Abraham Eisenstark

ROLE OF ESCHERICHIA COLI RPOS AND ASSOCIATED GENES IN DEFENSE AGAINST OXIDATIVE DAMAGE

The first phenotype described for mutations in the Escherichia coli rpoS gene was hypersensitivity to near-ultraviolet radiation and to its oxidative photoproduct, hydrogen peroxide. Initially named nur, this gene is now known to code for a sigma factor, and has acquired new names such as katF and rpoS. The role of its protein product (sigma-38) is to regulate a battery of genes as cells enter and rest in stationary phase. Some of the gene products are involved in protection against oxidants (e.g., catalases) and repair of oxidative damage (e.g., exonuclease III). Sigma-38 may also modulate transcription of certain growth phase genes, including hydroperoxidase I and glutathione reductase. Sigma-38 activity is regulated at transcriptional, translational, and protein stabilization levels. This review describes the complex mechanisms whereby sigma-38 controls various genes, the interaction of sigma-38 with other regulators, and a possible role of sigma-38 in bacterial virulence.

Role of rpoS (katF) in oxyR-independent regulation of hydroperoxidase I in Escherichia coli

We present evidence showing that rpoS (katF) is a regulator of katG gene transcription In an oxyR‐independent manner. Mutation of the rpoS gene in several different Escherichia coli strains caused a significant reduction in catalase HPI activity. In rpoSδoxyR double mutants, the level of HPI was considerably lower compared to the δoxyR parent strain, and was restored when transformed with an rpoS+ plasmid. Overproduction of HPI in oxyR suppressor strains was greatly diminished after inactivation of the rpoS gene and was accompanied by a substantial increase in sensitivity to menadione. Beta‐galactostdase expression from a katG::lacZ promoter was lower in rpoS strains compared to rpoS+ isogenic parents. Several δoxyR strains had detectable levels of katG transcription that was significantly diminished after rpoS gene inactivation.

The present state of phage taxonomy.

Characteristics of over 1,150 phages have been categorized into 17 groups, based upon morphology and nucleic acid content. A scheme is presented for cataloging phage data for computer storage, retriev

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium.

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.

Compartmentalization of catalases in Escherichia coli

Escherichia coli possess three catalase genes: katG codes for protein HPI and katE codes for HPII; katF is also needed for HPII but may be a positive regulatory gene for katE. We have assayed for HPI and HPII in the outer cell membrane, the periplasmic space, the inner membrane, and in the cytoplasm of E. coli. Following synthesis of catalase in the cytoplasm the active katG gene product (HPI) was found in the periplasmic and in the cytoplasmic membrane fractions. HPII remained in the cytoplasm.

DNA Amplification and Rearrangements in Archival Salmonella enterica Serovar Typhimurium LT2 Cultures

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.

Genomic Diversification among Archival Strains of Salmonella enterica Serovar Typhimurium LT7

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at -70 degrees C or in serovar Typhimurium LT2 stocked either at -70 degrees C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at -70 degrees C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.

Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells

BackgroundA collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes. Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy.ResultsExtensive and variable losses and a few gains of catabolic functions were observed by this standardized method. Thus, 95 catabolic reactions were scored in each of three plates in wells containing specific carbon and nitrogen substrates.ConclusionWhile the phenotype microarray did not reveal a distinct pattern of mutation among the archival isolates, the data did confirm that various isolates have used multiple strategies to survive in the archival environment. Data from the MacConkey plates verified the changes in carbohydrate metabolism observed in the Biolog™ system.

Role of RPOS Regulon in Resistance to Oxidative Stress and Near- UV Radiation In ΔOXYR Suppressor Mutants of ESCHERICHIA COLI

Escherichia coli delta oxyR mutants are hyper-sensitive to oxidative agents but this sensitivity is reversed to hyper-resistance in delta oxyR suppressor strains (delta oxyRsup; Greenberg, J.T. and Demple, B. 1988. EMBO J. 7:2611-2618). Also, delta oxyR mutants have increased mutation rates that are also reversed in delta oxyRsup. We now report that the rpoS regulon may have a role in determining hyper-resistance and loss of hyper-mutability of delta oxyRsup. Delta oxyRsup cells were also resistant to near-ultraviolet radiation (near-UV) and survived longer in stationary phase than delta oxyR cells. In delta oxyRsup cells elevated beta-galactosidase expression from a rpoS::lacZ promoter fusion and significant overproduction of RpoS protein was observed. These increases were accompanied by substantial elevation in transcription of rpoS-dependent genes as determined by beta-galactosidase expression from katE::lacZ, dps::lacZ, and xthA::lacZ promoters. Catalase HPI and HPII activities were also increased. When rpoS::Tn10 was transduced into delta oxyRsup, phenotypes switched back to hyper-sensitive, hyper-mutable and reduced catalases I and II. Individual delta oxyR colonies exhibited significant clonal variability in beta-galactosidase expression from rpoS::lacZ promoter. These results provide further evidence of the functional and regulatory overlap between two major anti-oxidant defense systems of bacteria.

Diversity of Phage Types among Archived Cultures of the Demerec Collection of Salmonella enterica serovar Typhimurium Strains

ABSTRACT The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.