Bacterial Adaptation and Horizontal Gene Transfer

• Bacteria adapt and evolve by modifying gene function through mutation or acquiring new genes via horizontal gene transfer (HGT).

Horizontal Gene Transfer (HGT)

• High-frequency exchange of large DNA sequences between species via HGT contributes significantly to genome variability and rapid pathogen evolution.
• DNA transferred by HGT is often stably maintained even without selective pressure.
• Genes for bacterial toxins and virulence factors evolve through HGT mediated by plasmids, transposons, and bacteriophages.
• HGT facilitates the acquisition of genes encoding antibiotic resistance, toxins, and other virulence factors.
• Acquired genes help bacteria evade the host's immune system, grow on varied nutrients, survive environmental conditions, and disseminate within the host.
• HGT allows traits to evolve separately before combining in a single bacterium to enhance virulence.
• Considerable HGT can occur outside human or animal environments, posing potential risks if genetic material evolves in unusual environments.

Mechanisms of Genetic Change and Diversification

Spontaneous Mutation

• Single nucleotide exchanges (SNPs) occur at rates ranging from 10−7 to 10−10 per nucleotide per generation.
• Recombination events like DNA duplication, insertions/deletions (indels), or rearrangements can occur at a rate of 10−4 to 10−6 mutations per cell per generation.
• Genetic changes affect biological activity or regulation, impacting growth, fitness, and virulence.
• Tandem duplication of a bacterial chromosome region can increase the amount of a regulatory protein, affecting gene expression.
• Stress-induced mutagenesis involves an error-prone DNA polymerase that introduces frequent mutations, enabling bacteria to cope with stress.

Phase Variation

• Changes in the expression of virulence proteins occur at high frequency compared to spontaneous mutations.
• Salmonella species can switch between making flagella with the type H1 flagellin protein to flagella with the type H2 flagellin protein, caused by a DNA invertase (Hin).
• Phase variation plays a role in the formation of diverse phenotypes within microbial communities in biofilms.
• Mycoplasma species, obligate intracellular pathogens, constantly alter the expression of their surface antigens through promoter inversion.
• Inversion of the promoter sequence can create a hairpin structure that acts as a transcriptional stop signal, controlling gene expression.
• In Bordetella pertussis, virulence gene expression is modulated through mutation of the coding sequence of a gene, bvgS.
• Slipped-strand misrepair at sites of repeated DNA sequences leads to different protein sequences or truncations from frameshifts.
• Opa proteins in Neisseria species undergo reversible phase variation through slipped-strand frameshifts.
• Lipooligosaccharide (LOS) in Neisseria species varies in surface structure due to slipped-strand misrepair in genes encoding glycosyltransferases and sialyltransferases.

Antigenic Variation

• Pathogenic microbes can avoid the host immune system by changing their surface antigens through gene-shuffling events.
• The pili of N. gonorrhoeae undergo phase and antigenic variation by recombination between different pilin genes.
• Recombination between conserved regions of pilS and pilE genes results in a new version of the pilE gene, producing a new pilin protein.
• Pilin recombination in Neisseria requires RecA protein and is a type of gene conversion.
• Phase and antigenic variation are major mechanisms of Neisseria pathogenesis, leading to repeated infections.
• Intergenic recombination occurs when recipient cells pick up extracellular DNA from donor cells, allowing pilS to recombine with pilE.
• Neisseria bacteria frequently undergo autolysis to release genomic DNA and are naturally transformable, enabling intergenic recombination.

