Two Bacterial Small Heat Shock Proteins, IbpA and IbpB, Form a Functional Heterodimer.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. ß-hairpins formed by strands ß5 and ß7 of IbpA or IbpB crystallin domains associate with the other one's ß-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.

DiagnoTop: A Computational Pipeline for Discriminating Bacterial Pathogens without Database Search.

Pathogen identification is crucial to confirm bacterial infections and guide antimicrobial therapy. Although MALDI-TOF mass spectrometry (MS) serves as foundation for tools that enable rapid microbial identification, some bacteria remain challenging to identify. We recently showed that top-down proteomics (TDP) could be used to discriminate closely related enterobacterial pathogens (Escherichia coli, Shigella, and Salmonella) that are indistinguishable with tools rooted in the MALDI-TOF MS approach. Current TDP diagnostic relies on the identification of specific proteoforms for each species through a database search. However, microbial proteomes are often poorly annotated, which complicates the large-scale identification of proteoforms and leads to many unidentified high-quality mass spectra. Here, we describe a new computational pipeline called DiagnoTop that lists discriminative spectral clusters found in TDP data sets that can be used for microbial diagnostics without database search. Applied to our enterobacterial TDP data sets, DiagnoTop could easily shortlist high-quality discriminative spectral clusters, leading to increased diagnostic power. This pipeline opens new perspectives in clinical microbiology and biomarker discovery using TDP.

In vitro assessment of two novel Cellulases from Trabulsiella odontotermitis for agricultural waste utilization.

BACKGROUND: The production of agricultural wastes still growing as a consequence of the population growing. However, the majority of these residues are under-utilized due their chemical composition, which is mainly composed by cellulose. Actually, the search of cellulases with high efficiency to degrade this carbohydrate remains as the challenge. In the present experiment, two genes encoding an endoglucanase (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21) were overexpressed in Escherichia coli and their recombinant enzymes (egl-FZYE and cel-FZYE, respectively) characterized. Those genes were found in Trabulsiella odontermitis which was isolated from the gut of termite Heterotermes sp. Additionally, the capability to release sugars from agricultural wastes was evaluated in both enzymes, alone and in combination. RESULTS: The results have shown that optimal pH was 6.0 and 6.5, reaching an activity of 1051.65 ± 47.78 and 607.80 ± 10.19 U/mg at 39 °C, for egl-FZYE and cel-FZYE, respectively. The Km and Vmax for egl-FZYE using CMC as substrate were 11.25 mg/mL and 3921.57 U/mg, respectively, whereas using Avicel were 15.39 mg/mL and 2314.81 U/mg, respectively. The Km and Vmax for cel-FZYE using Avicel as substrate were 11.49 mg/mL and 2105.26 U/mg, respectively, whereas using CMC the enzyme did not had activity. Both enzymes had effect on agricultural wastes, and their effect was improved when they were combined reaching an activity of 955.1 ± 116.1, 4016.8 ± 332 and 1124.2 ± 241 U/mg on corn stover, sorghum stover and pine sawdust, respectively. CONCLUSIONS: Both enzymes were capable of degrading agricultural wastes, and their effectiveness was improved up to 60% of glucose released when combined. In summary, the results of the study demonstrate that the recombinant enzymes exhibit characteristics that indicate their value as potential feed additives and that the enzymes could be used to enhance the degradation of cellulose in the poor-quality forage generally used in ruminant feedstuffs.

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification.

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide - endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

Electrostatic Frustration Shapes Folding Mechanistic Differences in Paralogous Bacterial Stress Response Proteins.

Paralogous proteins play a vital role in evolutionary adaptation of organisms and species divergence. One outstanding question is the molecular basis for how folding mechanisms differ in paralogs that not only exhibit similar topologies but also evolve under near-identical selection pressures. Here, we address this question by studying a paralogous protein pair from enterobacteria, Hha and Cnu, combining experiments, simulations and statistical modeling. We find that Hha is less stable and folds an order of magnitude slower than Cnu despite similar packing and topological features. Differences in surface charge-charge interactions, however, promote a N-terminal biased unfolding mechanism in Hha unlike Cnu that unfolds via the C terminus. Our work highlights how electrostatic frustration contributes to the population of heterogeneous native ensembles in paralogs and the avenues through which evolutionary topological constraints could be overcome by modulating charge-charge interactions.

