What substance will a bacterium produce when a human gene is added to its genome?

Bacterial Genes and Expression

The bacterial genome is the total collection of genes carried by a bacterium, both on its chromosome and on its extrachromosomal genetic elements, if any. Bacteria usually have only one copy of their chromosomes (they are thereforehaploid), whereas eukaryotes usually have twodistinct copies of each chromosome (they are therefore diploid). With only one chromosome, alteration of a bacterial gene (mutation) will have a more obvious effect on the cell. In addition, the structure of the bacterial chromosome is maintained by polyamines, such as spermine and spermidine, rather than by histones.

In addition to protein-structural genes (cistrons, which are coding genes), the bacterial chromosome contains genes for ribosomal and transfer ribonucleic acid (tRNA). Bacterial genes are often grouped intooperons or islands (e.g.,pathogenicity islands) that share function or coordinate their control. Operons with many structural genes arepolycistronic.

Bacteria also may containextrachromosomal genetic elements such asplasmids orbacteriophages (bacterial viruses). These elements are independent of the bacterial chromosome and in most cases can be transmitted from one cell to another.

Abstract

Bacterial genes are functional units of DNA that have their effects through a diffusible product. The final product may be an RNA molecule, or the RNA intermediate may be translated into protein, which then exerts the effect. The effect of the gene is manifested in a phenotype, an observable property of the organism. Bacterial gene names are usually related to the phenotype. Changes in the DNA sequence are mutations, which may be silent (having no effect on the phenotype) but most often result in changed properties. Different versions of a gene are alleles. Genes that are near to each other in the DNA sequence are frequently transferred together during recombination events and are then said to be linked. Gene structure includes start and stop signals, and genes may overlap. When an mRNA codes for more than one gene, the arrangement is called an operon. Interruptions in gene continuity are known as introns (removed from the RNA transcript) and inteins (removed from the translated polypeptide). For organisms without experimental tools for gene transfer, candidate protein-coding genes can be identified in a DNA sequence by the placement of start and stop signals, by amino acid composition of the inferred coding sequences (CDS), and by the base composition of DNA, especially codon usage. A protein with sequence very similar to that of one with known function may be annotated with that function in sequence databases.

Many bacterial virulence genes are subject to positive regulation by ‘two-component regulators’

These two-component regulators typically comprise two separate proteins (Fig. 2.10):

one acting as a sensor to detect environmental changes (such as alterations in temperature)

the other acting as a DNA-binding protein capable of activating (or repressing in some cases) transcription.

Bacteria may possess multiple two-component regulators recognizing different environmental stimuli. Thus, bacteria residing in more complex environments tend to carry increased numbers of two-component regulators.

InBordetella pertussis, the causative agent of whooping cough (seeCh. 20), a two-component regulator (encoded by the bvg locus) controls expression of a large number of virulence genes. The sensor protein, BvgS, is a cytoplasmic membrane-located histidine kinase, which senses environmental signals (temperature, Mg2+, nicotinic acid), leading to an alteration in its autophosphorylating activity. In response to positive regulatory signals such as an elevation in temperature, BvgS undergoes autophosphorylation and then phosphorylates, so activating the DNA-binding protein BvgA. BvgA then binds to the operators of the pertussis toxin operon and other virulence-associated genes and activates their transcription.

InStaphylococcus aureus, a variety of virulence genes are influenced by global regulatory systems, the best studied and most important of which is a two-component regulator termed accessory gene regulator (agr). Agr control is complex in that it serves as a positive regulator for exotoxins secreted late in the bacterial life cycle (post-exponential phase) but behaves as a negative regulator for virulence factors associated with the cell surface.

The control of virulence gene expression inV. cholerae is under the control of ToxR, a cytoplasmic membrane-located protein, which senses environmental changes. ToxR activates both the transcription of the cholera toxin operon and another regulatory protein, ToxT, which in turn activates the transcription of other virulence genes such as toxin-co-regulated pili, an essential virulence factor required for colonization of the human small intestine.

Overlapping, Frameshifted, and Nested Genes

In some cases, adjacent genes overlap and are translated in different frames from the same sequence. Usually the overlap is small. A significant minority of genes in operons overlap by one or four nucleotides for example: TAATG, where TAA is the stop for the upstream gene, and ATG is the start of the second; or GTGA, where GTG is the start of the downstream gene and TGA the stop for the upstream gene.

Numerous examples of ribosomal frameshifting have been described in viruses and insertion sequences as well as at least two conventional bacterial genes. Translating ribosomes ‘slip’ on the message at a defined location (called a ‘slippery sequence’) and continue translation in a frame different from the original one. This occurs with dnaX of E. coli, leading to expression of replication factor gamma. A subset of ribosomes fails to frameshift; these terminate translation at a stop codon not far away, resulting in translation of replication factor tau, so that there are two gene products.

In rare instances, two genes may overlap extensively: the IS5 insertion sequence expresses one protein from one strand and two others from the other strand. In this instance, the same sequence segment codes for two genes. This sort of overlap is more frequent in mobile elements and bacteriophage, which have presumably experienced evolutionary pressure to keep genomes small.

