Jonathan Caguiat - Academia.edu (original) (raw)

Papers by Jonathan Caguiat

Journal of Visualized Experiments

Micromachines

Cell-based biosensors harness a cell’s ability to respond to the environment by repurposing its s... more Cell-based biosensors harness a cell’s ability to respond to the environment by repurposing its sensing mechanisms. MerR family proteins are activator/repressor switches that regulate the expression of bacterial metal resistance genes and have been used in metal biosensors. Upon metal binding, a conformational change switches gene expression from off to on. The genomes of the multimetal resistant bacterial strains, Stenotrophomonas maltophilia Oak Ridge strain 02 (S. maltophilia 02) and Enterobacter sp. YSU, were recently sequenced. Sequence analysis and gene cloning identified three mercury resistance operons and three MerR switches in these strains. Transposon mutagenesis and sequence analysis identified Enterobacter sp. YSU zinc and copper resistance operons, which appear to be regulated by the protein switches, ZntR and CueR, respectively. Sequence analysis and reverse transcriptase polymerase chain reaction (RT-PCR) showed that a CueR switch appears to activate a S. maltophilia...

Journal of Biological Chemistry, 1989

Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chlorop... more Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chloroplast energy transducing complex (CF1.CF0) in spinach chloroplasts. This protein purifies by size exclusion chromatography as a 180-kDa aggregate and, thus, is probably composed of a trimer of beta polypeptides. The purified aggregate binds ADP to a high and a low affinity site with dissociation constants of 15 and 202 microM, respectively. Mg2+ is required for ADP to bind to both sites. Manganese binds to the protein in a cooperative manner to at least two sites with high affinity. The beta subunit preparation catalyzes Mg2+-dependent ATP hydrolysis at rates which are comparable to other subunit-deficient CF1 preparations and is increased by treatments known to activate the Mg2+-ATPase activity of CF1. However, Ca2+ is not an effective cofactor for this reaction and treatments which activate the Ca2+-ATPase of CF1 are either ineffective or inhibitory.

Journal of Bacteriology, 1999

Expression of the Tn 21 mercury resistance ( mer ) operon is controlled by a metal-sensing repres... more Expression of the Tn 21 mercury resistance ( mer ) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO ), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by ς 70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100- and 1,000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-...

Journal of Visualized Experiments, 2014

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), wh... more Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host's genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir + Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir + E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Proteome Science, 2009

Background: Enterobacter sp. YSU is resistant to several different heavy metal salts, including s... more Background: Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway. Results: Growth curves using M-9 minimal medium containing 40 mM selenite and 1 mM sulfate showed that Enterobacter sp. YSU grew when L-cysteine was present but died when it was absent. Differential protein expression analysis by two dimensional gel electrophoresis showed that CysK was present in cultures containing selenite and lacking L-cysteine but absent in cultures containing both selenite and L-cysteine. Additional RT-PCR studies demonstrated that transcripts for the sulfate permease genes, cysA, cysT and cysW, were down-regulated in the presence of L-cysteine. Conclusion: L-cysteine appeared to confer selenite resistance upon Enterobacter sp. YSU by decreasing the level of selenite transport into the cell through the non-specific pathway.

Journal of Biological Systems, 1998

A Fourier Transform of Equal Symbols (FTES) was applied as a spectral density analysis method to ... more A Fourier Transform of Equal Symbols (FTES) was applied as a spectral density analysis method to identify DNA bases that repeat at any frequency in selected protein-coding genes. The analysis especially focused on identification of bases responsible for the dominant signal at frequency f=1/3 found in all protein-coding genes. The study included homologous sequences from two gene families and multiple unrelated sequences from single organisms. No signal pattern or spectrum specifically characterized either gene family. However, the patterns of bases comprising the signal at f=1/3 suggested the presence of a genome-specific label for protein-coding genes from the same genome. Data suggest that three factors form the informational basis for the signal structure at f=1/3: (1) codon base positional bias; (2) codon preference; and (3) codon arrangement. Quantitative measure of the contribution of each base to the period-3 signal suggests a basis to distinguish protein-coding genes from di...

Current Microbiology, 2009

The Y-12 plant in Oak Ridge, TN, which manufactured nuclear weapons during World War II and the C... more The Y-12 plant in Oak Ridge, TN, which manufactured nuclear weapons during World War II and the Cold War, contaminated East Fork Poplar Creek with heavy metals. The multimetal resistant bacterial strain, Stenotrophomonas maltophilia Oak Ridge strain O2 (S. maltophilia O2), was isolated from East Fork Poplar Creek. Sequence analysis of 16s rDNA suggested that our working strain of S. maltophilia O2 was a strain of Enterobacter. Phylogenetic tree analysis and biochemical tests confirmed that it belonged to an Enterobacter species. This new strain was named Enterobacter sp. YSU. Using a modified R3A growth medium, R3A-Tris, the Hg(II), Cd(II), Zn(II), Cu(II), Au(III), Cr(VI), Ag(I), As(III), and Se(IV) MICs for a confirmed strain of S. maltophilia O2 were 0.24, 0.33, 5, 5, 0.25, 7, 0.03, 14, and 40 mM, respectively, compared to 0.07, 0.24, 0.8, 3, 0.05, 0.4, 0.08, 14, and 40 mM, respectively, for Enterobacter sp. YSU. Although S. maltophilia O2 was generally more metal resistant than Enterobacter sp. YSU, in comparison to Escherichia coli strain HB101, Enterobacter sp. YSU was resistant to Hg(II), Cd(II), Zn(II), Au(III), Ag(I), As(III), and Se(IV). By studying metal resistances in these two strains, it may be possible to understand what makes one microorganism more metal resistant than another microorganism. This work also provided benchmark MICs that can be used to evaluate the metal resistance properties of other bacterial isolates from East Fork Poplar Creek and other metal contaminated sites.

Journal of Bacteriology, 2004

The repressor-activator MerR that controls transcription of the mercury resistance ( mer ) operon... more The repressor-activator MerR that controls transcription of the mercury resistance ( mer ) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially t...