Horizontal Gene Transfer: Mobile Genetic Elements

Natural Transformation

• Bacteria exchange DNA through transformation, conjugation, or transduction.
• Natural transformation involves a recipient cell taking up DNA released from a donor cell.
• Progeny from natural transformation are called transformants.
• Most bacteria need artificial help such as chemical or electrical treatments to efficiently uptake DNA, making them "competent".
• Molecular biology relies on introducing recombinant DNA into chemically or electrocompetent bacteria.
• Some bacteria are naturally competent, including pathogens like Haemophilus influenzae and Streptococcus pneumoniae.
• Natural competence allows for the uptake and incorporation of homologous DNA from the environment into the chromosome.
• In 1928, Frederick Griffith's experiment with S. pneumoniae demonstrated natural transformation.
• Griffith observed pathogenic (S strain/smooth colonies) and nonpathogenic (R strain/rough colonies) varieties of S. pneumoniae.
• Mice injected with the S strain died; mice injected with heat-killed S strain or the R strain lived.
• Mice injected with live R strain mixed with heat-killed S strain died, and smooth colonies were recovered.
• Griffith concluded a "transforming principle" transferred the pathogenic trait to the live R strain, with the "transforming principle" later identified in 1944 as DNA by Oswald Avery, Colin MacLeod, and Maclyn McCarty.
• Many naturally competent bacteria have short (10 to 12 base pairs) DNA-uptake sequences (DUS) for DNA binding and uptake.
• Some bacteria have specific receptors recognizing their own DUS, limiting DNA uptake to their own species (e.g., N. gonorrhoeae: 5′-GCCGTCTCAA-3′).
• Other bacteria, such as Bacillus subtilis and S. pneumoniae, uptake any DNA, but only homologous DNA sequences are recombined.
• Competence is highly regulated, involving quorum-sensing signals and environmental factors like acidity.
• The quorum-sensing signal for S. pneumoniae is a small, linear peptide called competence stimulatory peptide.
• Competent bacteria initiate a gene expression cascade to produce competence proteins for DNA uptake and recombination.
• Some species, like V. cholerae and S. pneumoniae, undergo fratricide, killing other cells to release DNA for uptake by competent bacteria.
• DNA uptake across the inner membrane in Gram-negative bacteria is similar to Gram-positive bacteria, with conserved proteins involved in competence.
• Core competence proteins are homologous to type 2 secretion systems (T2SS) and type IV pili, suggesting an evolutionary relationship.
• In Gram-negative bacteria, DNA uptake requires DUS binding to type IV pili and transport into the periplasm through a secretin (PilQ) pore.
• ATP hydrolysis drives transport via pili retraction, pulling DNA into the periplasm where it interacts with the DNA receptor ComEA.
• Transport across the inner membrane involves nicking and degradation of one DNA strand and funneling the other through the ComEC transmembrane channel.
• Inside the cytoplasm, the DNA strand integrates into the chromosome through homologous recombination.
• In Gram-positive bacteria, double-stranded DNA binds nonspecifically to a type IV-like pseudopilus, which retracts to bring DNA to the ComEA receptor.
• The nuclease EndA degrades one DNA strand, while the other is transported across the cell membrane by the ComEC protein.
• In the cytoplasm, the single-stranded donor DNA recombines with a homologous region of the chromosomal DNA.
• H. pylori and C. jejuni use the ComB system for DNA uptake, which is related to type 4 secretion systems (T4SS) and the VirB/VirD system in Agrobacterium tumefaciens.

