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The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor λCII, thereby helping the
phage to lysogenize the host bacterium. λCIII, a small (54-residue) protein of unknown structure, also protects...
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... helical region makes up the core of the molecule, which was synthesized as the peptide CIIIC. The calculated values for secondary structural elements predicted from the sequence of CIII and as analyzed from the far-UV CD spectra of CIII and CIIIC (Fig. 1B and C) are shown in Table 1. The CD spectrum of CIII was also recorded under reducing conditions in the presence of -ME. ...
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The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence align...
Citations
... A strong association with a two-pore potassium channel plays its role in bacterial survival and invasion by both sustaining intracellular homeostasis and modulating the secretion of effectors by type III secretion systems [45]. The lamda phage-encoded CIII protein serves as an inhibitor of the FtsH protease, thereby assisting the phage in its propagation during the lytic lifecycle [46]. At the same time, FtsH proteolytic activity may also hydrolyze the MgtC virulence protein essential for successful proliferation in macrophages [47]; therefore, the inhibition of FtsH could promote the normal course of systemic infections. ...
Salmonella enterica is a bacterial pathogen known to cause gastrointestinal infections in diverse hosts, including humans and animals. Despite extensive knowledge of virulence mechanisms, understanding the factors driving host specificity remains limited. In this study, we performed a comprehensive pangenome-wide analysis of S. enterica to identify potential loci determining preference towards certain hosts. We used a dataset of high-quality genome assemblies grouped into 300 reference clusters with a special focus on four host groups: humans, pigs, cattle, and birds. The reconstructed pangenome was shown to be open and enriched with the accessory component implying high genetic diversity. Notably, phylogenetic inferences did not correspond to the distribution of affected hosts, as large compact phylogenetic groups were absent. By performing a pangenome-wide association study, we identified potential host specificity determinants. These included multiple genes encoding proteins involved in distinct infection stages, e.g., secretion systems, surface structures, transporters, transcription regulators, etc. We also identified antibiotic resistance loci in host-adapted strains. Functional annotation corroborated the results obtained with significant enrichments related to stress response, antibiotic resistance, ion transport, and surface or extracellular localization. We suggested categorizing the revealed specificity factors into three main groups: pathogenesis, resistance to antibiotics, and propagation of mobile genetic elements (MGEs).
... The expression of cro then began to suppress lacZ expression after pRM promoter (Additional file 1: Table S1) via binding to its binding site [25]. Additionally, cro expression has been shown to be strengthened by cII expression, which is inhibited to a degree because cII expression is still suppressed by endogenous Ftsh gene expression [26]. On the other hand, fermentation of lactose by the gut microbiota has been demonstrated to produce lactic acid and other short-chain fatty acids, leading to a pH drop within the colon, which would weaken patp2 and inhibit cI expression. ...
... Hence, ompA-lldD expression would gradually recover to a normal condition, producing a signal peptide [27], and L-LDH [28,29] would be translocated to the cell membrane to convert lactic acid to pyruvate in the periplasm. Additionally, the gradual recovery of cIII expression would unsuppress cII expression by inhibiting endogenous expression of Ftsh [26]. Unsuppressed cII expression would then strengthen cro expression, thus accelerating the inhibition of lacZ expression. ...
Background
Lactose malabsorption occurs in around 68% of the world’s population, causing lactose intolerance (LI) symptoms, such as abdominal pain, bloating, and diarrhea. To alleviate LI, previous studies have mainly focused on strengthening intestinal β-galactosidase activity while neglecting the inconspicuous drop in the colon pH caused by the fermentation of non-hydrolyzed lactose by the gut microbes. A drop in colon pH will reduce the intestinal β-galactosidase activity and influence intestinal homeostasis.
Results
Here, we synthesized a tri-stable-switch circuit equipped with high β-galactosidase activity and pH rescue ability. This circuit can switch in functionality between the expression of β-galactosidase and expression of L-lactate dehydrogenase in response to an intestinal lactose signal and intestinal pH signal, respectively. We confirmed that the circuit functionality was efficient in bacterial cultures at a range of pH levels, and in preventing a drop in pH and β-galactosidase activity after lactose administration to mice. An impact of the circuit on gut microbiota composition was also indicated.
Conclusions
Due to its ability to flexibly adapt to environmental variation, in particular to stabilize colon pH and maintain β-galactosidase activity after lactose influx, the tri-stable-switch circuit can serve as a promising prototype for the relief of lactose intolerance.
