Robert Villafane - Academia.edu (original) (raw)

Papers by Robert Villafane

Journal of Bacteriology, 1994

Mutations in the tailspike gene (gene 9) of Salmone1la typhimurium phage P22 have been used to id... more Mutations in the tailspike gene (gene 9) of Salmone1la typhimurium phage P22 have been used to identify amino acid interactions during the folding of a polypeptide chain. Since temperature-sensitive folding (tsf) mutations cause folding defects in the P22 tailspike polypeptide chain, it is likely that mutants derived from these and correcting the original tsf defects (second-site intragenic suppressors) identify interactions during the folding pathway. We report the isolation and identification of second-site revertants to tsf mutants.

Arch Virol, 1994

A structure/function study has been initiated for the E34bacteriophage proteins involved in lysog... more A structure/function study has been initiated for the E34bacteriophage proteins involved in lysogeny in Salmonella newington. Hydroxylamine and nitrosoguanidine mutagenesis of a wild type E34phage was used to generate clear plaque variants. Complementation analysis was used to define four genes involved in the phage lysogenic pathway. A relative mapping order has been established. In addition, a virulent mutant, E34vir82,which defines a repressor binding site has been isolated. edicated to the memory oCProCessor Harrison "

BMC Microbiology

Abstract Background The presence of prophages has been an important variable in genetic exchange ... more Abstract Background The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε34, a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes. Results The sequence shows that ε34 is a mosaically related member of the P22 branch of the lambdoid phages. Its sequence is compared with the known P22-like phages and several related but previously unanalyzed prophage sequences in reported bacterial genome sequences. Conclusion These comparisons indicate that there has been little if any genetic exchange within the procapsid assembly gene cluster with P22-like E. coli/Shigella phages that are have orthologous but divergent genes in this region. Presumably this observation reflects the fact that virion assembly proteins interact intimatel...

The Journal of biological chemistry, Jan 25, 1991

Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike p... more Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike protein have recently been isolated (Fane, B. and King, J. (1991) Genetics 127, 263-277). The starting folding defects were in missense polypeptide chains generated by host amino acid insertions at different amber mutant sites. Fragments of genes carrying the amber mutations with and without their independently isolated suppressor mutations were cloned and sequenced. The parental nonsense mutations were located at Q45, K122, E156, W202, W207, Y232, and W365. Their conformational suppressors were single amino acid substitutions at a limited set of sites, V84 greater than A, V331 greater than A, and A334 greater than V. The V331 greater than A or A334 greater than V suppressors were independently recovered starting with different mutant sites suggesting that they acted by some global or general mechanism. When the V331 greater than A and A334 greater than V mutations were crossed into well-...

1982. Cretaceous palacopositions of the Falkland Plateau relative to southern Africa using Mesozo... more 1982. Cretaceous palacopositions of the Falkland Plateau relative to southern Africa using Mesozoic seafloor spreading anomalies. Geophys. Jl R. astr. Soc., 71(3):567-579. Anomalies M0-M 10 in the Natal Valley confirm that seafloor spreading began simultaneously north and south of the Falkland Agulhas Fracture Zone. Other results date the change in early pole rotation at 105 Myr, final separation of the Falkland Plateau from Africa at 98.3 Myr, and the formation of the oceanic northern part of the Agulhas Plateau at 97.3-90.7 Myr. CSIR,

Protein Design and the Development of New Therapeutics and Vaccines, 1990

Open Journal of Biophysics, 2013

The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae an... more The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant sequence identity to the HMG-box family of proteins. A multiple sequence alignment of several α-domains and various structurally determined HMG-box domains have revealed that both domains possess very similar structural and functional residues. We found that the basic amino acids of the N-terminal loop, the intercalating hydrophobic residues of the first helix, and the hydrophobic residues required for interactions within the core of the protein are remarkably conserved in α-domains and HMG-box proteins. Our generated molecular models suggest that the first and third helix will be shorter and that the HMG-box core is not an isolated domain. The region beyond the conserved HMG-box motif contains an extended helical region for about 20 -30 amino acids. Structural models generated by comparative modeling and ab initio modeling reveal that this region will add two or more additional α-helices and will make significant contacts to helix III, II and I of the HMG-box core. We were able to illustrate how the extended α-domain would bind to DNA by merging of the α-domain and the LEF-1/DNA complex. The models we are reporting will be helpful in understanding how MATα1 binds to DNA with its partner MCM1 and activates transcription of α-specific genes. These models will also aid in future biophysical studies of MATα1 including the crystallization and structure determination. Transcription UAS's of α-specific and a-specific genes contain sequences that bind the MATα1/MCM1 and the MATα2/