Conjugation: Plasmids and Transposons

• Conjugation is a process where most Gram-negative and Gram-positive bacteria transfer DNA directly from one cell to another.
• In 1947, it was observed that mixing two different strains of E. coli resulted in new progeny strains, suggesting a mating process where DNA strands separate and one strand moves from donor to recipient via a mating bridge.
• The two single strands in each cell serve as replication templates to generate double-stranded DNA again.
• Progeny from conjugation are called transconjugants.
• Plasmids are extrachromosomal DNA that replicate independently of the chromosome.
• Plasmids frequently carry virulence genes, including those for antibiotic resistance, toxins, and adherence.
• Commensal bacteria in the gut can maintain plasmids and act as reservoirs for genetic traits that can spread to other bacteria.
• Some naturally occurring plasmids are not transmissible (nonconjugative), while others are self-transmissible or mobilizable.
• Self-transmissible plasmids encode genes for a transfer system (Tra system) necessary for DNA transfer from donor to recipient via conjugation.
• F (fertility)-factors are conjugative plasmids that replicate separately from the chromosome and transfer via conjugation.
• F-factors contain genes for plasmid replication, pili for cell-cell contact, DNA transfer, and insertion sequences.
• Some F-factors can contain complex transposons with antibiotic resistance genes, spreading antibiotic resistance upon transfer.
• An integrated F-factor is referred to as an episome.
• An integrated F-factor can aberrantly recombine out of the bacterial chromosome, including a segment of adjacent bacterial chromosome that is now included in the plasmid.
• Bacteria with an integrated F-factor are termed high-frequency recombination (Hfr) strains.
• In conjugative transposition, an integrative conjugative element (ICE) is initially integrated at a specific site in the bacterial chromosome and excises from the chromosome into its conjugative plasmid form.
• ICEs contain an oriT site, tra genes encoding a conjugative T4SS, a t4cp gene encoding an ATPase T4SS coupling protein (T4CP), raf genes encoding relaxosome accessory factors (RAFs), and a mob gene encoding a relaxase (Mob).
• Mob relaxase recognizes and nicks the oriT site, followed by RAFs binding to form a relaxosome complex.
• Resulting single-stranded DNA with relaxase at the end is funneled through T4SS to the recipient cell, recircularized, and replicated.
• In the recipient cell, site-specific recombination integrates the ICE plasmid into the bacterial chromosome at specific attachment sites.
• ICEs can carry cargo genes such as antibiotic resistance and virulence genes.
• Promiscuous transfer systems in conjugative plasmids and conjugative transposon ICEs can move DNA between unrelated species, significantly impacting antibiotic resistance.
• Mobilizable plasmids contain integrative mobilizable elements (IMEs) with an oriT site and a mob gene but lack tra genes.
• Mobilizable plasmids can replicate in cells lacking another conjugative helper plasmid but cannot be transferred by conjugation alone.
• In bacteria with a conjugative plasmid, Tra functions are provided in trans, allowing mobilizable plasmids to transfer to recipient cells, with such plasmids being useful for transferring cloned genes in biotechnology.
• Transposons (Tns or "jumping genes") are MGEs that move from one DNA location to another within the same bacterial cell where they can move between sites on bacterial chromosomes or between plasmids and the chromosome and vice versa.
• The smallest type of bacterial transposon, called an insertion sequence (IS), consists of a transposase gene that encode an enzyme that promotes their transposition, and inverted repeat sequences at the flanking ends, which are used to target sequences for insertion in target DNA.
• Upon insertion, the target DNA is duplicated as a direct repeat flanking the integrated IS.
• When an IS element inserts in the coding region of a gene, the gene is usually inactivated; thus, IS elements are not generally tolerated in essential genes.
• Composite (or complex) transposons consist of two IS elements flanking a central core region that contains cargo genes, including antibiotic resistance or virulence genes.
• IS elements and composite transposons move within the DNA of a single bacterial cell; unlike conjugative transposons (ICEs), which move between bacterial cells.
• Transposons and conjugative transposons create genetic and phenotypic diversity, transmitting antibiotic resistance and virulence genes within and between bacterial populations.

