Current Status of Food Shelf and Soup Kitchen Programs within the Phillips Neighborhood of Minneapolis, Minnesota.

Funded by a CURA Communiversity Personnel Grant

A terminal assessment of stages theory : introducing a dynamic states approach to entrepreneurship

Stages of Growth models were the most frequent theoretical approach to understanding entrepreneurial business growth from 1962 to 2006; they built on the growth imperative and developmental models of that time. An analysis of the universe of such models (N=104) published in the management literature shows no consensus on basic constructs of the approach, nor is there any empirical confirmations of stages theory. However, by changing two propositions of the stages models, a new dynamic states approach is derived. The dynamic states approach has far greater explanatory power than its precursor, and is compatible with leading edge research in entrepreneurship

Mycobacterium tuberculosis Rv3406 is a type II alkyl sulfatase capable of sulfate scavenging.

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb's single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase's role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases

The reaction catalyzed by Rv3406.

<p>The alpha carbon of an alkyl sulfate is oxidized by Rv3406 in the presence of α-ketoglutarate and spontaneously collapses to an aldehyde and sulfate, liberating CO₂ and succinate. The product formation was monitored using a coupled assay using LADH to reduce the aldehyde in a NADH dependent manner.</p

Biochemical characterization of Rv3406.

<p>(A) Rv3406 is an αKG and ascorbate dependent sulfatase. Black squares are the complete assay with Rv3406, 2-EHS, αKG, ascorbate and iron. Red triangles are without 2-EHS. Blue circles are without αKG. (B) Rv3406 is an iron dependent enzyme. (C) The rate of Rv3406 accelerates with the addition of ascorbate up to 1 mM. Rv3406 enzyme concentration was between 0.5 and 0.75 µM for all experiments.</p

Protein alignment of alkyl sulfatase enzymes with taurine dioxygenase enzymes.

<p><b>Enzymes in bold have been biochemically characterized.</b> (A) Alignment of disordered loop 1 where the red boxes are indicating the taurine binding residues in taurine dioxygenases and the analogous amino acids in alkyl sulfatase enzymes. (B) Alignment of disordered loop 2 where the red box is indicating the conserved phenylalanine in taurine dioxygenases and the analogous tyrosine in alkyl sulfate enzymes.</p

Rv3406 is essential in Mtb for growth on 2-EHS as the sole sulfur source.

<p>(A) Growth of Mtb strains using either 2-EHS alone or 2-EHS with sodium sulfate. (B) Growth of Mtb strains on <i>n</i>-heptyl sulfate or SDS. Data represents three biological replicates and error bars denote standard deviation. Asterisk indicates a p value of less than 0.005.</p