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2.5: Gene and Operon - Biology

2.5: Gene and Operon - Biology


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Genes

The entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide or RNA molecule. Thus, a gene contains additional sequence information beyond that which codes for the amino acids in a protein or the nucleotides in an RNA molecule. The gene also contains the DNA necessary to get a particular transcript made.

Transcription control regions can be remote to the coding region (on the order of Kb's or 10's of Kb's away).

Most prokaryotic genes lack introns (intervening DNA sequence). In prokaryotes, genes which encode proteins with relationships in a metabolic pathway form Operons - which produce polycistronic mRNA's.

Definitions: Operon and Promoter

  • An operon is in bacterial DNA, a cluster of contiguous genes transcribed from one promoter that gives rise to a polycistronic mRNA.
  • A Promoter is a DNA sequence to which RNA polymerase binds prior to initiation of transcription - usually found just upstream of the transcription start site of a gene

e.g. Trp Operon - involved in the biosynthesis of the amino acid tryptophan:

Figure 2.5.1: Chemical pathway of trp operon

Figure 2.5.2: Trp operon in DNA/RNA

A consequence of the arrangement of bacteria genes into operons is that the level of mRNA for each of the genes in the operon is exactly the same.

Ribosomes transcribe from the start of each gene, not only from the first gene.

Another consequence of the arrangement of bacteria genes into operons is that an upstream mutation (i.e. possibly inhibiting transcription) can prevent "downstream" genes from being transcribed and expressed. Most eukaryotic transcription units produce monocistronic mRNA's, (i.e. they encode only one protein). There is a fundamental difference in the translation processes of prokaryotes and eukaryotes:

  1. In prokaryotes ribosomes can bind at specific recognition sequences anywhere within the mRNA (called ribosome binding sites, or "Shine-Dalgarno" sites).
  2. In eukaryotes, ribosomes bind via the interaction with specifically modified 5' region (so called 5' cap site) of mRNA molecules.
  3. Most eukaryotic mRNA's are therefore monocistronic.
  • Mutations in simple eukaryotic transcription units affect only one protein.

Complex Eukaryotic Transcription Units

The primary RNA transcript encoded by complex transcription units can be splicedin more than one way. Because of the different processing possibilities, the exons (coding regions) in a single complex transcription unit can be linked in alternative ways, to yield different mRNAs and different proteins.

Figure 2.5.3: Complex transcription units

Transcriptional regulation

Successful survival requires adaptability and economy:

  1. The ability to switch from metabolizing one substrate to another as environmental resources change
  2. It would be an energetic waste to produce enzymes for a metabolic pathway which is not needed.

Induction versus Repression of Enzyme Synthesis

In E. coli certain enzymes are produced only when the cells are grown on certain substrates. This effect is called enzyme induction. For example, when cells are grown in the absence of a type of sugar known as a b ­galactoside (e.g. lactose) the cells contain very few molecules (~5 per cell) of the enzyme b-galactosidase (which cleaves lactose into glucose and galactose).

  • There is no need for this enzyme in the absence of lactose.
  • If lactose is added to E. coli, in a very short amount of time there are approximately 5000 molecules of b-galactosidase per cell (approximately ~1,000 fold induction).
  • If lactose is removed from the media synthesis of b-galactosidase stops.

A similar but opposite situation occurs in regard to the synthesis of tryptophan (the biosynthetic enzymes are contained in the trp operon). In this case production of the enzymes for tryptophan biosynthesis are rapidly shut down if tryptophan is present, in a process called repression. Repression is a transcriptional regulatory mechanism for commonly required gene products. Induction is a transcriptional regulatory mechanism for gene products which may be required under unusual or infrequent situations.


