How does bacteria transfer genetic information?

Signal Exchange in Early Stages of Rhizobium–Legume Interaction

Following progress in molecular genetics of Gram-negative bacteria, genetics of rhizobia developed in the 1980s. Rhizobial mutants defective for nodulation were isolated and the corresponding genes identified (nod, noe, and nol genes). Some of these genes, such as nodABCD, were found in most rhizobia and called common nod genes. Other genes, such as nodH and nodFE in Sinorhizobium meliloti, were found only in a limited number of rhizobial species and were called host-specific nod genes since mutations in these genes or their transfer to another rhizobium strain could result in changes in host range. Most nod genes (except nodD) are not transcribed when rhizobia are grown in culture medium. They are organized in operons whose expression requires flavonoid compounds secreted by the host plant root and a rhizobial NodD protein. The NodD proteins are transcriptional activators of the LysR family. They bind to conserved sequences located upstream of the nod operons, called nod boxes, and activate nod gene expression in the presence of flavonoids (Figure 1(a)). NodD proteins from different rhizobia differ in their specificity toward plant-produced flavonoids, providing a first level of host range control. Rhizobia can contain several nodD genes of different flavonoid specificity as well as other regulators of nod gene expression.

The biochemical function of the flavonoid-inducible nod genes remained elusive until it was found that they controlled the production by rhizobia of diffusible compounds called Nod factors (NFs), able to induce symbiotic responses on host plant roots. The structure of NFs produced by a number of rhizobia has been determined in the 1990s. In all cases, they are lipochitooligosaccharides made of a backbone of three to five N-acetylglucosamine residues N-acylated at the nonreducing end (Figure 1(b)). In addition, the oligosaccharide backbone can be substituted by chemical groups such as sulfate, fucose, and acetate which vary according to the rhizobial strain, as well as the length and degree of unsaturation of the acyl-chain. While the common nodABC genes determine the synthesis of the lipooligosaccharide core common to all NFs, the various substitutions are encoded by the host-specific nod genes. Some nod genes are also involved in NF secretion. The substitutions confer to NFs their specificity toward the legume host plants. For example, the sulfate on S. meliloti NFs is required for nodulation of Medicago plants.

Rhizobia (defined as bacteria able to form a nitrogen-fixing symbiosis with legume plants) are a very heterogeneous group that includes a number of species of alpha- and betaproteobacteria, groups that also include nonsymbiotic bacteria. The nod genes are often located on plasmids or symbiotic islands on chromosomes, facilitating horizontal transfer from strain to strain. Until recently, all rhizobia were thought to contain nod genes. However, sequencing the genome of photosynthetic Bradyrhizobium strains nodulating tropical legumes of the Aeschynomene genus showed that these strains did not contain nod genes, indicating that they can bypass NF signaling. Frankia bacteria, which can induce the formation of nitrogen-fixing nodules on nonleguminous plants called actinorhizal plants, are also often devoid of nod genes.

3.53.4.3 Synthetic Phage Therapy

Bacteriophage, or simply called phage, has played a central role in the development of molecular biology, bacterial genetics and providing the earliest tools for recombining DNA molecules, such as restriction enzymes and ligases. In the last decade the study of phage resistance mechnaisms has led to the discovery of one of the most important enabling technologies for SB since PCR; that is the CRISPR nuclease systems, which bacteria and archaea evolved as an adaptive defense to exogenous DNA. The recent identification of numerous other previously uncharacterized antiphage sytems may provide further breakthrough technologies for SB applications.45 In this historical context it is fitting then that SB is now applied to phage to perfect their use as therapeutic agents.

Phage was discovered independently by Frederik Twort and Félix D’Hérelle in the early part of the 20th century46,47 and soon after pursued as antimicrobiol agents to treat infectious diseases such as cholera and plague. In Eastern Europe phage therapy became common place and several renowned treatment centers exist to this day, for example, in Georgia and Poland. In the West, after some sporatic initial successes, phage therapy faded from use as antibiotics became widely available. Today with the rise of antimicrobial resistant pathogens, initially the so-called ESKAPE strains48 but now an even wider set of pathogens49 which are predicted to result in more deaths than cancer by 2050, there is renewed interest in phage therapy.

