Efficacy of Salmonella Bacteriophage S1 Delivered and Released by Alginate Beads in a Chicken Model of Infection.

Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.

A Cut above the Rest: Characterization of the Assembly of a Large Viral Icosahedral Capsid.

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C-terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

Associations between antibiotic resistance and bacteriophage resistance phenotypes in laboratory and clinical strains of Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages have received great attention as an alternative over antibiotics due to the host specificity. Therefore, this study was designed to evaluate the associations between bacteriophage-insensitive (BI) and antibiotic-resistant mutants of Salmonella Typhimurium strains. Bacteriophage-sensitive (BS) Salmonella enterica serovar Typhimurium ATCC 19585 (BSSTWT), ciprofloxacin-induced S. Typhimurium ATCC 19585 (BSSTCIP), S. Typhimurium KCCM 40253 (BSSTLAB), and clinically isolated multidrug-resistant S. Typhimurium CCARM 8009 (BSSTMDR) were used to induce the bacteriophage-insensitive mutants (BISTWT, BISTCIP, BISTLAB, and BISTMDR), which were characterized by measuring mutant frequency lysogenic induction, phage adsorption, antibiotic susceptibility, and differential gene expression. The numbers of BSSTWT, BSSTCIP, and BSSTLAB were reduced by P22 (>3 log), while the least lytic activity was observed for BSSTMDR, suggesting alteration in bacteriophage-binding receptors on the surface of multidrug-resistant strain. BSSTWT treated with P22 showed the large variation in the cell state (CV>40%) and highest mutant frequency (62%), followed by 25% for BSSTCIP. The least similarities between BSSTWT and BISTWT were observed for P22 and PBST-13 (<12%). The relative expression levels of bacteriophage-binding receptor-related genes (btuB, fhuA, fliK, fljB, ompC, ompF, rfaL, and tolC) were decreased in BISTCIP and BISTMDR. These results indicate that the bacteriophage resistance is highly associated with the antibiotic resistance. The findings in this study could pave the way for the application of bacteriophages as an alternative to control antibiotic-resistant bacteria.

Resistance mechanisms adopted by a Salmonella Typhimurium mutant against bacteriophage.

Bacteriophages have key roles in regulating bacterial populations in most habitats. A Salmonella Typhimurium mutant (N18) with impaired sensitivity to phage fmb-p1 was obtained and examined, the adsorption efficiency of fmb-p1 to N18 was reduced to 6%, compared to more than 97% for wild type S. Typhimurium CMCC50115. Reduced adsorption was accompanied by a reduction of 90% in the LPS content compared to wild type. Electron microscopy showed phage scattered around N18 with minimal engagement, while the phage were efficiently adsorbed to the wild type with tails oriented towards the bacterial surface. Evidence suggests fmb-p1 can slightly infect N18 and this does not give rise to an increase of phage titer. RT-qPCR data show that several Salmonella genes involved in lipopolysaccharide synthesis and five virulence related genes were down-regulated upon exposure of N18 to phage fmb-p1. In contrast, phage resistance related genes such as the SOS response, restriction-modification (RM), and Cas1 gene were up-regulated in N18. These data suggest that although inefficient adsorption and entry is the primary mechanism of resistance, transcriptional responses to phage exposure indicate that alternative resistance mechanisms against phage infection are also brought to bear, including digestion of phage nucleic acids and activation of the SOS. These findings may help develop strategies for biocontrol of Salmonella where multi-resistant bacteria are encountered or emerge in applications for food production, bioremediation or wastewater treatment.

The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi-like protein tail fibre.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Šresolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

PURPOSE: The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute - at the same time - to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. MATERIALS AND METHODS: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. RESULTS: Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. CONCLUSION: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition.

The ability of phages to infect specific bacteria has led to their exploitation as bio-tools for bacterial remediation and detection. Many phages recognize bacterial hosts via adhesin tips of their long tail fibers (LTFs). Adhesin sequence plasticity modulates receptor specificity, and thus primarily defines a phage's host range. Here we present the crystal structure of an adhesin (gp38) attached to a trimeric ß-helical tip (gp37) from the Salmonella phage S16 LTF. Gp38 contains rare polyglycine type II helices folded into a packed lattice, herein designated "PGII sandwich." Sequence variability within the domain is limited to surface-exposed helices and distal loops that form putative receptor-binding sites. In silico analyses revealed a prevalence of the adhesin architecture among T-even phages, excluding the archetypal T4 phage. Overall, S16 LTF provides a valuable model for understanding binding mechanisms of phage adhesins, and for engineering of phage adhesins with expandable or modulated host ranges.

Incidence and Genetic Bases of Nitrofurantoin Resistance in Clinical Isolates of Two Successful Multidrug-Resistant Clones of Salmonella enterica Serovar Typhimurium: Pandemic "DT 104" and pUO-StVR2.

