Integrated molecular, physiological and in silico characterization of two Halomonas isolates from industrial brine.

Two haloalkaliphilic bacteria isolated from industrial brine solutions were characterized via molecular, physiological, and in silico metabolic pathway analyses. Genomes from the organisms, designated Halomonas BC1 and BC2, were sequenced; 16S ribosomal subunit-based phylogenetic analysis revealed a high level of similarity to each other and to Halomonas meridiana. Both strains were moderate halophiles with near optimal specific growth rates (≥60 % µ max) observed over <0.1-5 % (w/v) NaCl and pH ranging from 7.4 to 10.2. Isolate BC1 was further characterized by measuring uptake or synthesis of compatible solutes under different growth conditions; in complex medium, uptake and accumulation of external glycine betaine was observed while ectoine was synthesized de novo in salts medium. Transcriptome analysis of isolate BC1 grown on glucose or citrate medium measured differences in glycolysis- and gluconeogenesis-based metabolisms, respectively. The annotated BC1 genome was used to build an in silico, genome-scale stoichiometric metabolic model to study catabolic energy strategies and compatible solute synthesis under gradients of oxygen and nutrient availability. The theoretical analysis identified energy metabolism challenges associated with acclimation to high salinity and high pH. The study documents central metabolism data for the industrially and scientifically important haloalkaliphile genus Halomonas.

Proteomic and systems biology analysis of the monocyte response to Coxiella burnetii infection.

Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and C. burnetii is considered a significant bioterror threat. C. burnetii occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of C. burnetii and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.

Proteomic and systems biology analysis of monocytes exposed to securinine, a GABA(A) receptor antagonist and immune adjuvant.

Securinine, a GABA(A) receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABA(A) receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5'-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca(2+) binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants.