CII Activity Research Articles

Abstract 14366: Pulmonary Hypertension-Induced RV Hypertrophy and Dysfunction is Associated With Reduced Activity and Abundance of Complex IV in Respiratory Supercomplexes

Introduction: PH results in RV hypertrophy, fibrosis and impaired function resulting in RV failure. PH is associated with impaired heart metabolism and mitochondrial respiration. Mitochondrial supercomplexes (mSC) are assemblies of multiple electron transport chain (ETC) complexes that consist of physically associated complex I, III and IV to enhance respiration. We determined if activity and/or abundance of mSC’s is altered in a rat model of PH. Methods: PH was induced in SD-rats by Sugen-Hypoxia (3 weeks) followed by normoxia (4 weeks) . Control (n=11) and PH (n=10) were subjected to echocardiography and isolation of LV and RV mitochondria. Clear native PAGE with equal amounts mitochondrial protein was used for SC abundance and activity with immunoblot or in gel activity with specific substrates. Results: PH animals had significantly reduced PAT and TAPSE, increased RV wall thickness and hypertrophy (RV/LVS). Western analysis of total protein fractions demonstrated reductions in representative CI, II, III, and IV proteins in RV indicating reduced mitochondrial content in PH. Assays with normalized mitochondrial protein showed activity (100+/- 3.6 vs 64.0+/-3.8 %, p<.01) and abundance (100+/-1.8 vs 49.3 +/-1.1, p=.02) of complex IV in mitochondrial SCs was severely reduced in PH animals compared to control. There were no differences in total CIV activity or abundance in lower order ETC assemblies (100 vs 97.5%, p=.78). TAPSE, PAT and RV Wall thickness significantly correlated with CIV SC activity (r=.71, .66, and -.80 respectively P<.01). CIII abundance was lower in SCs (P=.01) but total CIII unchanged. CI activity in SC assemblies was reduced in PH but this was not significant (100+/-12 vs 77.4 +/-8.2, p=.14). Overall levels of CI were unchanged. There were no changes in CII activity or abundance in RV or LV. In the LV, activity of CIV in SC was reduced in PH (100+/-6 vs 78+/-6, p=.02) but to a lesser extent than RV. There were also decreases in overall abundance of CIII and CIV assemblies in the LV in PH. Conclusion: The data support the concept that although mitochondrial abundance may be altered, there are also significant alterations associated with assembly and activity of mitochondrial SCs in the RV with pronounced differences in complex IV assembled into SCs.

Lung mitochondria adaptation to endurance training in rats

We elucidated the impact of eight weeks of endurance training on the oxidative metabolism of rat lungs. Adult 3.5-month-old male rats were randomly allocated to a treadmill training group or a sedentary group as control. In the lungs, endurance training raised the expression level of the oxygen sensors hypoxia inducible factor 1α (HIF1α) and lysine-specific demethylase 6A (KDM6A) as well as stimulated mitochondrial oxidative capacity and mitochondrial biogenesis, while lactate dehydrogenase activity was reduced. Endurance training enhanced antioxidant systems (the coenzyme Q content and superoxide dismutase) in lung tissue but decreased them (and uncoupling protein 2) in lung mitochondria. In the lung mitochondria of trained rats, the decreased Q content and Complex I (CI) activity and the enhanced cytochrome pathway activity (CIII + CIV) may account for the diminished Q reduction level, resulting in a general decrease in H2O2 formation by mitochondria. Endurance training enhanced oxidation of glutamate and fatty acids and caused opposite effects in functional mitochondrial properties during malate and succinate oxidation, which were related to reduced activity of CI and increased activity of CII, respectively. In addition, endurance training downregulated CI in supercomplexes and upregulated CIII in the CIII2+CIV supercomplex in the oxidative phosphorylation system. We concluded that the adaptive lung responses observed could be due to hypoxia and oxidative stress induced by strenuous endurance training.

Comparative analysis of the bioenergetics of human adenocarcinoma Caco-2 cell line and postoperative tissue samples from colorectal cancer patients.

The aim of this work was to explore the key bioenergetic properties for mitochondrial respiration in the widely-used Caco-2 cell line and in human colorectal cancer (HCC) postoperational tissue samples. Oxygraphy and metabolic control analysis (MCA) were applied to estimate the function of oxidative phosphorylation in cultured Caco-2 cells and HCC tissue samples. The mitochondria of Caco-2 cells and HCC tissues displayed larger functional activity of respiratory complex (C)II compared with CI, whereas in normal colon tissue an inverse pattern in the ratio of CI to CII activity was observed. MCA showed that the respiration in Caco-2 and HCC tissue cells is regulated by different parts of electron transport chain. In HCC tissues, this control is performed essentially at the level of respiratory chain complexes I-IV, whereas in Caco-2 cells at the level of CIV (cytochrome c oxidase) and the ATP synthasome. The differences we found in the regulation of respiratory chain activity and glycose index could represent an adaptive response to distinct growth conditions; this highlights the importance of proper validation of results obtained from in-vitro models before their extrapolation to the more complex in-vivo systems.

Lysis-lysogeny coexistence: prophage integration during lytic development.

The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic pathways. Although traditionally thought to be mutually exclusive, increasing evidence suggests that this lysis‐lysogeny decision is more complex than once believed, but exploring its intricacies requires an improved resolution of study. Here, with a newly developed fluorescent reporter system labeling single phage and E. coli DNAs, these two distinct pathways can be visualized by following the DNA movements in vivo. Surprisingly, we frequently observed an interesting “lyso‐lysis” phenomenon in lytic cells, where phage integrates its DNA into the host, a characteristic event of the lysogenic pathway, followed by cell lysis. Furthermore, the frequency of lyso‐lysis increases with the number of infecting phages, and specifically, with CII activity. Moreover, in lytic cells, the integration site attB on the E. coli genome migrates toward the polar region over time, leading to more spatial overlap with the phage DNA and frequent colocalization/collision of attB and phage DNA, possibly contributing to a higher chance for DNA integration.

Increased mitochondrial ATP production capacity in brain of healthy mice and a mouse model of isolated complex I deficiency after isoflurane anesthesia.

We reported before that the minimal alveolar concentration (MAC) of isoflurane is decreased in complex I-deficient mice lacking the NDUFS4 subunit of the respiratory chain (RC) (1.55 and 0.81 % at postnatal (PN) 22–25 days and 1.68 and 0.65 % at PN 31–34 days for wildtype (WT) and CI-deficient KO, respectively). A more severe respiratory depression was caused by 1.0 MAC isoflurane in KO mice (respiratory rate values of 86 and 45 at PN 22–25 days and 69 and 29 at PN 31–34 days for anesthetized WT and KO, respectively). Here, we address the idea that isoflurane anesthesia causes a much larger decrease in brain mitochondrial ATP production in KO mice thus explaining their increased sensitivity to this anesthetic. Brains from WT and KO mice of the above study were removed immediately after MAC determination at PN 31–34 days and a mitochondria-enriched fraction was prepared. Aliquots were used for measurement of maximal ATP production in the presence of pyruvate, malate, ADP and creatine and, after freeze-thawing, the maximal activity of the individual RC complexes in the presence of complex-specific substrates. CI activity was dramatically decreased in KO, whereas ATP production was decreased by only 26 % (p < 0.05). The activities of CII, CIII, and CIV were the same for WT and KO. Isoflurane anesthesia decreased the activity of CI by 30 % (p < 0.001) in WT. In sharp contrast, it increased the activity of CII by 37 % (p < 0.001) and 50 % (p < 0.001) and that of CIII by 37 % (p < 0.001) and 40 % (p < 0.001) in WT and KO, respectively, whereas it tended to increase that of CIV in both WT and KO. Isoflurane anesthesia increased ATP production by 52 and 69 % in WT (p < 0.05) and KO (p < 0.01), respectively. Together these findings indicate that isoflurane anesthesia interferes positively rather than negatively with the ability of CI-deficient mice brain mitochondria to convert their main substrate pyruvate into ATP.

Abstract 403: Mitochondrial Complex II is a Source of the Reserve Respiratory Capacity that is Regulated by Metabolic Sensors via Sirtuin 3

A cell’s survivability depends on its ability to meet its energy requirements. We hypothesized that the cells’ mitochondrial reserve respiratory capacity (RRC) is a critical component of its bioenergetics that can be utilized during an increase in energy demand, thereby, enhancing viability. Our goal was to identify the elements that regulate and contribute to the development of RRC and its involvement in cell survival. Our results show that development of RRC is dependent on metabolic substrate availability in a cell type-dependent manner. While the neonatal rat cardiac myocytes (NRCM) utilize glucose as the main substrate, developing a RRC [1.4-2.5 fold higher than basal oxygen consumption rate (OCR)] required fatty acids in addition to glucose. Accordingly, inhibition of either glucose or fatty acid oxidation separately, completely abrogated RRC, while having little impact on basal OCR, which is sustainable with either substrate or glutamate in the medium. Conversely, RRC was enhanced (1.4-1.8 fold) through increasing glucose oxidation via inhibiting pyruvate dehydrogenase kinase with dichloroacetate, or through increasing fatty acid oxidation via activation of AMP-activated kinase (AMPK). The latter was partly mediated through peroxisome proliferator-activated receptor alpha. These results suggested that RRC is an independently regulated entity of the cells’ bioenergetics. An electron flow activity assay revealed that the increase in RRC correlated with a specific increase in complex II (cII) activity. Inhibiting or disassembling holo cII completely abolished RRC, accompanied by a slight decrease in basal OCR (0.82-0.9 fold), thus confirming it as the source of RRC. Moreover, the development of RRC required Sirtuin (Sirt)3. Functionally, we show that enhancing RRC via fatty acid oxidation with 5-Aminoimidazole-4-carboxamide 1-β-Dribofuranoside in NRCM results in a burst of cII-dependent oxidative phosphorylation accompanied by reduced superoxide production and enhanced cell survival post-energy deprivation conditions. Thus, for the first time, we show that metabolic sensors increase the cells’ RRC via activating cII in a Sirt3-dependent manner, and that this mechanism can be exploited for increasing cell survival after hypoxia.

Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation.

Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

ROS-Triggered Phosphorylation of Complex II by Fgr Kinase Regulates Cellular Adaptation to Fuel Use

Electron flux in the mitochondrial electron transport chain is determined by the superassembly of mitochondrial respiratory complexes. Different superassemblies are dedicated to receive electrons derived from NADH or FADH2, allowing cells to adapt to the particular NADH/FADH2 ratio generated from available fuel sources. When several fuels are available, cells adapt to the fuel best suited to their type or functional status (e.g., quiescent versus proliferative). We show that an appropriate proportion of superassemblies can be achieved by increasing CII activity through phosphorylation of the complex II catalytic subunit FpSDH. This phosphorylation is mediated by the tyrosine-kinase Fgr, which is activated by hydrogen peroxide. Ablation of Fgr or mutation of the FpSDH target tyrosine abolishes the capacity of mitochondria to adjust metabolism upon nutrient restriction, hypoxia/reoxygenation, and T cell activation, demonstrating the physiological relevance of this adaptive response.

Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V

The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI–CIV) and the FoF1-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (ΔpH) and electrical charge (Δψ) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with Δψ depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24h) CI inhibition in HEK293 cells induces a proton-based Δψ hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O2 consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced Δψ depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which Δψ hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT.

SAT0040 Arthritis induced by newly developed anti-type II collagen mabs associated with antigen affinity

BackgroundPolyclonal antibodies or a combination of 4 or 5 mAbs specific for type II collagen (CII) induce severe arthritis in mice (CAIA model). The induction of CAIA is associated with...