The cyl Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Streptococcus Hemolytic Lipid, Granadaene

Group B Streptococcus (GBS) is a β-hemolytic, Gram-positive bacterium that commonly colonizes the female lower genital tract and is associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A major factor promoting GBS virulence is the β-hemolysin/cytolysin, which is cytotoxic to several host cells. We recently showed that the ornithine rhamnolipid pigment, Granadaene, produced by the gene products of the cyl operon, is hemolytic. Here, we demonstrate that heterologous expression of the GBS cyl operon conferred hemolysis, pigmentation, and cytoxicity to Lactococcus lactis, a model non-hemolytic Gram-positive bacterium. Similarly, pigment purified from L. lactis is hemolytic, cytolytic, and identical in structure to Granadaene extracted from GBS, indicating the cyl operon is sufficient for Granadaene production in a heterologous host. Using a systematic survey of phyletic patterns and contextual associations of the cyl genes, we identify homologs of the cyl operon in physiologically diverse Gram-positive bacteria and propose undescribed functions of cyl gene products. Together, these findings bring greater understanding to the biosynthesis and evolutionary foundations of a key GBS virulence factor and suggest that such potentially toxic lipids may be encoded by other bacteria.

Keywords: Gram-positive bacteria Group B Streptococcus bacterial toxin microbial evolution virulence factor.

Copyright © 2020 Armistead, Whidbey, Iyer, Herrero-Foncubierta, Quach, Haidour, Aravind, Cuerva, Jaspan and Rajagopal.

Figures

Complementation of L. lactis with…

Complementation of L. lactis with pcylX-K, but not empty plasmid vector, pEmpty, conferred…

Pigment extracted and purified from…

Pigment extracted and purified from L. lactis p cylX-K is identical to Granadaene…

Phyletic analysis suggests the cyl…

Phyletic analysis suggests the cyl operon evolved prior to the diversification of Gram-positive…


A GATA-dependent nkx-2.5 regulatory element activates early cardiac gene expression in transgenic mice

nkx-2.5 is one of the first genes expressed in the developing heart of early stage vertebrate embryos. Cardiac expression of nkx-2.5 is maintained throughout development and nkx-2.5 also is expressed in the developing pharyngeal arches, spleen, thyroid and tongue. Genomic sequences flanking the mouse nkx-2.5 gene were analyzed for early developmental regulatory activity in transgenic mice. Approximately 3 kb of 5' flanking sequence is sufficient to activate gene expression in the cardiac crescent as early as E7.25 and in limited regions of the developing heart at later stages. Expression also was detected in the developing spleen anlage at least 24 hours before the earliest reported spleen marker and in the pharyngeal pouches and their derivatives including the thyroid. The observed expression pattern from the -3 kb construct represents a subset of the endogenous nkx-2.5 expression pattern which is evidence for compartment-specific nkx-2.5 regulatory modules. A 505 bp regulatory element was identified that contains multiple GATA, NKE, bHLH, HMG and HOX consensus binding sites. This element is sufficient for gene activation in the cardiac crescent and in the heart outflow tract, pharynx and spleen when linked directly to lacZ or when positioned adjacent to the hsp68 promoter. Mutation of paired GATA sites within this element eliminates gene activation in the heart, pharynx and spleen primordia of transgenic embryos. The dependence of this nkx-2. 5 regulatory element on GATA sites for gene activity is evidence for a GATA-dependent regulatory mechanism controlling nkx-2.5 gene expression. The presence of consensus binding sites for other developmentally important regulatory factors within the 505 bp distal element suggests that combinatorial interactions between multiple regulatory factors are responsible for the initial activation of nkx-2.5 in the cardiac, thyroid and spleen primordia.


2.5: Gene and Operon - Biology

The observation that the N-terminal 23 residues of β-galactosidase can be replaced with other amino acids without affecting enzymatic activity enabled the production of fusions between the lacI and lacZ genes, which yielded a hybrid protein with both Lac repressor and β-galactosidase activities. This opened the way for the construction of a variety of lac fusions, resulting in the formation of hybrid genes that specify hybrid proteins. Nowadays, a number of commercially available vectors for constructing lacZ fusions in vitro exist. The sensitive detection of β-galactosidase activity with a colourless, soluble substrate, 5-bromo-4-chloro-3-indolyl-β- d -galactoside (X-Gal), which forms an insoluble blue dye upon hydrolysis, has made lacZ gene one of the most commonly used in vivo reporter genes. It provides a sensitive method of detecting genes that are subject to specific regulatory signals, of studying the localization of a protein to a given cellular compartment and of analyzing developmentally regulated genes, transgene expression, tissue-specific expression and other aspects involved in embryogenesis or developmental biology. The regulation, identification and localization of many proteins of various origins have been uncovered by using β-galactosidase activity of fusion proteins as a marker.