The use of phages has several advantages over antibiotics, primarily the potential to target specific strains and thereby leave the beneficial microbial community intact, and also the ability to overcome resistance by use of complementing phage cocktails, ‘training’ phages on selected host strains or simply isolating new phages against a pathogen. Conversely there are several hurdles to overcome and areas for improvment for phage therapy to become a reliable medicine and widely adopted in the West, such as; strong clinical trials with proper controls, reducing the time for identifying phage with the appropriate host range, overcoming phage resistance and exclusion mechansims of the target strain without the need for complex cocktails, circumventing unwanted immune responses to the phage particles and containment of generalized transduction of antibiotic resistance genes or bacterial virulence factors. Even in the case of successful killing of a target strain, the rapid lysis of a large number of bacteria and the concomitant release of endotoxins and superantigens may result in a strong infammatory response and an unfavourable clinical outcome.

The highly modular organization of phage genomes and the assembly of the phage structure as functional modules, such as tail fibers, spikes, tail tubes and capsid, makes phage ideal targets for SB approaches, in a sense phage genomes are already organized into BioBricks. In an early template for future synthetic phage design the filamentous phage Pf3 was modified to treat Pseudomonas aeruginosa infection in a mouse model.50 An export protein gene of Pf3 was replaced with a gene encoding the BglII restriction endonuclease, with the reasoning that (1) this gene replacement renders Pf3 non-replicative thereby introducing a containment strategy, (2) the phage can be stably propagated in a host containing the BglII methylase gene and (3) the BglII would catalyze double strand breaks in genomic DNA of the target strain for killing. An important finding of this study was that treatment of infected mice with the engineered phage Pf3R or with a lytic phage gave comparable survivability for mice challenged with a mimimum lethal dose of 3, but at a minimum lethal dose of 5 the survival rate was significantly better with Pf3R phage therapy. Analysis of serum cytokine levels indicated a reduced inflamatory response indicating that the better outcome for the Pf3R treatment group is due to the efficient killing of the target strain without lysis and endotoxin release.

The killing of P. aeruginosa by Pf3R relies on the phage host range to provide specificity of targeting as BglII restriction sites would be expected to be present in essentially all bacterial genomes. An improvement is suggested by the remarkable discovery of a bacteriophage which has obtained a CRISPR/Cas system, from an unknown source, for its own use.51 The phage-encoded CRISPR/Cas system is able to acquire new spacers and the CAS3 nuclease has been re-targeted to a chromosomal element that its host, Vibrio cholera, uses for innate immunity. Following this discovery, the Type II CRISPR system from Streptococcus pyogenes was engineered into M13 bacteriophage with spacers to target sequences for antibiotic resistance and virulence genes in Escherichia coli, the authors referring to these devices as RNA-guided nucleases (RGNs).52 Demonstrating the exquisite specificity of this system an RGN was able to discriminately kill a strain harboring a single nucleotide polymorphism in DNA gyrase which confers quinolone resistance. Further, in an artificial consortium of three bacterial strains they were able to kill selected strains (400–20,000 fold killing compared to controls) while leaving other consortium members intact. The specificity of CRISPR/Cas-mediated killing could expand phage therapy beyond targeting pathogens to precise modulation of human microbiomes, the composition of which has been implicated in the prognosis for some cancers and even neurological disorders such as autism, Parkinsons and Alzheimers vía the gut–brain axis.