... Kobiler et al. (2007) also suggested that oligomerization of cIII is required for its function as the HflB inhibitor. However, Halder et al. (2007) proposed that the cIII protein exists as a dimer under native conditions. Apart from the regulatory factors influencing the lysis-versuslysogenization decision known for many years, perhaps surprisingly, it is still possible to discover novel players in this game. ...
Bacteriophage λ, rediscovered in the early 1950s, has served as a model in molecular biology studies for decades. Although currently more complex organisms and more complicated biological systems can be studied, this phage is still an excellent model to investigate principles of biological processes occurring at the molecular level. In fact, very few other biological models provide possibilities to examine regulations of biological mechanisms as detailed as performed with λ. In this chapter, recent advances in our understanding of mechanisms of bacteriophage λ development are summarized and discussed. Particularly, studies on (i) phage DNA injection, (ii) molecular bases of the lysis-versus-lysogenization decision and the lysogenization process itself, (iii) prophage maintenance and induction, (iv), λ DNA replication, (v) phage-encoded recombination systems, (vi) transcription antitermination, (vii) formation of the virion structure, and (viii) lysis of the host cell, as published during several past years, will be presented.
... replicon that can exist either as a plasmid or integrated No gpC analogue in P22 gpNu3 gpNu3 gp8 gp8 Monomer/dimer/tetramer for gp8; monomer/dimer for gpNu3 gpB gpE gp1 gp5 Seen in reconstructions a gpNu3 gpB gp8 gp1 Scaffolds required for portal assembly into procapsid gp8 gp5 Scaffold-coat interaction not studied for l gpD gpD Dec Dec Both trimers; bind slightly different sites. a Note that P22 has no Dec, but its very close relative phage L does and L Dec binds to P22 capsids gpE gpE gp5 gp5 Both make icosahedral T¼7 head shells a gpU gpU No analogue in P22 gpD gpE Dec gp5 Dec is more discriminatory than D, i.e., unlike D, Dec does not occupy all of the quasiequivalent local threefold positions on the shell gp4 gp5 Unkown in l gp4 gp10 No gp10 analogue in l (but see gpFII above) gp4 gp9 No gp9 analogue in l (but see Stf below?) gp10 gp9 No gp9 analogue in l (but see Stf below?) gp10 gp10 No gp10 analogue in l (but see gpFII above) gp10 gp26 No gp10 or gp26 (?) analogue in l Xis? Xis Xis Monomer in l, but binds cooperatively so could interact on DNA (Abbani et al., 2007); multimer in P22 CIII CIII C3 C3 C3 is homologue of l CIII; l CIII is dimer (Halder et al., 2007); P22 interaction by homology with l CIII Cro Cro Cro Cro Both homodimers gpRz gpRz1 gp15 gp15 Predicted for P22 by homology a Not recognizably similar in amino acid sequence but very likely ancient homologues. ...
Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology.
... The in vitro assay for MalE-p25 cleavage by PopC was performed as described (Rolbetzki et al., 2008). In vitro degradation of His6-PopC/PopD-Strep, PopD-Strep and RpoH-His6 by GST-FtsH D or GST-FtsH Ec was performed as described (Halder et al., 2007). Briefly, purified proteins were dialysed against buffer P (50 mM Tris-acetate, 100 mM NaCl, 5 mM MgCl2, 25 mM Zn-acetate, 1.4 mM b-mercaptoethanol, pH 7.2). ...
Proteolytic cleavage of precursor proteins to generate intercellular signals is a common mechanism in all cells. In Myxococcus xanthus the contact-dependent intercellular C-signal is a 17 kDa protein (p17) generated by proteolytic cleavage of the 25 kDa csgA protein (p25), and is essential for starvation-induced fruiting body formation. p25 accumulates in the outer membrane and PopC, the protease that cleaves p25, in the cytoplasm of vegetative cells. PopC is secreted in response to starvation, therefore, restricting p25 cleavage to starving cells. We focused on identifying proteins critical for PopC secretion in response to starvation. PopC secretion depends on the (p)ppGpp synthase RelA and the stringent response, and is regulated post-translationally. PopD, which is encoded in an operon with PopC, forms a soluble complex with PopC and inhibits PopC secretion whereas the integral membrane AAA+ protease FtsH(D) is required for PopC secretion. Biochemical and genetic evidence suggest that in response to starvation, RelA is activated and induces the degradation of PopD thereby releasing pre-formed PopC for secretion and that FtsH(D) is important for PopD degradation. Hence, regulated PopC secretion depends on regulated proteolysis. Accordingly, p17 synthesis depends on a proteolytic cascade including FtsH(D) -dependent degradation of PopD and PopC-dependent cleavage of p25.
