Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.

Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1.

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CITREM is an emulsifier used in the food industry and contains citric acid esters of mono- and diglycerides (GCFE). It is generally recognized as safe but no publication on its digestibility under gastrointestinal conditions and impact on fat digestion was available. It was shown here that fatty acids are released from CITREM by gastric lipase, pancreatic lipase, pancreatic-lipase-related protein 2 and carboxyl ester hydrolase. A two-step in vitro digestion model mimicking lipolysis in the stomach and upper small intestine of term and preterm infants was then used to evaluate the digestibility of CITREM alone, CITREM-containing infant formula and fat emulsions, and isolated GCFE fractions. Overall, it was shown that fat digestion is not significantly changed by the presence of CITREM, and fatty acids contained in CITREM compounds are released to a large extent by lipases. Nevertheless, undigestible water-soluble compounds containing glycerol and citric acid units were identified, indicating that the ester bond between citric acid and glycerol is not fully hydrolyzed throughout the proposed digestion.

SummaryTerminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N-terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in E. coli. Biochemical characterization of gpA-ΔN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N-terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme -DNA maturation and DNA packaging -are discussed.

To obtain molecular insights into the action mode of antimicrobial activity of pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D 2 O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of pediocin, as registered by the amide I band, and phospholipid conformation, as revealed by the methylene s (CH 2 ) and carbonyl (CAO) stretching vibrations. The results show that no interaction between pediocin and DMPC occurs. By contrast, pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.Pediocin PA-1 is a 44-amino-acid (molecular mass, 4,629 Da; pI, 9.6) (35) class IIa bacteriocin (38) produced by Pediococcus acidilactici PAC 1.0 and naturally found in fermented sausages (37) or other meat and vegetable fermentations (5). Its sequence is highly homologous to other class IIa bacteriocins (59). The N-terminal 20-amino-acid half of pediocin is in the majority of cases polar or cationic and highly conserved, with the consensus sequence Y3-G4-N5-G6-V7. The C-terminal half (residues A21 to C44) is much less polar and less conserved, containing a hypothetical hydrophobic membrane interaction domain. Two disulfide bonds stabilize the peptide structure, the one between the residues 24 and 44 accounting for its broad range of antimicrobial activity (17). Because of its specific antimicrobial activity against the foodborne psychrotrophic pathogen Listeria monocytogenes, pediocin PA-1 can potentially serve as a nontoxic food preservative to improve the quality, naturalness, and safety (55) of dairy and meat products.The data for Listeria innocua death induced by pediocin PA-1 reveal that its activity maximum is reached near pD 6 and decreases at pD 7 and 8 (15, 30). A relation between activity and structure alterations has not yet been completely established. The loss of activity as a function of pD seems to be associated to a structural reorganization and a decrease in the protein flexibility that in turn results in an irreversible aggregation upon heating (30). The C-terminal region is first involved in the aggregation process, resulting in a total inactivation of the protein (30). The N-terminal region participates in the aggregation process in a second step. Pediocin can exist in a soluble and hydrophobic structural form (16). In an aqueous environment, pediocin presents an unordered structure as seen by circular dichroism (75) and Fourier transform infrared (FTIR) spectroscopy (30), whereas upon adsorption to vesicles, a structura...

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The Mycobacterium tuberculosis LipY protein, a prototype of the proline‐glutamic acid (PE) family, exhibits a triacylglycerol (TAG) hydrolase activity that contributes to host cell lipid degradation and persistence of the bacilli. LipY is found either as a full‐length intracytosolic form or as a mature extracellular form lacking the N‐terminal PE domain. Even though the contribution of the extracellular form in TAG consumption has been partly elucidated, very little information is available regarding the potential interactions of either full‐length LipY with the cytoplasmic membrane, or mature form LipY with the outer membrane. Herein, several LipY variants truncated in their N‐terminal domain were produced and biochemically characterized in lipid–protein interaction assays, using the monomolecular film technique and FTIR. Comparison of the catalytic activities of these recombinant proteins showed that LipY∆149, corresponding to the extracellular form of LipY lacking the PE domain, is more active than the full‐length protein. This confirms previous studies reporting that the PE domain negatively modulates the TAG hydrolase activity of LipY. Lipid–protein interaction studies indicate that the PE domain anchors LipY onto membrane lipids. Consistent with these findings, we show that LipY∆149 is loosely associated with the mycobacterial cell wall, and that this interaction is mediated by the sole lipase domain. Overall, our results bring new information regarding the molecular mechanisms by which LipY either binds and hydrolyses host cell lipids or degrades TAG, the major source of lipids within mycobacterial intracytosolic lipid inclusions.