This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Young, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Young, R.

 Previous Article  |  Next Article 

Journal of Bacteriology, May 2002, p. 2572-2575, Vol. 184, No. 10
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.10.2572-2575.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Genetics of Bacteria and Phages, 2001

Ry Young*

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128


arrow
INTRODUCTION
 
The Molecular Genetics of Bacteria and Phages annual meeting was held at the University of Wisconsin—Madison (31 July to 5 August). For more than 50 years this meeting has covered a broad spectrum of topics pertaining to genetics, physiology, cell biology, and development of bacteria and bacteriophages. In the early years of this meeting, from the 1950s through the early 1970s, participants described and debated many of the seminal discoveries that led to the basic principles of molecular genetics. The meeting, with its well-deserved reputation for informality, engagement, and rigor, features short research talks by graduate students, postdocs, and faculty, often serving as the forum for the national debut of many of today's established investigators in the molecular biology of prokaryotes.


arrow
GOLDEN ANNIVERSARY FOR P1, P2, AND LAMBDA
 
This year's meeting was especially notable for marking the 50th anniversary of the published discoveries of the classic bacteriophages lambda, P1, and P2 (1, 2). To the delight and edification of a mostly much younger audience, Giuseppe Bertani (CalTech) reflected on the circumstances that led him to the discovery of P1 and P2 as prophages of the "Lisbon strain" of Escherichia coli, to his decision to work on the large plaque-forming P2 rather than on the tiny-plaque-forming P1 (Fig. 1A), and to the formulation of the original LB (Luria-Bertani) medium. Bertani and fellow conferees Waclaw Szybalski (lambda) (University of Wisconsin Medical School) and Abe Eisenstark (P22 and Salmonella) (University of Missouri) represented more than 150 years of accumulated expertise and productivity in the molecular genetics of phages and bacteria (Fig. 1B), and judging from the vigor of their participation at the meeting, they are still going strong.



View larger version (99K):
[in this window]
[in a new window]
  		FIG. 1. (A) First published image of plaques from P1 (left) and P2 (right) (adapted from reference 1 with permission of the publisher). (B) More than 150 years of experience in phage and bacterial genetics, and still going strong. Left, Abe Eisenstark; center, Giuseppe Bertani; right, Waclaw Szybalski.


arrow
STERNBERG AWARD GOES TO LARA-TEJERO
 
The golden anniversary of P1 was a fitting setting for the presentation of the annual Sternberg Award. This award honors the life of Nat Sternberg, who developed P1 into a sophisticated molecular system that has dramatically influenced both basic molecular biology and biotechnology. The Sternberg Award is given to the author of the doctoral dissertation judged most representative of Nat's widely recognized standards for innovation and insight. Maria Lara-Tejero is the recipient of the 2001 Nat Sternberg Award. Lara-Tejero, who conducted her thesis research in the laboratory of Jorge Galan (Yale), studied Campylobacter jejuni cytolethal distending toxin (CDT). Lara-Tejero and colleagues showed that CDT induces fatal cell cycle arrest by acting as a type I endonuclease that cleaves the chromosome of intoxicated cells. CDT is composed of three subunits: CdtA, CdtB, and CdtC. CdtB alone, but not CdtA and CdtC, is capable of inducing chromatin cleavage when injected into tissue culture cells. On the other hand, only the holotoxin (CdtABC) can cause chromatin cleavage when incubated with tissue culture cells, suggesting that CdtA and CdtC act to deliver CdtB into the cytosol of eukaryotic cells.

This remarkable and unexpected mechanism for cytotoxicity set the tone for the meeting. A few of the other highlights are described below. There were more than 80 talks in the 10 platform sessions, and many more posters in addition, making it impossible to give a useful summary in the space available here. Rather, the presentations mentioned below were chosen to be merely representative of the depth and breadth of interesting science discussed at the 2001 meeting. (Where necessary, the speaker is identified by the name of the sponsoring principal investigator and institution in parentheses.)


arrow
HOST-PATHOGEN INTERACTIONS
 
In the opening session, S. Mazmanian (O. Schneewind; University of Chicago) reported the identification of a second sortase (SrtB) in Staphylococcus aureus. Previous work had identified sortase (SrtA), an enzyme that anchors surface proteins to the cell wall of gram-positive bacteria and cleaves sorting signals at an LPXTG motif. SrtB functions analogously for cell wall proteins with a different motif, NPQTN. A srtB mutant is defective in the persistence of animal infections. srtB is part of an iron-regulated locus called iron-responsive surface determinants (isd), which also contains genes for a ferrichrome transporter and surface proteins with NPQTN and LPXTG motifs and appears to be involved in a novel mechanism of iron acquisition important for pathogenesis. L. Cheng (O. Schneewind) reported on Yersinia enterocolitica export of Yop proteins via a type III secretion pathway active in low-calcium medium. Mutations in yopN (lcrE), yscB, sycN, or tyeA make the cells "calcium blind." A model was proposed in which YopN transport serves as a regulatory mechanism for the activity of the type III pathway. According to this model, the binding of a YscB/SycN complex or TyeA to YopN facilitates or inhibits the initiation of YopN into the type III pathway, respectively. In contrast, TyeA binding to YopN in the bacterial cytoplasm prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.


arrow
CELL SURFACES, SECRETION, AND IMPORT
 
J.-F. Collet (J. Bardwell; University of Michigan) addressed the question of how the periplasmic disulfide bond isomerase DsbC is itself maintained in a reduced state at its characteristic C-X-X-C motif, despite the presence of the protein oxidant DsbB, known to oxidize the cysteine residues in the C-X-X-C in DsbA. Apparently, it is the dimeric state of DsbC that prevents it from being a substrate of DsbB; mutant DsbC unable to dimerize is, like the monomeric DsbA protein, by default oxidized by DsbB.

The functional architecture of biofilms was the subject of a talk by G. O'Toole (Dartmouth Medical School), who described the fluid-filled channels permeating biofilm macrocolonies as necessary for nutrient and waste product diffusion. To keep these channels free of invading bacteria, it is proposed that rhamnolipid surfactants disrupt the unwanted cell-cell and cell-surface interactions.

Natural transformation by some bacteria is an ancient phenomenon heretofore considered to be interesting only at the DNA uptake level, considering the tendency of naturally transformable cells to release DNA upon spontaneous lysis. In recent years, various pilus structures have been implicated in natural transformation, but until now it was generally thought they captured DNA released by lysis of other cells. This story has now taken a surprising turn. H. Hamilton (J. Dillard; University of Wisconsin Medical School) presented evidence that fragments of gonococcal chromosomal DNA are actually secreted into the medium through a type IV secretion system. The export system is encoded by a 60-kb pathogenicity island bearing multiple genes with similarity to the F conjugal transfer system.


arrow
CHAPERONES, HEAT SHOCK, AND PROTEIN DEGRADATION
 
The session on chaperones, heat shock, and protein degradation reflected the current excitement in understanding the intracellular regulatory roles of protein remodeling complexes. B. Burton (T. Baker; MIT) presented work solving a long-standing question: how does ClpX, the regulatory subunit of the ClpXP ring protease, remodel the Mu transposase-DNA complex to allow disassembly? It was shown that ClpX unfolds or partially unfolds a single subunit of the tetramer, which probably triggers a conformational change in the rest of the protein that facilitates disassembly. Another aspect of ClpX function was revealed by J. Flynn (T. Baker; MIT), who is studying the 11-amino-acid SsrA tag, which is attached to incomplete proteins to target them for degradation. It turns out that binding determinants in the SsrA tag are arranged so that ClpX and its ribosome-associated targeting factor, SspB, can bind synergistically. However, binding determinants of SspB and ClpA overlap so that binding is mutually exclusive, suggesting that ClpAP may be shunted off to deal with free protein substrates, leaving ClpXP to interact with nascent proteins in need of degradation. Finally, C. Dartigalongue (S. Raina; University of Geneva) and J. Collier (P. Bouloc; CNRS, Orsay) both reported preliminary characterization of YaeL, a recently detected membrane-embedded Zn2+ metalloprotease. YaeL is a member of the RIP (regulated intramembrane proteolysis) family, conserved from bacteria to humans. The plethora of dramatic phenotypes associated with loss or overexpression of this protease suggests that it plays as important a role in E. coli as the other members of this protease class do in higher organisms.