In this study, the incidence and genetic bases of nitrofurantoin resistance were established for clinical isolates of two successful clones of Salmonella enterica serovar Typhimurium, the pandemic "DT 104" and the pUO-StVR2 clone. A total of 61 "DT 104" and 40 pUO-StVR2 isolates recovered from clinical samples during 2008-2014 and assigned to different phage types, were tested for nitrofurantoin susceptibility. As previously shown for older isolates, all newly tested pUO-StVR2 isolates were highly resistant to nitrofurantoin (minimal inhibitory concentration [MIC] of 128 µg/ml), while 42.6%, 24.6%, and 32.8% of the "DT 104" isolates were susceptible, showed intermediate resistance or were highly resistant, with MICs of 8, 64, and 128 µg/ml, respectively. The genetic bases of nitrofurantoin resistance were established by PCR amplification and sequencing of the nfsA and nfsB genes encoding oxygen-insensitive nitroreductases. pUO-StVR2 isolates shared identical alterations in both nfsA (IS1 inserted into the coding region) and nfsB (in frame duplication of two codons). "DT 104" isolates with intermediate or high resistance had a missense mutation affecting the start codon of nfsA, while a single resistant isolate carried an additional frameshift mutation affecting nfsB. Complementation studies, performed with wild-type nfsA and nfsB, cloned independently and together into low and high copy-number vectors, confirmed NfsA and NfsB as responsible for nitrofurantoin toxicity. The same alterations persisted along time in isolates of each clone belonging to different phage types. Accordingly, changes leading to nitrofurantoin resistance have probably occurred before phage type diversification.

Inferring bacteriophage infection strategies from genome sequence: analysis of bacteriophage 7-11 and related phages.

BACKGROUND: Analyzing regulation of bacteriophage gene expression historically lead to establishing major paradigms of molecular biology, and may provide important medical applications in the future. Temporal regulation of bacteriophage transcription is commonly analyzed through a labor-intensive combination of biochemical and bioinformatic approaches and macroarray measurements. We here investigate to what extent one can understand gene expression strategies of lytic phages, by directly analyzing their genomes through bioinformatic methods. We address this question on a recently sequenced lytic bacteriophage 7 - 11 that infects bacterium Salmonella enterica. RESULTS: We identify novel promoters for the bacteriophage-encoded σ factor, and test the predictions through homology with another bacteriophage (phiEco32) that has been experimentally characterized in detail. Interestingly, standard approach based on multiple local sequence alignment (MLSA) fails to correctly identify the promoters, but a simpler procedure that is based on pairwise alignment of intergenic regions identifies the desired motifs; we argue that such search strategy is more effective for promoters of bacteriophage-encoded σ factors that are typically well conserved but appear in low copy numbers, which we also verify on two additional bacteriophage genomes. Identifying promoters for bacteriophage encoded σ factors together with a more straightforward identification of promoters for bacterial encoded σ factor, allows clustering the genes in putative early, middle and late class, and consequently predicting the temporal regulation of bacteriophage gene expression, which we demonstrate on phage 7-11. CONCLUSIONS: While MLSA algorithms proved highly useful in computational analysis of transcription regulation, we here established that a simpler procedure is more successful for identifying promoters that are recognized by bacteriophage encoded σ factor/RNA polymerase. We here used this approach for predicting sequence specificity of a novel (bacteriophage encoded) σ factor, and consequently inferring phage 7-11 transcription strategy. Therefore, direct analysis of bacteriophage genome sequences is a plausible first-line approach for efficiently inferring phage transcription strategies, and may provide a wealth of information on transcription initiation by diverse σ factors/RNA polymerases.

A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

Magnetostrictive particle based biosensors for in situ and real-time detection of pathogens in water.

Biosensors for in situ detection of pathogenic bacteria in liquid are developed using magnetostrictive particles (MSP) as the sensor platform. The sensing elements used are phage E2 against Salmonella typhimurium, monoclonal antibody against Listeria monocytogenes, polyclonal antibody against Escherichia coli, and polyclonal antibody against Staphylococcus aureus, respectively. These biosensors were characterized in cultures with different populations ranging from 5 × 10(1) to 5 × 10(8) cfu/mL. It is found that the MSP-based biosensors work well in water and have a rapid response with a response time in minutes, which makes the MSP-based sensors suitable for in situ and real-time detection of pathogenic bacteria in liquid. The experimental results show that all MSP-phage and MSP-antibody biosensors in size of 1.0 mm × 0.3 mm × 15 µm exhibit a detection limit better than 100 cfu/mL. Based on the Hill plot, it is concluded that each bacterial cell is bound onto the sensor surface through about four-to-five sites. When the cultures with low population (<10(6) cfu/mL) are tested, both MSP-phage and MSP-antibody sensors exhibit the similar response. However, the phage-MSP sensors exhibit a higher capability in the capture of target bacterial cell.

Long tail fibres of the novel broad-host-range T-even bacteriophage S16 specifically recognize Salmonella OmpC.