Structure of a fucose-rich polysaccharide derived from EPS produced by Kosakonia sp. CCTCC M2018092 and its application in antibacterial film.

A novel exopolysaccharide (EPS) with high molecular weight (3.65 × 105 Da) and film-forming ability was produced by the strain Kosakonia sp. CCTCC M2018092. Partially acid hydrolyzed EPS (AH-EPS) with high content of fucose was prepared and exhaustively characterized. The molecular weight of AH-EPS was determined to be 3.47 × 104 Da. GC-MS and HPLC analyses indicated that AH-EPS is composed of L-fucose, d-glucose, D-galactose, D-glucuronic acid and pyruvic acid in the molar ratio of 2.03:1.00:1.18:0.64:0.67. Chemical and NMR analyses revealed that AH-EPS is an anionic heteropolysaccharide, with a major linkage structural motif as follows. Utilizing AH-EPS as reducing and stabilizing agent, silver nanoparticles (AH-EPS@Ag NPs) with uniform size (diameter about 20 nm) were synthesized through a green method. A hybrid film containing EPS and AH-EPS@Ag NPs was further prepared, and its antibacterial effectiveness to Staphylococcus aureus was confirmed. Taken together, this work revealed the structural characteristics of a novel fucose-rich polysaccharide, with good potential in developing new biodegradable antibacterial film.

Identification of novel mobile colistin resistance gene mcr-10.

Mobile colistin resistance (mcr) genes represent an emerging challenge. Here we describe a novel mcr gene, mcr-10, on an IncFIA plasmid of an Enterobacter roggenkampii clinical strain. mcr-10 has the highest nucleotide identity (79.69%) with mcr-9 and encodes MCR-10 with 82.93% amino acids identical to MCR-9. mcr-10 confers 4-fold increase in colistin MIC (from 1 to 4 mg/L) when cloned into a colistin-susceptible E. roggenkampii strain. By screening GenBank, mcr-10 was found in various Enterobacteriaceae species of countries in four continents, suggesting that this gene has widely spread. MCR-10 shows 79.04% to 83.67% amino acid identity and highly conserved predicted protein structures with chromosomally encoded MCR-like phosphoethanolamine transferases (designated MCR-B here) of various Buttiauxella species. MCR-10, MCR-9 and MCR-B proteins may, therefore, originate from a common ancestor. mcr-10 was adjacent to a site-specific recombinase-encoding gene and was bracketed by IS903 and may be mobilized by site-specific recombination or composite transposon. Our results indicate that mcr-10 is a novel plasmid-borne colistin resistance gene and warrants immediate monitoring and further studies.

Three chlorotoluene-degrading bacterial strains: Differences in biodegradation potential and cell surface properties.

Pollution of the environment with chlorinated aromatic compounds is a problem of increasing importance, which has stimulated the search for efficient methods for the remediation of contaminated soil and water. Additionally, for better understanding of the significance of bioavailability to biodegradation, investigation of the cell surface properties is necessary. Hence, this study concerns the properties and possible application, in chlorotoluene removal, of three newly isolated environmental bacterial strains from the genera Pseudomonas, Raoultella and Rahnella. The results show the differences in the biochemical profiles of the isolated strains, their cellular fatty acid composition and their hemolytic properties. However, all three strains exhibit high biodegradation potential, degrading not less than 60% of each monochlorotoluene isomer in 21-day experiments. What is more, observations of changes in the cell surface properties indicate the possible adaptation mechanisms of the strains that enable efficient biodegradation of hydrophobic pollutants such as monochlorotoluenes.

A close look on the effect of polyethylene glycol on the levansucrase thermostability: a case study of Brenneria sp. levansucrase.