arrow
DNA REPLICATION AND REPAIR
 
Recently, it has become clear that replication forks can fail, requiring a restart mediated by special pathways, some of which involve homologous recombination. S. Dasgupta (K. Nordstrom; Uppsala University) presented the first direct measurements of the frequency of replication fork arrest in E. coli, using synchronized dnaC(Ts) mutants that cannot restart replication forks at the restrictive temperature. Surprisingly, about 20% of the cells failed to complete replication at the restrictive temperature, indicating that replication fork failure is much more frequent than previously believed.

New features of the process of stationary-phase mutation, in which a subset of bacterial cells in stationary phase are thought to undergo a very high frequency of mutation, were presented. All of the results support a model in which stationary-phase mutations depend on recombination-dependent DNA replication, initiated from double-strand breaks. R. Ponder (S. Rosenberg; Baylor College of Medicine) showed that an engineered double-strand break could stimulate stationary-phase mutation by 2 or 3 orders of magnitude. Stationary-phase mutation was previously shown to occur when a plasmid contained the transfer system of the F plasmid, suggesting that a nick at oriT can sometimes be converted into a double-strand break (when the engineered double-strand break is not provided).

K. Dudas (K. Kreuzer; Duke University Medical School) reported that the initiation of DNA replication in bacteriophage T4 differs between early and late in infection. Early replication depends on R-loop structures that are formed at replication origins, while at late times of infection, replication becomes completely dependent on recombination proteins and is thought to involve the assembly of replication complexes onto D loops. A key role in this switch is played by a late protein, the UvsW helicase, which unwinds origin R loops and activates recombination-dependent replication, perhaps by favoring the formation of D loops.


arrow
POSTTRANSCRIPTIONAL CONTROL
 
J. Hager (U. Jakob; University of Michigan) described a mutational analysis of FtsJ, a heat shock protein recently shown to be a member of the conserved RNA methyltransferase family, leading to a suggested renaming as RrmJ. Defects in FtsJ (RrmJ) lead to ribosomal instability under heat shock stress. Despite its sequence and structural similarity to vaccinia virus VP39, the best-characterized RNA methyltransferase, the 23S rRNA binding site appears to be significantly different in FtsJ (RrmJ). K. Gerdes (University of Southern Denmark) described studies on the physiology associated with the RelE-RelB toxin-antitoxin system. RelE function is stimulated when the Lon protease, activated by amino acid starvation, degrades RelB; surprisingly, RelE activation causes a generalized repression of protein synthesis but no cell death. This raises a new perspective for the biological role of the so-called addiction systems, some of which may now turn out to be global regulators rather than addiction systems purely dedicated to maintenance of episomal determinants.


arrow
CELL DIVISION AND DEVELOPMENT
 
Commitment to sporulation in Bacillus subtilis is subject to several checkpoints to ensure that the DNA is replicated and segregated. One checkpoint, activated by defects in replication initiation, results in induction of a small protein, Sda, that inhibits autophosphorylation of two of the histidine kinases required for activation of the master regulator Spo0A. W. Burkholder (A. Grossman; MIT) reported that expression of sda is also induced by DNA damage and by blocking replication fork elongation. A mechanism for these responses is now apparent, involving DnaA and LexA binding sites upstream of sda. The model is that DnaA, which is negatively regulated by the active replication complex, acts positively at its binding site, followed by a failure in replication initiation or elongation, whereas LexA repression at its site is abolished by damage-activated RecA-mediated proteolysis. Collaborator G. King (University of Connecticut Health Center) reported on nuclear magnetic resonance studies that have revealed a solution structure for Sda. Structure-based mutagenesis of Sda indicates that a patch of conserved surface residues on Sda are required for kinase binding and inhibition.