Virology, 2007

Macrophages are recognized cellular compartments involved in HIV infection; however, the extent t... more Macrophages are recognized cellular compartments involved in HIV infection; however, the extent to which precursor monocytes are infected in vivo and its significance remains poorly understood. Our aim was to analyze the contribution of monocytes to HIV infection in vivo. PCR assays did not detect HIV-1 proviral DNA in monocytes of HAART-suppressed patients. Monocyte-derived macrophages from individuals under suppressive HAART did not show evidence of harboring HIV, thereby, minimizing the possibility of infection by the integration of sequestered virus after differentiation. These results suggest that the infection of permissive monocytes is directly related to the success of HAART (p<0.001). HIV-1 env was characterized from patients under sub-optimal HAART and hence, with infected monocytes. Sequence analyses showed a consistent relationship between monocytes and plasma virus. Altogether, we found that in suppressive HAART, neither monocytes nor Monocyte-derived macrophages-harbored HIV.

American Journal of Microbiological Research, 2013

The P22 tailspike protein is an intensely studied protein whose structure and sequence has been d... more The P22 tailspike protein is an intensely studied protein whose structure and sequence has been described. However, a study, describing important protein interactions related to its function at the N-terminal domain, has been lacking. The P22 tailspike protein (TSP) consists of three identical polypeptide chains of 666aa. The first 108 of the 666aa in the P22 TSP form a trimeric N-terminal domain (NTD). Each of the three chains of the trimeric NTD contributes to the formation of a dome-like structure. Our studies suggest that a short stretch of amino acids located within the first fifteen amino acids of the P22 TSP NTD is critical for the stability of the dome structure formed by the first 108aa of the P22 TSP NTD. The first 23aa are located within this dome-like structure and have been dubbed the "stem" of the NTD. Although amino acid residues in the first 15aa (lower stem) are critical, deletion analysis and in vitro assembly studies implicate the rest of the stem in additional stabilizing interactions. Our studies implicate a common protein-protein interaction motif made up of interchain hydrophobic contacts between adjacent chains

Advances in Microbiology, 2014

The P22 phage system is an intensely studied model system. Studies have ranged from biochemical a... more The P22 phage system is an intensely studied model system. Studies have ranged from biochemical analysis of basic life processes to the use of this phage for phage therapy. The phage tailspike protein (TSP) has itself been the subject of intensive studies over the past fifty years. The P22 TSP is essential for initiation of the infection process and instrumental as the last protein assembled onto the phage particle structure to complete its assembly. It has also been the subject for many structural studies including cryoelectron microscopic analysis and photophysical studies. It has been a model for in vivo and in vitro protein folding including analysis using P22 TSP temperaturesensitive for folding mutations (tsf). Recently the structure and function of the N-terminal domain (NTD), including some aspects of the structural stability of the P22 TSP NTD (aa1-aa108), are being genetically dissected. This report strongly supports the notion that two amino acids, not localized to the internal NTD dome stem, are important in the structural stability of the P22 TSP NTD.

Methods in Molecular Biology, 2009

Recent studies have established that the most abundant life form, that of phages, has had major i... more Recent studies have established that the most abundant life form, that of phages, has had major influence on the biosphere, bacterial evolution, bacterial genome, and lateral gene transmission. Importantly the phages have served and continue to serve as valuable model systems. Such studies have led to a renewed interest and activity in the study of phages and their genomes. In order to determine the details of the involvement of phages in these important processes and activities, it is critical to assign specific functions to the phage gene products. The initial functional and gene assignments can be made by general mutagenesis of the phage genomes and of these specific gene products. A very informative mutagenic protocol that has found renewed interest is that using hydroxylamine. This mutagenic protocol has been used to obtain gene mutations involved in the lysogenic cycle of the Salmonella enterica serovar Anatum var. 15+ phage ε 34 (hereafter phage ε 34 ) and to isolate conditional lethal mutants of phage ε 34 . A similar protocol using plasmid is also described. A plate complementation method is presented to determine quickly the number of genes which are present in the population of mutations isolated from hydroxylamine mutagenesis.