Phage Transduction

• Transduction is a mechanism where bacteria exchange DNA through transfer by bacterial viruses (bacteriophages, or "phages").
• Research in 1926 showed a filterable agent from scarlet fever isolates could convert non-inducing strains of Streptococcus pyogenes into strains that could induce scarlet fever.
• The diphtheria toxin toxA gene was demonstrated in 1951 to be encoded on a β-bacteriophage from Corynebacterium diphtheriae.
• Many toxins and virulence factors are encoded on integrated bacteriophages in Gram-negative and Gram-positive bacteria.
• Transduction allows a bacterium to acquire new DNA segments, typically 20 to 100 kilobases.
• Lytic phages replicate their genetic material, synthesize phage structural proteins, and lyse host bacterial cells to escape.
• Temperate phages have a lytic phase and a lysogenic phase, employing site-specific recombination to integrate into the bacterial chromosome.
• During the lytic phase, viral proteins assemble into capsids and tails, and the viral genome is packaged into virions.
• A phage-encoded holin inserts into the bacterial cytoplasmic membrane, forming a pore for lysin transport to digest peptidoglycan and cause cell lysis.
• Temperate (lysogenic) phages integrated into the bacterial chromosome are called prophages.
• A lysogenic phage produces a repressor protein to prevent excision from the bacterial chromosome, except under stress.
• Lysogenic phages can revert to the lytic phase, directing the lytic phase once out of the bacterial chromosome.
• Prophages can confer new biochemical and virulence properties on host bacteria.
• Defective prophages are inactive but continue to express virulence genes.
• Virulence gene-encoding prophages are a form of pathogenicity island.
• Generalized transducing phages transfer a segment of bacterial chromosomal DNA during a phage lytic phase.
• During the lytic phase, a phage-encoded nuclease degrades the bacterial chromosome into pieces of the same size as the phage genome.
• Phage DNA is packaged into capsid phage heads by a "head-full" mechanism based on DNA size.
• Occasionally, a segment of the bacterial chromosome is packaged instead of phage DNA, injecting DNA into recipient bacteria.
• Delivered bacterial DNA segments recombine into the recipient's chromosome via RecA-dependent homologous recombination.
• In generalized transduction, mutations and genes are transferred from one bacterium to another.
• Specialized transducing phages contain an almost complete phage genome plus one or two bacterial genes adjacent to the phage attachment site.
• During lysogeny, the phage genome circularizes, undergoing site-specific recombination into a specific DNA sequence in the bacterial chromosome.
• During the transition to the lytic phase, most phage genomes excise precisely and are packaged into phage capsid heads.
• Aberrant phages package genes in a bacterial chromosome adjacent to the phage attachment site, infecting and lysogenizing other bacteria.
• This form of transduction is "specialized" because only bacterial genes adjacent to the attachment site are accidentally packaged.

Control of Horizontal Gene Transfer

Toxin-Antitoxin (TA) Systems

• Acquisition of a large extrachromosomal piece of DNA, like a plasmid, places a metabolic burden on the cell in terms of energy, machinery, and cellular effects of the expressed gene products.
• To maintain the plasmid once acquired, many plasmids carry a two-gene operon encoding a toxin-antitoxin (TA) system.
• The toxin is a protein or peptide that inhibits an essential cellular function, causing growth arrest or bacterial death.
• The antitoxin is a regulatory small RNA (sRNA) that blocks toxin mRNA translation, an RNA that binds to toxin protein, or peptide that binds tightly to the toxin, neutralizing its toxic activity.
• The antitoxin serves as a bacterial "immunity" factor, protecting the bacterium from the toxin's action.
• As long as the bacterium retains the plasmid with the TA system, both toxin and antitoxin continue to be expressed, and the antitoxin prevents the toxin from working.
• If the plasmid is lost during replication, the bacterial progeny cannot make any more toxin or antitoxin.
• Since the toxin is more stable than the antitoxin, the released toxin will block bacterial growth or kill the bacterium.
• Besides plasmid maintenance, TA systems on plasmids, bacteriophages, and chromosomes also play additional roles in bacterial population survival and persistence under stressful conditions.

Restriction-Modification (RM) Systems

• Most (90%) bacteria and archaea harbor restriction-modification (RM) systems to prevent acquisition of foreign DNA.
• RM systems are comprised of two cognate genes; one encodes a restriction enzyme, and the other a modification enzyme.
• The restriction enzyme is a nuclease that cleaves double-stranded DNA at a specific recognition site.
• The modification enzyme is a site-specific DNA methylase that modifies DNA at the sequence recognized by its cognate restriction enzyme.
• DNA methylation prevents the restriction nuclease from cleaving the DNA.
• The specific double-stranded DNA sequence recognized by an RM pair is usually 4 to 8 base pairs in length and frequently is found in bacterial or phage genomes.
• Invading foreign double-stranded DNA that has not been methylated at the restriction recognition site will be degraded by the restriction enzyme.
• A mutation that inactivates the methylase of an RM pair is lethal.
• Phages have evolved ways to evade RM systems, including modifying bases, encoding phage proteins that mask restriction sites, encoding a phage methylase, or encoding phage proteins that neutralize or sequester the RM system nucleases.
• Unmethylated foreign DNA that enters recipient cells as single strands through natural transformation or conjugation will be cleaved by restriction enzymes.