During the past few decades, transgenic mice bearing lacZ reporter gene have been used extensively to analyze gene expression patterns through the developmental stages of the life cycle in different tissues. To obtain transgenic mice, the lacZ gene is fused to the promoter of a targeted gene, then DNA is prepared and microinjected into fertilized mouse eggs. The expression of the transgene bearing β-galactosidase activity can easily be visualized in situ in tissue sections by using the chromogenic substrate X-Gal, or the recently introduced different fluorogenic substrates that allow sensitive and precise localizations.

The explosion of genome sequence data revealed that a large fraction of the open reading frames encoded by these genomes has no known biological function. A major challenge remains in finding efficient new techniques to investigate the function of these genes, an approach known as ‘functional genomics’, that include analysis of protein–protein interactions, that are central to most biological processes. At present, the most powerful approach used to select or screen for protein–protein interactions is based on two-hybrid methods. Originally developed by Fields and Song in 1989, at present several yeast or bacterial two-hybrid systems have been described in general the two putative protein partners are fused to two interacting polypeptides, such as transcription factors or complementary domains of a specific protein. In most systems, the readout of hybrid protein association is coupled to screanable or to selectable phenotypes, one of the most sensitive ones being LacZ expression. See also Genetic Engineering: Reporter Genes, and Protein Domain Fusion

Α-Complementation

Perhaps the most widely exploited property of β-galactosidase is the phenomenon called α-complementation. This complementation involves noncovalent reassociation of complementary fragments of the β-galactosidase subunit polypeptide chain, which then reassemble into an enzymatically active tetrameric structure. In the case of α-complementation, restoration of enzyme activity occurs when the N-terminal 60 residues of β-galactosidase interact in vivo or in vitro with an inactive lacZ mutant (α-acceptor) which lacks codons 11–41. This phenomenon was used to develop new cloning vectors for identifying bacterial colonies containing recombinant DNA in 1977 the lac regulatory region and the 60 first codons of the lacZ gene were inserted into the DNA of phage M13. Bacteria, producing the α-acceptor protein, when infected with the phage yield active β-galactosidase by complementation and form blue plaques in the presence of X-Gal. Insertion of foreign DNA into the α-region of M13 interferes with α-complementation, giving rise to recombinants that form colourless plaques. This simple test has made cloning in M13 a routine procedure.

Intracistronic complementation of the lacZ gene has recently been adapted for use in eukaryotic cells. Complementation of relevant lacZ mutants in mammalian cells has been shown to permit analysis of cell fusion and detection of co-localized interacting proteins within single intact cells. Intracistronic complementation of β-galactosidase has also been used for direct assessment of specific protein dimerization interactions in a biologically relevant context. See also Use of Animal Cells and Display Methods for the Production of Human Monoclonal Antibodies

Lac repressor as a tool

The capacity of Lac repressor to bind efficiently to the operator and be released from it by inducer has frequently been used to modulate gene expression, or to select homozygous negative mutants in eukaryotic cells. The modular structure of the Lac repressor allows creation of fusion proteins, including eukaryotic nuclear localization domains and transcriptional activation domains to both the N- and C-terminus of repressor without significant disruption of specific DNA binding. This property of the Lac repressor has been used to screen complex peptide libraries for direct interaction with a given receptor. The peptides fused to the C-terminus of the Lac repressor can still bind to the operator with high efficiency. This linkage allows enrichment for specific peptide ligands in the random population of peptides by affinity purification of the peptide–repressor–operator complexes with an immobilized receptor.

The commercially available Big Blue transgenic mouse mutation detection system provides a powerful approach for direct analysis of spontaneous and chemically induced mutations in vivo . The Big Blue mouse is transgenic for three genetic elements of the lactose operon: the lacI gene, the operator and the lacZ gene. The lacI gene is the target of mutagenesis. Cells with a mutated lacI gene produce defective Lac repressor, therefore β-galactosidase is synthesized and can be easily detected. Cells with unmutated lacI gene produce active repressor and no β-galactosidase is produced. This system has been widely used to study the effects of chemical carcinogens on mutation frequencies. See also Experimental Organisms Used in Genetics, and Proteins: Affinity Tags

Given the wide range of applications to which the lac operon has been associated, one can anticipate that further developments will contribute to our understanding of many features of cellular regulation and organization.


