A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.     

Insertion into the Mycobacterium smegmatis genome of the aph gene through lysogenization with the temperate mycobacteriophage Ms6

A derivative of the temperate mycobacteriophage Ms6 containing the aph gene from transposon Tn5 was constructed. In the transductants the aph gene was integrated in the bacterial genome. The aph gene is stably maintained in the absence of positive selection after more than 150 generations. The results presented in this report show that Ms6 can be used as a vehicle for the integration of foreign DNA into the Mycobacterium smegmatis genome.     

Functional analysis of a putative holin-like peptide-coding gene in the genome of Bacillus licheniformis AnBa9

BhlA, a putative holin-like protein of Bacillus licheniformis AnBa9 expressed in Escherichia coli BL21(DE3) showed antibacterial activity against several gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Micrococcus luteus. Deletion analysis of bhlA suggests that a hydrophobic transmembrane domain, BhlATM is essential for antibacterial activity. Though the minimum inhibitory concentration (MIC) of BhlA was sevenfold lower than BhlATM, transmembrane domain deleted construct (BhlA∆TM) had no antibacterial activity. The expression of BhlA in E. coli was found to be toxic to cells. Therefore, the bhlA was cloned in yeast surface display vector pYD1 and expressed in Saccharomyces cerevisiae. The surface displayed yeast showed inhibition of several gram-positive bacteria. This recombinant yeast expressing BhlA may be used as biodrug for efficient control of multiple drug-resistant bacterial infections.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.     

Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria

Background  

Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNA^ala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNA^ala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNA^ala genes.     

Functional analysis of prokaryotic SELB proteins

Since the discovery of selenocysteine as the 21st amino acid considerable progress has been made in elucidating the system responsible for its insertion into proteins. Elongation factor SELB, whose amino-terminal part shows homology to EF-Tu, was found to be the key component mediating delivery of selenocysteyl-tRNA(Sec) to the ribosomal A site. It exhibits a distinct tertiary structure comprising binding sites for guanosine nucleotides, the cognate tRNA, an mRNA secondary structure (SECIS element) and presumably ribosomal components. The kinetics of interaction of SELB with its ligands have been studied in detail. GDP was found to bind with about 20-fold lower affinity than GTP and to be in rapid exchange, which obviates the need for a guanosine nucleotide exchange factor. The affinity of SELB for the SECIS element is in the range of 1 nM and further increases upon binding of selenocysteyl-tRNA(Sec) to the protein. This supports the model that SELB forms a tight quaternary complex on the SECIS element which is loosened after insertion of the tRNA into the ribosomal A site and the concomitant hydrolysis of GTP.     

Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system

The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308-7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An Escherichia coli mutant containing the blyAB locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of sheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in B. burgdorferi dramatically stimulated blyAB expression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.     

Functional analysis of glucose-dependent insulinotropic polypeptide fusion proteins

To generate functional fluorescently tagged glucose-dependent insulinotropic polypeptide (GIP), a series of GIP expression constructs were devised. These included G1 (complete preprohormone), G2 (lacking the C-terminal extension), G3 (lacking both N- and C-terminal extensions), G4 (G2 fused to green fluorescent protein, GFP), and G5 (G3 fused to GFP). Expression of G5 in bacteria generated immunopositive GIP together with GFP fluorescence, while G4 generated only fluorescence without immunoreactivity. Transfection of NIH3T3 cells with cDNAs of G1, G3, G5, but not G2, G4, and EGFP, resulted in immunologically detectable GIP formation, although fluorescence could be detected in the latter two. GIP as well as GIP-GFP secreted by NIH3T3 cells significantly stimulated intracellular cAMP accumulation and Ca(2+) mobilization in SaOS2 cells. The GIP receptor antagonist GIP(7-30) abolished these responses. These results suggest that a GIP-GFP fusion protein seven times larger than the native peptide retains function and may be used as an in vivo probe to detect GIP receptor distribution and to explore GIP's biological roles.     

MSTF: a domain involved in bacterial metallopeptidases and surface proteins, mycobacteriophage tape-measure proteins and fungal proteins

Here we report a novel domain, MSTF (domain involved in bacterial metallopeptidases, surface proteins and other proteins, also present in mycobacteriophage tape-measure proteins and fungal proteins), which is present in bacteria, phages and fungi. MSTF is about 67-94 amino acids in length with one HxDHxH motif and some highly conserved residues including His, Gly, Ala and Asp. Secondary structure prediction indicated that this domain contains two alpha-helices and one beta-sheet. Identification of MSTF will provide an opportunity to develop new strategies to combat pathogenic microorganisms, especially Mycobacterium tuberculosis.     

Functional analysis of two lebocin-related proteins from Manduca sexta

Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1-4 that are located close to the C-termini. In addition, we found that synthetic Leb-B(22-48) peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.     

Weird fingers: Functional analysis of WIP domain proteins

WIP proteins form a plant specific subfamily of C2H2 zinc finger (ZF) proteins. In this study, we functionally characterized the WIP domain, which consists of four ZF motifs, and discuss molecular functions for WIP proteins. Mutations in each of the ZFs lead to loss of function of the TT1/WIP1 protein in Arabiopsis thaliana. SV40 type nuclear localisation signals were detected in two of the ZFs and functionally characterized using GFP fusions as well as new mutant alleles identified by TILLING. Promoter swap experiments showed that selected WIP proteins are partially able to take over TT1 function. Activity of the AtBAN promoter, a potential TT1 target, could be increased by the addition of TT1 to the TT2-TT8-TTG1 regulatory complex.     

Comparative analysis of the base composition and codon usages in fourteen mycobacteriophage genomes

To study the possible codon usage and base composition variation in the bacteriophages, fourteen mycobacteriophages were used as a model system here and both the parameters in all these phages and their plating bacteria, M. smegmatis had been determined and compared. As all the organisms are GC-rich, the GC contents at third codon positions were found in fact higher than the second codon positions as well as the first + second codon positions in all the organisms indicating that directional mutational pressure is strongly operative at the synonymous third codon positions. Nc plot indicates that codon usage variation in all these organisms are governed by the forces other than compositional constraints. Correspondence analysis suggests that: (i) there are codon usage variation among the genes and genomes of the fourteen mycobacteriophages and M. smegmatis, i.e., codon usage patterns in the mycobacteriophages is phage-specific but not the M. smegmatis-specific; (ii) synonymous codon usage patterns of Barnyard, Che8, Che9d, and Omega are more similar than the rest mycobacteriophages and M. smegmatis; (iii) codon usage bias in the mycobacteriophages are mainly determined by mutational pressure; and (iv) the genes of comparatively GC rich genomes are more biased than the GC poor genomes. Translational selection in determining the codon usage variation in highly expressed genes can be invoked from the predominant occurrences of C ending codons in the highly expressed genes. Cluster analysis based on codon usage data also shows that there are two distinct branches for the fourteen mycobacteriophages and there is codon usage variation even among the phages of each branch.     

Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti

The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti.     

Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1

The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131st proline residue of the wild-type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N-terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor.     

Functional analysis of expressed peptides that bind yeast STE proteins

Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.     

Functional analysis of candidate ABC transporter proteins for sitosterol transport

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.