BACKGROUND: To increase the low residual activity of levansucrase during long-time processing, an enhancement of its weak thermostability is needed. Here, the effect of metal ions and polyethylene glycol (PEG) on the thermostability of levansucrase from Brenneria sp. EniD312 were studied and evaluated. The residual activity was determined and the protein structure was evaluated by circular dichroism spectrum, fluorescence intensity (FI), and surface hydrophobicity (S0 ). RESULTS: As a result of incubation with 10% (w/v) PEG 4000, the enzyme activity was increased by 1.24-fold. After incubation with 5% PEG 4000 for 6 h, the residual activity at 35 and 45 °C was decreased to 55% and 60% of the initial activity, with an increase of 1.2- and 3.3-fold than the wild-type enzyme. Furthermore, the random coil content of enzyme was decreased from 53% of the wild-type enzyme to 33.9% of the PEG pre-incubated enzyme. Additionally, the FI was maximally increased and the S0 was decreased from 117 to 69. CONCLUSION: All of these results suggested that after incubation with PEG 4000, the secondary and tertiary structure of wild-type enzyme could be greatly maintained and then its thermostability could be increased. This study was the first report on the enhancement of levansucrase thermostability by PEG incubation and might be a good guideline to other researches on levansucrase. © 2019 Society of Chemical Industry.

The efficacy of 2 phytases on inositol phosphate degradation in different segments of the gastrointestinal tract, calcium and phosphorus digestibility, and bone quality of broilers.

The anti-nutritional effects of dietary inositol phosphates (IP6 through IP3) have been recognized in broiler chickens; however, inositol hexaphosphate (IP6) is more potent than the lower IP esters. The efficacies of 2 commercial phytases, a Buttiauxella sp. phytase (BSP) and a Citrobacter braakii phytase (CBP) at 500 and 1,000 FTU/kg, were studied on IP6-3 concentrations in the crop, proventriculus + gizzard, and distal ileum digesta, and ileal IP6 disappearance in broilers at day 22. Apparent ileal P and Ca digestibility, and bone quality at days 22 and 33 were also measured. Female Ross 308 broilers (n = 1,890; 30 birds × 7 diets × 9 replicates) were fed corn-soy-based crumbled diets. The 7 diets included a primary breeder recommendation-based positive control diet (PC); the PC marginally reduced in available P by 0.146% and Ca by 0.134% of the diet, (NC1) or moderately reduced by 0.174 and 0.159% of the diet, respectively (NC2). Other diets were the NC1 + BSP or CBP at 500 FTU/kg (NC1+500BSP and NC1+500CBP) and the NC2 + BSP or CBP at 1,000 FTU/kg (NC2+1,000BSP and NC2+1,000CBP). Each of the NC1 and NC2 had distal ileum IP6 disappearance similar to that of PC, but each had lower P digestibility and the majority of measured bone quality parameters than the PC. The ileal IP6 levels were decreased by 52.0 and 32.7% for NC1+500BSP and NC1+500CBP, respectively, relative to NC1 and by 73.6 and 50.9% for NC2+1,000BSP and NC2+1,000CBP, respectively, relative to NC2 (P < 0.001), with a similar effect for distal ileum IP6 disappearance. Overall, phytase in the NC diets increased P digestibility, and femur breaking strength and cortical bone mineral density at days 22 and 33. Overall, each of the phytases at each dose degraded IP6-3 across the gastrointestinal tract segments to increase P digestibility and the P and Ca utilization in bone. However, dietary BSP at 1,000 FTU/kg was most effective. Supplemental phytase degrades phytate to decrease the anti-nutritional effects in a dose- and phytase-dependent manner.

Long-term effects of Buttiauxella sp. phytase on performance, eggshell quality, apparent ileal Ca and P digestibility, and bone properties of white egg layers.

Adequate dietary Ca and available phosphorus (avP) are essential to long-term egg production and bone health in laying hens. The effects of dietary Ca and avP levels and Buttiauxella sp. phytase (BSP) were studied in Lohmann LSL Lite hens from 30 to 70 wk of age (woa). Hens (n = 456; 4 per cage) were fed either a primary breeder recommendation-based diet (positive control; PC); the PC with avP and Ca levels reduced by 0.146 and 0.134% of the diet, respectively, without (NC) or with 300 FTU/kg BSP (NC+BSP). Egg production, BW, feed intake, FCR, and eggshell quality from 30 to 70 woa, and apparent ileal digestibility of P (AIDP) and Ca (AIDCa), and bone quality at 32, 48, and 70 woa were measured. The avP and Ca levels in the NC diet were not clinically deficient, as most parameters were unaffected by diet. Hen BW from 34 to 70 woa tended to be 2.9% greater (P = 0.076) for PC and NC+BSP compared to NC. Mid-diaphysis cortical bone mineral content (CBMC) tended to be 10% and 9% higher (P = 0.065) in the NC+BSP hens than in NC hens at 48 and 70 woa, respectively. AIDP of NC+BSP was 24% greater (P = 0.034) than of NC at 32 woa and tended to be 18% greater (P = 0.082) than AIDP of PC at 48 woa, and 25% lower than of NC and PC at 70 woa (P = 0.028). AIDCa was 25% lower for NC+BSP than PC at 48 woa only (P = 0.037). The avP and Ca sufficiency in the NC diet limited the opportunity to determine a phytase effect. Although the supplemental BSP tended to increase BW and 48 and 70 woa CBMC, and increased 32 woa AIDP, the efficacy of BSP could not be determined due to the lack of an NC effect on most parameters. Commercial laying hens can maintain health and productivity at lower than recommended levels of dietary Ca and avP; phytase supplementation may allow for even further reductions.

