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The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.

The phage λ

SummaryThe CI and Cro repressors of bacteriophage λ create a bistable switch between lysogenic and lytic growth. In λ lysogens, CI repressor expressed from thePRMpromoter blocks expression of the lytic promotersPLandPRto allow stable maintenance of the lysogenic state. When lysogens are induced, CI repressor is inactivated and Cro repressor is expressed from the lyticPRpromoter. Cro repressor blocksPRMtranscription and CI repressor synthesis to ensure that the lytic state proceeds. RexA and RexB proteins, like CI, are expressed from thePRMpromoter in λ lysogens; RexB is also expressed from a second promoter,PLIT, embedded inrexA.Here we show that RexA binds CI repressor and assists the transition from lysogenic to lytic growth, using both intact lysogens and defective prophages with reporter genes under control of the lyticPLandPRpromoters. Once lytic growth begins, if the bistable switch does return to the immune state, RexA expression lessens the probability that it will remain there, thus stabilizing the lytic state and activation of the lyticPLandPRpromoters. RexB modulates the effect of RexA and may also help establish phage DNA replication as lytic growth ensues.

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3’→5’ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

Genetic elements in the bacteriophage λ immunity region contribute to stable maintenance and synchronous induction of the integrated E. coli prophage. There is a bistable switch between lysogenic and lytic growth that is orchestrated by the CI and Cro repressors acting on the lytic (P(L) and P(R)) and lysogenic (P(RM)) promoters, referred to as the Genetic Switch. Other less well-characterized elements in the phage immunity region include the P(LIT) promoter and the immunity terminator, T(IMM). The P(LIT) promoter is repressed by the bacterial LexA protein in λ lysogens. LexA repressor, like the λ CI repressor, is inactivated during the SOS response to DNA damage, and this regulation ensures that the P(LIT) promoter and the lytic P(L) and P(R) promoters are synchronously activated. Proper RexA and RexB protein levels are critical for the switch from lysogeny to lytic growth. Mutation of P(LIT) reduces RexB levels relative to RexA, compromising cellular energetics and causing a 10-fold reduction in lytic phage yield. The RexA and RexB proteins interact with themselves and each other in a bacterial two-hybrid system. We also find that the transcription terminator, T(IMM), is a Rho-independent, intrinsic terminator. Inactivation of T(IMM) has minimal effect on λ lysogenization or prophage induction.

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of &lambda, Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known inEscherichia colithat the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wild-type Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphicftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling,ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring.IMPORTANCEAll cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit inEscherichia coli, but its role in regulatingE. colicell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

Transcription and translation are coupled processes in bacteria. A role of transcription elongation cofactor NusG in coupling has been suggested by in vitro structural studies. NMR revealed association of the NusG carboxy-terminal domain with S10 (NusE), implying a direct role for NusG as a bridge linking RNAP and the lead ribosome. Here we present the first in vitro and in vivo evidence of full-length NusG association with mature 70S ribosomes. Binding did not require accessory factors in vitro. Mutating the NusG:S10 binding interface at NusG F165 or NusE M88 and D97 residues weakened NusG:S10 association in vivo and completely abolished it in vitro, supporting the specificity of this interaction. Mutations in the binding interface increased sensitivity to chloramphenicol. This phenotype was suppressed by rpoB*35, an RNAP mutation that reduces replisome-RNAP clashes. We propose that weakened NusG:S10 interaction leads to uncoupling when translation is inhibited, with resulting RNAP backtracking, replication blocks, and formation of lethal DNA double-strand breaks.

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli. Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.

Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli. In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts' threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Furdependent, switch-like activation instead of a graded response to iron deprivation. Israel Science Foundation [514415 to J.S.]; Feinberg Foundation Visiting Faculty Program ( to J.M.-G.); MICINN (Spain) [FIS2012-32349 to J.M.-G.]; Intramural Research Program of the National Institutes of Health (to D.L.C.); National Cancer Institute (to D.L.C.); Center for Cancer Research (to D.L.C.); Siegfried and Irma Ullman Professorial Chair ( to J. S.). Funding for open access charge: Israel Science Foundation.