Contents

The term "operon" was first proposed in a short paper in the Proceedings of the French Academy of Science in 1960. [9] From this paper, the so-called general theory of the operon was developed. This theory suggested that in all cases, genes within an operon are negatively controlled by a repressor acting at a single operator located before the first gene. Later, it was discovered that genes could be positively regulated and also regulated at steps that follow transcription initiation. Therefore, it is not possible to talk of a general regulatory mechanism, because different operons have different mechanisms. Today, the operon is simply defined as a cluster of genes transcribed into a single mRNA molecule. Nevertheless, the development of the concept is considered a landmark event in the history of molecular biology. The first operon to be described was the lac operon in E. coli. [9] The 1965 Nobel Prize in Physiology and Medicine was awarded to François Jacob, André Michel Lwoff and Jacques Monod for their discoveries concerning the operon and virus synthesis.

Operons occur primarily in prokaryotes but also in some eukaryotes, including nematodes such as C. elegans and the fruit fly, Drosophila melanogaster. rRNA genes often exist in operons that have been found in a range of eukaryotes including chordates. An operon is made up of several structural genes arranged under a common promoter and regulated by a common operator. It is defined as a set of adjacent structural genes, plus the adjacent regulatory signals that affect transcription of the structural genes. 5 [11] The regulators of a given operon, including repressors, corepressors, and activators, are not necessarily coded for by that operon. The location and condition of the regulators, promoter, operator and structural DNA sequences can determine the effects of common mutations.

Operons are related to regulons, stimulons and modulons whereas operons contain a set of genes regulated by the same operator, regulons contain a set of genes under regulation by a single regulatory protein, and stimulons contain a set of genes under regulation by a single cell stimulus. According to its authors, the term "operon" is derived from the verb "to operate". [12]

An operon contains one or more structural genes which are generally transcribed into one polycistronic mRNA (a single mRNA molecule that codes for more than one protein). However, the definition of an operon does not require the mRNA to be polycistronic, though in practice, it usually is. [5] Upstream of the structural genes lies a promoter sequence which provides a site for RNA polymerase to bind and initiate transcription. Close to the promoter lies a section of DNA called an operator.

All the structural genes of an operon are turned ON or OFF together, due to a single promoter and operator upstream to them, but sometimes more control over the gene expression is needed. To achieve this aspect, some bacterial genes are located near together, but there is a specific promoter for each of them this is called gene clustering. Usually these genes encode proteins which will work together in the same pathway, such as a metabolic pathway. Gene clustering helps a prokaryotic cell to produce metabolic enzymes in a correct order. [13]

An operon is made up of 3 basic DNA components:

    – a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should be used for messenger RNA creation – and, by extension, control which proteins the cell produces.
  • Operator – a segment of DNA to which a repressor binds. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon. [14] The main operator (O1) in the lac operon is located slightly downstream of the promoter two additional operators, O2 and O3 are located at -82 and +412, respectively. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes. – the genes that are co-regulated by the operon.

Not always included within the operon, but important in its function is a regulatory gene, a constantly expressed gene which codes for repressor proteins. The regulatory gene does not need to be in, adjacent to, or even near the operon to control it. [15]

An inducer (small molecule) can displace a repressor (protein) from the operator site (DNA), resulting in an uninhibited operon.

Alternatively, a corepressor can bind to the repressor to allow its binding to the operator site. A good example of this type of regulation is seen for the trp operon.

Control of an operon is a type of gene regulation that enables organisms to regulate the expression of various genes depending on environmental conditions. Operon regulation can be either negative or positive by induction or repression. [14]

Negative control involves the binding of a repressor to the operator to prevent transcription.

  • In negative inducible operons, a regulatory repressor protein is normally bound to the operator, which prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to the repressor and changes its conformation so that it is unable to bind to the operator. This allows for expression of the operon. The lac operon is a negatively controlled inducible operon, where the inducer molecule is allolactose.
  • In negative repressible operons, transcription of the operon normally takes place. Repressor proteins are produced by a regulator gene, but they are unable to bind to the operator in their normal conformation. However, certain molecules called corepressors are bound by the repressor protein, causing a conformational change to the active site. The activated repressor protein binds to the operator and prevents transcription. The trp operon, involved in the synthesis of tryptophan (which itself acts as the corepressor), is a negatively controlled repressible operon.