CRISPR-Cas Systems

• CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and their associated Cas proteins) are another form of bacterial immunity against acquisition of foreign DNA.
• CRISPR-Cas modules are found in large multigene operons in most archaea and many bacteria and block invading genetic elements, such as bacteriophages and plasmids.
• Each CRISPR locus is comprised of DNA direct repeat sequences of 23 to 50 base pairs in length that are separated by similar-sized, nonrepetitive DNA spacer sequences that have sequence identity with DNA sequences from plasmids or bacteriophages.
• Alternating direct repeats (palindromes) and unique spacer sequences (CRISPRs) can range from 2 to several hundred within a single CRISPR locus and specify the targets of CRISPR interference, which are foreign DNA to which the bacterium has previously been exposed and is now protected against if it invades again.
• A set of cas genes immediately precedes or follows the CRISPR locus and encodes the Cas protein machinery.
• The Cas protein machinery is responsible for processing and mediating the CRISPR-mediated interference activity, resulting in degradation of foreign DNA (or in some cases RNA).
• There are three main types of CRISPR-Cas systems, defined on the basis of their signature Cas protein with nuclease activity (Cas3, Cas9, or Csm/Cmr, respectively).
• The crRNP complexes of type I and type III systems contain multiple Cas subunits involved in assembly of the crRNP and surveillance, whereas the type II system has only one Cas9 protein plus a transactivating crRNA (tracrRNA).
• Extensive comparative genome studies have revealed considerable diversity in CRISPR-Cas systems, so it is important to bear in mind there are many subtypes and variants of the three main types.

Type 6 Secretion Systems (T6SS)

• The type 6 secretion system (T6SS) is a bacterial nanomachine found in a wide variety of Gram-negative bacteria.
• Bacteria use their T6SS apparatus to antagonize adjacent eukaryotic or bacterial cells by ejecting a toxin-coated "spear".
• This spear penetrates the membrane of adjacent cells, delivering toxic proteins.
• In certain Pseudomonas species, the T6SS has a special posttranslational regulatory system, sensing exogenous membrane perturbations, and builds its T6SS machine to aim its toxic spears directly at the source.
• One type of membrane perturbation recognized by this system is the mating pair complex formation associated with T4SS-mediated DNA conjugation.
• When a conjugative donor cell attempts to conjugate DNA into these T6SS-positive Pseudomonas recipient bacteria, the donor cell is frequently met with a lethal T6SS counterattack.

Pathogenicity Islands and Pathogen Evolution

Properties of Pathogenicity Islands (PAIs)

• Studies of E. coli strains involved in urinary and intestinal diseases revealed that the hly genes, encoding the pore-forming toxin α-hemolysin, were located on large distinct chromosomal DNA regions (genomic islands) with a different % G+C content and codon usage than the rest of the bacterial chromosome.
• The % G+C content of these DNA regions was found to be 41%, whereas the overall content of the E. coli chromosome is 51%.
• Additional virulence genes, encoding P fimbriae, a toxin called cytotoxic necrotizing factor-1, and other virulence-associated proteins, were also located within these regions.
• These regions were flanked by direct repeats of 16 to 18 base pairs, indicating that these DNA sequences had been acquired through HGT.
• In the early 1990s, these DNA segments of the genome containing one or more virulence genes acquired through HGT were named pathogenicity islands (PAIs).
• Comparing a large number of genome sequences has revealed the importance of PAIs in the diversification of strains within a bacterial species.
• The genomes of most bacterial pathogens contain multiple PAIs, whereas their nonpathogenic counterparts do not.
• Many PAIs appear by sequence analysis to be prophages or remnants thereof, but some appear to be integrated conjugative elements.
• PAIs can constitute as much as 20% of a bacterial genome.
• PAIs are usually inserted into defined locations within highly conserved DNA regions on the chromosome, such as phage attachment (att) sites, bacterial tRNA genes (leuX, selC), or insertion sequences (direct repeats).
• Many PAIs are still part of active mobile DNA elements (plasmids, ICEs, transposons, or phages) or of mobilizable DNA elements and have a tendency to excise from the chromosome at frequencies as high as 10−4 to 10−5 or to undergo duplications and amplifications.
• PAIs that are still mobilizable (plasmids, transposons, or prophages) carry genes needed for transmission.
• Many PAIs encode genes that provide the bacteria with a selective advantage under certain environmental conditions, such as new pili for altered adhesion, means for acquiring iron and other nutrients, novel surface structures such as modified LPS for increased serum resistance, capsular biosynthesis to prevent phagocytosis, or delivery of proteins that enhance bacterial invasion, modulate intracellular signaling processes, or dampen immune responses.
• Genes encoding most bacterial protein toxins are located on PAIs.
• Lysogenic prophages carrying toxin genes are thought to serve as a major natural reservoir for toxin genes in bacterial populations.
• HGT of toxin genes in natural environments may account for the prevalence of related toxins among diverse pathogens.
• The cholera toxin gene (ctx), which is phage encoded in Vibrio cholerae, is closely related to the heat-labile enterotoxin genes (elt or etx) found in different strains of E. coli that cause diarrhea.
• Shiga toxin-related (stx) genes in Shigella and E. coli strains and the closely related botulinum or tetanus neurotoxin (bot or tet) genes are found in different strains and species of Clostridium.
• Phages containing stx from Shigella and E. coli can be transmitted not only between different bacteria in the intestine of humans and other animals but also in external aquatic environments.
• The diverse locations of the PAIs encoding Clostridium botulinum and Clostridium tetani neurotoxin genes illustrate the degree of HGT that has contributed to their evolution.