An ideal synthetic phage platform could be one in which host range binding is engineered to be very broad while specificity of strain targeting is provided by the CRISPR/Cas payload. In this way phage could be readily deployed for treatments, without a new platform having to be isolated ad hoc for each pathogen. Further the broad host range combined with CRISPR arrays targeting several antibiotic resistance or virulence genes could allow the use of presumptive phage therapy, that is before pathogen identification. Host range extension strategies include forward genetic screens to identify phage receptors and required host factors,53 mining of prophage receptor binding protein (RBP) sequences from bacterial genomes and rebooting of synthetic phage which could, for example, encode receptor binding protein RBP libraries for HTS.54,55 The masking of receptors by capsules can be overcome by expressing exopolysaccharide hydrolyzing enzymes56 and other enzymes to degrade biofilms57 while other masking mechanisms and phase variation in receptor expression can be overcome by phage engineered with several tail fibers containing different RBPs or RBPs to non-canonical highly conserved cell surface targets. Bacteria deploy numerous anti-phage systems, principle ones being the innate immunity of restriction-modification and the adaptive immunity of CRISPR/CAS, but conversely so have phage counter-evolved multiple strategies to defeat these systems such as using non-canonical nucleotides in their DNA, having fewer restriction sites or hyper methylating their genomes and delivering proteins that inhibit restriction enzymes or enhance methylating enzymes of the host.

The synthetic phage platform described here would avoid the need for phage cocktails for which the regulatory approval may be more complex. Remaining targets for phage engineering are generic to many biologics, such as stability and response of the immune system. Phagocytic cells in particular are responsible for clearing phage particles from the circulatory system. Long-circulating phage mutants were obtained by a serial passage technique58 and were found to be mutated in the major capsid protein. Later a single amino acid change, also in a capsid protein, introduced by direct genetic manipulation resulted in a 13,000 to 16,000-fold increased capacity for the phage to remain in the mouse circulatory system.59 Other parameters to improve phage as therapeutic agents, such as production, formulation and route of administration probably fall outside the scope of SB activities, but the lessons learned from previous work with native phages will also apply to SB engineered phages.

Over the last decade with the renewed interest in phage therapy and sporadic reports of successful individual patient cases there have been attempts to run controlled clinical trials which has resulted in no significant adverse effects, but efficacy still not emphatically demonstrated. It will be an exciting next article of the phage therapy story, which began over 100 years ago, to see SB engineered phage enter clinical trials.

Bacteria and Viruses

Bacteria are single-celled, microscopic organisms. Most have a cell membrane and all lack membrane-bound organelles, including a nucleus. The bacterial genetic material is a single, circular molecule of DNA not arranged into a chromosome. Bacteria can have several shapes (e.g., rod shaped; filamentous; spiral shaped). Many bacteria cause disease by producing toxins. Bacterial infections that cause human illness can be prevented by vaccines or can be cured by antibiotics. A virus is a tiny, noncellular particle composed of a nucleic acid core (DNA or RNA) and a protein coat. Viruses are parasitic and reproduce only within a host cell. Some viral-caused human illnesses can be prevented by vaccination, but viruses are not harmed by antibiotics. First we discuss some of the sexually transmitted diseases caused by bacteria and then those caused by viruses. Other kinds of organisms causing STDs, such as fungi, protozoa, and invertebrates, are also mentioned.

A Bacterial Models

Although higher plant cells may have more in common with the fungi or the blue-green algae than with bacteria, we have chosen to compare them with the nonfilamentous bacteria, the Eubacteriales. The amenability of the nonfilamentous bacteria to various genetic and cultural manipulations has resulted in the development of a great variety of selection schemes. Major advances in bacterial genetics have been made with Escherichia coli K12; consequently, some people tend to equate bacterial genetics with E. coli. However, the physiological genetics of bacteria is enriched with information derived from representatives of different genera. Aside from a minimal number of relevant references to other systems, we find sufficient examples that illustrate diverse selection schemes among studies of two groups of gram-negative bacteria: the coliform bacteria and the fluorescent pseudomonads, soil bacteria. The advantages of these bacteria as objects for physiological genetic studies include their ability to grow rapidly with a generation time of under 30 min, to grow as a uniform suspension of single cells, and to form a discrete colony of cells from a single cell on agar-solidified medium. Concomitant with the latter characteristic is their amenability to replica plating.