Operons can also be positively controlled. With positive control, an activator protein stimulates transcription by binding to DNA (usually at a site other than the operator).

  • In positive inducible operons, activator proteins are normally unable to bind to the pertinent DNA. When an inducer is bound by the activator protein, it undergoes a change in conformation so that it can bind to the DNA and activate transcription.
  • In positive repressible operons, the activator proteins are normally bound to the pertinent DNA segment. However, when an inhibitor is bound by the activator, it is prevented from binding the DNA. This stops activation and transcription of the system.

The lac operon of the model bacterium Escherichia coli was the first operon to be discovered and provides a typical example of operon function. It consists of three adjacent structural genes, a promoter, a terminator, and an operator. The lac operon is regulated by several factors including the availability of glucose and lactose. It can be activated by allolactose. Lactose binds to the repressor protein and prevents it from repressing gene transcription. This is an example of the derepressible (from above: negative inducible) model. So it is a negative inducible operon induced by presence of lactose or allolactose.

Discovered in 1953 by Jacques Monod and colleagues, the trp operon in E. coli was the first repressible operon to be discovered. While the lac operon can be activated by a chemical (allolactose), the tryptophan (Trp) operon is inhibited by a chemical (tryptophan). This operon contains five structural genes: trp E, trp D, trp C, trp B, and trp A, which encodes tryptophan synthetase. It also contains a promoter which binds to RNA polymerase and an operator which blocks transcription when bound to the protein synthesized by the repressor gene (trp R) that binds to the operator. In the lac operon, lactose binds to the repressor protein and prevents it from repressing gene transcription, while in the trp operon, tryptophan binds to the repressor protein and enables it to repress gene transcription. Also unlike the lac operon, the trp operon contains a leader peptide and an attenuator sequence which allows for graded regulation. [16] This is an example of the corepressible model.

The number and organization of operons has been studied most critically in E. coli. As a result, predictions can be made based on an organism's genomic sequence.

One prediction method uses the intergenic distance between reading frames as a primary predictor of the number of operons in the genome. The separation merely changes the frame and guarantees that the read through is efficient. Longer stretches exist where operons start and stop, often up to 40–50 bases. [17]

An alternative method to predict operons is based on finding gene clusters where gene order and orientation is conserved in two or more genomes. [18]

Operon prediction is even more accurate if the functional class of the molecules is considered. Bacteria have clustered their reading frames into units, sequestered by co-involvement in protein complexes, common pathways, or shared substrates and transporters. Thus, accurate prediction would involve all of these data, a difficult task indeed.

Pascale Cossart's laboratory was the first to experimentally identify all operons of a microorganism, Listeria monocytogenes. The 517 polycistronic operons are listed in a 2009 study describing the global changes in transcription that occur in L. monocytogenes under different conditions. [19]


2.5: Gene and Operon - Biology

Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon (Figure 1). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized.

Figure 1. The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed.

The trp operon includes three important regions: the coding region, the trp operator and the trp promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just before the coding region is the transcriptional start site . The promoter sequence, to which RNA polymerase binds to initiate transcription, is before or “upstream” of the transcriptional start site. Between the promoter and the transcriptional start site is the operator region.

The trp operator contains the DNA code to which the trp repressor protein can bind. However, the repressor alone cannot bind to the operator. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes the shape of the repressor protein to a form that can bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding to the promoter and transcribing the downstream genes.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator, the polymerase can transcribe the enzyme genes, and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is said to be negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators .


Cloning and structural analysis of the full-length cytolethal distending toxin (cdt) gene operon from Campylobacter lari

Polymerase chain reaction (PCR) amplicons (approximately 2.5 kbp) encoding a cdt gene operon and two partial and putative open reading frames (ORFs) were identified in six urease-negative (UN) Campylobacter lari isolates using a new PCR primer pair constructed in silico. Three closely spaced and putative ORFs for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic p-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtA and cdtB and a TAA for cdtC. Interestingly, an overlap of four nucleotides was detected between cdtA and cdtB and the non-coding region of six base pairs occurring between cdtB and cdtC. The start codons for the three cdt genes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtA gene, six in cdtB and two in cdtC among the seven isolates (including C. lari RM2100), no polymorphic sites occurred in the putative promoters, hypothetically intrinsic transcription terminator and the three ribosome binding sites among the seven isolates. All nine amino acid residues specific for both Escherichia coli cdtB and mammalian DNase I were completely conserved in the cdtB gene locus in the 26 C. lari isolates, as well as in C. jejuni and C. coli. No PCR amplicons were generated with urease-positive thermophilic campylobacters (UPTC n=10) using the primer pair.


