Iron-Utilization System in Vibrio vulnificus M2799

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

Effect of Spermidine on Biofilm Formation in Escherichia coli K-12

ABSTRACT Polyamines are essential for biofilm formation in Escherichia coli, but it is still unclear which polyamines are primarily responsible for this phenomenon. To address this issue, we constructed a series of E. coli K-12 strains with mutations in genes required for the synthesis and metabolism of polyamines. Disruption of the spermidine synthase gene (speE) caused a severe defect in biofilm formation. This defect was rescued by the addition of spermidine to the medium but not by putrescine or cadaverine. A multidrug/spermidine efflux pump membrane subunit (MdtJ)-deficient strain was anticipated to accumulate more spermidine and result in enhanced biofilm formation compared to the MdtJ+ strain. However, the mdtJ mutation did not affect intracellular spermidine or biofilm concentrations. E. coli has the spermidine acetyltransferase (SpeG) and glutathionylspermidine synthetase/amidase (Gss) to metabolize intracellular spermidine. Under biofilm-forming conditions, not Gss but SpeG plays a major role in decreasing the too-high intracellular spermidine concentrations. Additionally, PotFGHI can function as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions. Last, we report here that, in addition to intracellular spermidine, the periplasmic binding protein (PotD) of the spermidine preferential ABC transporter is essential for stimulating biofilm formation. IMPORTANCE Previous reports have speculated on the effect of polyamines on bacterial biofilm formation. However, the regulation of biofilm formation by polyamines in Escherichia coli has not yet been assessed. The identification of polyamines that stimulate biofilm formation is important for developing novel therapies for biofilm-forming pathogens. This study sheds light on biofilm regulation in E. coli. Our findings provide conclusive evidence that only spermidine can stimulate biofilm formation in E. coli cells, not putrescine or cadaverine. Last, ΔpotD inhibits biofilm formation even though the spermidine is synthesized inside the cells from putrescine. Since PotD is significant for biofilm formation and there is no ortholog of the PotABCD transporter in humans, PotD could be a target for the development of biofilm inhibitors.

Esters of Glucose-2-Phosphate: Occurrence and Chemistry

Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by Agrobacterium fabrum C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic Agrobacterium radiobacter K84 strain used as a biocontrol agent by competing with Agrobacterium fabrum C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the Agrobacterium agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.

Actinobacillus utilizes a binding-protein dependent ABC transporter to acquire Vitamin B6

Bacteria require high efficiency uptake systems to survive and proliferate in nutrient limiting environments, such as those found in the host. The ABC transporters at the bacterial plasma membrane provide a mechanism for transport of many substrates. We recently demonstrated that an AfuABC operon, previously annotated as encoding a ferrous iron uptake system, is in fact a cyclic hexose/heptose-phosphate transporter with high selectivity and specificity for these metabolites. In this study, we examine a second operon containing a periplasmic binding protein discovered in Actinobacillus for its potential role in nutrient acquisition. Using electron density obtained from the crystal structure of the periplasmic binding protein we modeled a pyridoxal-5’-phosphate (P5P/PLP/Vitamin B6) ligand into the atomic resolution electron density map. The identity of the Vitamin B6 bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from A. pleuropneumoniae into an E. coli K-12 strain that was devoid of a key enzyme required for Vitamin B6 synthesis. The growth of this strain at low levels of Vitamin B6 supports the role of this newly identify operon in Vitamin B6 uptake.

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

The evolving story of AtzT, a periplasmic binding protein

Atrazine is an s-triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s-triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.