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.

Transcriptional slippage is a class of error in which ribonucleic acid (RNA) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (DNA) template. This phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. The bacteriophage λ N protein reduces transcriptional slippage within actively growing cells and in vitro. N appears to stabilize the RNA/DNA hybrid, particularly at the 5′ end, preventing loss of register between transcript and template. This report provides the first evidence of a protein that directly influences transcriptional slippage, and provides a clue about the molecular mechanism of transcription termination and N-mediated antitermination.

Synthetic single-strand oligonucleotides (oligos) with homology to genomic DNA have proved to be highly effective for constructing designed mutations in targeted genomes, a process referred to as recombineering. The cellular functions important for this type of homologous recombination have yet to be determined. Towards this end, we have identified Escherichia coli functions that process the recombining oligo and affect bacteriophage λ Red-mediated oligo recombination. To determine the nature of oligo processing during recombination, each oligo contained multiple nucleotide changes: a single base change allowing recombinant selection, and silent changes serving as genetic markers to determine the extent of oligo processing during the recombination. Such oligos were often not incorporated into the host chromosome intact; many were partially degraded in the process of recombination. The position and number of these silent nucleotide changes within the oligo strongly affect both oligo processing and recombination frequency. Exonucleases, especially those associated with DNA Polymerases I and III, affect inheritance of the silent nucleotide changes in the oligos. We demonstrate for the first time that the major DNA polymerases (Pol I and Pol III) and DNA ligase are directly involved with oligo recombination.

Cell-to-cell variations in protein abundance, called noise, give rise to phenotypic variability between isogenic cells. Studies of noise have focused on stochasticity introduced at transcription, yet the effects of post-transcriptional regulatory processes on noise remain unknown. We study the effects of RyhB, a small-RNA of Escherichia coli produced on iron stress, on the phenotypic variability of two of its downregulated target proteins, using dual chromosomal fusions to fluorescent reporters and measurements in live individual cells. The total noise of each of the target proteins is remarkably constant over a wide range of RyhB production rates despite cells being in stress. In fact, coordinate downregulation of the two target proteins by RyhB reduces the correlation between their levels. Hence, an increase in phenotypic variability under stress is achieved by decoupling the expression of different target proteins in the same cell, rather than by an increase in the total noise of each. Extrinsic noise provides the dominant contribution to the total protein noise over the total range of RyhB production rates. Stochastic simulations reproduce qualitatively key features of our observations and show that a feed-forward loop formed by transcriptional extrinsic noise, an sRNA and its target genes exhibits strong noise filtration capabilities.

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader , M. B. ; Costantino , N. ; Elkins , C. A. ; Chen , C. Y. ; Patel , I. ; Makusky , A. J. ; Choy , J. S. ; Court , D. L. ; Markey , S. P. ; Kowalak , J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011 , 10 , M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography-tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion., IMPORTANCE Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously, in vitro studies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here, in vivo experiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway in E. coli.

Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex.

Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here, we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Summary RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family represented by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the family. Previously, we have shown that RNase III uses two catalytic sites to create the 2-nucleotide (nt) 3′ overhangs in its products. Here, we present three crystal structures of RNase III in complex with double-stranded RNA, demonstrating how Mg2+ is essential for the formation of a catalytically competent protein–RNA complex, how the use of two Mg2+ ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modelled a protein–substrate complex and a protein–reaction intermediate (transition state) complex on the basis of the crystal structures. Together, the crystal structures and the models suggest a stepwise mechanism for RNase III to execute the phosphoryl transfer reaction.