Virus Genes, 2000

A distinguishing feature of many microorganisms, belonging to the Gram negative group of bacteria... more A distinguishing feature of many microorganisms, belonging to the Gram negative group of bacteria, is the presence of the lipopolysaccharide on their cell surface. Salmonella is a prominent member of this group of bacteria. Many Salmonella phages use the LPS as the initial receptor in the infection process and they can distinguish subtle changes in the LPS molecules. The phage protein that is responsible for recognition of these cells is the tail or tailspike protein (TSP). Those TSPs, which use LPS as a receptor, are prokaryotic LPS-binding proteins. As an initial step in using phage TSPs as model systems for a detailed molecular genetic analysis of protein-LPS interactions, a comparison of two phages and their TSPs from two different Salmonella bacterial viruses (phages), Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15+ phage e 34 , is being carried out. This present study shows significant viral protein homology between many viral structural proteins from these two phages including their TSPs. Significantly this report suggests a general structural motif for part of the TSP of phages and suggests that a more detailed comparative analysis of these TSPs is warranted.

Journal of Molecular Biology, 1990

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalto... more The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene I protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein.

Journal of Molecular Biology, 1988

Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the f... more Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. We report here the location and amino acid substitutions for 24 independent tsf mutants. The distribution of these and previously identified mutations is distinctly non-random; all of the 32 unambiguous sites of tsf mutations are located in the central 350 residues of the 666 residue tailspike polypeptide chain. No ts mutation has been found among the N-terminal 140 amino acids, and none among the C-terminal 170 amino acids. Since the physiological defect in these mutants is the destabilization of an early intermediate in the folding pathway, the localization of the mutants suggests that the central region of the chain is critical for formation or stabilization of this early intermediate. The majority of amino acids that served as sites for the tsf mutations were hydrophilic residues. Sixty percent of the replacements of these residues represented charge changes. This probably reflects the selection for mutant sites at the mature protein surface where the substitutions can be best tolerated without interfering with function. None of the sites of tsf mutations were at aromatic residues, and only one proline site was found. Substitutions at these residues may cause lethal folding defects which are not recovered as tsf mutants. The local sequences at tsf sites resemble those reported for turns. Structural studies identify beta-sheet as the dominant secondary structure. These mutations may disrupt the formation of conformational features of beta-sheets which are repeated, such as turns, associations between pairs of strands, or sheet/sheet packing interactions. Such a model accounts for the occurrence of tsf mutations with similar defective phenotypes at multiple positions along the chain.

Gene, 2007

To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, ... more To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, a molecular genetic approach has been employed, using phage as a model system. The phage ε 34 is a Salmonella phage whose tailspike protein (TSP) uses the host LPS as its initial host cell receptor. Previous studies indicated that there was a similarity between the well-studied tail protein of Salmonella phage P22 and the ε 34 . This study reports the identification of the gene for the ε 34 TSP as well as its initial characterization. In addition, some aspects of the structure of the ε 34 TSP have been deduced.

BMC Microbiology, 2008

The presence of prophages has been an important variable in genetic exchange and divergence in mo... more The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε 34 , a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes.

Archives of Virology, 2005

To study the interaction between lipopolysaccharide and protein, a comparative approach was emplo... more To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding regions in some tail proteins) may play a critical element role in the classification of Salmonella viruses. * Currently there has been a resurgence of work on phages . This resurgence is partly fueled by data supporting their importance in disease, potential therapy, horizontal gene transfer and as model systems . A comparison of functionally related proteins has often led to the identification of the functional roles of particular amino acids in polypeptide chains. To better understand how some proteins bind and hydrolyze lipopolysaccharides (LPS), as modeled by the bacterial virus (phage) P22 tailspike protein (TSP) and its

Archives of Virology, 1994

A structure/function study has been initiated for the a34 bacteriophage proteins involved in lyso... more A structure/function study has been initiated for the a34 bacteriophage proteins involved in lysogeny in Salmonella newington. Hydroxylamine and nitrosoguanidine mutagenesis of a wild type a34 phage was used to generate clear plaque variants. Complementation analysis was used to define four genes involved in the phage lysogenic pathway. A relative mapping order has been established. In addition, a virulent mutant, e34vir82, which defines a repressor binding site has been isolated.

