Joseph Cleary - Academia.edu (original) (raw)

Papers by Joseph Cleary

Research paper thumbnail of Low-ash xanthan gum

Research paper thumbnail of Genetically purified gellan gum

Research paper thumbnail of PHB-free gellan gum broth

Research paper thumbnail of Genetically purified gellan gum

Research paper thumbnail of Recombinant DNA plasmid for xanthan gum synthesis

Research paper thumbnail of The Dna Replication Origin Region of the Enterobacteriaceae

The Initiation of Dna Replication, 1981

Research paper thumbnail of Identification, genetic and biochemical analysis of genes involved in synthesis of sugar nucleotide precursors of xanthan gum

Journal of general microbiology, 1993

A genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for x... more A genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for xanthan polysaccharide synthesis (xps) was undertaken. Seven xps DNA regions were isolated after conjugation of chemically induced non-mucoid mutants with a genomic library of X. campestris DNA. No overlapping segments between regions were detected, based on physical mapping, indicating the unlinked character of these regions. Clones complementing several different mutants belonging to the same region contained overlapping segments of X. campestris chromosomal DNA. Complementation and biochemical analysis, and DNA mapping were used to identify and characterize xpsZZZ, ZV and VZ DNA regions. Mutants in these three regions were able to synthesize both lipid intermediates and xanthan gum in vitro when sugar nucleotides were provided as substrates. HPLC analysis of the intracellular sugar nucleotide content showed that the XpsIII group comprises two different classes of mutants : XpsIIIA, def...

Research paper thumbnail of Dna Initiation Determinants of Bacteriophage M13 and of Chimeric Derivatives Carrying Foreign Replication Determinants

The Initiation of Dna Replication, 1981

Research paper thumbnail of Molecular modeling suggests induced fit of Family I carbohydrate-binding modules with a broken-chain cellulose surface

Protein Engineering Design and Selection, 2007

Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, ... more Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, their molecular mode of action on cellulose is not well understood. These enzymes act to detach and hydrolyze cellodextrin chains from crystalline cellulose in a processive manner, and the carbohydrate-binding module (CBM) is thought to play an important role in this process. Understanding the interactions between the CBM and cellulose at the molecular level can assist greatly in formulating selective mutagenesis experiments to confirm the function of the CBM. Computational molecular dynamics was used to investigate the interaction of the CBM from Trichoderma reesei cellobiohydrolase I with a model of the (1,0,0) cellulose surface modified to display a broken chain. Initially, the CBM was located in different positions relative to the reducing end of this break, and during the simulations it appeared to translate freely and randomly across the cellulose surface, which is consistent with its role in processivity. Another important finding is that the reducing end of a cellulose chain appears to induce a conformational change in the CBM. Simulations show that the tyrosine residues on the hydrophobic surface of the CBM, Y5, Y31 and Y32 align with the cellulose chain adjacent to the reducing end and, importantly, that the fourth tyrosine residue in the CBM (Y13) moves from its internal position to form van der Waals interactions with the cellulose surface. As a consequence of this induced change near the surface, the CBM straddles the reducing end of the broken chain. Interestingly, all four aromatic residues are highly conserved in Family I CBM, and thus this recognition mechanism may be universal to this family.

Research paper thumbnail of Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence

Proceedings of the National Academy of Sciences, 1983

The chromosomal replication origin (oriC) of Vibrio harveyi has been isolated on a plasmid and sh... more The chromosomal replication origin (oriC) of Vibrio harveyi has been isolated on a plasmid and shown to function as an origin inEscherichia coli. The nucleotide sequence of the V. harveyi oriC was determined. From a comparison of this sequence with oriC sequences of five enteric bacteria, we derived a consensus sequence of bacterial origins that function in E. coli This consensus sequence identifies 122 positions within oriC where nucleotide substitutions can occur without loss of origin function. These positions are clustered rather than scattered. Four interrelated nine-base-pair repeats and eight of the dam methylation G-A-T-C sites are conserved in the consensus sequence. Very few relative insertion-deletion changes occur, and these are localized to one region of oriC. The genes for three polypeptides linked to the V. harveyi oriC were identified by using in vitro protein synthesis directed by deletion derivative plasmid templates. One of these genes, coding for a 58,000 Mr polypeptide and located 3.0 kilobase pairs from the V. harveyi oriC region, is lethal to E. coli when many copies (approximately 40 per cell) are present (high copy lethal or HCL gene). In addition, nucleotide sequence analysis showed that a different gene, the gid gene to the left of oriC, is highly conserved between E. coli and V. harveyi, whereas the coding region to the right of oriC is much less conserved.