Laboratory validation of a KPC-producing strain identification method based on the detection of a specific 11,109 Da peak via Maldi-Tof-Vitek MS in an endemic area.

Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Vitek MS system is a useful technique to identify bacteria strains isolated in clinical samples. In this paper, we applied this method to KPC-producing Enterobacteriaceae detection through the determination of a specific 11,109 (±8) Da peak. We assayed the presence, specificity and reliability of this peak on routine workflow through the analysis of 183 Enterobacteriaceae strains isolated from clinical samples and characterized by classical approaches. The peak was detected in 95.5% (129/135) of carbapenemase-producing strains spectra compared with the 48 extended spectrum beta-lactamase producing controls strains, which all lacked this peak. Hence, this 11,109 Da peak determination showed a Positive Predictive Value (PPV) of 100% and a Negative Predictive Value (NPV) of 94.4%. The characterization of this specific peak in a MALDI-TOF Vitek MS system might be considered a valuable tool to reveal KPC-producing Enterobacteriaceae especially in KPC endemic region.

Comparison between Vitek MS, Bruker Biotyper, Vitek2, and API20E for differentiation of species of the genus Raoultella.

Rapid and reliable identification of microorganisms in the clinical laboratory is essential for an early and accurate diagnosis guiding timely therapy. However, conventional methods are sometimes unreliable and show controversial outcomes. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a rapid and reliable method for identification of bacteria and fungi isolated from clinical samples. Members of the genus Raoultella are increasingly recognized as clinically relevant. There are difficulties in their identification at the species level since sequencing the 16S rRNA or the rpoB genes does not show conclusive results. The aim of this study has been to compare two MALDI-TOF MS systems (Vitek MS and Bruker Biotyper) with Vitek2 and API20E systems for differentiation of Raoultella species. A collection of 97 clinical isolates of Raoultella species was identified with Vitek MS, in parallel with Vitek2 and API, and finally with Bruker Biotyper. Among the two most widely used MALDI-TOF MS platforms, results obtained with Vitek MS were slightly superior to those obtained with the Bruker Biotyper system, with sensitivities and specificities of 98.9/57.9% and 98.8/37.0%, respectively. The current commercial phenotypic identification systems are not optimized for the identification of Raoultella species. Our results indicate that MALDI-TOF-based identification is more accurate and sensitive than that provided by phenotypic methods.

Insights into multiple use water enterobacteria from the Brazilian semi-arid: the first approach by pyrosequencing

Thousands of people living in semi-arid regions face problems of drought and loss of water quality. In addition, high incidence of acute diarrheal diseases related to water consumption has been responsible for a high number of deaths and high economic costs for human health. Many of the diseases can be caused by the presence of enterobacteria in reservoirs that serve for multiple purposes. This study aimed to confirm the presence of potentially harmful bacteria, which was highlighted in other articles, and to reveal non-identified genera by culture-dependent methods and pyrosequencing. Twenty-three genera of the Enterobacteriaceae family were detected, with emphasis on Escherichia genus and confirmation of the presence of species such as Salmonella enterica and Enterobacter cloacae. The abundance of heterotrophic prokaryotes and the physical and chemical data show an expected average for this type of environment due to the numbers historically presented in previous articles. The unprecedented detection of the presence of some potentially pathogenic species can alert and raise awareness of the populations that use stored water in the semi-arid regions. Consequently, as a result of the peculiar characteristics of reservoirs under this climate influence, there is a cosmopolitanism of enterobacteria that may be related to the alarming numbers of infections from Waterborne Diseases.

Impact of short-incubation MALDI-TOF MS on empiric antibiotic therapy in bloodstream infections caused by Pseudomonas aeruginosa, Enterococcus spp. and AmpC-producing Enterobacteriaceae.

The impact of the short-incubation matrix-assisted laser desorption ionization time-of-flight (si-MALDI-TOF) mass spectrometry technique was evaluated in the treatment of bloodstream infections (BSIs) caused by Pseudomonas aeruginosa, Enterococcus spp., and Amp-C producing Enterobacteriaceae. A total of 124 bacteremia episodes were divided into 2 groups: i) si-MALDI-TOF group (n = 69) and ii) control group (n = 55). Identification by si-MALDI-TOF resulted in 12.8% increase in cases receiving appropriate antibiotic treatment within 48 h from blood culture draw. The importance of the rapid identification was emphasized in BSIs caused by enterococci (n = 62), where si-MALDI-TOF led to appropriate antibiotic treatment in 87.9% of cases (versus control group 65.5%, P = 0.036). Implementation of si-MALDI-TOF technology for microbial identification was associated with increased proportion of patients receiving effective antibiotic treatment within 48 h from blood culture draw. The effect was most significant in BSIs caused by enterococcal species and in a subgroup of immunosuppressed patients.

Daqu Fermentation Selects for Heat-Resistant Enterobacteriaceae and Bacilli.

Daqu is a spontaneous solid-state cereal fermentation used as saccharification and starter culture in Chinese vinegar and liquor production. The evolution of microbiota in this spontaneous fermentation is controlled by the temperature profile, which reaches temperatures from 50 to 65°C for several days. Despite these high temperatures, mesophilic Enterobacteriaceae (including Cronobacter) and bacilli are present throughout Daqu fermentation. This study aimed to determine whether Daqu spontaneous solid-state fermentation selects for heat-resistant variants of these organisms. Heat resistance in Enterobacteriaceae is mediated by the locus of heat resistance (LHR). One LHR-positive strain of Kosakonia cowanii was identified in Daqu, and it exhibited higher heat resistance than the LHR-negative K. cowanii isolated from malted oats. Heat resistance in Bacillus endospores is mediated by the spoVA2mob operon. Out of 10 Daqu isolates of the species Bacillus licheniformis, Brevibacillus parabrevis, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velezensis, 5 did not contain spoVA2mob, 3 contained one copy, and 2 contained two copies. The presence and copy number of the spoVA2mob operon increased the resistance of spores to treatment with 110°C. To confirm the selection of LHR- and spoVA2mob-positive strains during Daqu fermentation, the copy numbers of these genetic elements in Daqu samples were quantified by quantitative PCR (qPCR). The abundance of LHR and the spoVA2mob operon in community DNA relative to that of total bacterial 16S rRNA genes increased 3-fold and 5-fold, respectively, during processing. In conclusion, culture-dependent and culture-independent analyses suggest that Daqu fermentation selects for heat-resistant Enterobacteriaceae and bacilli.IMPORTANCE Daqu fermentations select for mobile genetic elements conferring heat resistance in Enterobacteriaceae and bacilli. The locus of heat resistance (LHR), a genomic island conferring heat resistance in Enterobacteriaceae, and the spoVA2mob operon, conferring heat resistance on bacterial endospores, were enriched 3- to 5-fold during Daqu fermentation and maturation. It is therefore remarkable that the LHR and the spoVA2mob operon are accumulated in the same food fermentation. The presence of heat-resistant Kosakonia spp. and Bacillus spp. in Daqu is not of concern for food safety; however, both genomic islands are mobile and transferable to pathogenic bacteria or toxin-producing bacteria by horizontal gene transfer. The identification of the LHR and the spoVA2mob operon as indicators of fitness of Enterobacteriaceae and bacilli in Daqu fermentation provides insights into environmental sources of heat-resistant organisms that may contaminate the food supply.

Switching Protein Conformational Substates by Protonation and Mutation.

Protein modules that regulate the availability and conformational status of transcription factors determine the rapidity, duration, and magnitude of cellular response to changing conditions. One such system is the single-gene product Cnu, a four-helix bundle transcription co-repressor, which acts as a molecular thermosensor regulating the expression of virulence genes in enterobacteriaceae through modulation of its native conformational ensemble. Cnu and related genes have also been implicated in pH-dependent expression of virulence genes. We hypothesize that protonation of a conserved buried histidine (H45) in Cnu promotes large electrostatic frustration, thus disturbing the H-NS, a transcription factor, binding face. Spectroscopic and calorimetric methods reveal that H45 exhibits a suppressed p Ka of ∼5.1, the protonation of which switches the conformation to an alternate native ensemble in which the fourth helix is disordered. The population redistribution can also be achieved through a mutation H45V, which does not display any switching behavior at pH values greater than 4. The Wako-Saitô-Muñoz-Eaton (WSME) statistical mechanical model predicts specific differences in the conformations and fluctuations of the fourth and first helices of Cnu determining the observed pH response. We validate these predictions through fluorescence lifetime measurements of a sole tryptophan, highlighting the presence of both native and non-native interactions in the regions adjoining the binding face of Cnu. Our combined experimental-computational study thus shows that Cnu acts both as a thermo- and pH-sensor orchestrated via a subtle but quantifiable balance between the weak packing of a structural element and protonation of a buried histidine that promotes electrostatic frustration.

Efficient synthesis of a 6-deoxy-talose containing tetrasccharide found in Franconibacter helveticus LMG23732T.

Synthesis of the 6-deoxy-talose (6-dTal) containing tetrasaccharide, naturally found in Franconibacter helveticus LMG23732T, has been described. The synthetic method utilized an allyloxyethylidene group for protecting the 1-OH and 2-OH groups of rhamnopyranose and a redox reaction for synthesizing 6-deoxy talose, which eventually formed a disaccharide containing α-Glcp-(1→2)-6dTalp configured glycosidic bonds using a [2 + 2] synthetic strategy.

Physicochemical properties of a high molecular weight levan from Brenneria sp. EniD312.

A high molecular weight levan was produced by a novel levansucrase and some properties of this polymer were investigated. The levan exhibited a poroid microstructure as well as series of individual ellipsoidal or spheroidal particles. The weight-average molecular weight (M¯w) of the levan was determined to be 1.41×108Da. In a 0.1% solution, the levan showed a mean diameter of 176nm, while in a 1% solution the diameter was 182nm. The decomposition temperature was determined to be 216.67°C, with an endothermic peak at 147.41°C and a melting enthalpy of 76.9J/g. The small angle X-ray diffraction pattern showed a distinctive peak pattern between 15° and 40° (2q). The levan solution showed a shear-thinning behaviour. These results suggest this levan could be a good additive in the food processing industry, as well as an important bio-based material in the medicinal or chemical industry.

Coliform Bacteria for Bioremediation of Waste Hydrocarbons.

Raw, domestic sewage of Kuwait City contained about 106 ml-1 colony forming units of Enterobacter hormaechei subsp. oharae (56.6%), Klebsiella spp. (36%), and Escherichia coli (7.4%), as characterized by their 16S rRNA-gene sequences. The isolated coliforms grew successfully on a mineral medium with crude oil vapor as a sole source of carbon and energy. Those strains also grew, albeit to different degrees, on individual n-alkanes with carbon chains between C9 and C36 and on the individual aromatic hydrocarbons, toluene, naphthalene, phenanthrene, and biphenyl as sole sources of carbon and energy. These results imply that coliforms, like other hydrocarbonoclastic microorganisms, oxidize hydrocarbons to the corresponding alcohols and then to aldehydes and fatty acids which are biodegraded by ß-oxidation to acetyl CoA. The latter is a well-known key intermediate in cell material and energy production. E. coli cells grown in the presence of n-hexadecane (but not in its absence) exhibited typical intracellular hydrocarbon inclusions, as revealed by transmission electron microscopy. Raw sewage samples amended with crude oil, n-hexadecane, or phenanthrene lost these hydrocarbons gradually with time. Meanwhile, the numbers of total and individual coliforms, particularly Enterobacter, increased. It was concluded that coliform bacteria in domestic sewage, probably in other environmental materials too, are effective hydrocarbon-biodegrading microorganisms.