Journal of Bacteriology, 1994

Mutations in the tailspike gene (gene 9) of Salmone1la typhimurium phage P22 have been used to id... more Mutations in the tailspike gene (gene 9) of Salmone1la typhimurium phage P22 have been used to identify amino acid interactions during the folding of a polypeptide chain. Since temperature-sensitive folding (tsf) mutations cause folding defects in the P22 tailspike polypeptide chain, it is likely that mutants derived from these and correcting the original tsf defects (second-site intragenic suppressors) identify interactions during the folding pathway. We report the isolation and identification of second-site revertants to tsf mutants.

Arch Virol, 1994

A structure/function study has been initiated for the E34bacteriophage proteins involved in lysog... more A structure/function study has been initiated for the E34bacteriophage proteins involved in lysogeny in Salmonella newington. Hydroxylamine and nitrosoguanidine mutagenesis of a wild type E34phage was used to generate clear plaque variants. Complementation analysis was used to define four genes involved in the phage lysogenic pathway. A relative mapping order has been established. In addition, a virulent mutant, E34vir82,which defines a repressor binding site has been isolated. edicated to the memory oCProCessor Harrison "

BMC Microbiology

Abstract Background The presence of prophages has been an important variable in genetic exchange ... more Abstract Background The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε34, a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes. Results The sequence shows that ε34 is a mosaically related member of the P22 branch of the lambdoid phages. Its sequence is compared with the known P22-like phages and several related but previously unanalyzed prophage sequences in reported bacterial genome sequences. Conclusion These comparisons indicate that there has been little if any genetic exchange within the procapsid assembly gene cluster with P22-like E. coli/Shigella phages that are have orthologous but divergent genes in this region. Presumably this observation reflects the fact that virion assembly proteins interact intimatel...

The Journal of biological chemistry, Jan 25, 1991

Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike p... more Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike protein have recently been isolated (Fane, B. and King, J. (1991) Genetics 127, 263-277). The starting folding defects were in missense polypeptide chains generated by host amino acid insertions at different amber mutant sites. Fragments of genes carrying the amber mutations with and without their independently isolated suppressor mutations were cloned and sequenced. The parental nonsense mutations were located at Q45, K122, E156, W202, W207, Y232, and W365. Their conformational suppressors were single amino acid substitutions at a limited set of sites, V84 greater than A, V331 greater than A, and A334 greater than V. The V331 greater than A or A334 greater than V suppressors were independently recovered starting with different mutant sites suggesting that they acted by some global or general mechanism. When the V331 greater than A and A334 greater than V mutations were crossed into well-...

1982. Cretaceous palacopositions of the Falkland Plateau relative to southern Africa using Mesozo... more 1982. Cretaceous palacopositions of the Falkland Plateau relative to southern Africa using Mesozoic seafloor spreading anomalies. Geophys. Jl R. astr. Soc., 71(3):567-579. Anomalies M0-M 10 in the Natal Valley confirm that seafloor spreading began simultaneously north and south of the Falkland Agulhas Fracture Zone. Other results date the change in early pole rotation at 105 Myr, final separation of the Falkland Plateau from Africa at 98.3 Myr, and the formation of the oceanic northern part of the Agulhas Plateau at 97.3-90.7 Myr. CSIR,

Protein Design and the Development of New Therapeutics and Vaccines, 1990

Open Journal of Biophysics, 2013

The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae an... more The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant sequence identity to the HMG-box family of proteins. A multiple sequence alignment of several α-domains and various structurally determined HMG-box domains have revealed that both domains possess very similar structural and functional residues. We found that the basic amino acids of the N-terminal loop, the intercalating hydrophobic residues of the first helix, and the hydrophobic residues required for interactions within the core of the protein are remarkably conserved in α-domains and HMG-box proteins. Our generated molecular models suggest that the first and third helix will be shorter and that the HMG-box core is not an isolated domain. The region beyond the conserved HMG-box motif contains an extended helical region for about 20 -30 amino acids. Structural models generated by comparative modeling and ab initio modeling reveal that this region will add two or more additional α-helices and will make significant contacts to helix III, II and I of the HMG-box core. We were able to illustrate how the extended α-domain would bind to DNA by merging of the α-domain and the LEF-1/DNA complex. The models we are reporting will be helpful in understanding how MATα1 binds to DNA with its partner MCM1 and activates transcription of α-specific genes. These models will also aid in future biophysical studies of MATα1 including the crystallization and structure determination. Transcription UAS's of α-specific and a-specific genes contain sequences that bind the MATα1/MCM1 and the MATα2/

