Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA.

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Sinomicrobium pectinilyticum sp. nov., a pectinase-producing bacterium isolated from alkaline and saline soil, and emended description of the genus Sinomicrobium.

A Gram-reaction-negative, non-spore-forming strain, designated 5DNS001(T), was isolated from soil of an ancient salt-extracting facility in China. Analysis of the almost-complete 16S rRNA gene sequence of the bacterium suggested that it belongs to the genus Sinomicrobium in the family Flavobacteriaceae. It exhibited highest 16S rRNA gene sequence similarity with Sinomicrobium oceani SCSIO 03483(T) (96.3 %), but less than 93 % sequence similarity with members of the genera Imtechella, Zhouia and Joostella and other recognized members of the family Flavobacteriaceae. The strain was able to hydrolyse pectin and starch by producing pectinase and α-amylase. The DNA G+C content of the strain was 42.6 mol%. The major respiratory quinone was MK-6. The major polar lipid detected in the strain was phosphatidylethanolamine. The dominant cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω6c/C16 : 1ω7c). Based on phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, a novel species, Sinomicrobium pectinilyticum, is proposed. The type strain is 5DNS001(T) ( = CGMCC1.11000(T) = KCTC23776(T)).

An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system.

N-acetyl-D-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl-D-glucosamine 2-epimerase and N-acetyl-D-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l(-1)) Neu5Ac was produced from 200 mM N-acetyl-D-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes.

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications.

BACKGROUND: Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. RESULTS: A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. CONCLUSIONS: The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

Pulp mill wastewater sediment reveals novel methanogenic and cellulolytic populations.

Pulp mill wastewater generated from wheat straw is characterized as high alkalinity and very high COD pollution load. A naturally developed microbial community in a pulp mill wastewater storage pool that had been disused were investigated in this study. Owing to natural evaporation and a huge amount of lignocellulose's deposition, the wastewater sediment contains high concentrations of organic matters and sodium ions, but low concentrations of chloride and carbonate. The microbiota inhabiting especially anaerobic community, including methanogenic arhcaea and cellulolytic species, was studied. All archaeal sequences fall into 2 clusters of family Halobacteriaceae and methanogenic archaeon in the phylum Euryarchaeota. In the methanogenic community, phylogenetic analysis of methyl coenzyme M reductase A (mcrA) genes targeted to novel species in genus Methanoculleus or novel genus of order Methanomicrobiales. The predominance of Methanomicrobiales suggests that methanogenesis in this system might be driven by the hydrogenotrophic pathway. As the important primary fermenter for methane production, the cellulolytic community of enzyme GHF48 was found to be dominated by narrower breadth of novel clostridial cellulase genes. Novel anoxic functional members in such extreme sediment provide the possibility of enhancing the efficiency of anoxic treatment of saline and alkaline wastewaters, as well as benefiting to the biomass transformation and biofuel production processes.

Metabolic versatility of halotolerant and alkaliphilic strains of Halomonas isolated from alkaline black liquor.

Wheat straw black liquor is a notorious pulp mill wastewater with very high pH and pollution load. Two halotolerant and alkaliphilic bacteria, designated as Halomonas sp. 19-A and Y2, were isolated from wheat straw black liquor and shown to be able to use guaiacol, vanillin, dibenzo-p-dioxin, biphenyl and fluorene, as sole carbon and carbazole as sole carbon and nitrogen source at pH 9.5 and in the presence of 10% NaCl. The two strains produced carboxymethylcellulase (CMCase), xylanase, lipase, amylase, and pullulanase. High activities of CMCase, xylanase, and amylase were observed at pH 5.0-11.0 and NaCl concentrations of 0-15%. The metabolic versatility of these Halomonas strains even under extreme pH and salinity conditions makes them promising agents for bioremediation and industrial processes.