Research paper thumbnail of Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13

Proceedings of the National Academy of Sciences, 1980

The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained... more The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid lasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected pol Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare tranucing phages carrying singstranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmi containing a 270-nucleotide fragment from the gene H-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequebce coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M3-infected polA cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA. The intergenic region (1) of the closely related filamentous phages M13, fd, and fi contains the origins for the replication of both the viral (+) and complementary (-) strands of the phage DNA (2-4). The region is 507 nucleotides in length and is located between genes II and IV on the phage chromosome (6). The central portion of the intergenic region has been shown to contain: (i) the site of action of the gene II protein , the strand-specific endonuclease required for viral strand replication (8), and (ii) the sequence of the RNA primer used to initiate complementary strand DNA synthesis (9).

Research paper thumbnail of Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant

Research paper thumbnail of Interactions of the complete cellobiohydrolase I from Trichodera reesei with microcrystalline cellulose Iβ

Cellulose, 2008

We describe the construction of a model complex of the cellobiohydrolase I (CBH I) cellulase from... more We describe the construction of a model complex of the cellobiohydrolase I (CBH I) cellulase from Trichoderma reesei bound to a cellulose microfibril in an aqueous environment for use in molecular dynamics (MD) simulations. Preliminary characterization from the initial phases of an MD simulation of this complex is also described. The linker sequence between the two globular domains was found to be quite flexible, and the oligosaccharides bound to this linker were found to prefer to be splayed like the spokes in a wheel due to their hydration requirements. The overall conformations of the two globular domains remained stable in the simulations, although both underwent changes in their orientations.

Research paper thumbnail of Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Iβ: the enzyme–substrate complex

Carbohydrate Research, 2009

Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei ce... more Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand cellobiohydrolase function, new simulations have been conducted using CHARMM of cellobiohydrolase I (CBH I) from T. reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.

Research paper thumbnail of Low-ash xanthan gum

Research paper thumbnail of Genetically purified gellan gum

Research paper thumbnail of PHB-free gellan gum broth

Research paper thumbnail of Genetically purified gellan gum

Research paper thumbnail of Recombinant DNA plasmid for xanthan gum synthesis

Research paper thumbnail of The Dna Replication Origin Region of the Enterobacteriaceae

The Initiation of Dna Replication, 1981

Research paper thumbnail of Identification, genetic and biochemical analysis of genes involved in synthesis of sugar nucleotide precursors of xanthan gum

Journal of general microbiology, 1993

A genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for x... more A genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for xanthan polysaccharide synthesis (xps) was undertaken. Seven xps DNA regions were isolated after conjugation of chemically induced non-mucoid mutants with a genomic library of X. campestris DNA. No overlapping segments between regions were detected, based on physical mapping, indicating the unlinked character of these regions. Clones complementing several different mutants belonging to the same region contained overlapping segments of X. campestris chromosomal DNA. Complementation and biochemical analysis, and DNA mapping were used to identify and characterize xpsZZZ, ZV and VZ DNA regions. Mutants in these three regions were able to synthesize both lipid intermediates and xanthan gum in vitro when sugar nucleotides were provided as substrates. HPLC analysis of the intracellular sugar nucleotide content showed that the XpsIII group comprises two different classes of mutants : XpsIIIA, def...

Research paper thumbnail of Dna Initiation Determinants of Bacteriophage M13 and of Chimeric Derivatives Carrying Foreign Replication Determinants

The Initiation of Dna Replication, 1981

Research paper thumbnail of Molecular modeling suggests induced fit of Family I carbohydrate-binding modules with a broken-chain cellulose surface

