Phage Φ80 Research Articles

A FhuA mutant of Escherichia coli is infected by phage T1-independent of TonB

Infection of Escherichia coli K-12 by phages T1 and φ80 requires the FhuA outer membrane protein and the TonB protein. Mutations in the N-terminal globular domain close to the predicted channel in the β-barrel of FhuA were created. The FhuAΔ107–111 N104K K110D L111P mutant and the FhuA(L 109DPNGLK 110) insertion mutant were sensitive to phage T1, but nearly resistant to phage φ80. FhuAΔ107–111 N104K K110D L111P mediated phage T1 infection in a tonB mutant without formation of TonB-independent phage T1 host-range mutants. The FhuA mutants showed no altered sensitivity to phage T5. Although the phages share overlapping binding sites in FhuA, the structural alterations elicited by the mutations resulted in very different phage sensitivities. In the FhuA deletion mutant, the TonB requirement for phage T1 infection was partially bypassed.

An efficient heat-inducible Bacillus subtilis bacteriophage φ105 expression and secretion system for the production of the Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP)

The Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP) has been shown to be a potent inhibitor of class A β-lactamases including the Escherichia coli TEM-1 β-lactamase ( K i=0.6 nM). A heat-inducible BLIP expression system was constructed based on a derivative of Bacillus subtilis phage φ105. The recombinant BLIP produced by this system was secreted to the culture medium, purified to homogeneity, and fully active. We have shown that the signal peptide of BLIP functions well in B. subtilis to secrete BLIP out of the cells, which facilitates purification. The absence of a His-tag also avoids the activity and structure of BLIP being altered. An unprecedented high yield of recoverable protein in culture supernatant (3.6 mg of >95% pure BLIP/l culture) was achieved by a simple purification protocol. We have developed an efficient production process in which the culture time before heat-induction was 3–4 h and the culture supernatant could be collected 5 h after induction. This total time of 8–9 h is considered to be very short compared to that of the native S. clavuligerus culturing (60–70 h). We achieved a very efficient BLIP production rate of 0.8–0.9 mg/l/h. Heterologous gene expression was tightly controlled and no production of BLIP was observed before heat-induction, suggesting that cell density can be further increased to improve enzyme yield.

Interaction of a Putative Transcriptional Regulatory Protein and the Thermo-inducible cts-52 Mutant Repressor in the Bacillus subtilis Phage φ105 Genome

A 144 amino acid residue cts-52 mutant repressor (mtcφ105) located in the EcoRI-F immunity region ( immF) of Bacillus subtilis phage φ105 is involved in the control mechanism of a thermo-inducible expression system. Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor. On the basis of the protein sequence alignment, a DNA-binding α helix–β turn–α helix (HTH) motif was identified in the N-terminal region (residues 18–37) of the repressor as well as in the polypeptide of ORF4 (residues 22–41). In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. To study their DNA binding properties, the wild-type repressor (wtcφ105) and the mutant repressor mtcφ105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays. Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR). Our data showed that a temperature shift does not influence the wtcφ105–operators' binding affinity, while the binding of mtcφ105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtcφ105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures. mRNA levels monitored by real-time RT-PCR indicated a suppression of mtcφ 105 expression, but a stimulation of ORF4 transcription after thermo-induction. Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P M and P R within the immF region.

Mutant analysis of the Escherichia coli FhuA protein reveals sites of FhuA activity.

The FhuA outer membrane protein of Escherichia coli actively transports ferrichrome, albomycin, and rifamycin CGP 4832, and confers sensitivity to microcin J25, colicin M, and the phages T1, T5, and phi80. Guided by the FhuA crystal structure and derived predictions on how FhuA might function, mutants were isolated in the cork domain (residues 1 to 160) and in the beta-barrel domain (residues 161 to 714). Deletion of the TonB box (residues 7 to 11) completely inactivated all TonB-dependent functions of FhuA. Fixation of the cork to turn 7 of the barrel through a disulfide bridge between introduced C27 and C533 residues abolished ferrichrome transport, which was restored by reduction of the disulfide bond. Deletion of residues 24 to 31, including the switch helix (residues 24 to 29), which upon binding of ferrichrome to FhuA undergoes a large structural transition (17 A) and exposes the N terminus of FhuA (TonB box) to the periplasm, reduced FhuA transport activity (79% of the wild-type activity) but conferred full sensitivity to colicin M and the phages. Duplication of residues 23 to 30 or deletion of residues 13 to 20 resulted in FhuA derivatives with properties similar to those of FhuA with a deletion of residues 24 to 31. However, a frameshift mutation that changed QSEA at positions 18 to 21 to KKAP abolished almost completely most of FhuA's activities. The conserved residues R93 and R133 among energy-coupled outer membrane transporters are thought to fix the cork to the beta-barrel by forming salt bridges to the conserved residues E522 and E571 of the beta-barrel. Proteins with the E522R and E571R mutations were inactive, but inactivity was not caused by repulsion of R93 by R522 and R571 and of R133 by R571. Point mutations in the cork at sites that move or do not move upon the binding of ferrichrome had no effect or conferred only slightly reduced activities. It is concluded that the TonB box is essential for FhuA activity. The TonB box region has to be flexible, but its distance from the cork domain can greatly vary. The removal of salt bridges between the cork and the barrel affects the structure but not the function of FhuA.

