Andrew Johnson, a pioneer in the development of the amphibian axolotl as a model to study the early stages of metazoan development, died 15th September 2021. Known as 'AJ' by his family, and by his friends and colleagues, his older sister Pam referred to him as an unstoppable 'force of nature' who at the age of 9 or 10 said to her, 'I'm going to become a professor'. Here, we reflect on AJ's life and work, paying particular attention to his studies on the establishment of primordial germ cells in vertebrates.

Purpose The purpose of this paper is to establish a supported and validated reference point for understanding how Nicodemus, Kansas warrants significant inclusion in both the history of management and current entrepreneurship education. Design/methodology/approach This paper consists of a literature review to identify the salient historical and theoretical importance of the community of Nicodemus, Kansas. The research was conducted by reviewing and couching the research in this context. Findings Nicodemus, born out of an entrepreneurial spirit of newly liberated Black Americans, is the surviving entrepreneurial force for the African-American efforts in the western US expansion efforts in the later 1800s. The community, courage and cooperative views of the settlers were instrumental in overcoming a variety of hardships inherent in the location, society and time period to not only survive but also deliver growth and success. Nicodemus personified cultural pride and self-reliance, which fueled personal and commercial success. Practical implications The cooperative advantage is justified to be included in discussions of American management history, taught in the entrepreneurship curriculum and used by practitioners. Social implications Collective courage and cooperative advantage used by Nicodemus carries implications for how modern Black communities can advance their economic and social agendas. Originality/value Coverage of Black contributions management and entrepreneurship is scant, but a Black Enlightenment period has recently changed that scholars have recently begun to cover these significant moments in the literature (Prieto and Phipps, 2019). We argue that Nicodemus as an entrepreneurial community serves as a case study that needs contextualization in this Black Enlightenment era and holds pertinent implications for modern Black communities.

Genetic recombination is a fundamental process in biology that operates continually to shape and reshape the genomes of all organisms. It rearranges genes or parts of genes both within and between chromosomes, limits the divergence of repeated DNA sequences, guides the proper segregation of chromosomes at cell division, and promotes repair of damaged DNA. It provides therefore a potent evolutionary force that serves both to promote genetic diversity and to conserve genetic identity. Recent years have seen remarkable progress in our understanding of recombination reactions. Much of this progress stems from the pioneering genetic and molecular analysis initiated by A. J. Clark and P. Howard-Flanders some 30 years ago when the first recombination gene, recA, was identified in Escherichia coli and shown to be involved in repair of DNA damage (29, 74). RecA is the most crucial component for the homologous (also known as “general”) recombination reaction, and the study of its properties over the last 15 years has laid the foundations for in vitro analysis. The number of genes linked with recombination has now grown substantially. Many have been studied in detail, and the point has been reached where we can begin to understand the sequence of protein-DNA interactions that give rise to recombinants in crosses or which lead to repair of chromosomal damage. Homologs and analogs of E. coli genes have been discovered in a wide range of bacteria and viruses. More recently, RecA-like proteins have been discovered in yeasts and higher eukaryotes, including humans, which suggests that a similar reaction mechanism operates in all organisms (159, 204, 205). The genetics and biochemistry of homologous recombination have been the subject of excellent recent reviews (30, 92, 246). We focus here on recombination during genetic exchange in E. coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) and on related aspects of DNA repair. Recombinational exchanges associated with other types of DNA rearrangement (site specific, transpositional) are reviewed elsewhere in this volume (see chapters 124, 125, and 140). Aspects of recombination leading to chromosomal rearrangements are reviewed in detail in chapter 120, and recombinational repair is discussed in further detail in chapter 121.

Chromosome duplication initiates via the assembly of replication forks at defined origins. Forks proceed in opposite directions until they fuse with a converging fork. Recent work highlights that fork fusions are highly choreographed both in pro- and eukaryotic cells. The circular Escherichia coli chromosome is replicated from a single origin (oriC), and a single fork fusion takes place in a specialised termination area opposite oriC that establishes a fork trap mediated by Tus protein bound at ter sequences that allows forks to enter but not leave. Here we further define the molecular details of fork fusions and the role of RecG helicase in replication termination. Our data support the idea that fork fusions have the potential to trigger local re-replication of the already replicated DNA. In ΔrecG cells this potential is realised in a substantial fraction of cells and is dramatically elevated when one fork is trapped for some time before the converging fork arrives. They also support the idea that the termination area evolved to contain such over-replication and we propose that the stable arrest of replication forks at ter/Tus complexes is an important feature that limits the likelihood of problems arising as replication terminates.

Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Delta rep Delta uvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Delta rep Delta uvrD lethality. Thus replicationtranscription conflicts can be alleviated by the partial sacrifice of amechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p) ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p) ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced ‘adaptive’ mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair.

Over the previous decade the collection of countries known as the BRIC nations has worked together to advance social and economic agendas. These four nations represent unique challenges and immense opportunities for MNC’s searching to expand their business into these countries. This paper reviews these opportunities and challenges in the context of the fertilizer industry and provides an overall outlook for this international market. Recommendations are provided for how an international business might succeed in each of the BRIC nations.

RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair.

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.

Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit several origins to replicate each chromosome.

RecG is a DNA translocase that helps to maintain genomic integrity. Initial studies suggested a role in promoting recombination, a possibility consistent with synergism between recG and ruv null alleles and reinforced when the protein was shown to unwind Holliday junctions. In this article we describe novel suppressors of recG and show that the pathology seen without RecG is suppressed on reducing or eliminating PriB, a component of the PriA system for replisome assembly and replication restart. Suppression is conditional, depending on additional mutations that modify ribosomal subunit S6 or one of three subunits of RNA polymerase. The latter suppress phenotypes associated with deletion of priB, enabling the deletion to suppress recG. They include alleles likely to disrupt interactions with transcription anti-terminator, NusA. Deleting priB has a different effect in ruv strains. It provokes abortive recombination and compromises DNA repair in a manner consistent with PriB being required to limit exposure of recombinogenic ssDNA. This synergism is reduced by the RNA polymerase mutations identified. Taken together, the results reveal that RecG curbs a potentially negative effect of proteins that direct replication fork assembly at sites removed from the normal origin, a facility needed to resolve conflicts between replication and transcription.

Background Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I. Results In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli , suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG. Conclusions Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.

RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC.

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.

Replication of the Escherichia coli chromosome usually initiates at a single origin (oriC) under control of DnaA. Two forks are established and move away in opposite directions. Replication is completed when these meet in a broadly defined terminus area half way around the circular chromosome. RecG appears to consolidate this arrangement by unwinding D-loops and R-loops that PriA might otherwise exploit to initiate replication at other sites. It has been suggested that without RecG such replication generates 3' flaps as the additional forks collide and displace nascent leading strands, providing yet more potential targets for PriA. Here we show that, to stay alive, cells must have either RecG or a 3' single-stranded DNA (ssDNA) exonuclease, which can be exonuclease I, exonuclease VII, or SbcCD. Cells lacking all three nucleases are inviable without RecG. They also need RecA recombinase and a Holliday junction resolvase to survive rapid growth, but SOS induction, although elevated, is not required. Additional requirements for Rep and UvrD are identified and linked with defects in DNA mismatch repair and with the ability to cope with conflicts between replication and transcription, respectively. Eliminating PriA helicase activity removes the requirement for RecG. The data are consistent with RecG and ssDNA exonucleases acting to limit PriA-mediated re-replication of the chromosome and the consequent generation of linear DNA branches that provoke recombination and delay chromosome segregation.

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched DNAs in vitro, including Holliday junctions, replication forks, D-loops and R-loops. Coupled with the reported pleiotropy of recG mutations, this broad range of potential targets has made it hard to pin down what the protein does in vivo, though roles in recombination and replication fork repair have been suggested. However, recent studies suggest that RecG provides a more general defence against pathological DNA replication. We have postulated that this is achieved through the ability of RecG to eliminate substrates that the replication restart protein, PriA, could otherwise exploit to re-replicate the chromosome. Without RecG, PriA triggers a cascade of events that interfere with the duplication and segregation of chromosomes. Here we review the studies that led us to this idea and to conclude that RecG may be both a specialist activity and a general guardian of the genome.