... An internal amphipathic helix in CIII and oligomerization are important for inhibition of FtsH [89,90]. The amphipathic helix might be used for recruiting to FtsH but the termini seem to be involved in degradation initiation [91]. Stabilized CII induces the expression of the CI repressor for entry into lysogeny. ...
Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA(+) proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed.
... Other factors such as λCIII and the host hfl proteins that influence the lysis-lysogeny switching affect the stability of CII in one way or the other. λCIII promotes lysogeny by acting as a general inhibitor of E. coli HflB that degrades CII [16]. ...
The lysis-lysogeny decision in the temperate coliphage λ is influenced by a number of phage proteins (CII and CIII) as well as host factors, viz. Escherichia coli HflB, HflKC and HflD. Prominent among these are the transcription factor CII and HflB, an ATP-dependent protease that degrades CII. Stabilization of CII promotes lysogeny, while its destabilization induces the lytic mode of development. All other factors that influence the lytic/lysogenic decision are known to act by their effects on the stability of CII. Deletion of hflKC has no effect on the stability of CII. However, when λ infects ΔhflKC cells, turbid plaques are produced, indicating stabilization of CII under these conditions.
We find that CII is stabilized in ΔhflKC cells even without infection by λ, if CIII is present. Nevertheless, we also obtained turbid plaques when a ΔhflKC host was infected by a cIII-defective phage (λcIII67). This observation raises a fundamental question: does lysogeny necessarily correlate with the stabilization of CII? Our experiments indicate that CII is indeed stabilized under these conditions, implying that stabilization of CII is possible in ΔhflKC cells even in the absence of CIII, leading to lysogeny.
We propose that a yet unidentified CII-stabilizing factor in λ may influence the lysis-lysogeny decision in ΔhflKC cells.
... No homologues are known from Archaea, and only six from Bacteria, including three from Family I75 contains the bacteriophage lambda CIII protein, which has been shown to be an inhibitor of the metalloendopeptidase FtsH (family M41). Degradation of the phage CII protein by FtsH pushes the phage towards lysis rather than lysogeny, so inhibition of FtsH retains the phage in the lysogenic state [111]. Homologues are known from E. coli and Salmonella enterica (Enterobacteriaceae). ...
In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment.
... Bacteriophages have two alternative modes of development, a quiescent phase where the phage genome is incorporated into the host genome (lysogeny) and a lytic phase, where phage particles are released from the infected cell. The host peptidase FtsH (M41.001) pushes the phage towards lysis by degrading the phage protein CII, and by inhibiting FtsH with CIII, the phage can persist in the lysogenic stage [59]. ...
The MEROPS website (http://merops.sanger.ac.uk) includes information on peptidase inhibitors as well as on peptidases and their substrates. Displays have been put in place to link peptidases and inhibitors together. The classification of protein peptidase inhibitors is continually being revised, and currently inhibitors are grouped into 67 families based on comparisons of protein sequences. These families can be further grouped into 38 clans based on comparisons of tertiary structure. Small molecule inhibitors are important reagents for peptidase characterization and, with the increasing importance of peptidases as drug targets, they are also important to the pharmaceutical industry. Small molecule inhibitors are now included in MEROPS and over 160 summaries have been written.
... The mutant cII genes were subcloned into pET15b at the NdeI and BamHI sites, to change the antibiotic selection marker from kanamycin to ampicillin. The resulting plasmids were derivatives of pAB412 (Halder et al., 2007) which carries the wild-type cII gene (Supplementary Fig. S3c). ...
... Complementation assays. The in vivo activity of His 6 -tagged native and mutant CII was tested by complementation assays following the method described by Halder et al. (2007), as described below. E. coli BL21 (DE3) cells containing pAB412 (carrying the native cII gene) or any of the plasmids pKP301 to pKP306 (carrying one of the mutated cII genes, see Supplementary Table S1) were used as hosts. ...
The CII protein of the temperate bacteriophage lambda is the decision-making factor that determines the viral lytic/lysogenic choice. It is a homotetrameric transcription activator that recognizes and binds specific direct repeat sequences TTGCN(6)TTGC in the lambda genome. The quaternary structure of CII is held by a four-helix bundle. It is known that the tetrameric organization of CII is necessary for its activity, but the molecular mechanism behind this requirement is not known. By specific site-directed mutagenesis of hydrophobic residues in the alpha4 helix of CII that constitutes the four-helix bundle, we found that residues leu70, val74 and leu78 were crucial for maintaining the tetrameric structure of the protein. When any of these residues was substituted by a polar one, CII lost its activity and failed to promote lysogeny. This loss of activity was accompanied by the inability of CII to form tetramers, to bind DNA or to activate transcription.
