blood feeders, leeches from the Haementeria genus (Hirudinida: Glossiphoniidae) have established a symbiotic association with bacteria harbored intracellularly in esophageal bacteriomes. Previous genome sequence analyses of these endosymbionts revealed co-divergence with their hosts, a strong genome reduction, and a simplified metabolism largely dedicated to the production of B vitamins, which are nutrients lacking from a blood diet. 'Candidatus Providencia siddallii' has been identified as the obligate nutritional endosymbiont of a monophyletic clade of Mexican and South American Haementeria spp. However, the Haementeria genus includes a sister clade of congeners from Central and South America, where the presence or absence of the aforementioned symbiont taxon remains unknown. In this work, we report on a novel bacterial endosymbiont found in a representative from this Haementeria clade. We found that this symbiont lineage has evolved from within the Pluralibacter genus, known mainly from clinical but also environmental strains. Similarly to Ca. Providencia siddallii, the Haementeria-associated Pluralibacter symbiont displays clear signs of genome reduction, accompanied by an A+T-biased sequence composition. Genomic analysis of its metabolic potential revealed a retention of pathways related to B vitamin biosynthesis, supporting its role as a nutritional endosymbiont. Finally, comparative genomics of both Haementeria symbiont lineages suggests that an ancient Providencia symbiont was likely replaced by the novel Pluralibacter one, thus constituting the first reported case of nutritional symbiont replacement in a leech without morphological changes in the bacteriome. IMPORTANCE: Obligate symbiotic associations with a nutritional base have likely evolved more than once in strict blood-feeding leeches. Unlike those symbioses found in hematophagous arthropods, the nature, identity, and evolutionary history of these remains poorly studied. In this work, we further explored obligate nutritional associations between Haementeria leeches and their microbial symbionts, which led to the unexpected discovery of a novel symbiosis with a member of the Pluralibacter genus. When compared to Providencia siddallii, an obligate nutritional symbiont of other Haementeria leeches, this novel bacterial symbiont shows convergent retention of the metabolic pathways involved in B vitamin biosynthesis. Moreover, the genomic characteristics of this Pluralibacter symbiont suggest a more recent association than that of Pr. siddallii and Haementeria. We conclude that the once-thought stable associations between blood-feeding Glossiphoniidae and their symbionts (i.e., one bacteriome structure, one symbiont lineage) can break down, mirroring symbiont turnover observed in various arthropod lineages.

Biodegradation pathway and mechanism of tri (2-chloropropyl) phosphate by Providencia rettgeri.

Tri (2-chloropropyl) phosphate (TCPP) was an emerging contaminant of global concern because of its frequent occurrence, potential toxic effects, and persistence in the environment. Microbial degradation might be an efficient and safe removal method, but limited information was available. In this study, Providencia rettgeri was isolated from contaminated sediment and showed it could use TCPP as unique phosphorus source to promote growth, and decompose 34.7% of TCPP (1 mg/L) within 5 days. The microbial inoculation and the initial concentration of TCPP could affect the biodegradation efficient. Further study results indicated that TCPP decomposition by Providencia rettgeri was mainly via phosphoester bond hydrolysis, evidenced by the production of bis (2-chloropropyl) phosphate (C6H13Cl2PO4) and mono-chloropropyl phosphate (C3H8ClPO4). Both intracellular and extracellular enzymes could degrade TCPP, but intracellular degradation was dominant in the later reaction stage, and the presence of Cu2+ ions had a promoting effect. These findings developed novel insights into the potential mechanism of TCPP microbial degradation.

Structural basis for plazomicin antibiotic action and resistance.

The approval of plazomicin broadened the clinical library of aminoglycosides available for use against emerging bacterial pathogens. Contrarily to other aminoglycosides, resistance to plazomicin is limited; still, instances of resistance have been reported in clinical settings. Here, we present structural insights into the mechanism of plazomicin action and the mechanisms of clinical resistance. The structural data reveal that plazomicin exclusively binds to the 16S ribosomal A site, where it likely interferes with the fidelity of mRNA translation. The unique extensions to the core aminoglycoside scaffold incorporated into the structure of plazomicin do not interfere with ribosome binding, which is analogously seen in the binding of this antibiotic to the AAC(2')-Ia resistance enzyme. The data provides a structural rationale for resistance conferred by drug acetylation and ribosome methylation, i.e., the two mechanisms of resistance observed clinically. Finally, the crystal structures of plazomicin in complex with both its target and the clinically relevant resistance factor provide a roadmap for next-generation drug development that aims to ameliorate the impact of antibiotic resistance.