We report isolation and characterization of the novel T4-like Salmonella bacteriophage vB_SenM-S16. S16 features a T-even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full-length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects 'rough' Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.

Tail morphology controls DNA release in two Salmonella phages with one lipopolysaccharide receptor recognition system.

Bacteriophages use specific tail proteins to recognize host cells. It is still not understood to molecular detail how the signal is transmitted over the tail to initiate infection. We have analysed in vitro DNA ejection in long-tailed siphovirus 9NA and short-tailed podovirus P22 upon incubation with Salmonella typhimurium lipopolysaccharide (LPS). We showed for the first time that LPS alone was sufficient to elicit DNA release from a siphovirus in vitro. Crystal structure analysis revealed that both phages use similar tailspike proteins for LPS recognition. Tailspike proteins hydrolyse LPS O antigen to position the phage on the cell surface. Thus we were able to compare in vitro DNA ejection processes from two phages with different morphologies with the same receptor under identical experimental conditions. Siphovirus 9NA ejected its DNA about 30 times faster than podovirus P22. DNA ejection is under control of the conformational opening of the particle and has a similar activation barrier in 9NA and P22. Our data suggest that tail morphology influences the efficiencies of particle opening given an identical initial receptor interaction event.

A conserved acetyl esterase domain targets diverse bacteriophages to the Vi capsular receptor of Salmonella enterica serovar Typhi.

A number of bacteriophages have been identified that target the Vi capsular antigen of Salmonella enterica serovar Typhi. Here we show that these Vi phages represent a remarkably diverse set of phages belonging to three phage families, including Podoviridae and Myoviridae. Genome analysis facilitated the further classification of these phages and highlighted aspects of their independent evolution. Significantly, a conserved protein domain carrying an acetyl esterase was found to be associated with at least one tail fiber gene for all Vi phages, and the presence of this domain was confirmed in representative phage particles by mass spectrometric analysis. Thus, we provide a simple explanation and paradigm of how a diverse group of phages target a single key virulence antigen associated with this important human-restricted pathogen.

Identification of host receptor and receptor-binding module of a newly sequenced T5-like phage EPS7.

The virulent bacteriophage EPS7 active against a number of Salmonella serovar and Escherichia coli strains, isolated from the local sewage in Korea, belongs to the family Siphoviridae. The ESP7 genome constitutes a linear double-stranded DNA of 111 382 bp. DNA sequencing and genomic analysis of EPS7 showed that it belongs to the phage T5 family. We identified the EPS7 genes involved in DNA repair, replication, viral structure and bacterial lysis by comparing the EPS7 genome with that of T5. In contrast, the tail genes encoding for putative host receptor-binding protein and the putative receptor-blocking lipoprotein precursor of EPS7 exhibit high homologies with the corresponding gene products of BF23, another member of the T5-family. BF23 binds to BtuB, a surface receptor in the host and involved in vitamin B12 uptake, but its infection is independent of TonB. By constructing a series of deletion mutants in Salmonella and in E. coli and studying phage infection in the mutant hosts, we showed that BtuB is also the host receptor of the phage EPS7. Whether EPS7 infection depends on TonB needs to be further studied.

Interaction of the Gifsy-1 Xis protein with the Gifsy-1 attP sequence.

The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.

Simple colorimetric microplate test of phage lysis in Salmonella enterica.

A simple microplate method, based on conversion of tetrazolium to formazan, was devised for rapidly assessing Salmonella survival after phage treatment. Results were easily interpretable. Monitoring with a microplate reader was useful, but not required. The method was used in defining phage-Salmonella interactions for selection of phage biocontrol cocktails.

Genetic and molecular analysis of GogB, a phage-encoded type III-secreted substrate in Salmonella enterica serovar typhimurium with autonomous expression from its associated phage.

Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S.enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2-inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process.

Temperature sensitive mutation in the 38 kDa minor structural protein gene of phage MB78 interferes with phage morphogenesis.

Temperature sensitive mutation in the gene for the 38 kDa minor structural protein of the phage MB78, a virulent phage of Salmonella enterica serovar typhimurium, interferes with phage development at restrictive temperature. Electron microscopy of particles produced at non-permissive temperature indicated that the particles formed are tailless. Two types of particles are seen: (i) empty capsids, which are not perfect icosahedral (ii) icosahedral particles filled with DNA. The gene for the 38 kDa protein is located in the SalIG fragment of the phage genome. Nucleotide sequence of the SalIG fragment of MB78 as well as its temperature sensitive mutant has been determined and analysed. Such analysis indicated that in the mutant the codon GCA has been changed to GTA resulting in substitution of alanine at position 75 of the protein by valine (A75V). This makes the protein thermolabile. Our results suggest that normal functioning of this 38 kDa protein is necessary for attachment of tail fibre to the capsid. Or in other words, this 38 kDa protein is involved in phage morphogenesis.