Virology, 2007

Macrophages are recognized cellular compartments involved in HIV infection; however, the extent t... more Macrophages are recognized cellular compartments involved in HIV infection; however, the extent to which precursor monocytes are infected in vivo and its significance remains poorly understood. Our aim was to analyze the contribution of monocytes to HIV infection in vivo. PCR assays did not detect HIV-1 proviral DNA in monocytes of HAART-suppressed patients. Monocyte-derived macrophages from individuals under suppressive HAART did not show evidence of harboring HIV, thereby, minimizing the possibility of infection by the integration of sequestered virus after differentiation. These results suggest that the infection of permissive monocytes is directly related to the success of HAART (p<0.001). HIV-1 env was characterized from patients under sub-optimal HAART and hence, with infected monocytes. Sequence analyses showed a consistent relationship between monocytes and plasma virus. Altogether, we found that in suppressive HAART, neither monocytes nor Monocyte-derived macrophages-harbored HIV.

American Journal of Microbiological Research, 2013

The P22 tailspike protein is an intensely studied protein whose structure and sequence has been d... more The P22 tailspike protein is an intensely studied protein whose structure and sequence has been described. However, a study, describing important protein interactions related to its function at the N-terminal domain, has been lacking. The P22 tailspike protein (TSP) consists of three identical polypeptide chains of 666aa. The first 108 of the 666aa in the P22 TSP form a trimeric N-terminal domain (NTD). Each of the three chains of the trimeric NTD contributes to the formation of a dome-like structure. Our studies suggest that a short stretch of amino acids located within the first fifteen amino acids of the P22 TSP NTD is critical for the stability of the dome structure formed by the first 108aa of the P22 TSP NTD. The first 23aa are located within this dome-like structure and have been dubbed the "stem" of the NTD. Although amino acid residues in the first 15aa (lower stem) are critical, deletion analysis and in vitro assembly studies implicate the rest of the stem in additional stabilizing interactions. Our studies implicate a common protein-protein interaction motif made up of interchain hydrophobic contacts between adjacent chains

Advances in Microbiology, 2014

The P22 phage system is an intensely studied model system. Studies have ranged from biochemical a... more The P22 phage system is an intensely studied model system. Studies have ranged from biochemical analysis of basic life processes to the use of this phage for phage therapy. The phage tailspike protein (TSP) has itself been the subject of intensive studies over the past fifty years. The P22 TSP is essential for initiation of the infection process and instrumental as the last protein assembled onto the phage particle structure to complete its assembly. It has also been the subject for many structural studies including cryoelectron microscopic analysis and photophysical studies. It has been a model for in vivo and in vitro protein folding including analysis using P22 TSP temperaturesensitive for folding mutations (tsf). Recently the structure and function of the N-terminal domain (NTD), including some aspects of the structural stability of the P22 TSP NTD (aa1-aa108), are being genetically dissected. This report strongly supports the notion that two amino acids, not localized to the internal NTD dome stem, are important in the structural stability of the P22 TSP NTD.

Methods in Molecular Biology, 2009

Recent studies have established that the most abundant life form, that of phages, has had major i... more Recent studies have established that the most abundant life form, that of phages, has had major influence on the biosphere, bacterial evolution, bacterial genome, and lateral gene transmission. Importantly the phages have served and continue to serve as valuable model systems. Such studies have led to a renewed interest and activity in the study of phages and their genomes. In order to determine the details of the involvement of phages in these important processes and activities, it is critical to assign specific functions to the phage gene products. The initial functional and gene assignments can be made by general mutagenesis of the phage genomes and of these specific gene products. A very informative mutagenic protocol that has found renewed interest is that using hydroxylamine. This mutagenic protocol has been used to obtain gene mutations involved in the lysogenic cycle of the Salmonella enterica serovar Anatum var. 15+ phage ε 34 (hereafter phage ε 34 ) and to isolate conditional lethal mutants of phage ε 34 . A similar protocol using plasmid is also described. A plate complementation method is presented to determine quickly the number of genes which are present in the population of mutations isolated from hydroxylamine mutagenesis.