Caloric restriction restores the chronological life span of the goa1 null mutant of Candida albicans in spite of high cell levels of ROS

The Candida albicans Goa1p is required for mitochondrial functions. In a strain lacking GOA1 (GOA31), respiration, mitochondrial membrane potential, complex I (CI) activity of the electron transport chain, and ATP synthesis are significantly decreased. A shortened chronological life span (CLS) of GOA31 occurs in 2% glucose that is associated with an increase in cell reactive oxidant species (ROS) and apoptosis. We now show that caloric restriction (CR) in media containing 0.5% glucose instead of 2% glucose-SC extends the CLS to the level of parental and gene-reconstituted strains. Paradoxically, ROS levels in GOA31 far exceed those of control strains in 0.5% glucose and, as a consequence, increased lipid peroxidation occurs even though CLS is restored. Microarray analysis was used to characterize transcriptional changes during CR in GOA31. We found that CR shifts cells of all strains to a non-glucose carbon metabolism (β-oxidation). Our model of ROS formation in GOA31 follows the paradigm that the generation of oxygen radicals from β-oxidation of cell lipids via FADH2 (CII) and NADH (CI) creates an unfavorable cellular FADH2/NADH ratio that causes a transient overload in CII activity resulting in excess free cell radicals. In GOA31 the CI and peroxisomal dysfunctions increase the levels of ROS compared to control strains. Recovery from high levels of ROS may be associated with an increase in iron and sugar transporters, as well as an anti-stress response that includes the SOD1 and GPX1. Thus, CR creates a favorable growth environment, but cells of GOA31 must overcome a high but transient ROS production.

The mitochondrial respiratory chain has a critical role in the antiviral process in Coxsackievirus B3-induced myocarditis

Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5±1.4 × 104 plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4±0.8 × 107 p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.

Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision

The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic α helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell.

Quantitative kinetic analysis of the bacteriophage λ genetic network

The lysis-lysogeny decision of bacteriophage lambda has been a paradigm for a developmental genetic network, which is composed of interlocked positive and negative feedback loops. This genetic network is capable of responding to environmental signals and to the number of infecting phages. An interplay between CI and Cro functions suggested a bistable switch model for the lysis-lysogeny decision. Here, we present a real-time picture of the execution of lytic and lysogenic pathways with unprecedented temporal resolution. We monitor, in vivo, both the level and function of the CII and Q gene regulators. These activators are cotranscribed yet control opposite developmental pathways. Conditions that favor the lysogenic response show severe delay and down-regulation of Q activity, in both CII-dependent and CII-independent ways. Whereas CII activity correlates with its protein level, Q shows a pronounced threshold before its function is observed. Our quantitative analyses suggest that by regulating CII and CIII, Cro plays a key role in the ability of the lambda genetic network to sense the difference between one and more than one phage particles infecting a cell. Thus, our results provide an improved framework to explain the longstanding puzzle of the decision process.

Role of C-Terminal Residues in Oligomerization and Stability of λ CII: Implications for Lysis-Lysogeny Decision of the Phage

A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

The bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the “lysis-versus-lysogeny” decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA+ or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII.

The phage lambda CII transcriptional activator carries a C-terminal domain signaling for rapid proteolysis.

ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the lambda CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the lambda CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage lambda carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein.

Stability of CII is a key element in the cold stress response of bacteriophage lambda infection.

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

Cleavage of the cII protein of phage lambda by purified HflA protease: control of the switch between lysis and lysogeny.

The activity of the cII protein of phage lambda is probably the critical controlling factor in the choice of the lytic or lysogenic pathway by an infecting virus. Previous work has established that cII activity is regulated through the turnover of cII protein; the products of the hflA and hflB loci of Escherichia coli are needed for a degradative reaction, and lambda cIII functions in stabilizing cII. By using the cloned hflA locus, we have purified a cII-cleaving enzyme that we term HflA. Purified HflA contains three polypeptides; at least two of the subunits are products of the hflA region, and the third is probably a cleavage product of the larger of these two hflA-encoded polypeptides. The HflA protease activity cleaves cII to small fragments. We conclude that the switch between lambda developmental pathways involves regulated cleavage of cII by the specific protease HflA.