Characterization of a High-affinity Complex Between the Bacterial Outer Membrane Protein FhuA and the Phage T5 Protein pb5

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as β-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Φ80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 °C; (ii) thermal denaturation of the complex occurred at 85 °C, while pb5 and FhuA were denatured at 45 °C and 74 °C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

Comparative analysis of the genomes of the temperate bacteriophages φ11, φ12 and φ13 of Staphylococcusaureus 8325

The genomes of the three temperate bacteriophages contained in the chromosome of Staphylococcusaureus 8325 have been extracted from the sequence database and analyzed. φ11, φ12 and φ13 are members of the same lytic group but different serogroups and consequently co-habitate the same host cell. Their genomes are approximately 42 kb to 45kb and contain about 90 ORFs of at least 50 codons. Of these, about 50 have similarities to known genes or to genes of other staphylococcal phages. Each of the phages clusters within a homology group that share large regions of sequence identity while intergroup homology is comparatively low. The arrangement of genes on the chromosomes of the three phages is similar and consistent with current modular theory of phage gene organization. The replicated genomes appear to be packaged by different mechanisms. Phage φ11 and φ12 have been found to contain sequences consistent with pac-site phages while φ13 has sequences consistent with cos-site phages. The attBsite for φ11 is located in an intergenic region of the S. aureus chromosome while φ12 and φ13 integrate into specific genes. The φ12 att-site is within an unknown gene, but the φ13 att-site is within the β-toxin gene. In contrast to the other two phages, φ13 also introduces the staphylokinase gene (sak) and a second gene related to expression of fib.

In vivo synthesis of the periplasmic domain of TonB inhibits transport through the FecA and FhuA iron siderophore transporters of Escherichia coli.

The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage phi80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuADelta5-160 was also inhibited by TonB(33-239), although FhuADelta5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.

Robustness of a gene regulatory circuit.

Complex interacting systems exhibit system behavior that is often not predictable from the properties of the component parts. We have tested a particular system property, that of robustness. The behavior of a system is termed robust if that behavior is qualitatively normal in the face of substantial changes to the system components. Here we test whether the behavior of the phage lambda gene regulatory circuitry is robust. This circuitry can exist in two alternative patterns of gene expression, and can switch from one regulatory state to the other. These states are stabilized by the action at the O(R) region of two regulatory proteins, CI and Cro, which bind with differential affinities to the O(R)1 and O(R)3 sites, such that each represses the synthesis of the other one. In this work, this pattern of binding was altered by making three mutant phages in which O(R)1 and O(R)3 were identical. These variants had the same qualitative in vivo patterns of gene expression as wild type. We conclude that the behavior of the lambda circuitry is highly robust. Based on these and other results, we propose a two-step pathway, in which robustness plays a key role, for evolution of complex regulatory circuitry.

Normal induction of the SOS response in Bacillus subtilis is prevented by the mutant repressor from phage φ105 cts23

The presence of the Φ105 cts23 mutant prophage in Bacillus subtilis induces a series of pleiotropic effects that could be ascribed to an anti-SOS activity. In order to circumvent the phage function responsible for this phenomenon, the cts23 mutant repressor was cloned and sequenced. The isolated repressor reduced the survival capacity of the host cells after mitomycin C or nalidixic acid treatments and lowered the spontaneous reversion frequency. When SOS induction kinetics were studied, low or null induction of the damage-inducible din22:: LacZ fusion was observed. In contrast, the presence of the wild-type prophage amplified the SOS response. Sequencing of the mutant repressor revealed that the cts23 mutation is a T→C transition affecting the 5′ closest codon to one of the two reported DNA binding domains.

A heat-inducible Bacillus subtilis bacteriophage Φ105 expression system for the production of the protective antigen of Bacillus anthracis

The protective antigen of Bacillus anthracis is the major protective immunogen in the current human vaccine. A heat-inducible protective antigen expression system was constructed based on a derivative of Bacillus subtilis phage Φ105. The recombinant protein produced by this system protected immunised animals against challenge with spores of B. anthracis. Gene instability and protease activity of the host strain contributed to the low level of recoverable protein in culture supernatant (∼2 mg l −1).

