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 .

	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 .

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.

	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 .

	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 .

	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 .

	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 .

	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 .

	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 .

	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 .

	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 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 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 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 .

	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 .

	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 .

	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 .

	ACKNOWLEDGMENTS

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

	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 .

	REFERENCES