Sensitive and rapid detection of cytolethal distending toxin encoding genes in Providencia alcalifaciens.

Cytolethal distending toxins (CDTs) produced by P. alcalifaciens are considered as potential virulence factors. A loop-mediated isothermal amplification (LAMP) method for the detection of cdtA, cdtB, and cdtC genes was established which showed high specificity and strong sensitivity. The LAMP assay showed a detection threshold was 3.13 pg/µl within 40 min.

A neurotransmitter produced by gut bacteria modulates host sensory behaviour.

Animals coexist in commensal, pathogenic or mutualistic relationships with complex communities of diverse organisms, including microorganisms1. Some bacteria produce bioactive neurotransmitters that have previously been proposed to modulate nervous system activity and behaviours of their hosts2,3. However, the mechanistic basis of this microbiota-brain signalling and its physiological relevance are largely unknown. Here we show that in Caenorhabditis elegans, the neuromodulator tyramine produced by commensal Providencia bacteria, which colonize the gut, bypasses the requirement for host tyramine biosynthesis and manipulates a host sensory decision. Bacterially produced tyramine is probably converted to octopamine by the host tyramine ß-hydroxylase enzyme. Octopamine, in turn, targets the OCTR-1 octopamine receptor on ASH nociceptive neurons to modulate an aversive olfactory response. We identify the genes that are required for tyramine biosynthesis in Providencia, and show that these genes are necessary for the modulation of host behaviour. We further find that C. elegans colonized by Providencia preferentially select these bacteria in food choice assays, and that this selection bias requires bacterially produced tyramine and host octopamine signalling. Our results demonstrate that a neurotransmitter produced by gut bacteria mimics the functions of the cognate host molecule to override host control of a sensory decision, and thereby promotes fitness of both the host and the microorganism.

Selective adsorption of palladium and platinum from secondary wastewater using Escherichia coli BL21 and Providencia vermicola.

It is important to recover precious metals from secondary wastewater because of their low crustal abundance. The selective adsorption of palladium (Pd) and platinum (Pt) ions from secondary wastewater, which contains a large amount aluminium and sodium ions, was investigated using Escherichia coli BL21 (BL21), genetically modified E. coli BL21 (EC20) and Providencia vermicola (P. V.). The results demonstrated that P.V., BL21 and EC20 cells took 95.9%, 88.2% and 97.5% of Pd ions, and 64.8%, 93.2% and 100% of Pt ions form industrial wastewater, respectively. All three bacterial biomass could be reused for Pd adsorption with a second adsorption efficiency of > 85%, specifically, the EC20 cells could absorb 93.8% of Pd ions from wastewater. SEM-EDS and XPS analyses confirmed the occurrence of Pd and Pt on the surface of wastewater-absorbed biomass. The shift in FTIR spectrum implied that functional groups, such as hydroxyl, amino, carboxyl and phosphate groups, were involved in wastewater adsorption.

Discovery of a novel native bacterium of Providencia sp. with high biosorption and oxidation ability of manganese for bioleaching of heavy metal contaminated soils.