FhuA, an Escherichia coli outer membrane protein with a dual function of transporter and channel which mediates the transport of phage DNA

FhuA ( M r = 78 9010) is an Escherichia coli outer membrane protein which transports the ferric siderophore ferrichrome and is the receptor for phage T5, φ80 and T1 and for colicin M. FhuA was purified chromatographically in non-ionic detergent (octyl glucoside). The circular dichroism spectrum indicates that FhuA is essentially organized in β-strands like the majority of proteins of the outer membrane of Gram-negative bacteria. The structural parameters of FhuA were assessed from size exclusion chromatography, sedimentation equilibrium and velocity experiments. FhuA is monomeric in solution and functional since binding of phage T5 causes the release of the phage genome, a double-stranded DNA of 121 000 base pairs, into the surrounding medium. Planar lipid bilayer experiments showed that the FhuA transporter is converted into high conductance channel upon binding of phage T5. FhuA was reconstituted into large unilamellar vesicles (mean diameter 125 nm). Cryo-electron microscopy and fluorescence experiments, using a DNA intercalant YO-PRO 1, showed that binding of T5 to FhuA triggers the transfer of the phage genome into the proteoliposomes without altering their morphology. Two models can account for these observations, which apply both to in vitro and in vivo DNA transport. The simplest model supposes that the naked DNA is transported through the FhuA channel. Alternatively transfer of DNA might be mediated by pb2, the protein forming the phage straight fiber pb2 would insert either directly in the membrane or inside the FhuA channel.

Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.

Structure of a genome region of the Lactobacillus gasseri temperate phage φadh covering a repressor gene and cognate promoters

By sequencing the DNA regions which flank the intG gene encoding integrase of the temperate Lactobacillus ( Lb.) gasseri bacteriophage φadh, a continuous sequence of 6590 bp was established. It encompasses five newly identified ORFs, of which four are located upstream, and one ( orfC) downstream of intG. Proteins corresponding to the expected products of the intG upstream coding regions, orfA (33 kDa), orf2 (14 kDa), rad (12.1 kDa), and tec (7.9 kDa), were identified by in vitro expression of subcloned DNA fragments. Rad shares homology with transcription regulators, including SinR of Bacillus species and the repressor of phage φ105. The gp orf2 is similar to predicted products of topologically equivalent coding regions of the Lactococcus lactis phage TP901-1 and the B. subtilis phage φ105. Promoters for the divergently oriented rad and tec genes were mapped within the 435-bp region between them and specify overlapping transcripts with extended 5′-untranslated sequences. As shown with lacZ fusions, Rad repressed transcription from the tec and rad promoters 20- and 5-fold, respectively. In Lb. gasseri, weak expression of cloned rad was sufficient to mediate immunity towards φadh.

Cloning and characterization of the exbB-exbD-tonB locus of Pasteurella haemolytica A1

A recombinant plasmid (pMG1) carrying Pasteurella haemolytica A1 DNA which complements a tonB mutation of Escherichia coli has been isolated. E. coli tonB metE which carries pMG1 exhibits growth kinetics in the presence of vitamin B 12 similar to that of the wild-type host. In addition, the complemented E. coli is susceptible to killing by bacteriophage φ80 and colicin B. Analysis of the nucleotide sequence in the complementing DNA showed that it codes for three genes in the order of exbB-exbD-tonB. This genetic organization has been reported in Haemophilus influenzae, H. ducreyi, Pseudomonas putida and Vibrio cholerae, and may represent a separate lineage of evolution from that of the Enterobacteriaceae in which tonB is unlinked with the accessory genes exbB and exbD. A comparison of the DNA flanking the exbB-exbD-tonB locus in P. haemolytica A1 and H. influenzae showed that the flanking regions are completely different between the two organisms.

Characterization of an insertion in the phage φ105 genome that blocks host Bacillus subtilis lysis and provides strong expression of heterologous genes

A defective prophage vector, φ105MU331, for high-level protein overproduction in Bacillus subtilis, was derived by random insertion of a lacZ reporter gene. The site of insertion not only provided efficient inducible transcription of heterologous genes, but also prevented lysis of the host cell. The region of the insertion in φ105MU331 lies close to the right cohesive end of φ105. DNA sequence analysis revealed that this region of φ105 somewhat resembles the lysis cassette of various phages, including λ. The site of insertion lies in a possible ‘holin’ gene, which could explain the block in host cell lysis. Dual promoters apparently responsible for the strong inducible transcription lie in an untranslated region just upstream from the putative holin gene. This region is probably equivalent to the site of the major late promoter and antiterminator of the lambdoid phages. The sequence features could, thus, account for the useful properties of the φ105MU331 vector system.

