Mutational analysis of the TonB1 energy coupler of Pseudomonas aeruginosa

Siderophore-mediated iron transport in Pseudomonas aeruginosa is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the tonB1 gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both P. aeruginosa and Escherichia coli. Appending it to the N terminus of the nonfunctional (in P. aeruginosa) Escherichia coli TonB protein (TonB(Ec)) rendered TonB(Ec) weakly active in P. aeruginosa and did not compromise the activity of this protein in E. coli. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in P. aeruginosa and E. coli. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB(Ec) function in E. coli, was shown here to be essential for TonB1 activity in E. coli but not in P. aeruginosa. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both P. aeruginosa and E. coli, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in E. coli but not in P. aeruginosa. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in P. aeruginosa. Several TonB1-TonB(Ec) chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in P. aeruginosa, although chimeras possessing an E. coli C terminus were active in E. coli.     

Differential effects of mutations in tonB1 on intrinsic multidrug resistance and iron acquisition in Pseudomonas aeruginosa

Loss of tonB1 adversely affects iron acquisition and intrinsic multidrug resistance in Pseudomonas aeruginosa. Several mutations in tonB1 compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-à-vis their contribution to drug resistance only.     

The Evolution of the Ribonucleotide Reductases: Much Ado About Oxygen

Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.     

The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5

The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.     

Identification of a new gene responsible for the oxygen tolerance in aerobic life of Streptococcus mutans

Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.     

Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids

N2-fixation by Rhizobium-legume symbionts is of major ecological and agricultural importance, responsible for producing a substantial fraction of the biosphere's nitrogen. On the basis of 15N-labelling studies, it had been generally accepted that ammonium is the sole secretion product of N2-fixation by the bacteroid and that the plant is responsible for assimilating it into amino acids. However, this paradigm has been challenged in a recent 15N-labelling study showing that soybean bacteroids only secrete alanine. Hitherto, nitrogen secretion has only been assessed from in vitro 15N-labelling studies of isolated bacteroids. We show that both ammonium and alanine are secreted by pea bacteroids. The in vitro partitioning between them will depend on whether the system is open or closed, as well as the ammonium concentration and bacteroid density. To overcome these limitations we identified and mutated the gene for alanine dehydrogenase (aldA) and demonstrate that AldA is the primary route for alanine synthesis in isolated bacteroids. Bacteroids of the aldA mutant fix nitrogen but only secrete ammonium at a significant rate, resulting in lower total nitrogen secretion. Peas inoculated with the aldA mutant are green and healthy, demonstrating that ammonium secretion by bacteroids can provide sufficient nitrogen for plant growth. However, plants inoculated with the mutant are reduced in biomass compared with those inoculated with the wild type. The labelling and plant growth studies suggest that alanine synthesis and secretion contributes to the efficiency of N2-fixation and therefore biomass accumulation.     

Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.     

Spinotrapezius muscle microcirculatory function: effects of surgical exteriorization

Intravital microscopy facilitates insights into muscle microcirculatory structural and functional control, provided that surgical exteriorization does not impact vascular function. We utilized a novel combination of phosphorescence quenching, microvascular oxygen pressure (microvascular PO(2)), and microsphere (blood flow) techniques to evaluate static and dynamic behavior within the exposed intact (I) and exteriorized (EX) rat spinotrapezius muscle. I and EX muscles were studied under control, metabolic blockade with 2,4-dinitrophenol (DNP), and electrically stimulated conditions with 1-Hz contractions, and across switches from 21 to 100% and 10% inspired O(2). Surgical preparation did not alter spinotrapezius muscle blood flow in either I or EX muscle. DNP elevated muscle blood flow approximately 120% (P < 0.05) in both I and EX muscles (P > 0.05 between I and EX). Contractions reduced microvascular PO(2) from 30.4 +/- 4.3 to 21.8 +/- 4.8 mmHg in I muscle and from 33.2 +/- 3.0 to 25.9 +/- 2.8 mmHg in EX muscles with no difference between I and EX. In each O(2) condition, there was no difference (each P > 0.05) in microvascular PO(2) between I and EX muscles (21% O(2): I = 37 +/- 1; EX = 36 +/- 1; 100%: I = 62 +/- 5; EX = 51 +/- 9; 10%: I = 20 +/- 1; EX = 17 +/- 2 mmHg). Similarly, the dynamic behavior of microvascular PO(2) to altered inspired O(2) was unaffected by the EX procedure [half-time (t(1/2)) to 100% O(2): I = 23 +/- 5; EX = 23 +/- 4; t(1/2) to 10%: I = 14 +/- 2; EX = 16 +/- 2 s, both P > 0.05]. These results demonstrate that the spinotrapezius muscle can be EX without significant alteration of microvascular integrity and responsiveness under the conditions assessed.