Heavy metal removal from contaminated soils is a long-term challenging problem important for global economics, environment, and human health. Marine and freshwater-originated Mn(II)-oxidizing bacteria are considered as the promising bioremediation agents for environmental applications. However, practical application of soil-originated Mn(II)-oxidizing bacteria remains to be developed for contaminated soil remediation. In this work, the Mn(II) biosorption/oxidation mechanism of a new soil-originated bacterium and its bioleaching efficiency of heavy metals from soils was studied in detail. First, we found, isolated and identified a new highly Mn(II)-tolerant bacterial strain Providencia sp. LLDRA6 from heavy metal-contaminated soils. Next, strain LLDRA6 demonstrated its high Mn(II) biosorption capacity in aqueous solution. Then, Mn(II) adsorption by LLDRA6 was largely proven to be a synergistic effect of (i) Mn(II) precipitation on the cell surface, (ii) oxidation of Mn(II) into BioMnOx on the cell surface, and (iii) intracellular accumulation of insoluble MnCO3. Finally, combination bioleaching by the bacterium of Providencia sp. LLDRA6 and its formed BioMnOx was proposed to develop a potential environment-friendly and cost-effective technique to remediate severely heavy metal-contaminated soils. The bioleaching tests demonstrated that the combination of Providencia sp. LLDRA6 and BioMnOx exhibited an excellent removal efficiency for heavy metals of Pb (81.72%), Cr (88.29%), Cd (90.34%), Cu (91.25%), Mn (56.13%), and Zn (59.83%) from contaminated soils, resulting in an increase of removal efficiency in the range of 1.68-26.4% compared to Providencia sp. LLDRA6 alone. Moreover, the bacterial leachate facilitated the residual fraction of metals to transform into the easily migratory fractions in soils. These findings have demonstrated that strain LLDRA6 has high adsorption ability to remove heavy metals from contaminated soils, thus providing a promising bio-adsorbent for environmental bioremediation.

Providencia entomophila sp. nov., a new bacterial species associated with major olive pests in Tunisia.

Bioprospection for potential microbial biocontrol agents associated with three major insect pests of economic relevance for olive cultivation in the Mediterranean area, namely the olive fly, Bactrocera oleae, the olive moth, Prays oleae, and the olive psyllid, Euphyllura olivina, led to the isolation of several strains of readily cultivable Gram-negative, rod-shaped bacteria from Tunisian olive orchards. Determination of 16S ribosomal RNA encoding sequences identified the bacteria as members of the taxonomic genus Providencia (Enterobacterales; Morganellaceae). A more detailed molecular taxonomic analysis based on a previously established set of protein-encoding marker genes together with DNA-DNA hybridization and metabolic profiling studies led to the conclusion that the new isolates should be organized in a new species within this genus. With reference to their original insect association, the designation "Providencia entomophila" is proposed here for this hypothetical new taxon.

Structure elucidation and gene cluster characterization of the O-antigen of Vibrio cholerae O68 containing (2S,4R)-2,4-dihydroxypentanoic acid.

The O-polysaccharide (O-antigen, OPS) of Vibrio cholerae O68 was studied using chemical analyses and 1D and 2D NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the OPS was established: where Dhpa indicates (2S,4R)-2,4-dihydroxypentanoic acid existing mainly in the form of 1,4-lactone. Recently, (2R,4S)- and (2R,4R)-isomers of Dhpa have been found in the OPS of Providencia alcalifaciens O8 and O31, respectively. Functions of genes in the O-antigen gene cluster of Vibrio cholerae O68 were predicted according to the OPS structure established. These data provide a molecular basis for classification of V. cholerae strains.

Exploring the glyphosate-degrading characteristics of a newly isolated, highly adapted indigenous bacterial strain, Providencia rettgeri GDB 1.

This study explored the characteristics of a newly isolated glyphosate (GLYP)-degrading bacterium Providencia rettgeri GDB 1, for GLYP bioremediation. Due to the serial selection pressure of high GLYP concentrations for enriched isolation, this highly tolerant GLYP biodegrader shows very promising capabilities for GLYP removal (approximately 71.4% degradation efficiency) compared to previously reported strains. High performance liquid chromatography analyses showed aminomethylphosphonic acid (AMPA) rather than sarcosine (SAR) to be the sole intermediate of GLYP decomposition via the AMPA formation pathway. Moreover, GLYP biodegradation was biochemically favorable in aerobic cultures due to its strong growth-associated characteristics. To the best of our knowledge, this is the first report to indicate that bacterial strains in the Providencia genus could demonstrate highly promising GLYP-degrading characteristics in environments with high GLYP contents.

Molecular dynamics simulations investigate the pathway of substrate entry active site of rhomboid protease.

Rhomboid proteases can catalyze peptide bond cleavage and participate in abundant biological processes encompassing all branches of life; however, the pathway for substrate entry into its active site remains ambiguous. Here, the two possible pathways are preliminarily determined through molecular dynamics: One pathway is between Tm2 and Tm5, and the other is between Loop3 and Loop5. Then, the umbrella sampling simulations are performed to investigate the more feasible pathway for substrate entry. The results show that free energy barriers along the two pathways are similar; in the pathway 1, Trp236 and Trp157 as pivotal residues are responsible for the rotation of substrate in the binding process; in the pathway 2, among some important residues, the residue His150 plays an important role in substrate entry. Further, combining with previous experiment results, it is concluded that the substrate is inclined to enter into the active site along pathway 2. Our results are important for further understanding the function and catalysis mechanism of rhomboid proteases. Communicated by Ramaswamy H. Sarma.