An efficient expression and secretion system based on Bacillus subtilis phage φ105 and its use for the production of B. cereus β-lactamase I

A novel expression system based on the Bacillus subtilis bacteriophage φ105 has been developed to permit the highlevel synthesis and secretion of β-lactamase I (Blal) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of β-galactosidase (βGal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of φ 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blal gene from B. cereus resulted in the production of up to 500 μg of secreted Blal per ml of culture supernatant.

A novel Bacillus subtilis expression vector based on bacteriophage φ105

We have developed a novel expression vector based on the bacteriophage φ105, and employed it for the production of mutant β-lactamases in Bacillus subtilis. Expression of the β-lactamase-encoding gene was low when cloned into the prophage under the control of its own promoter. However, expression was considerably elevated when the gene was inserted into the phage genome in the same orientation as phage transcription. A defective φ105 vector was constructed with a deletion removing a region needed for cell lysis, and with a mutation in the immunity repressor, rendering it temperature sensitive. Production of β-lactamase could then be induced by a shift in temperature and without concomitant cell lysis, facilitating purification of the protein from the culture supernatant. This phage has considerable potential for development as a vector for controllable production of heterologous proteins in B. subtilis.

φ80 transcription antitermination complex: A 15 kDa host-coded protein interacts with the gene N transcription antitermination protein of bacteriophage φ80

We purified the transcription antitermination complex of φ80 (φ80-pN complex). This complex had a molecular weight of approx. 42,000, and the subunits of this complex could be separated by SDS polyacrylamide gel electrophoresis. The complex was composed of two different polypeptides, one was the φ80 N gene product (φ80-pN) with a molecular weight of approx. 12,000, and the other was a host-coded protein with a molecular weight of approx. 15,000. The densitometric analysis after SDS polyacrylamide gel electrophoresis showed that the molar ratio of two proteins was 2 to 1 (φ80-pN: 15kDa), and the complex was assumed to be a trimer. Bacteriophage φ80 could grow in nusA , nusC , and nusE mutants of Escherichia coli , but failed to grow in a nusB mutant. The molecular weight of the nusB gene product is calulated to be 15,689 from the sequencing data. These results suggest that the partner of the N gene product is possibly a host-coded nusB gene product.

Purification and in vitro DNA-binding Specificity of the Bacillus subtilis Phage ϕ105 Repressor

The Bacillus subtilis phage phi 105 repressor, a lambda repressor-like transcriptional regulatory protein, was overproduced in Escherichia coli and purified to near homogeneity in order to examine its in vitro DNA-binding properties. The active form of repressor appears to be a tetramer. DNase I protection experiments demonstrate that repressor can specifically bind to six distinct sites, all located within the phi 105 EcoRI-F immunity region (immF). Three of these sites had been identified earlier as functional operators by genetic analysis. They share a common 14-base pair, asymmetric core sequence, 5'-GACGGAAATACAAG-3', termed OR. The three additional sites show significant homology with OR. For an individual binding site, hydroxyl-radical footprinting reveals symmetrical repressor-DNA interactions established via one side of the helix. Dimethyl sulfate protection indicates that guanines at the conserved OR base pair positions 1, 3, and 4 may participate in sequence-specific interactions with repressor in agreement with a previously proposed recognition model. However, since the OR sequence is not symmetrical with respect to this GNCG motif, at present it remains difficult to completely understand the molecular basis of this interaction.

Two forms of RecA-single-stranded DNA-adenosine 5'-O-(3-thiotriphosphate) complexes with different activities for cleavage of phage phi 80 cI repressor.

The kinetics of cleavage of the phage phi 80 cI repressor by Escherichia coli RecA protein were studied. The rate of cleavage in the presence of single-stranded DNA (ssDNA) and either adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ATP or dATP is very low in the first hour at 37 degrees C and then increases sharply as incubation continues. The initial rate of cleavage of the repressor is greatly increased by incubating the RecA protein with ssDNA prior to addition of ATP gamma S and the repressor. However, when ATP gamma S is present during preincubation of RecA protein with ssDNA, the stimulatory effect of preincubation is greatly reduced. This difference in the effect of preincubation in two different conditions can be explained by formation of RecA-ssDNA-ATP gamma S complexes with different activities for cleavage of the repressor. The active complex is formed by binding of ATP gamma S to a complex of RecA protein and ssDNA. However, when the RecA protein binds to ATP gamma S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor.