Medha Bhagwat

Functional heterogeneity of RpoS in stress tolerance of enterohemorrhagic Escherichia coli strains.

ABSTRACT The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher β-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogens survival in food processing environments, as well in the hosts stomach and intestine.

IDENTIFICATION OF RESIDUES OF T4 RNASE H REQUIRED FOR CATALYSIS AND DNA BINDING

Bacteriophage T4 RNase H, which removes the RNA primers that initiate lagging strand fragments, has a 5′- to 3′-exonuclease activity on DNA·DNA and RNA·DNA duplexes and an endonuclease activity on flap or forked DNA structures (Bhagwat, M., Hobbs, L. J., and Nossal, N. J. (1997) J. Biol. Chem. 272, 28523–28530). It is a member of the RAD2 family of prokaryotic and eukaryotic replication and repair nucleases. The crystal structure of T4 RNase H, in the absence of DNA, shows two Mg2+ ions coordinated to the amino acids highly conserved in this family. It also shows a disordered region proposed to be involved in DNA binding (Mueser, T. C., Nossal, N. G., and Hyde, C. C. Cell (1996) 85, 1101–1112). To identify the amino acids essential for catalysis and DNA binding, we have constructed and characterized three kinds of T4 RNase H mutant proteins based on the possible roles of the amino acid residues: mutants of acidic residues coordinated to each of the two Mg2+ ions (Mg2+-1: D19N, D71N, D132N, and D155N; and Mg2+-2: D157N and D200N); mutants of conserved basic residues in or near the disordered region (K87A and R90A); and mutants of residues with hydroxyl side chains involved in the hydrogen bonding network (Y86F and S153A). Our studies show that Mg2+-1 and the residues surrounding it are important for catalysis and that Lys87 is necessary for DNA binding.

The 5′-Exonuclease Activity of Bacteriophage T4 RNase H Is Stimulated by the T4 Gene 32 Single-stranded DNA-binding Protein, but Its Flap Endonuclease Is Inhibited

Bacteriophage T4 RNase H is a 5′- to 3′-nuclease that has exonuclease activity on RNA·DNA and DNA·DNA duplexes and can remove the pentamer RNA primers made by the T4 primase-helicase (Hollingsworth, H. C., and Nossal, N. G. (1991) J. Biol. Chem. 266, 1888–1897; Hobbs, L. J., and Nossal, N. G. (1996)J. Bacteriol. 178, 6772–6777). Here we show that this exonuclease degrades duplex DNA nonprocessively, releasing a single oligonucleotide (nucleotides 1–4) with each interaction with the substrate. Degradation continues nonprocessively until the enzyme stops 8–11 nucleotides from the 3′-end of the substrate. T4 gene 32 single-stranded DNA-binding protein strongly stimulates the exonuclease activity of T4 RNase H, converting it into a processive nuclease that removes multiple short oligonucleotides with a combined length of 10–50 nucleotides each time it binds to the duplex substrate. 32 protein must bind on single-stranded DNA behind T4 RNase H for processive degradation. T4 RNase H also has a flap endonuclease activity that cuts preferentially on either side of the junction between single- and double-stranded DNA in flap and fork DNA structures. In contrast to the exonuclease, the endonuclease is inhibited completely by 32 protein binding to the single strand of the flap substrate. These results suggest an important role for T4 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle.

Comparative analysis of transcriptional regulatory elements of glutamate‐dependent acid‐resistance systems of Shigella flexneri and Escherichia coli O157:H7

The ability to withstand an acid-challenge of pH 2.5 or less by Shigella flexneri is a necessary trait for virulence and is generally believed to be restricted to the stationary-phase of growth. Earlier reports indicated the glutamate-dependent acid-resistance (GDAR) system of S. flexneri is under the regulation of rpoS, the gene encoding alternative sigma factor that is induced in the stationary-growth phase. The present study reports that unlike Escherichia coli O157:H7, S. flexneri cells when grown in minimal medium, require acid-induction in the stationary-growth phase for a functional GDAR. When grown on complex medium at pH 5.5, GDAR of S. flexneri was vigorous compared to the cells grown at pH 7.5. No acid-induction was required for the stationary phase E. coli cells grown on either minimal or complex growth media. Distinct differences in the gadA, gadBC, gadE, and hdeA (but not in rpoS) transcript levels were observed in the stationary-growth phase cells between the two pathogens grown on minimal medium. Additionally, rpoS-independent acid-induction of GDAR in the logarithmic growth phase that has been recently observed in E. coli strains [FEMS Microbiol. Lett. 227 (2003) 39-45] was not detected in the S. flexneri rpoS mutant. Although some differences in the DNA sequence at the upstream regulatory elements of gadBC were noticed, they do not appear to be significant and involvement of additional regulators in S. flexneri is anticipated, which also may explain the observed differences in the GDAR of two enteric pathogens.

