Molecular Dissection of Phage Endolysin: AN INTERDOMAIN INTERACTION CONFERS HOST SPECIFICITY IN LYSIN A OF MYCOBACTERIUM PHAGE D29*

Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages.     

Coevolution of the ATPase ClpV,the Sheath Proteins TssB and TssC,and the Accessory Protein TagJ/HsiE1 Distinguishes Type VI Secretion Classes

The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA⁺ ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one of the classes. Using bacterial two-hybrid approaches, we showed that the P. aeruginosa homolog HsiE1 interacts strongly with ClpV1. We then resolved the crystal structure of HsiE1 in complex with the N terminus of HsiB1, a TssB homolog and component of the contractile sheath. Phylogenetic analysis confirmed that these differences distinguish T6SS classes that resulted from a functional co-evolution between TssB, TssC, TagJ/HsiE, and ClpV. The interaction of TagJ/HsiE with the sheath as well as with ClpV suggests an alternative mode of disassembly in which HsiE recruits the ATPase to the sheath.     

The episodic evolution of fibritin: traces of ancient global environmental alterations may remain in the genomes of T4‐like phages

The evolutionary adaptation of bacteriophages to their environment is achieved by alterations of their genomes involving a combination of both point mutations and lateral gene transfer. A phylogenetic analysis of a large set of collar fiber protein (fibritin) loci from diverse T4‐like phages indicates that nearly all the modular swapping involving the C‐terminal domain of this gene occurred in the distant past and has since ceased. In phage T4, this fibritin domain encodes the sequence that mediates both the attachment of the long tail fibers to the virion and also controls, in an environmentally sensitive way, the phage's ability to infect its host bacteria. Subsequent to its distant period of modular exchange, the evolution of fibritin has proceeded primarily by the slow vertical divergence mechanism. We suggest that ancient and sudden changes in the environment forced the T4‐like phages to alter fibritin's mode of action or function. The genome's response to such episodes of rapid environmental change could presumably only be achieved quickly enough by employing the modular evolution mechanism. A phylogenetic analysis of the fibritin locus reveals the possible traces of such events within the T4 superfamily's genomes.     

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus

Abstract The putative ribosome binding sites preceding 32 of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H genes were compared. A highly conserved consensus sequence for the ribosome binding sites of LL-H genes was inferred, GAAAGGAG. This study included the characterization of the last nucleotides of the 3'-end of the 16S rRNA molecule from L. delbrueckii subsp. lactis and its comparison to the ribosome binding site consensus sequence.     

Function and structure of microvirid phage α3 genome. DNA sequence of H gene and properties of missense H mutant

The nucleotide sequence of wild-type α3 H gene and its surrounding region was determined and compared with those of ?X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.     

Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: analysis of mutant UvsX proteins from infected cells

Summary The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5 end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsX-am64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frame-shift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable.     

Isolation and genetic characterization of new uvsW alleles of bacteriophage T4

Summary TheuvsW gene of bacteriophage T4 is required for wild-type levels of recombination, for normal survival and mutagenesis after UV irradiation, and for wild-type resistance to hydroxyurea. Additionally,uvsW mutations restore the arrested DNA synthesis caused by mutations in any of several genes that block secondary initiation (recombination-primed replication, the major mode of initiation at late times), but only partially restore the reduced burst size. AuvsW deletion mutation was constructed to establish the null-allele phenotype, which is similar but not identical to the phenotype of the canonicaluvsW mutation, and to demonstrate convincingly that theuvsW gene is non-essential (althoughuvsW mutations severely compromise phage production). In an attempt to uncouple the diverse effects ofuvsW mutations, temperature-sensitiveuvsWts mutants were isolated. Recombination and replication effects were partially uncoupled in these mutants, suggesting distinct and separable roles foruvsW in the two processes. Furthermore, the restoration of DNA synthesis but not recombination in the double mutantsuvsW uvsX anduvsW uvsY prompts the hypothesis that the restored DNA synthesis is not recombinationally initiated.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.     

Bacteriophage T4 DNA polymerase determines the amount and specificity of ultraviolet mutagenesis

Summary Ultraviolet mutagenesis in bacteriophage T4 proceeds via error-prone repair (EPR) and requires the functional integrity of the uvsWXY system which mediates genetic recombination, recombinational repair, and mutability by diverse DNA damaging agents. Current opinion holds that mutagens acting through EPR generate DNA damage which blocks the progress of the replication complex and that EPR consists of the facilitated bypass of such inaccurate, damaged templates. This notion predicts that the T4 DNA polymerase (encoded by gene 43) mediates EPR in UV irradiated phage T4. This prediction is verified by the discovery that gene 43 mutations often enhance or reduce UV mutagenesis (which is scored by the induction of r mutants) and sometimes change its specificity.