Heavy water-labeled Raman spectroscopy reveals carboxymethylcellulose-degrading bacteria and degradation activity at the single-cell level.

Biodegradation of cellulose-rich substrates is an indispensable process for soil carbon replenishment in various ecological niches. Biodegradation of cellulose has been studied extensively via an enzyme assay to quantify the amount of cellulase with a view to identify effective cellulose degraders. However, a bulk enzyme assay undermines the role of physiological heterogeneity between cells; it is therefore imperative to opt out for a more effective method such as single-cell Raman spectroscopy combined with heavy water (D2O) to reveal active cellulose degraders. Cellular incorporation of D2O-derived D produces a new C-D Raman band which can act as a quantitative indicator of microbial activity. In this study, metabolic responses of seven cellulose-degrading bacteria to carboxymethylcellulose (CMC) and glucose were evaluated via the C-D Raman band. On the basis of % C-D, CMC was demonstrated to be most efficiently metabolized by Bacillus velezensis 2a-9 and Providencia vermicola 5a-9(b). Metabolic activity between individual cells of B. velezensis and P. vermicola towards CMC ranged from approximately 8 to 27% and 6 to 16%, respectively, clearly indicating heterogeneous degradation activities among isogenic populations. Linear correlation between % C-D and specific endoglucanase activity validated Raman results on CMC-degrading activity. Also, % C-D obtained from bacteria cultivated with only glucose was around 60% higher than that obtained from CMC, indicating the preference of bacteria for simple sugar glucose than CMC. In conclusion, Raman spectroscopy combined with heavy water is a sensitive analytical technique to reveal cellulose degraders and their degrading activities.

The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion.

Providencia stuartii is the most common Providencia species capable of causing human infections. Currently P. stuartii is involved in high incidence of urinary tract infections in catheterized patients. The ability of bacteria to swarm on semisolid (viscous) surfaces and adhere to and invade host cells determines the specificity of the disease pathogenesis and its therapy. In the present study we demonstrated morphological changes of P. stuartii NK cells during migration on the viscous medium and discussed adhesive and invasive properties utilizing the HeLa-M cell line as a host model. To visualize the interaction of P. stuartii NK bacterial cells with eukaryotic cells in vitro scanning electron and confocal microscopy were performed. We found that bacteria P. stuartii NK are able to adhere to and invade HeLa-M epithelial cells and these properties depend on the age of bacterial culture. Also, to invade the host cells the infectious dose of the bacteria is essential. The microphotographs indicate that after incubation of bacterial P. stuartii NK cells together with epithelial cells the bacterial cells both were adhered onto and invaded into the host cells.

Efficient synthesis of ß-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase.

Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of ß-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, ßF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16 h to 2-2.5 h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcat/Km)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcat/Km)Ps values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcat/Km)Ps/(kcat/Km)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the ßF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.

CntA oxygenase substrate profile comparison and oxygen dependency of TMA production in Providencia rettgeri.

CntA oxygenase is a Rieske 2S-2Fe cluster-containing protein that has been previously described as able to produce trimethylamine (TMA) from carnitine, gamma-butyrobetaine, glycine betaine, and in one case, choline. TMA found in humans is exclusively of bacterial origin, and its metabolite, trimethylamine oxide (TMAO), has been associated with atherosclerosis and heart and renal failure. We isolated four different Rieske oxygenases and determined that there are no significant differences in their substrate panels. All three had high activity toward carnitine/gamma-butyrobetaine, medium activity toward glycine betaine, and very low activity toward choline. We tested the influence of low oxygen concentrations on TMA production in CntA-containing Providencia rettgeri cell cultures and discovered that this process, although dependent on the amount of oxygen, is still feasible in environments with 1 and 0.2% oxygen, which is comparable to oxygen levels in some parts of the digestive system.

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.

Metallothionein assisted periplasmic lead sequestration as lead sulfite by Providencia vermicola strain SJ2A.