Addiction modules, with a stable toxin and an unstable antitoxin, were originally discovered on low-copy-number bacterial plasmids and prophages and stabilize plasmids by killing plasmid-free cells. Paradoxically, however, many such modules have been found on the bacterial chromosome. H. Engelberg-Kulka (Hebrew University-Hadassah Medical School) reported that one of these systems, mazEF, previously shown to trigger cell death in response to antibiotics that block translation, is also responsible for thymineless death, an unexplained phenomenon that dates to the early days of bacterial genetics.


arrow
BACTERIOPHAGE DEVELOPMENT AND HOST INTERACTIONS
 
Although the historical perspective of G. Bertani (see above) was a hard act to follow, the speakers in the session on bacteriophage development and host interactions continued the tradition that phage biology can still provide fundamental new perspectives on subjects of general interest. For example, A. Poteete (University of Massachusetts Medical School) and S. Hayes (University of Saskatchewan) both reported on the phage lambda Rap protein, a nuclease that can cut three- and four-stranded DNA junctions that are thought to be intermediates in recombination. Although it is encoded by a gene in the dispensable nin region of the phage genome, Rap plays an important role in the recombination pathway that leads to concatemeric DNA molecules that are the optimum substrates for DNA packaging. Evidence was presented suggesting that if the host RecBCD system is replaced by the phage Red genes, Rap can efficiently replace RuvC. D. Pawloski (G. Koudelka; SUNY Buffalo) reported that the RecA-mediated autocleavage activity of the phage 434 repressor is enhanced by the presence of specific operator DNA and its induced dimerization of the repressor. This result raises the possibility that LexA-like repressors may undergo differential cleavage depending on the specific operator sequence.

R. King (R. Weisberg; NIH) reported studies on the molecular mechanism of transcription antitermination in the lambdoid phage HK022. This phage accomplishes specific antitermination to effect delayed early transcription, as in the classic N-mediated antitermination paradigm of phage lambda; however, it does so without a trans-acting protein factor, depending instead on the cis-acting putL and putR RNA elements. Remarkably, certain mutations in the Zn2+ finger domain in the N-terminal region of the RpoC (ß') subunit of RNA polymerase, although silent in terms of cell growth, abolish antitermination by putL but not by putR. These and other results suggest that the Zn2+ finger domain recognizes the put structures and indicate that this domain may have a general role in transcriptional termination.


arrow
TRANSCRIPTION
 
All the talks in the session on transcription illustrated the rapid pace of advance in our understanding of RNA polymerase at a fundamental mechanistic level, and the richness of the molecular details provided means only a few can be mentioned here. For example, R. Ebright (Rutgers) presented results of FRET measurements between an extensive set of probes in {sigma}70 and reference points in ß and ß'; the results permitted detailed structural modeling of RNA polymerase holoenzyme and the RNA polymerase-promoter-open complex. K. Geszvain (R. Landick; University of Wisconsin) described RNA polymerase mutants that define a key contact in the {sigma}70-core interface of the holoenzyme. A hydrophobic patch on the so-called flap-tip helix of the ß subunit was reported to be necessary for initiation at -35 element-dependent promoters, but not at so-called extended -10 promoters that do not require the -35 element. Also, G. Bar-Nahum (E. Nudler; NYU Medical Center) described in vitro studies that suggest that a subpopulation of transcribing RNA polymerase molecules do not release {sigma}70. This surprising finding raises the prospect of novel regulatory strategies for transcription and will likely stimulate new work on the topic. Another surprising finding described by D. Jin (NIH) was that the bacterial Swi2/Snf homolog, RapA, is capable of stimulating transcription of compacted DNA templates in an ATP-dependent fashion. J. Hernandez (SUNY Buffalo) presented evidence that, in initiating transcription complexes, short hairpins in the nontemplate DNA strand extrude and facilitate promoter escape. G. Koudelka reported that efficiency of repression by bacteriophage 434 repressor is affected by sequences in the -10 region that control the kinetics of transcription initiation. Overall, this session proved that new insights into the mechanism of transcription will continue to go in unexpected directions, despite the detailed knowledge already available on the subject.