Arsenic Contents and Its Biotransformation in the Marine Environment

Kiran Kalia , Devang B. Khambholja , in Handbook of Arsenic Toxicology , 2015

28.6.2 Marine Bacteria

Microbe–mediated arsenic metabolism has played a major role in the ecology of arsenic, which affects the parameters of mobility and bioavailability, as well as toxicity with in the marine environment. Bacteria implicated in the processes of biotransformation readily metabolize arsenic and participate in various metabolic functions including detoxification, anaerobic respiration, methylation, and assimilation. Marine bacteria have the ability to transform inorganic arsenic species to various methylated and/or complex organic forms of arsenic [47] , and also have the capacity to decompose complex organoarsenicals into inorganic arsenic [75] . To survive with arsenic compounds in their environment, microbes have developed several metabolic pathways mediated by their genetic makeup and their products [88] . The genes involved in arsenic biotransformation are aox/aos genes (arsenite oxidation, recently named aio genes), arr genes (anaerobic arsenate respiration), and ars genes (reduction and methylation). The schematic model of arsenic biotransformation in bacterial cells is summarized in Figure 28–3 [3] . Since arsenate and arsenite may act as analogues of phosphate and glycerol, respectively, they enter the microbial cells via phosphate (Pst/Pit) transporters and glyceroporin (Glp) membrane proteins, respectively [88] . Cellular arsenite uptake was also observed via hexose transporters [90] or glucose permease GLUT1 [91] . The intracellular arsenite serves as an inducer to bind the regulatory protein ArsR (encoded by arsR gene) leading to its conformational changes and removal of previously bound regulatory protein to the operator site of the ars operon ( Figure 28–3 ). The removal of the ArsR regulatory protein leads to the transcription of ars genes. The best characterized mechanism of arsenic detoxification in bacteria involves the reduction of arsenate to arsenite, mediated by arsenate reductase encoded by the arsC gene. Arsenite is further extruded by a membrane-associated efflux pump encoded by the arsB gene or sequestered in the intracellular compartments. Other genes like arsD and arsA (encoded for ATPase) are found along with arsC and arsB in the majority of prokaryotes [92] . The arsenic tolerance mechanism is conferred by the ars operon, which is located either on plasmid or chromosome in different bacterial species. Bacteria from the genera Halomonas and Acinetobacter isolated from Mandovi and Zuari estuarine water systems in Goan territory, India, have been shown to harbor arsA, arsB, and arsC genes on their plasmid [93] . Arsenite is formed by reduction of arsenate, either excreted from the cell or methylated to form various methylated arsenicals mediated by ArsM (S-adenosylmethyltransferase) encoded by the arsM gene, which are further pumped out of the cell by an unidentified transporter.

Figure 28–3 . Arsenic biotransformation in prokaryotic cells. (A) Respiratory arsenate reductase (Arr) is involved in the reduction of As(V). (B) Arsenite oxidase (Aso/Aox) is responsible for oxidation of As(III). (C) S-adenosylmethyltransferase (ArsM) is responsible for methylation of As(III) to produce methylated arsenicals as the end product. (D) Genes (arsRDABC) and proteins involved in the uptake of As(III) and As(V) (GlpF and Pst/Pit transporter), reduction of As(V) (ArsC), extrusion of As(III) (ArsAB), regulation (ArsRD) by arsenic-resistant organisms.