Lead resistant Providencia vermicola strain SJ2A was isolated from the waste of a battery manufacturing industry which could tolerate upto 3.0mM lead nitrate in the minimal medium. Interestingly, this isolate showed presence of a plasmid borne metallothionein gene, bmtA that matched significantly (96%) with that of Pseudomonas aeruginosa. Scanning electron micrographs of bacterial cells exposed to lead revealed a unique alteration in the cell morphology from rods to long inter-connected filaments. On the other hand, electron dispersive X-ray spectroscopy (EDX) clearly indicated no significant lead adsorption therefore, we speculated intracellular sequestration in this bacterial strain. Transmission electron micrographs of the bacterial cells exposed to lead evidently demonstrated periplasmic sequestration of lead which was also supported by Fourier transformed infrared spectroscopic (FTIR) analysis. The bacterium internalised 155.12mg Pb2+/g biomass as determined by atomic absorption spectroscopy. Subsequently, the accumulated lead was identified as lead sulfite by X-ray diffraction studies. Therefore P. vermicola strain SJ2A has potential to bioremediate lead contaminated environmental sites.

Reversible Unfolding of Rhomboid Intramembrane Proteases.

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.

Nitrogen removal by Providencia rettgeri strain YL with heterotrophic nitrification and aerobic denitrification.

Providencia rettgeri strain YL shows the capability of nitrogen removal under sole aerobic conditions. By using isotope ratio mass spectrometry, (15)N-labelled N2O and N2 were detected in aerobic batch cultures containing [Formula: see text], [Formula: see text] or [Formula: see text]. Strain YL converted [Formula: see text], [Formula: see text] and [Formula: see text] to produce more N2O than N2 in the presence of [Formula: see text]. An (15)N isotope tracing experiment confirmed that the nitrogen removal pathway of strain YL was heterotrophic nitrification-aerobic denitrification. The optimal treatment conditions for nitrogen removal were pH of 8, C/N ratio of 12, temperature of 25°C and shaking speed of 105 rpm. A continuous aerobic bioreactor inoculated with strain YL was developed. With an influent [Formula: see text] concentration of 90-200 mg/L, the [Formula: see text] removal efficiency ranged from 80% to 97% and the total nitrogen removal efficiency ranged from 72% to 95%. The nitrogen balance in the continuous bioreactor revealed that approximately 35-52% of influent [Formula: see text] was denitrified aerobically to form gaseous nitrogen. These findings show that the P. rettgeri strain YL has potential application in wastewater treatment for nitrogen removal under sole aerobic conditions.

Copper-resistant bacteria reduces oxidative stress and uptake of copper in lentil plants: potential for bacterial bioremediation.

For effective microbe-assisted bioremediation, metal-resistant plant growth-promoting bacteria (PGPB) must facilitate plant growth by restricting excess metal uptake in plants, leading to prevent its bio-amplification in the ecosystem. The aims of our study were to isolate and characterize copper (Cu)-resistant PGPB from waste water receiving contaminated soil. In addition, we investigated the phytotoxic effect of copper on the lentil plants inoculated with copper-resistant bacteria Providencia vermicola, grown in copper-contaminated soil. Copper-resistant P. vermicola showed multiple plant growth promoting characteristics, when used as a seed inoculant. It protected the lentil plants from copper toxicity with a considerable increase in root and shoot length, plant dry weight and leaf area. A notable increase in different gas exchange characteristics such as A, E, C i , g s , and A/E, as well as increase in N and P accumulation were also recorded in inoculated plants as compared to un-inoculated copper stressed plants. In addition, leaf chlorophyll content, root nodulation, number of pods, 1,000 seed weight were also higher in inoculated plants as compared with non-inoculated ones. Anti-oxidative defense mechanism improved significantly via elevated expression of reactive oxygen species -scavenging enzymes including ascorbate peroxidase, superoxide dismutase, catalase, and guaiacol peroxidase with alternate decrease in malondialdehyde and H2O2 contents, reduced electrolyte leakage, proline, and total phenolic contents suggesting that inoculation of P. vermicola triggered heavy metals stress-related defense pathways under copper stress. Overall, the results demonstrated that the P. vermicola seed inoculation confer heavy metal stress tolerance in lentil plant which can be used as a potent biotechnological tool to cope with the problems of copper pollution in crop plants for better yield.