Another strategy used in several instances is to initiate translation at two different locations in the same frame, resulting in a full-length protein (from the first initiation site) and an N-terminal truncation. In the best-known examples, such as the Tn5 transposase and Inh protein, the truncated protein acts to inhibit or otherwise regulate the activity of the full-length protein. Because the functions are significantly different, this can be considered two genes coded by the same sequence.

Susceptibility Genes

The inheritance of CD and UC cannot be described by a simple Mendelian genetics model. Multiple genes are involved and different genes confer susceptibility, disease specificity, and phenotype. A landmark study combining data from more than 75,000 cases of CD and UC, controls, and results from 15 genome-wide association (GWA) studies reported a total of 163 loci that may confer increased risk for the development of IBD.40,41 Thirty of these loci are specific to CD and 23 to UC. These loci are thought to account for 13.6% of the disease variance of CD and 7.5% of the disease variance of UC. Interestingly, 113 of the 163 loci are associated with other immune diseases, most strongly with psoriasis and ankylosing spondylitis. Susceptibility loci for IBD are also shared with primary immunodeficiencies and mycobacterial infections. These results suggest that rather than being separate diseases, CD and UC are part of the same spectrum of disease. They also suggest that many immune diseases and even susceptibility to certain infections may be part of a disease continuum that reflects immune response to environmental triggers.

Due to genetic associations related to disease location, a genotype-phenotype association study proposed that IBD be considered as 3 conditions rather than 2: ileal CD, colonic CD, and UC.42 In addition, genome-wide genetic correlation between PSC and UC was significantly greater than that between PSC and CD.43

GWA studies have identified genes associated with susceptibility to mycobacterial infections, such as leprosy and tuberculosis.Mycobacterium tuberculosis susceptibility genes includeVDR, which encodes the vitamin D receptor, providing a possible link with epidemiologic data that negatively associate risk of CD with sunlight and vitamin D exposure.41

The findings of GWA studies in CD and IBD generally support a connection between disease susceptibility and host interactions with microbes. This is exemplified in the first described susceptibility locus for CD. TheNOD2 (Nucleotide-bindingOligomerizationDomain containing 2) gene, also known asCARD15 (CAspase-RecruitmentDomain 15) was identified in 2001.44,45 The allelic variants most commonly associated with CD in European and American populations include one frameshift insertion leading to early truncation of the protein (Leu1007fsinsC) and 2 missense mutations (Arg702Trp, Gly908Arg). Carriage of disease-associated allelic variants on both chromosomes confers an odds ratio for CD of 17.1 (95% confidence interval [CI], 10.7 to 27.2), whereas heterozygotes have an odds ratio of 2.5 (95% CI, 2.0 to 2.9) for the disease.46 Genetic polymorphisms ofNOD2/CARD15 have been associated with younger onset, ileal location of disease, and increased likelihood of stricture formation.46 It has been estimated that as many as 20% to 30% of patients with CD have abnormalNOD2/CARD15. Nevertheless, penetrance ofNOD2/CARD15 is not more than 5% of individuals bearing 2 copies of disease-associated polymorphisms, and roughly 0.5% in heterozygous persons,47 indicating that disease-related allelic variants of the gene may be found in a large number of people who do not have CD.

Gene Clusters

In bacteria, genes of proteins that are involved in a common metabolic pathway are often clustered to allow synchronous regulation. If two genes are adjacent to each other in several analyzed genomes, especially in distantly related organisms, it is suggested that proteins encoded by these genes may participate in the same or related metabolic pathways. If the function of one of these genes is known, it helps to determine the function of the second protein. Analysis of bacterial SelR homologs reveals that genes encoding SelR homologs are often located immediately upstream or downstream of the MsrA gene (Fig. 5B).

APPENDIX 25.2 Parallels Among Guanosine Triphosphate-Binding Proteins

EF-Tu/Ts, elongation factor Tu/Ts; ER, endoplasmic reticulum; GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEFs, guanine nucleotide exchange factors; RGS, regulators of G-protein signaling; SRP, signal recognition particle; WASp, Wiskott-Aldrich syndrome protein.

3.4.3 Operon predictions

In bacteria, genes are often transcribed in polycistronic messenger RNA; thus, several consecutive genes can be under the control of only one promoter. A set of cotranscribed genes is defined as an operon. The existence of operons provides a way for bacteria to ensure that expression of genes participating to the same biological process is coordinated (Price, Huang, Arkin, & Alm, 2005). While the existence of operons can help researchers identify related functions (Overbeek, Fonstein, D'Souza, Pusch, & Maltsev, 1999), the inability to predict correctly operons can pose a problem when trying to computationally predict promoter regions in genome sequences. Indeed, large regions containing other coding or transcribed sequences (small RNA, etc.) may separate a gene from its promoter. In addition, the systematic experimental determination of the operon structure of one genome is not trivial. Therefore, this information is not available for most sequenced bacterial genomes (the most extensive datasets available are for E. coli //regulondb.ccg.unam.mx/ and B. subtilis //dbtbs.hgc.jp/).