arrow
BACTERIAL GENOME: STRUCTURE, RECOMBINATION, AND TRANSPOSITION
 
Genome-scale high-density hybridization experiments assessing the transcription of all chromosomal genes are among the most exciting developments to emerge from genomics research. Two presentations illustrated the versatility of genome-scale array hybridization by addressing questions that lie outside of the typical applications of the technique. C. Rosenow (Affymetrix) presented an analysis of the boundaries of complete transcriptional units in E. coli, and K.Wassarman (University of Wisconsin) described a genomics-based study using microarrays to identify small functional RNAs whose coding regions are conserved among bacteria but that were previously unannotated in the E. coli genome. Both projects used high-density oligonucleotide arrays that contained probes both within and between annotated features of the genome. By examining the hybridization patterns for probes adjacent to expressed genes, Rosenow was able to define the 5' and 3' untranslated regions for many E. coli operons. He has applied this technique to RNA from a variety of growth conditions and defined a set of transcriptional units that can be compared to bioinformatics-based predictions about operon structure and locations of regulatory elements. Similarly, Wassarman reported that the annotated genome overlooked some coding features, including small open reading frames that encode peptides, and functional small RNAs. This work has nearly tripled the number of known small RNAs in E. coli.


arrow
SIGNALING AND GLOBAL CIRCUITS
 
The final session of the meeting primarily focused on the mechanisms used by cells to activate gene expression in response to external and internal stimuli and featured talks that illustrated the fascinating diversity in the mechanisms of signal transduction. For example, work was reported by C. Rosario (R. Bender; University of Michigan) on the LysR ortholog NAC (nitrogen assimilation control) protein, which has both positive and negative regulatory roles. The results suggested that differences in the DNA binding sites for NAC induce conformational changes in the protein that may cause it to form DNA loops in some cases but not others. Probably the most surprising results on two-component regulatory systems were reported by L. Zhou (B. Wanner; Purdue), who concluded that none of the two-component regulatory systems identified in E. coli is essential for growth. This is based on the phenotypic characterization of null mutations constructed in each two-component pair.


arrow
AND ON TO 2002
 
Overall, as in each of more than a half-century of its predecessors, the 2001 meeting demonstrated the amazing diversity of the systems, structures, and mechanisms underlying the living cell, the power of prokaryotic molecular genetics to get at these fundamental principles, and the resourcefulness and insight of the investigators who have chosen this as their field of scientific endeavor.


arrow
ACKNOWLEDGMENTS
 
The organizers (Tania Baker, MIT; Tom Silhavy, Princeton; and Robert Landick, University of Wisconsin) wish to acknowledge the generous support of Epicentre Corporation.


arrow
FOOTNOTES
 
* Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128. Phone: (979) 845-2087. Fax: (979) 862-4718. E-mail: ryland{at}tamu.edu. Back


arrow
REFERENCES
 
    1
  1. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62:293-297.[Free Full Text]
    2. 2
  2. Lederberg, E. M. 1951. Lysogenicity in E. coli K-12. Genetics 36:560.

Journal of Bacteriology, May 2002, p. 2572-2575, Vol. 184, No. 10
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.10.2572-2575.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Young, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Young, R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»