Searching NCBI's dbSNP Database

The Single‐Nucleotide Polymorphism database (dbSNP) is a variation database at the National Center for Biotechnology Information (NCBI). It is a public repository of submitted nucleotide variations and is part of NCBIs search and retrieval system Entrez. This unit describes two basic protocols to search dbSNP effectively, one to perform a text‐based search and another to perform a sequence‐based search. The unit also describes one of the result display formats called GeneView to obtain information about all submitted SNPs in a particular gene. Curr. Protoc. Bioinform. 32:1.19.1‐1.19.18.

Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies.

Gene expression studies require high quality messenger RNA (mRNA) in addition to other factors such as efficient primers and labeling reagents. To prevent RNA degradation and to improve the quality of gene array expression data, several commercial reagents have become available. We examined a conventional hot-phenol lysis method and RNA stabilization reagents, and generated comparative gene expression profiles from Escherichia coli cells grown on minimal medium. Our data indicate that certain RNA stabilization reagents induce stress responses and proper caution must be exercised during their use. We observed that the laboratory reagent (phenol/EtOH, 5:95, v/v) worked efficiently in isolating high quality mRNA and reproducibility was such that reliable gene expression profiles were generated. To assist in the analysis of gene expression data, we wrote a number of macros that use the most recent gene annotation and process data in accordance with gene function. Scripts were also written to examine the occurrence of artifacts, based on GC content, length of the individual open reading frame (ORF), its distribution on plus and minus DNA strands, and the distance from the replication origin.

PSI-BLAST Tutorial

PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool) derives a position-specific scoring matrix (PSSM) or profile from the multiple sequence alignment of sequences detected above a given score threshold using protein-protein BLAST. This PSSM is used to further search the database for new matches, and is updated for subsequent iterations with these newly detected sequences. Thus, PSI-BLAST provides a means of detecting distant relationships between proteins. In this chapter, we discuss practical aspects of using PSI-BLAST and provide a tutorial on how to uncover distant relationships between proteins and use them to reach biologically meaningful conclusions.

Methods and tools for comparative genomics of foodborne pathogens.

A comparison of genome sequences and of encoded proteins with the database of existing annotated sequences is a useful approach to understand the information at the genome level. Here we demonstrate the utility of several DNA and protein sequence comparison tools to interpret the information obtained from several genome projects. Comparisons are presented between closely related strains of Escherichia coli commensal isolates, different isolates of O157:H7, and Shigella spp. It is expected that comparative genome analysis will generate a wealth of data to compare pathogenic isolates with varying levels of pathogenicity, which in turn may reveal mechanisms by which the pathogen may adapt to a particular nutrient supply in certain foods. These genome sequence analysis tools will strengthen foodborne pathogen surveillance and subsequent risk assessment to enhance the safety of the food supply.

Using BLAT to Find Sequence Similarity in Closely Related Genomes

The BLAST-Like Alignment Tool (BLAT) is used to find genomic sequences that match a protein or DNA sequence submitted by the user. BLAT is typically used for searching similar sequences within the same or closely related species. It was developed to align millions of expressed sequence tags and mouse whole-genome random reads to the human genome at a higher speed. It is freely available either on the Web or as a downloadable stand-alone program. BLAT search results provide a link for visualization in the University of California, Santa Cruz (UCSC) Genome Browser, where associated biological information may be obtained. Three example protocols are given: using an mRNA sequence to identify the exon-intron locations and associated gene in the genomic sequence of the same species, using a protein sequence to identify the coding regions in a genomic sequence and to search for gene family members in the same species, and using a protein sequence to find homologs in another species.

Maturation of Bacteriophage T4 Lagging Strand Fragments Depends on Interaction of T4 RNase H with T4 32 Protein Rather than the T4 Gene 45 Clamp

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5′-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278–305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2–10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.