Thioarsenicals, structural analogues of oxyarsenicals in which sulfur replaces oxygen, are formed by exposure of oxyarsenicals to hydrogen sulfide [94] . Arsenic species reported to form thio species include MMA, DMA, arsenosugars, dimethylarsinoyl ethanol, and dimethylarsinoyl acetate. These species are thought to be formed in the presence of hydrogen sulfide during anaerobic degradation of seaweed [65] . Studies have shown that anaerobic microflora from human feces or mouse cecum and gastrointestinal tracts of marine organisms may convert DMA(V) into thiolated arsenic compounds such as dimethylthioarsenate and trimethylarsine sulfide [95–97] . Marine sediments and hydrothermal waters are rich in sulfur, where the reducing conditions and relatively high pH may favor the formation of thiomethylated arsenicals by anaerobic sedimentary bacteria. So far none of the bacterial species in the marine environment have been identified for their ability to synthesize thiomethylated arsenicals, either from inorganic arsenic or from methylated forms.

Oxidation of arsenite to arsenate mediated by arsenite oxidase, encoded by aox/aos/aoi genes, was observed in bacteria and archaeans as the detoxification mechanism. The toxicity of arsenic depends on its oxidation state, while arsenite is 100 times more toxic than arsenate in most biological systems [98] . Acinetobacter junii SeaH-As6s and Marinobacter sp. SeaH-As6w, isolated from coastal sediment and sea water, respectively, from Gwangyang Bay, Republic of Korea, were found to oxidize arsenite to arsenate.

Apart from the operon-mediated detoxification mechanism, Bacillus sp. strain XZM002 has shown an additional arsenic tolerance mechanism by changing its shape to reduce its surface area to survive in the presence of high arsenic concentrations in its environment. The initial rod shape was altered to oval and then to circular form with gradual increase in extracellular arsenic concentration. The circular form will have the least surface area for arsenic uptake and thus will exhibit less toxicity [99] .

Marine bacteria have the main role in the biogeochemical arsenic cycle of decomposing complex organoarsenicals into their simple organic forms or even into inorganic arsenic forms [75,100] . Two bacterial strains of the group Vibrio/Aeromonas were isolated from coastal sediments, and efficiently decomposed arsenobetaine in aerobic conditions to dimethylarsinic acid. The addition of sediment itself in the media, as the source of arsenobetaine-decomposing microorganisms, has shown the decomposition pattern of arsenobetaine to trimethylarsine to inorganic arsenic [75] . The oxidation and demethylation of methylated arsenicals by bacteria has been studied in marine waters [101] .

Arsenic mobility in the environment was significantly enhanced by bacterial cell-mediated arsenate reduction. Two different pathways for arsenate reduction were observed in microorganisms encoded by ars and arr systems. The arsenate reductase encoded by ars genes encodes cytoplasmic arsenate reductase associated with a detoxification mechanism, whereas membrane/periplasmic arsenate reductase encoded by arr genes were associated with cellular respiration [102–104] .


Operon

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Operon, genetic regulatory system found in bacteria and their viruses in which genes coding for functionally related proteins are clustered along the DNA. This feature allows protein synthesis to be controlled coordinately in response to the needs of the cell. By providing the means to produce proteins only when and where they are required, the operon allows the cell to conserve energy (which is an important part of an organism’s life strategy).

A typical operon consists of a group of structural genes that code for enzymes involved in a metabolic pathway, such as the biosynthesis of an amino acid. These genes are located contiguously on a stretch of DNA and are under the control of one promoter (a short segment of DNA to which the RNA polymerase binds to initiate transcription). A single unit of messenger RNA (mRNA) is transcribed from the operon and is subsequently translated into separate proteins.

The promoter is controlled by various regulatory elements that respond to environmental cues. One common method of regulation is carried out by a regulator protein that binds to the operator region, which is another short segment of DNA found between the promoter and the structural genes. The regulator protein can either block transcription, in which case it is referred to as a repressor protein or as an activator protein it can stimulate transcription. Further regulation occurs in some operons: a molecule called an inducer can bind to the repressor, inactivating it or a repressor may not be able to bind to the operator unless it is bound to another molecule, the corepressor. Some operons are under attenuator control, in which transcription is initiated but is halted before the mRNA is transcribed. This introductory region of the mRNA is called the leader sequence it includes the attenuator region, which can fold back on itself, forming a stem-and-loop structure that blocks the RNA polymerase from advancing along the DNA.

The operon theory was first proposed by the French microbiologists François Jacob and Jacques Monod in the early 1960s. In their classic paper they described the regulatory mechanism of the lac operon of Escherichia coli, a system that allows the bacterium to repress the production of enzymes involved in lactose metabolism when lactose is not available.