Computational tools to predict operons in genomic sequences have been developed to resolve this problem (Brouwer, Kuipers, & van Hijum, 2008). The main sources of information used by these algorithms are experimental evidence, regulatory sequences, intergenic distances, functional relation, or phylogenetic conservation. However, it appears that a small intergenic distance is by far the best indicator to predict if two consecutive genes are cotranscribed (Brouwer et al., 2008). Operon predictions for many sequenced bacterial genomes are available (//csbl1.bmb.uga.edu/OperonDB/DOOR.php, Mao, Dam, Chou, Olman, & Xu, 2009, //www.microbesonline.org/operons/, Price et al., 2005).

Cellular Antiterminators

While some bacterial genes are translated efficiently and are thus protected from Rho, expression of poorly translated (e.g., horizontally transferred) operons, as well as of ribosomal and tRNA genes, depends on antitermination factors. A λ N-like antitermination mechanism is utilized in E. coli ribosomal RNA (rRNA) operons, which are not translated and thus could be targeted by Rho, even though their extensive secondary structure inhibits Rho binding. A ribosomal antitermination complex is composed of a nut-like RNA element and several Nus proteins; it is yet unclear which protein is a key N-like factor in this complex, but a ribosomal protein S4 has been proposed as a candidate.

Many chromosomal genes of foreign origin encode important protein functions, for example, lipopolysaccharide (LPS) core and capsule biosynthesis enzymes. E. coli RfaH, a sequence-specific paralog of a universally conserved general elongation factor NusG, dramatically activates expression of these operons. RfaH counteracts strong Rho-mediated polarity by three mechanisms. First, RfaH reduces RNAP pausing, which is a prelude to Rho-dependent termination. Second, RfaH excludes NusG, which acts together with Rho to facilitate RNA release, from binding to RNAP. Third, RfaH may facilitate the ribosome recruitment to these mRNAs, which lack canonical ribosome-binding sites, and maintain a tight link between the leading RNAP and the trailing ribosome, leaving no room for Rho.

NusG homologs are present in all living organisms and play several roles in transcription regulation. First, together with two key mobile RNAP domains, they form a continuous clamp around the DNA, preventing pausing and termination and thus increasing an overall RNAP processivity. Second, they interact with other factors to form large nucleoprotein complexes that can regulate expression of many genes; bacterial NusG promotes transcription–translation coupling, whereas its eukaryotic Spt5 homologs interact with chromatin remodeling complexes and RNA capping, splicing, and polyadenylation machineries. Third, NusG homologs can be targeted to a subset of operons via sequence-specific interactions with DNA; RfaH recruitment requires the presence of a 12 nt ops DNA element in the leader regions of its target operons.

I. Introduction

As with other bacterial genes, the expression of most virulence genes seems to be regulated. This is usually rationalized as a mechanism that avoids inappropriate expression of virulence traits while ensuring their rapid expression when they are required. It implies that bacteria have the means to know when to express the genes and when not to. Consequently, those wishing to understand the control of gene expression must consider both the mechanisms of gene regulation and the means by which bacteria interpret their internal and external environments.

Most of the pioneering work on bacterial gene regulation was carried out in Escherichia coli K-12 and its near relatives, using as model systems genes coding for carbohydrate utilization and other traits with no obvious role in bacterial virulence. Nevertheless, the lessons learned from these studies have proved to be extremely valuable and in many cases are applicable to virulence genes.

In general terms, genes can be regulated positively (activated) or negatively (repressed), and in E. coli repression seems to be the chief method by which gene expression is controlled [1]. Genes can be regulated at transcription or translation, or posttranslationally. Regulation at the transcriptional level, and in particular at the level of transcriptional initiation, is the most efficient method for the obvious reason that it is better to control a complex process at its point of origin rather than further downstream. It is clear that bacteria have invested heavily in transcriptional control mechanisms, and many of these are emerging as control elements in virulence genes.

Thinking about bacterial gene regulation has been influenced very strongly by the operon model, in which a dedicated regulatory protein controls simultaneously the expression of a number of sequential genes that are transcribed as a polycistronic message. The operon represents a simple mechanism for coordination of gene expression, and this concept is critical for useful insights into the means by which the transcriptional profile of the entire cell is modulated as the bacterium experiences environmental change. Grouping several operons or individual genes under the command of a common regulatory protein produces a regulon, with all of the members being coregulated in response to a common signal (Fig. 1). Allowing regulon members to belong to more than one regulon produces a networking of regulons, with genes responding to distinct, yet overlapping signals. The challenge of understanding the complexities of these higher levels of coordination is emerging as a key issue in the new era of microbiology that follows determination of the genome sequences of many bacteria. It is within this regulatory complex that virulence genes are located. Many possess dedicated regulators and also display sensitivity to regulatory inputs that are shared with many of the “housekeeping” genes of the cell. For this reason, an appreciation of bacterial gene regulatory mechanisms in general is a prerequisite for understanding how virulence gene expression is controlled.