Virus Genes, 2000

A distinguishing feature of many microorganisms, belonging to the Gram negative group of bacteria... more A distinguishing feature of many microorganisms, belonging to the Gram negative group of bacteria, is the presence of the lipopolysaccharide on their cell surface. Salmonella is a prominent member of this group of bacteria. Many Salmonella phages use the LPS as the initial receptor in the infection process and they can distinguish subtle changes in the LPS molecules. The phage protein that is responsible for recognition of these cells is the tail or tailspike protein (TSP). Those TSPs, which use LPS as a receptor, are prokaryotic LPS-binding proteins. As an initial step in using phage TSPs as model systems for a detailed molecular genetic analysis of protein-LPS interactions, a comparison of two phages and their TSPs from two different Salmonella bacterial viruses (phages), Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15+ phage e 34 , is being carried out. This present study shows significant viral protein homology between many viral structural proteins from these two phages including their TSPs. Significantly this report suggests a general structural motif for part of the TSP of phages and suggests that a more detailed comparative analysis of these TSPs is warranted.

Journal of Molecular Biology, 1990

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalto... more The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene I protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein.

Journal of Molecular Biology, 1988

Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the f... more Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. We report here the location and amino acid substitutions for 24 independent tsf mutants. The distribution of these and previously identified mutations is distinctly non-random; all of the 32 unambiguous sites of tsf mutations are located in the central 350 residues of the 666 residue tailspike polypeptide chain. No ts mutation has been found among the N-terminal 140 amino acids, and none among the C-terminal 170 amino acids. Since the physiological defect in these mutants is the destabilization of an early intermediate in the folding pathway, the localization of the mutants suggests that the central region of the chain is critical for formation or stabilization of this early intermediate. The majority of amino acids that served as sites for the tsf mutations were hydrophilic residues. Sixty percent of the replacements of these residues represented charge changes. This probably reflects the selection for mutant sites at the mature protein surface where the substitutions can be best tolerated without interfering with function. None of the sites of tsf mutations were at aromatic residues, and only one proline site was found. Substitutions at these residues may cause lethal folding defects which are not recovered as tsf mutants. The local sequences at tsf sites resemble those reported for turns. Structural studies identify beta-sheet as the dominant secondary structure. These mutations may disrupt the formation of conformational features of beta-sheets which are repeated, such as turns, associations between pairs of strands, or sheet/sheet packing interactions. Such a model accounts for the occurrence of tsf mutations with similar defective phenotypes at multiple positions along the chain.

Gene, 2007

To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, ... more To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, a molecular genetic approach has been employed, using phage as a model system. The phage ε 34 is a Salmonella phage whose tailspike protein (TSP) uses the host LPS as its initial host cell receptor. Previous studies indicated that there was a similarity between the well-studied tail protein of Salmonella phage P22 and the ε 34 . This study reports the identification of the gene for the ε 34 TSP as well as its initial characterization. In addition, some aspects of the structure of the ε 34 TSP have been deduced.

BMC Microbiology, 2008

The presence of prophages has been an important variable in genetic exchange and divergence in mo... more The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε 34 , a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes.

Archives of Virology, 2005

To study the interaction between lipopolysaccharide and protein, a comparative approach was emplo... more To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding regions in some tail proteins) may play a critical element role in the classification of Salmonella viruses. * Currently there has been a resurgence of work on phages . This resurgence is partly fueled by data supporting their importance in disease, potential therapy, horizontal gene transfer and as model systems . A comparison of functionally related proteins has often led to the identification of the functional roles of particular amino acids in polypeptide chains. To better understand how some proteins bind and hydrolyze lipopolysaccharides (LPS), as modeled by the bacterial virus (phage) P22 tailspike protein (TSP) and its

Archives of Virology, 1994

A structure/function study has been initiated for the a34 bacteriophage proteins involved in lyso... more A structure/function study has been initiated for the a34 bacteriophage proteins involved in lysogeny in Salmonella newington. Hydroxylamine and nitrosoguanidine mutagenesis of a wild type a34 phage was used to generate clear plaque variants. Complementation analysis was used to define four genes involved in the phage lysogenic pathway. A relative mapping order has been established. In addition, a virulent mutant, e34vir82, which defines a repressor binding site has been isolated.