The Escherichia coli product of the suhB gene, SuhB, is an inositol monophosphatase (IMPase) that is best known as a suppressor of temperature-sensitive growth phenotypes in E. coli. To gain insights into these biological diverse effects, we determined the structure of the SuhB R184A mutant protein. The structure showed a dimer organization similar to other IMPases, but with an altered interface suggesting that the presence of Arg-184 in the wild-type protein could shift the monomer-dimer equilibrium toward monomer. In parallel, a gel shift assay showed that SuhB forms a tight complex with RNA polymerase (RNA pol) that inhibits the IMPase catalytic activity of SuhB. A variety of SuhB mutant proteins designed to stabilize the dimer interface did not show a clear correlation with the ability of a specific mutant protein to complement the DeltasuhB mutation when introduced extragenically despite being active IMPases. However, the loss of sensitivity to RNA pol binding, i.e. in G173V, R184I, and L96F/R184I, did correlate strongly with loss of complementation of DeltasuhB. Because residue 184 forms the core of the SuhB dimer, it is likely that the interaction with RNA polymerase requires monomeric SuhB. The exposure of specific residues facilitates the interaction of SuhB with RNA pol (or another target with a similar binding surface) and it is this heterodimer formation that is critical to the ability of SuhB to rescue temperature-sensitive phenotypes in E. coli.

Recombineering, in vivo genetic engineering using the bacteriophage lambda Red generalized recombination system, was used to create various modifications of a multicopy plasmid derived from pBR322. All genetic modifications possible on the Escherichia coli chromosome and on bacterial artificial chromosomes (BACs) are also possible on multicopy plasmids and are obtained with similar frequencies to their chromosomal counterparts, including creation of point mutations (5-10% unselected frequency), deletions and substitutions. Parental and recombinant plasmids are nearly always present as a mixture following recombination, and circular multimeric plasmid molecules are often generated during the recombineering.

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.

Bacteriophage λ is a temperate bacteriophage, meaning that it can reproduce and develop either in a lytic or lysogenic state. When λ infects its bacterial host Escherichia coli , the phage may develop lytically, causing cell lysis with the release of hundreds of progeny virus, or it may abort

Background Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. Results We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5′ end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5′ G-dC base pair and the 3′ end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5′ untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. Conclusions In E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0666-5) contains supplementary material, which is available to authorized users.

The phage HK022 Nun protein excludes phage λ by binding nascent λ p L and p R transcripts at nutL and nutR , respectively, and inducing transcription termination just downstream of these sites. Termination is more efficient at nutL than at nutR . One difference between nutL and nutR is the presence of RNase III processing sites (rIII) located immediately promoter distal to λ nutL . We found that deletion of rIII dramatically reduced Nun transcription arrest in vitro but had little effect on termination in vivo. However, consistent with the in vitro results, overexpression of a transcript carrying nutL and rIII efficiently titrated Nun, allowing λ to grow on a strain that expressed Nun, whereas a transcript carrying only nutL or nutL- rIII with nucleotides 97 to 141 deleted was ineffective. Rnc70, an RNase III mutant that binds but does not cleave rIII, also prevented Nun-mediated λ exclusion. We propose that rIII enhances the on-rate of Nun at nutL , stimulating Nun-mediated arrest in vitro. We have shown that a specific element in rIII, i.e., box C (G 89 GUGUGUG), strongly enhances arrest on rIII + templates. Nun-rIII interactions do not stimulate Nun termination in vivo, presumably because formation of the Nun- nutL complex is normally not rate-limiting in the cell. In contrast to Nun, N is not occluded by Rnc70 and is not efficiently titrated by a nutL -rIII transcript.

SummaryMembers of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3′ overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members.

Summary Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III•dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.

The lysogenic state of bacteriophage λ is exceptionally stable yet the prophage is readily induced in response to DNA damage. This delicate epigenetic switch is believed to be regulated by two proteins; the lysogenic maintenance promoting protein CI and the early lytic protein Cro. First, we confirm, in the native configuration, the previous observation that the DNA loop mediated by oligomerization of CI bound to two distinct operator regions ( O L and O R ), increases repression of the early lytic promoters and is important for stable maintenance of lysogeny. Second, we show that the presence of the cro gene might be unimportant for the lysogenic to lytic switch during induction of the λ prophage. We revisit the idea that Cro's primary role in induction is instead to mediate weak repression of the early lytic promoters.

