Divergent Evolution of the Activity and Regulation of the Glutamate Decarboxylase Systems in Listeria monocytogenes EGD-e and 10403S: Roles in Virulence and Acid Tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GAD_i), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GAD_i system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GAD_i/GAD_e). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GAD_i but not GAD_e the results indicate that the GAD_i system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.     

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter

OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 Å resolution. The structure shows the phosphate bound in a tripodal mode to the metal ions whereby two of the oxygen atoms of PO₄ are terminally bound to each metal ion and a third oxygen bridges the two metal ions, thus displacing the μOH in the active site. In silico modelling demonstrates that the phosphate moiety of a reaction product, e.g. diethyl phosphate, may bind in the same orientation, positioning the diethyl groups neatly into the substrate binding pocket close to the metal center. Thus, similar to the binuclear metallohydrolases urease and purple acid phosphatase the tripodal arrangement of PO₄ is interpreted in terms of a role of the μOH as a reaction nucleophile.     

Nisin inducible production of listeriolysin O in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000

Background  

Listeria monocytogenesis a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactisNZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.     

Cytokinin-induced apoptotic nuclear changes in cotyledons of Solanum aviculare and Lycopersicon esculentum

Cytokinin-induced nuclear changes have been observed in epidermal and sub-epidermal layers of cells in cotyledons of Lycopersicon esculentum and Solanum aviculare which compare closely to those observed during apoptosis in mammalian, insect and nematode species. The nuclei, which show invagination of the nuclear membrane, margination and eventual breakdown of the chromatin, are positive with the TUNEL reaction. Such changes are not induced by treatment of the cotyledons with 2,4-dichlorophenoxyacetic acid. In contrast, mesophyll cells induced to form vessels (programmed cell death) by treatment with 2,4-dichlorophenoxyacetic acid together with either 6-benzylamino-purine or zeatin, retain their diploid nuclei until the end of differentiation.     

A quantitative cytochemical study of UDP-d-glucose: NAD-oxidoreductase (E.C. 1.1.1.22) activity during stelar differentiation in Pisum sativum L. cv Meteor

Summary A quantitative cytochemical assay for UDP-d-glucose dehydrogenase (UDPGD) activity employing scanning and integrating microdensitometry has been revised and applied to a study of this enzmye during the initiation of secondary cell wall biosynthesis during the formation of primary vascular tissues in roots of Pisum sativum L. cv Meteor. The reaction involves the use of NBT as final electron acceptor and is inhibited 10-fold by either 10 mM UDP-d-xylose or 25 mM UDP-d-glucuronic acid, two molecules involved in feed-back inhibition of UDPGD activity in vivo. UDPGD is a far-from equilibrium enzyme representing a flux-generating step in the biosynthesis of precursors for hemicelluloses involved in secondary cell wall construction, and can be demonstrated to increase sharply in activity in cells of the developing primary vascular elements. This changed activity occurs 18–20 cells back from the root cap junction and coincides with the first cells containing the activated programme for secondary cell wall synthesis.