Early Recognition of Antimicrobial Drug Resistance

Indeed, the new ‘miracle drugs’ represented such an effective therapy against major bacterial pathogens that it was long a widely held opinion by experts, health policymakers, and patients that infectious diseases now were under complete therapeutic control. Until the mid 1950s, penicillin was generally available without prescription, which probably contributed to extensive drug misuse. Unfortunately, antibiotics are still sold over the counter in many countries, and the widespread belief in this ‘magical bullet’ still creates inappropriate expectations for an antibiotic solution regardless of the nature of the infection. Inappropriate prescription patterns by physicians to play safe, the common practice of prescribing antibiotics for minor and self-limiting bacterial infections as well as for viral infections such as acute bronchitis and the common cold, is well documented in many countries and settings. Overprescribing antibiotics has been encouraged by the respective industries since the 1950s through advertising such medicines as a means to increase efficacy in a physicians practice.

Antimicrobial resistance (AMR), after having been described by Ehrlich in 1907, had an inconspicuous career for decades being a phenomenon observed in laboratories of experimental pharmacology and bacterial genetics. The mass application of sulphonamides during World War Two resulted in it becoming a clinical phenomenon. Unsurprisingly, it came to accompany the application of penicillin almost from the outset, and it was even mentioned as a typically occurring phenomenon by its inventors. As early as 1945, in an interview with The New York Times, Fleming warned that the misuse of penicillin could lead to selection of resistant forms of bacteria. In his view, resistance to penicillin could occur in two ways: either through strengthening of the bacterial cell wall which the drug destroyed, or through selection of bacteria expressing mutant proteins capable of degrading penicillin.

Furthermore, patients failing to complete the full course of antibiotic treatment may also contribute to the development of resistant bacteria, a situation that is of particular relevance to tuberculosis. If tubercle bacilli are attacked with a subtherapeutic dosage failing to completely eliminate them, the surviving bacteria, those most resistant to treatment, are left alone to proliferate, thus causing a subsequent infection that is harder to treat.

Barber and Rozwadowska-Dowzenko (1948) reported that in one U.K. hospital in 1946, 14% of staphylococcal strains isolated from sick patients were penicillin resistant. Just a couple of years later, the same hospital reported resistant stains in 59 out of 100 patients with staphylociccal infections. These early observations can now be explained by studies of the exceptional genetic plasticity of bacteria. A vast number of specific genes and gene products are involved in the phenotypical expression of AMR, but the mechanisms can basically be sorted into three broad categories (Table 1).

Table 1. Microbiological mechanisms for antimicrobial drug resistance

MechanismExample1. The drug is modified or destroyed before it can reach the bacterial targetBeta-lactamases in staphylococci, gonococci and H. influenzaeAminoclycoside resistance in staphylococci and enterococci2. Access to the bacterial target is reduced by reduced permeability into the cell or active export out of the cellTetracycline resistance in enteric bacteriaKarbapenem resistance in P. aeruginosa (one of several mechanisms)3. The Bacterial target is modified by mutations or acquisition of new genes leading to reduced affinity for the antibioticMethicillin resistance in staphylococci (e.g., MRSA)Penicillin resistance in pneumococciVancomycin resistance in enterococciRifampicin resistance in TB

Resistance can develop through point mutations in preexisting genes or re-assortment of genes from different sources, and the completed genetic determinant will often be localized on a plasmid or another kind of mobile element which can be transferred between strains of the same species and also between different species. The capacity of AMR development is further enhanced by the short generation times and environmental adaptability of bacteria. With few exceptions, the microbial world has proved its ability to generate resistance against all antibiotics developed over the last 70 years. A first glimpse of what lay ahead was a pandemic of resistant Staphylococcus from 1953 onwards. The rise of staph 80/81 – named after the phages employed in its diagnosis – played an important role in the growth of clinical microbiology as a field. More generally it inspired much of the critical research into antibiotics application ever since.

What are the 3 methods of genetic transfer in bacteria?

How does gene transfer occur in bacteria?

How is the genetic information being transferred?