The lysis–lysogeny decision of bacteriophage λ has been a paradigm for a developmental genetic network, which is composed of interlocked positive and negative feedback loops. This genetic network is capable of responding to environmental signals and to the number of infecting phages. An interplay between CI and Cro functions suggested a bistable switch model for the lysis–lysogeny decision. Here, we present a real-time picture of the execution of lytic and lysogenic pathways with unprecedented temporal resolution. We monitor, in vivo , both the level and function of the CII and Q gene regulators. These activators are cotranscribed yet control opposite developmental pathways. Conditions that favor the lysogenic response show severe delay and down-regulation of Q activity, in both CII-dependent and CII-independent ways. Whereas CII activity correlates with its protein level, Q shows a pronounced threshold before its function is observed. Our quantitative analyses suggest that by regulating CII and CIII, Cro plays a key role in the ability of the λ genetic network to sense the difference between one and more than one phage particles infecting a cell. Thus, our results provide an improved framework to explain the longstanding puzzle of the decision process.

We report identification of the Escherichia coli ybhE gene as the pgl gene that encodes 6-phosphogluconolactonase. A tentative identification was first made based on the known approximate location of the pgl gene and the similarity of the presumptive ybhE-encoded protein sequence to a known Pgl enzyme. To test this notion, the ybhE gene was deleted and replaced with a drug marker. Like previously characterized pgl mutants, the ybhE deletion mutant had a Blu − phenotype (dark-blue staining with iodine due to accumulation of starch after growth on minimal maltose) and demonstrated impaired growth on minimal glucose medium when combined with a pgi mutation. Biochemical assay of crude extracts for 6-phosphogluconolactonase enzymatic activity showed that ybhE encodes this activity. The ybhE gene was transferred from the E. coli chromosome to an expression vector. This ybhE clone complemented both the precise deletion of the ybhE gene and a larger deletion, pgl Δ 8 , for the Blu − phenotype and for phosphogluconolactonase activity, confirming that ybhE is the pgl gene. A newly observed phenotype of pgl strains is a lowered frequency of appearance of Bgl + mutants that can utilize the β-glucoside salicin. This is likely due to poor growth of Bgl + pgl strains on salicin due to the accumulation of 6-phosphogluconolactone.

Summary Bacteriophage λ N and bacterial Nus proteins together with a unique site NUT in the leader of the early viral N gene transcript bind RNA polymerase (RNAP) and form a highly processive antitermination complex; N bound at NUT also represses N translation. In this study, we investigate whether N and NUT cause N translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the N-modified transcription complex for their effect on N-mediated translation repression. We show that nus and nut mutations that in combination destabilize multiple interactions in the antitermination complex prevent N-mediated translation repression. Likewise, transcription of the nut-N region by T7 RNAP, which does not lead to the assembly of an effective antitermination complex when N is supplied, eliminates translation repression. We also demonstrate that a unique mutant β subunit of RNAP reduces N-mediated translation repression, and that overexpression of transcription factor NusA suppresses this defect. We conclude that the N-modified RNAP transcription complex is necessary to repress N translation.

The bacteriophage lambda (lambda) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective lambda prophage. Here we describe the generation of a mini-lambda DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E. coli strain, including the DH10B-carrying BACs or PACs. The mini-lambda DNA integrates into the bacterial chromosome as a defective prophage. In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA. We describe here the use of the mini-lambda recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone. In addition, using the mini-lambda, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker. The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-lambda as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering.

A phage λ-based recombination system, Red, can be used for high-efficiency mutagenesis, repair, and engineering of chromosomal or episomal DNA in vivo in Escherichia coli . When long linear double-stranded DNA with short flanking homologies to their targets are used for the recombination, the λ Exo, Beta, and Gam proteins are required. The current model is: ( i ) Gam inhibits the host RecBCD activity, thereby protecting the DNA substrate for recombination; ( ii ) Exo degrades from each DNA end in a 5′ → 3′ direction, creating double-stranded DNA with 3′ single-stranded DNA tails; and ( iii ) Beta binds these 3′ overhangs to protect and anneal them to complementary sequences. We have tested this model for Red recombination by using electroporation to introduce overlapping, complementary oligonucleotides that when annealed in vivo approximate the recombination intermediate that Exo should create. Using this technique we found Exo-independent recombination. Surprisingly, a similarly constructed substrate with 5′ overhangs recombined more efficiently. This 5′ overhang recombination required both Exo and Beta for high levels of recombination and the two oligonucleotides need to overlap by only 6 bp on their 3′ ends. Results indicate that Exo may load Beta onto the 3′ overhang it produces. In addition, multiple overlapping oligonucleotides were successfully used to generate recombinants in vivo , a technique that could prove useful for many genetic engineering procedures.

The N-terminal arginine-rich motif of phage HK022 Nun protein binds to NUT sequences in phage λ nascent transcripts and induces transcription termination. Interactions between the Nun C terminus and RNA polymerase as well as the DNA template are required for termination. We have isolated Nun C-terminal point and deletion mutants that are unable to block transcription. The mutants bind NUT RNA and inhibit translation of the λ N gene. Thus HK022 excludes λ both by terminating transcription on the phage chromosome and by preventing translation of the essential λ N gene. Like N autoregulation, translation repression by Nun requires host RNaseIII deficiency ( rnc ) or a mutation in the RNaseIII processing site (rIII) located between NUTL and the beginning of the N coding sequence. Our data support the idea that Nun bound at NUTL causes steric interference with ribosome attachment to the nearby N coding sequence. Two models, Nun acting alone or in complex with host proteins, are discussed.

The Bacillus subtilis bex gene complemented the defect in an Escherichia coli era mutant. The Bex protein showed 39% identity and 67% similarity to the E. coli Era GTPase. In contrast to era , bex was not essential in all strains. bex mutant cells were elongated and filled with diffuse nucleoid material. They grew slowly and exhibited severely impaired spore formation.

We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the pL promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells. Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed. This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication. These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population. The cell division block is a result of the transient expression of the lambda kil gene. The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene. The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them. Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block. The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth.

ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the λ CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the λ CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage λ carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein.

This chapter describes the present state of recombineering in Escherichia coli and details the essential elements of the system. The success of this technology has encouraged its development for use in other organisms. The chapter discusses that goal and some obstacles that must be overcome in order to fully utilize recombineering in other prokaryotes. It emphasizes on the biochemistry of the proteins used for recombineering, the genetic manipulations possible, and the relative efficiencies of each. Several different types of genetic construction can be engineered by recombineering, and the desired genetic product dictates the type of DNA substrate used in the recombineering reaction. The following four types of substrates have been used successfully in laboratories: PCR products; short, partially dsDNA created by annealing ss-oligos; gapped linear plasmid DNA; and ssDNA oligonucleotides. Exo and Beta are needed to process the PCR product prior to its incorporation into the chromosome, and Gam is needed to prevent the degradation of the linear dsDNA by the RecBCD nuclease and possibly by the SbcCD nuclease. Recombineering in more distantly related bacteria will be facilitated by the identification and characterization of Red-like functions in phages from those organisms. Other requirements for the ready use of recombineering are those in common with other modes of genetic analysis, such as the development of systems permitting regulated gene expression and means of easily introducing DNA into the organism under study.

Background: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg 2+ -dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1–2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. Results: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn 2+ . The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg 2+ ion, which provides the first glimpse of RNase III in action. Conclusions: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn 2+ binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.