Pathogen Evolution in Quantum Leaps

• Point mutations, genomic rearrangements, and antigenic variation lead to slow adaptive evolutionary changes, but acquisition of a single PAI can convert a nonpathogenic bacterium into a pathogen in a single step.
• One of the first demonstrations of this was that of the phage-encoded diphtheria toxin.
• Nontoxigenic strains of C. diphtheriae are avirulent and indeed are often found colonizing the upper respiratory tract.
• Conversion into virulent toxigenic strains can occur by acquisition of the toxin gene via phage transduction.
• Plasmids, phages, and transposons are thus means for rapid evolutionary change.
• Since gene clusters in PAIs are acquired as a unit in a single HGT integration event, virulence genes carried on PAIs found in different bacterial genera have remarkable sequence similarity and are often found arranged in the same order.
• Stepwise acquisition of PAIs can lead to progressive increase in virulence and the rapid emergence of new pathogens.
• Two large PAIs have been identified in Salmonella, each contributing to a specific step in the course of infection.
• The Salmonella pathogenicity island-1 (SPI-1) encodes about 25 genes, including a type-3 protein secretion system and various effector proteins delivered into the mammalian host cells via the SPI-1 type-3 secretion system and confers the ability to invade epithelial cells.
• The Salmonella pathogenicity island-2 (SPI-2) encodes about 15 genes, including a second type-3 protein secretion system, a two-component regulatory system, and other effector proteins, conferring the ability to survive within macrophages and cause systemic infections.
• Acquisition of these two PAIs was critical in the development of Salmonella as an intracellular pathogen because these PAIs enable Salmonella to invade host cells, evade host defenses, and cause systemic infections.
• SPI-1 is present in strains from all subgroups of S. enterica, but SPI-2 is not found in Salmonella bongori strains, which are of intermediate virulence and are thought to represent an intermediate step in Salmonella evolution.
• There is tremendous diversity within bacterial species, as well as the more traditional view of differences between bacterial species.
• Two different clinical isolates that are the same bacterial species based on 16S rRNA gene sequence analysis can show considerable differences in the numbers and types of PAIs, lysogenic bacteriophages, and extrachromosomal elements they contain.
• These differences underlie the fact that single bacterial species cause a multitude of infections.
• Bacterial pathogens actually have a much larger gene pool, called a supragenome or pan-genome, than what is in the chromosome of any single bacterial cell.
• HGT, genome plasticity, and the existing diversity of the available gene pool make bacterial adaptation inevitable in the face of changing environmental and stress conditions and are a major driving force of pathogen evolution and the emergence of the so-called superbugs.