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Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo.To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1?74 mM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7?5 mmol per spleen and 88?4 mmol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.Nitrogen-fixing bacteria contain the nitrogenase enzyme complex, which catalyzes the reduction of atmospheric nitrogen to ammonium. The nitrogenase complex consists of two enzymes, dinitrogenase, an ␣ 2 ␤ 2 tetramer of the nifK and nifD gene products, and dinitrogenase reductase, an ␣ 2 dimer of the nifH gene product. Dinitrogenase reductase transfers electrons, in an ATP-dependent manner, to dinitrogenase, which contains the site of substrate reduction. This process is very energy demanding and thus is controlled at the transcriptional level and in some systems also at the posttranslational level. Transcriptional control of nif gene expression is found in all nitrogen-fixing bacteria studied and is best characterized in Klebsiella pneumoniae (24). Posttranslational control of nitrogenase activity has been detected in a number of nitrogenfixing bacteria, including Rhodospirillum rubrum (10, 23) and Rhodobacter capsulatus (14, 31) (both purple nonsulfur photosynthetic bacteria), Chromatium vinosum (a purple sulfur bacterium) (11), and Azospirillum brasilense (a microaerobic bacterium) (7,12,37,38).The nitrogenase posttranslational modification system is best characterized in R. rubrum. In response to environmental conditions such as darkness or introduction of a fixed nitrogen source like ammonium, an ADP-ribose group from NAD is covalently attached to arginine 101 on one subunit of the dinitrogenase reductase dimer (15). This modification disrupts electron transfer between din...

Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state.Mono-ADP-ribosylation of proteins is observed widely among eucaryotic and procaryotic systems (3,22,25,35). The transfer of ADP-ribose from NAD to an amino acid acceptor is catalyzed by an ADP-ribosyltransferase enzyme. Several bacterial toxins such as diphtheria and cholera toxins have this transferase activity and thus can modify their eucaryotic target proteins and thereby disrupt important cellular mechanisms (11,17,24).Several nitrogen-fixing bacteria utilize this mechanism of posttranslational modification to control nitrogenase activity. The nitrogenase enzyme complex catalyzes the reduction of atmospheric nitrogen to ammonium. This is a very energyintensive reaction, in that at least 16 ATP molecules are hydrolyzed per nitrogen molecule reduced. Therefore, Rhodospirillum rubrum (21, 30), Azospirillum brasilense (5, 10, 38, 39), Rhodobacter capsulatus (12, 29), and Chromatium vinosum (7) posttranslationally inactivate nitrogenase through an ADP-ribosylation system under energy-limiting conditions or when a source of fixed nitrogen is available.The nitrogenase complex consists of dinitrogenase, an ␣ 2 ␤ 2 tetramer which contains the site for substrate reduction, and dinitrogenase reductase, a homodimer which transfers electrons to dinitrogenase. The nitrogenase complex is posttranslationally regulated by the ADP-ribosylation of arginine 101 (in R. rubrum) of one subunit of the dinitrogenase reductase dimer (30). This modification interrupts the docking and the electron transfer between dinitrogenase reductase and dinitrogenase, thus resulting in the loss of nitrogenase activity (26).The transfer of ADP-ribose from NAD onto dinitrogenase reductase is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT) (19). In the phot...

At the University of Wisconsin-La Crosse, we have undertaken a program to integrate the study of bioinformatics across the undergraduate life science curricula. Our efforts have included incorporating bioinformatics exercises into courses in the biology, microbiology, and chemistry departments, as well as coordinating the efforts of faculty within those departments. Here, we assess student confidence in solving and ability to solve bioinformatics-related problems. Assessment data show increases in student performance on bioinformatics-related problems and more confidence in solving such problems with increased exposure to the field of bioinformatics. Additionally, the faculty perceive an increased awareness of the applications of bioinformatics among the students in their courses. The combination of three different assessment tools, a student self-assessment of learning, a content exam, and faculty survey, was an effective and efficient approach for evaluating this multi-departmental program.