Protein Engineering Design and Selection, 2007

Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, ... more Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, their molecular mode of action on cellulose is not well understood. These enzymes act to detach and hydrolyze cellodextrin chains from crystalline cellulose in a processive manner, and the carbohydrate-binding module (CBM) is thought to play an important role in this process. Understanding the interactions between the CBM and cellulose at the molecular level can assist greatly in formulating selective mutagenesis experiments to confirm the function of the CBM. Computational molecular dynamics was used to investigate the interaction of the CBM from Trichoderma reesei cellobiohydrolase I with a model of the (1,0,0) cellulose surface modified to display a broken chain. Initially, the CBM was located in different positions relative to the reducing end of this break, and during the simulations it appeared to translate freely and randomly across the cellulose surface, which is consistent with its role in processivity. Another important finding is that the reducing end of a cellulose chain appears to induce a conformational change in the CBM. Simulations show that the tyrosine residues on the hydrophobic surface of the CBM, Y5, Y31 and Y32 align with the cellulose chain adjacent to the reducing end and, importantly, that the fourth tyrosine residue in the CBM (Y13) moves from its internal position to form van der Waals interactions with the cellulose surface. As a consequence of this induced change near the surface, the CBM straddles the reducing end of the broken chain. Interestingly, all four aromatic residues are highly conserved in Family I CBM, and thus this recognition mechanism may be universal to this family.

Research paper thumbnail of Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence

Proceedings of the National Academy of Sciences, 1983

The chromosomal replication origin (oriC) of Vibrio harveyi has been isolated on a plasmid and sh... more The chromosomal replication origin (oriC) of Vibrio harveyi has been isolated on a plasmid and shown to function as an origin inEscherichia coli. The nucleotide sequence of the V. harveyi oriC was determined. From a comparison of this sequence with oriC sequences of five enteric bacteria, we derived a consensus sequence of bacterial origins that function in E. coli This consensus sequence identifies 122 positions within oriC where nucleotide substitutions can occur without loss of origin function. These positions are clustered rather than scattered. Four interrelated nine-base-pair repeats and eight of the dam methylation G-A-T-C sites are conserved in the consensus sequence. Very few relative insertion-deletion changes occur, and these are localized to one region of oriC. The genes for three polypeptides linked to the V. harveyi oriC were identified by using in vitro protein synthesis directed by deletion derivative plasmid templates. One of these genes, coding for a 58,000 Mr polypeptide and located 3.0 kilobase pairs from the V. harveyi oriC region, is lethal to E. coli when many copies (approximately 40 per cell) are present (high copy lethal or HCL gene). In addition, nucleotide sequence analysis showed that a different gene, the gid gene to the left of oriC, is highly conserved between E. coli and V. harveyi, whereas the coding region to the right of oriC is much less conserved.

Research paper thumbnail of Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13

Proceedings of the National Academy of Sciences, 1980

The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained... more The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid lasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected pol Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare tranucing phages carrying singstranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmi containing a 270-nucleotide fragment from the gene H-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequebce coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M3-infected polA cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA. The intergenic region (1) of the closely related filamentous phages M13, fd, and fi contains the origins for the replication of both the viral (+) and complementary (-) strands of the phage DNA (2-4). The region is 507 nucleotides in length and is located between genes II and IV on the phage chromosome (6). The central portion of the intergenic region has been shown to contain: (i) the site of action of the gene II protein , the strand-specific endonuclease required for viral strand replication (8), and (ii) the sequence of the RNA primer used to initiate complementary strand DNA synthesis (9).

Research paper thumbnail of Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant

Research paper thumbnail of Interactions of the complete cellobiohydrolase I from Trichodera reesei with microcrystalline cellulose Iβ

Cellulose, 2008

We describe the construction of a model complex of the cellobiohydrolase I (CBH I) cellulase from... more We describe the construction of a model complex of the cellobiohydrolase I (CBH I) cellulase from Trichoderma reesei bound to a cellulose microfibril in an aqueous environment for use in molecular dynamics (MD) simulations. Preliminary characterization from the initial phases of an MD simulation of this complex is also described. The linker sequence between the two globular domains was found to be quite flexible, and the oligosaccharides bound to this linker were found to prefer to be splayed like the spokes in a wheel due to their hydration requirements. The overall conformations of the two globular domains remained stable in the simulations, although both underwent changes in their orientations.

Research paper thumbnail of Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Iβ: the enzyme–substrate complex

Carbohydrate Research, 2009

Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei ce... more Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand cellobiohydrolase function, new simulations have been conducted using CHARMM of cellobiohydrolase I (CBH I) from T. reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.